Note: Descriptions are shown in the official language in which they were submitted.
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COMPOUNDS AND METHODS FOR REGULATING APOPTOSIS, AND METHODS
OF MAKING AND SCREENING FOR COMPOUNDS THAT REGULATE
APOPTOSIS
FIELD OF THE INVENTION
The present invention relates generally to the field of cell physiology, and
more
particularly, to apoptosis or programmed cell death, a process whereby
developmental or
environmental stimuli activate a genetically programmed cascade of events that
results in cell
death. Specifically, the invention relates to the regula~on of apoptosis,
including the
regulation of apoptosis resulting in cell survival, compounds therefor, and
methods of making
.and screening for such compounds. More specifically, the invention relates to
mutants of
Bcl-XLBcI-2 Associated Cell Death Regulator polypeptides ("BAD"), methods of
making
such mutants, and methods of screening for compounds that promote, induce,
inhibit, or
modulate apoptosis, or promote, induce, inhibit, or modulate cell survival.
BACKGROUND OF THE INVENTION
Throughout the life of an organism, there is a constant progression of
cellular
development. New cells are continually being produced, through such mechanisms
as mitosis
and meiosis, to replace old or damaged cells that are targeted for destruction
due to disease,
injury, extracellular cues and internal instructions. This cellular life-death
balance is a critical
feature not only in normal animal development, but also in pathogenesis.
Indeed, diseases
such as cancer and autoimmune disorders are associated with decreased cell
death, whereas
AIDS and neurodegenerative disorders are associated with increased cell death
(Thompson,
1995).
Programmed cell death, or apoptosis, is one manner in which cells that are no
longer
needed or that no longer function normally can be eliminated. Apoptosis is
believed to be a
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process actively regulated by the environment in which cells live. This
process is critical to
the normal development of all multicellular organisms and to the maintenance
of homeostasis
within such organisms (Raff, 1992). Moreover, apoptosis is vital in the
defense against viral
infection and in preventing the development of carcinogenesis. Amongst
multicellular
organisms, the apoptotic pathway leading to cell death is highly conserved
(Hengartner,
1994).
Many molecules that regulate the apoptotic pathway have been identified,
including
both positive regulators (agonists) and negative regulators (antagonists).
Such regulators are
often members of the same family of polypeptides, and can have roles important
in the
extracellular, cell surface, and/or intracellular steps of the apoptotic
pathway (Oltvai and
Korsmeyer, 1994). One such family of polypeptides, constituting an
intracellular checkpoint
in the apoptotic pathway, is the Bcl-2 family of polypeptides ("Bcl-2
family"). This
important family of apoptotic regulators can be divided into two classes:
those that suppress
cell death (apoptotic antagonists) (e.g., Bcl-2, Bcl-XL, MCL-1, and A1) and
those that appear
to promote apoptosis (apoptotic agonists) (e.g., BAX, BAK, Bcl-Xs, and BAD).
The first
member of the Bcl-2 family to be identified was Bcl-2, a cell death inhibitor
encoded by the
bcl-2 proto-oncogene, initially isolated from cells of a follicular lymphoma
(Bakhshi et al.,
1985; Tsujimoto et al., 1985; Cleary and Sklar, 1985). Bcl-2 is a 26 kD
integral membrane
polypeptide localized to the mitochondria that extends or promotes the
survival of many
different cell types by inhibiting apoptosis induced by a variety of cell
death-inducing stimuli
(Korsmeyer, 1992).
The Bcl-2 family contains members that are structurally and functionally
related to
Bcl-2, and is defined by polypeptides having amino acid sequence homology to
one or more
of four conserved motifs, termed Bcl-homology (BH1, BH2, BH3, and BH4) domains
(for
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reviews see Reed, 1997 and Chittenden, 1998). The Bcl-homology domains have
been shown
to be important in the formation of homodimers and heterodimers, both among
and between
Bcl-2 family members.
The functional characteristics of these proteins vary, depending on their
dimerization
partners (Yin et al., 1994; Boyd et al., 1995; Chittenden et al., 1995; Farrow
and Brown,
1996). The dimerization status of the proteins has been shown to depend on the
intracellular
concentrations of the particular family members, and is directly related to
whether a cell will
respond to an apoptotic signal (Oltvai and Korsmeyer, 1994). Moreover, the
formation of
dimers between cell death promoters and cell death inhibitors is competitive.
For example,
both Bcl-2 and Bcl-XL enhance cellular survival, while BAD and BAX promote
cell death
(Oltvai et al., 1993). However, when BAD is overexpressed, it counters the
death inhibitory
activity of Bcl-XL, and to a lesser extent Bcl-2, by the formation of
heterodimers. It is
thought that BAD competes with BAX for binding to Bcl-XL. Therefore, when the
intracellular levels of BAD increase, there is a sequestration of Bcl-XL in
BAD:BcI-XL
1 S heterodimers, and a concomitant increase in the amount of free BAX present
in the cell as
BAX monomers or BAX:BAX homodimers. The increased levels of BAX monomer and
homodimers, in turn, promote cellular susceptibility to apoptosis (Korsmeyer,
U.S. Patent No.
5,856,445).
BAD (Bcl-X~/Bcl-2 Associated Cell Death Regulator polypeptide) is a cell death
promoter distantly related to Bcl-2. It has been sequenced and shown to share
identity with
Bcl-2 only within the BH3 domain. BAD is an unique pro-apoptotic member of the
Bcl-2
family in that its function is regulated by phosphorylation (Yang et al.,
1995), suggesting an
important connection between extracellular apoptosis regulatory agents,
intracellular
signaling pathways and the function of this Bcl-2 family member. As discussed
above, BAD
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is believed to play a role in the apoptotic signaling pathway through an
association with Bcl-2
family members, chiefly the cell death inhibitors Bcl-X~ and Bcl-2 (Yang et
al., 1995). Upon
being dephosphorylated, BAD is active and forms heterodimers with Bcl-XL and
Bcl-2,
thereby displacing BAX and promoting cell death. The death-promoting activity
of BAD can
S be inhibited by the phosphorylation of either of two serine residues,
corresponding to the
serines at position 112 and position 136 in the amino acid sequence of murine
BAD (SEQ ID
N0:2). Upon phosphorylation of either of these two sites, BAD no longer binds
Bcl-X~ or
Bcl-2 and instead, is thought to be bound by the phosphoserine-binding protein
14-3-3,
thereby allowing Bcl-X~ and Bcl-2 to perform their anti-apoptotic functions
(Zha et al.,
1996).
The interaction of murine BAD and the cytosol polypeptide 14-3-3 was
discovered
using a GST-BAD fusion polypeptide having a heart muscle kinase (HMK) motif.
The fusion
polypeptide was labeled with y32P-ATP in vitro and used as a probe to screen
an oligo (dT)-
primed day-16 mouse embryonic EXlox cDNA expression library (Blanar and
Rutter, 1992).
Thereby two independent clones, each encoding a polypeptide of the tau form
(i) of 14-3-3
that bound to the BAD fusion polypeptide, were isolated (Nielsen, 1991).
The members of the 14-3-3 family, identified in at least seven mammalian
isoforms,
are highly conserved and ubiquitously expressed. They bind to and regulate a
variety of
proteins, including a number of proteins involved in signal transduction.
Family members
recognize and bind sequences containing a conserved phosphoserine motif
(Muslin et al.,
1996).
Muslin et al. identified a number of polypeptides, including BAD, that contain
this
motif and postulated that if these other polypeptides were appropriately
phosphorylated, 14-3-
3 would bind them as well. However, they did not perform any experiments to
determine
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whether 14-3-3 in fact binds phosphorylated BAD, nor did they discuss possible
physiological consequences of such binding. Moreover, there was no discussion
of other
potential BAD serine phosphorylation sites. Only the general suggestion that
14-3-3 might
interact with polypeptides to perform an essential chaperone function was
advanced.
Korsmeyer also suggested a likely role for 14-3-3 as a chaperone or protective
binding
polypeptide. In the case of BAD, it was suggested that 14-3-3 might facilitate
the
translocation of phosphorylated BAD from the mitochondrial membrane to
cytosolic
compartments, sequestering it therein (U.S. Pat. No. 5,856,445).
Thus far, BAD is the only known pro-apoptotic member of the Bcl-2 family whose
function is regulated by phosphorylation. The serine/threonine kinase Akt, a
downstream
effector of PI 3-kinase, phosphorylates murine BAD on the serine at position
136 (also called
"Ser136" or "serine-136"), thereby preventing murine BAD from associating with
Bcl-2 or
Bcl-XL, and freeing these proteins to promote cell survival. (Datta et al.,
1997; del Peso et
al., 1997).
In addition, murine BAD is phosphorylated at the serine at position 112 (also
called
"Ser112" or "serine-112"). Although phosphorylation of Ser136 was sufficient
to prevent
BAD from binding to Bcl-XL, phosphorylation of Ser112 appeared critical for
cellular
survival in some cell types but not in others (Zha et al., 1996; Datta et al.,
1997). Several
candidate enzymes have been proposed to be responsible for the phosphorylation
of Ser112,
including PKA, c-Raf, and MEK (Harada et al., 1999; Wang and Reed, 1998;
Scheid and
Duronio, 1998). However, the discoveries of the present invention indicate
that Serl 12 is, at
best, a minor site of phosphorylation by PKA. Similarly, Akt does not appear
to
phosphorylate BAD on Serl 12.
S
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Some disease conditions may be related to the development of a defective down-
regulation (i.e., inhibition or modulation) of apoptosis in the affected
cells. For example,
neoplasias may result, at least in part, from an apoptosis-resistant state in
which cell
proliferation signals inappropriately exceed cell death signals and apoptosis
is thereby down-
s regulated. Furthermore, some DNA viruses such as Epstein-Barr virus, African
swine fever
virus and adenovirus, parasitize the host's cellular machinery to drive their
own replication
and at the same time inhibit or modulate apoptosis, thereby repressing cell
death and allowing
the host cell to continue reproducing the virus. Moreover, certain other
disease conditions
such as lymphoproliferative conditions, arthritis, inflammation, autoimmune
diseases, and
cancers, including drug-resistant cancers, may result from a down-regulation
(e.g., inhibition
or modulation) of apoptosis. In such disease conditions it would be desirable
to promote or
induce apoptosis. By manipulating members of the signal transduction cascade
that trigger
apoptosis, one could selectively induce apoptosis. For example, BAD could be
altered to
promote or induce its binding to Bcl-XL and/or Bcl-2 and, thereby, diminish
(or inhibit or
1 S modulate) the cell-survival promoting activity of these cell death
inhibitors.
Conversely, in certain disease conditions it would be desirable to inhibit or
modulate
apoptosis, for example, in the treatment of immunodeficiency diseases,
including AIDS,
senescence, neurodegenerative disease, ischemic and reperfusion cell death,
infertility, and
wound-healing. In such cases, BAD could be altered to block its ability to
bind Bcl-XL and/or
Bcl-2 and, thereby, promote or induce the cell death repressor, or anti-
apoptotic, activity of
these cell death inhibitors.
Accordingly, it would be desirable to identify novel compositions and methods
that
could be used to modulate the binding of BAD to members of the Bcl-2 family,
such as Bcl-
X,_, and Bcl-2, and thereby induce, promote, inhibit or modulate apoptosis, or
induce,
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promote, inhibit, or modulate cell survival, and to utilize these novel
compositions and
methods as a basis for treatment of disease conditions involving either
inappropriate
inhibition or inappropriate acceleration of cell death.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a novel phosphorylation site
of BAD
that is regulated differentially from other known phosphorylation events of
BAD. The novel
phosphorylation site is at the serine at position 155 (also called "Ser155" or
"serine-155") of
SEQ ID N0:2 of a murine BAD ("longer murine BAD") that corresponds to the
serine at
position 118 (also called "Ser118" and "serine-118") of SEQ ID NO:1 of a human
BAD, and
the serine at position 113 (also called "Serl 13" or Serine-113") of SEQ ID
N0:3 of a murine
BAD ("shorter murine BAD"). A particularly significant aspect of the present
invention
relates to the discovery that the phosphorylation of BAD at the novel
phosphorylation site
renders BAD unable to bind to Bcl-X~, and promotes, induces, or modulates cell
survival
(and/or inhibits or modulates apoptosis). Moreover, the phosphorylation of BAD
at the novel
site is not dependent on the activation of PI 3-kinase and Akt. Rather, the
phosphorylation of
BAD at the novel phosphorylation site is regulated by the cAMP-dependent
protein kinase
("PKA"). For example, the phosphorylation of endogenous BAD at Ser155 in Rat-1
fibroblasts and several tumor cell lines, such as A275 and A431, can be
induced by growth
factors at physiological levels in a PKA-dependent manner, but not in a PI 3-
kinase dependent
and Akt-dependent manner. Consistent with these findings, cells treated with L-
epinephrine, a
G-protein-coupled receptor ligand that induces elevation of intracellular cAMP
levels, exhibit
phosphorylation of BAD at Ser155. In addition, the present invention relates
to the discovery
that, both in vitro and in vivo, Ser155 is the only major site of BAD
phosphorylation by PKA,
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and that the phosphorylation of BAD at Ser155 contributes significantly to the
overall
phosphorylation of BAD.
The present invention further relates to the discovery that BAD, having a
mutation
wherein Ser155 is changed to an alanine, promotes, agonizes, induces or
modulates apoptotic
S activity in HeLa cells, as compared to the naturally-occurring or wild-type
mammalian BAD.
Similarly, the elevation of the level of intracellular cAMP promotes, induces,
or modulates
cell survival (and/or inhibits or modulates apoptosis) in HeLa cells
expressing the naturally-
occurring or wild-type mammalian BAD, but not in HeLa cells expressing the BAD
having a
mutation at Ser155. In contrast, the change of serine 155 to an aspartic acid,
which mimics
the phospho-Ser155 BAD, showed no apoptotic activity when expressed in cells.
Accordingly, the object of the present invention is to provide compositions
and
methods that regulate apoptosis and/or cell survival (apoptotic agonists and
antagonists), for
example, by promoting, inducing, inhibiting, or modulating apoptosis, or
promoting,
inducing, inhibiting, or modulating cell survival, and to provide methods of
making and
screening for such compositions.
More specifically, an object of the present invention is to provide novel
compounds
and methods that can alter the phosphorylation of BAD and/or binding of BAD to
members
of the Bcl-2 family, such as Bcl-XL and Bcl-2, and thereby regulate apoptosis
and/or cell
survival, for example, by promoting, inducing, inhibiting, or modulating
apoptosis, or
promoting, inducing, inhibiting, or modulating cell survival, and to provide
methods of
making and screening for such compounds. Another object of the present
invention is to
provide methods that utilize such novel compounds as a basis for treatment of
disease
conditions involving either inappropriate inhibition or inappropriate
acceleration of cell death.
Such novel compounds include, for example, polypeptides and polynucleotides,
fragments of
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full-length polypeptides and polynucleotides, including mutants and homologs
thereof, and
chemical compounds, including peptide mimetics.
An embodiment of the present invention provides isolated or synthetic
polypeptides
comprising an amino acid sequence of a mutant BAD, or fragments of said
isolated or
synthetic polypeptides comprising a less than full-length amino acid sequence
of a mutant
BAD, having cell death promoting activity. The amino acid sequence of the
isolated or
synthetic polypeptides of a mutant BAD, or said fragments, wherein the amino
acid sequence
of said mutant BAD, or said fragment, is identical to or substantially
identical to either SEQ
ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, contains a domain that is substantially
identical to
a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not
contain a
serine at a position corresponding to position 118 of SEQ ID NO:1, position
155 of SEQ ID
N0:2, or position 113 of SEQ ID N0:3. The position corresponding to position
118 of SEQ
ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is
determined by
alignment of the amino acid sequence of the isolated or synthetic polypeptides
of the mutant
BAD, or said fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3,
respectively.
More particularly, an embodiment of the present invention provides isolated or
synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or
fragments of
an isolated or synthetic polypeptide comprising a less than full-length amino
acid sequence of
a mutant BAD, having cell death promoting activity, and/or having the ability
to bind Bcl-XL
and/or Bcl-2. The binding of said isolated or synthetic polypeptides
comprising an amino
acid sequence of a mutant BAD, or said fragments, may occur, for example,
through a domain
that is substantially identical to a BH3 domain of a naturally-occurnng or
wild-type
mammalian BAD. The amino acid sequence of the isolated or synthetic
polypeptides of a
mutant BAD, or said fragments, is derived from a naturally-occurring or wild-
type
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mammalian BAD, contains a domain that is substantially identical to a BH3
domain of a
naturally-occurring or wild-type mammalian BAD, and does not contain a serine
at a position
corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or
position
113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by alignment
of the
amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD,
or said
fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
An embodiment of the present invention provides isolated or synthetic
polypeptides
comprising an amino acid sequence of a mutant BAD, or fragments of an isolated
or synthetic
polypeptide comprising a less than full-length amino acid sequence of a mutant
BAD, having
cell death promoting activity. The amino acid sequence of the isolated or
synthetic
polypeptides of a mutant BAD, or said fragments, is derived from a naturally-
occurring or
wild-type mammalian BAD, contains a domain that is substantially identical to
a BH3 domain
of a naturally-occurring or wild-type mammalian BAD, and contains an alanine
at a position
corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or
position
113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by alignment
of the
amino acid sequence of the isolated or synthetic polypeptides of the mutant
BAD, or said
fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
An embodiment of the present invention provides isolated or synthetic
polypeptides
comprising an amino acid sequence of a mutant BAD, or fragments of an isolated
or synthetic
polypeptide comprising a less than full-length amino acid sequence of a mutant
BAD, having
cell death promoting activity. The amino acid sequence of the isolated or
synthetic
polypeptides of a mutant BAD, or said fragments, is derived from a naturally-
occurring or
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wild-type mammalian BAD, contains a domain that is substantially identical to
a BH3 domain
of a naturally-occurring or wild-type mammalian BAD, and does not contain a
glycine at a
position corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID
N0:2, or
position 113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ
ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by
alignment
of the amino acid sequence of the isolated or synthetic polypeptides of the
mutant BAD, or
said fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
An embodiment of the present invention provides isolated or synthetic
polypeptides
comprising an amino acid sequence of a mutant BAD, or fragments of an isolated
or synthetic
polypeptide comprising a less than full-length amino acid sequence of a mutant
BAD, having
cell death promoting activity. The amino acid sequence of the isolated or
synthetic
polypeptides of a mutant BAD, or said fragments, is derived from a naturally-
occurnng or
wild-type mammalian BAD, contains a domain that is substantially identical to
a BH3 domain
of a naturally-occurnng or wild-type mammalian BAD, and does not contain an
alanine at a
position corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID
N0:2, or
position 113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ
ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by
alignment
of the amino acid sequence of the isolated or synthetic polypeptides of a
mutant BAD, or said
fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
More particularly, an embodiment of the present invention provides isolated or
synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or
fragments of
an isolated or synthetic polypeptide comprising a less than full-length amino
acid sequence of
a mutant BAD, having cell death promoting activity. The amino acid sequence of
the isolated
or synthetic polypeptides of a mutant BAD, or said fragments, comprises the
amino acid
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sequence corresponding to positions 103-123 of SEQ ID NO:1, positions 140-160
of SEQ ID
N0:2, or positions 98-118 of SEQ ID N0:3, contains a domain that is
substantially identical
to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does
not
contain a serine at a position corresponding to position 118 of SEQ ID NO:1,
position 155 of
SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position corresponding to
position 118
of SEQ ID NO: l, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3
is
determined by alignment of the amino acid sequence of the isolated or
synthetic polypeptides
of the mutant BAD, or said fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID
N0:3,
respectively.
An embodiment of the present invention provides isolated or synthetic
polypeptides
comprising an amino acid sequence of a mutant BAD, or fragments of an isolated
or synthetic
polypeptide comprising a less than full-length amino acid sequence of a mutant
BAD, having
cell death promoting activity. The amino acid sequence of the isolated or
synthetic
polypeptides of a mutant BAD, or said fragments, contains a domain that is
substantially
identical to a BH3 domain of a naturally-occurnng or wild-type mammalian BAD,
wherein
the amino acid sequence of the naturally-occurnng or wild-type mammalian BAD
is SEQ ID
NO:1, SEQ ID N0:2, or SEQ ID N0:3, and does not contain a serine at a position
corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or
position
113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by alignment
of the
amino acid sequence of the isolated or synthetic polypeptides of the mutant
BAD, or said
fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
Another embodiment of the present invention provides methods for making
polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-
occurring or
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wild-type mammalian BAD, or said fragments comprising a less than full-length
amino acid
sequence of a naturally-occurring or wild-type mammalian BAD. The methods
comprise first
selecting an amino acid sequence of a naturally-occurring or wild-type
mammalian BAD, or
selecting a less than full-length amino acid sequence of a naturally-occurnng
or wild-type
mammalian BAD, comprising a BH3 domain substantially identical to the BH3
domain
encoded by the amino acids at positions 114-122 of SEQ ID NO:1, positions 151-
159 of SEQ
ID N0:2, or positions 109-117 of SEQ ID N0:3. The BH3 domain of the naturally-
occurnng
or wild-type mammalian BAD, or the BH3 domain of said fragment, is identified
by
alignment of the amino acid sequence of the naturally-occurring or wild-type
mammalian
BAD, or the amino acid sequence of said fragment, to SEQ ID NO:1, SEQ ID N0:2,
or SEQ
ID N0:3, respectively. Second, the amino acid of the amino acid sequence of
the naturally-
occurring or wild-type mammalian BAD, or said fragment, at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3 is changed to an amino acid other than serine. Thereby, polypeptides of a
mutant BAD,
or said fragments, comprising the amino acid sequence of a naturally-occurring
or wild-type
mammalian BAD having a mutation of the amino acid corresponding to position
118 of SEQ
ID NO: l, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3, are
made.
In particular, an embodiment of the present invention provides methods for
making
polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-
occurnng or
_ wild-type mammalian BAD, or said fragments comprising a less than full-
length amino acid
sequence of a naturally-occurnng or wild-type mammalian BAD. The methods
comprise first
selecting an amino acid sequence of a naturally-occurring or wild-type
mammalian BAD, or
selecting a less than full-length amino acid sequence of a naturally-occurring
or wild-type
mammalian BAD, comprising a BH3 domain substantially identical to the BH3
domain
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encoded by the amino acids at positions 114-122 of SEQ ID NO:1, positions 151-
159 of SEQ
ID N0:2, or positions 109-117 of SEQ ID N0:3. The BH3 domain of the naturally-
occurring
or wild-type mammalian BAD, or the BH3 domain of said fragment, is identified
by
alignment of the amino acid sequence of the naturally-occurnng or wild-type
mammalian
S BAD, or the amino acid sequence of said fragment, to SEQ ID NO:1, SEQ ID
N0:2, or SEQ
ID N0:3, respectively. Second, the amino acid of the amino acid sequence of
the naturally-
occurring or wild-type mammalian BAD, or said fragment, at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3 is changed to alanine.
Also in particular, an embodiment of the present invention provides methods
for
making polypeptides of a mutant BAD comprising an amino acid sequence of the
naturally-
occurring or wild-type mammalian BAD encoded by SEQ ID NO:1, SEQ ID N0:2, or
SEQ
ID N0:3, or said fragments comprising a less than full-length amino acid
sequence of the
naturally-occurnng or wild-type mammalian BAD encoded by SEQ ID NO:1, SEQ ID
N0:2,
or SEQ ID N0:3. The methods comprise first selecting an amino acid sequence of
a
naturally-occurnng or wild-type mammalian BAD, or selecting a less than full-
length amino
acid sequence of a naturally-occurring or wild-type mammalian BAD, comprising
a BH3
domain substantially identical to the BH3 domain encoded by the amino acids at
positions
114-122 of SEQ ID NO:1, positions 151-159 of SEQ ID N0:2, or positions 109-117
of SEQ
ID N0:3. The BH3 domain of the naturally-occurnng or wild-type mammalian BAD,
or the
BH3 domain of said fragment, is identified by alignment of the amino acid
sequence of the
naturally-occurring or wild-type mammalian BAD, or the amino acid sequence of
said
fragment, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. Second,
the amino
acid of the amino acid sequence of the naturally-occurring or wild-type
mammalian BAD, or
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said fragment, at a position corresponding to position 118 of SEQ ID NO:1,
position 155 of
SEQ ID N0:2, or position 113 of SEQ ID N0:3 is changed to an amino acid other
than
serene.
Another embodiment of the present invention provides methods for making
polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-
occurring or
wild-type mammalian BAD, or fragments comprising a less than full-length amino
acid
sequence of a naturally-occurnng or wild-type mammalian BAD. The methods
comprise first
selecting an amino acid sequence of a naturally-occurnng or wild-type
mammalian BAD, or
selecting a less than full-length amino acid sequence of a naturally-occurring
or wild-type
mammalian BAD, comprising a BH3 domain substantially identical to the BH3
domain
encoded by the amino acids at positions 114-122 of SEQ ID NO:1, positions 151-
159 of SEQ
ID N0:2, or positions 109-117 of SEQ ID N0:3. The BH3 domain of the naturally-
occurring
' or wild-type mammalian BAD, or the BH3 domain of said fragment, is
identified by
alignment of the amino acid sequence of the naturally-occurnng or wild-type
mammalian
BAD, or the amino acid sequence of said fragment, to SEQ ID NO:1, SEQ ID N0:2,
or SEQ
ID N0:3, respectively. Second the amino acid of the amino acid sequence of the
naturally-
occurring or wild-type mammalian BAD, or said fragment, at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3 is changed to an amino acid other than serine. Third, the polypeptide of
the mutant
BAD, or said fragment, is expressed in a host cell, wherein the host cell is
transformed with a
polynucleotide comprising the amino acid sequence of the mutant BAD, or
comprising the
amino acid sequence of said fragment, respectively.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes apoptosis. The methods comprise
first contacting a
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candidate drug with a sample comprising a mammalian BAD, or fragment of a
mammalian
BAD, and a kinase, to form a reacted fraction. The mammalian BAD, or said
fragment,
comprises an amino acid sequence containing a serine at a position
corresponding to position
118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID
N0:3. The
position of the serine is identified by alignment of the amino acid sequence
of the mammalian
BAD, or said fragment, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3,
respectively. The
kinase has phosphorylation activity capable of phosphorylating the mammalian
BAD.
Second, the reacted fraction is compared to a control fraction to~determine
whether the
candidate drug inhibits the phosphorylation activity of the kinase and,
thereby, has activity
that promotes apoptosis, by assaying for the amount of the mammalian BAD, or
said
fragment, that is unphosphorylated at the identified serine in the reacted
fraction as compared
to a control fraction. Alternatively, the reacted fraction is compared to a
control fraction to
determine whether the candidate drug inhibits the phosphorylation activity of
the kinase and,
thereby, has activity that promotes apoptosis, by assaying the isolated
fraction as compared to
a control fraction, for an amount of the mammalian BAD, or said fragment, that
is bound to
Bcl-X~ and/or Bcl-2. Preferably, treatment of the control fraction is
essentially identical to
that of the reacted fraction, except the mammalian BAD, or said fragment, in
the control
fraction is not contacted with the candidate drug.
Another embodiment of the present invention provides methods of inducing
apoptosis
in a cell expressing a mammalian BAD, or fragment of a mammalian BAD. The
methods
comprise first preparing a culture containing a cell line expressing the
mammalian BAD, or
said fragment. The mammalian BAD, or said fragment, comprises an amino acid
sequence
containing a serine at a position corresponding to position 118 of SEQ ID
NO:1, position 155
of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of the serine is
identified by
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alignment of the amino acid sequence of the mammalian BAD, or said fragment,
to SEQ ID
NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. Second, the cultured cells
are contacted
with an extracellular agent, and/or an intracellular agent is induced in the
cultured cells, to
form a reacted fraction, wherein the extracellular and/or the intracellular
agent is capable of
inhibiting the phosphorylation activity of a kinase in the cell that is
capable of
phosphorylating the serine at position 118 of SEQ ID NO:1, position 155 of SEQ
ID N0:2, or
position 113 of SEQ ID N0:3. An example of such an extracellular or
intracellular agent is
inhibitor H89, wherein H89 inhibits the phosphorylation activity of an
intracellular kinase.
Kinase inhibition can also be achieved, for example, by the binding of a
polypeptide or a
polynucleotide to the kinase, thereby inhibiting the phosphorylation activity
of the kinase, or
by the binding of a polypeptide or polynucleotide to a polynucleotide that
encodes the kinase,
thereby preventing the expression of the kinase. An example of such a kinase
is PKA, the
cyclic AMP (cAMP)-dependent protein kinase. Alternatively, the cultured cells
are contacted
with an extracellular agent, and/or an intracellular agent is induced in the
cultured cells,
wherein the extracellular and/or intracellular agent is capable of activating
the phosphatase
activity of a phosphatase in the cell that is capable of dephosphorylating the
mammalian
BAD, or said fragment, that is phosphorylated at the serine corresponding to
position 118 of
SEQ ID NO: l, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3.
Third,
treated cells in the reacted fraction are compared with untreated control
cells to determine
whether apoptosis is induced in the treated cells by assaying for the amount
of the mammalian
BAD, or said fragment, that is unphosphorylated and/or dephosphorylated in
each.
Alternatively, the treated cells in the reacted fraction are compared to
untreated control cells
to determine whether apoptosis is induced in the treated cells at a higher
level by monitoring
indicia of apoptosis in each. Preferably, treatment of the control cells is
essentially identical
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to that of the treated cells, except the control cells are not contacted with
an extracellular
agent and/or an intracellular agent is not induced in the control cells.
Another embodiment of the present invention provides methods of assaying a
candidate compound for phosphatase activity capable of dephosphorylating a
mammalian
BAD, or fragment of a mammalian BAD, at a serine at a position in the amino
acid sequence
of the mammalian BAD, or said fragment, corresponding to position 118 of SEQ
ID NO:1,
position 1 SS of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of
the serine is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. The methods
comprise first
contacting the candidate compound with the mammalian BAD, or said fragment, to
form a
reacted fraction, wherein the mammalian BAD, or said fragment, is de-
phosphorylated at the
specified serine. Second, the reacted fraction is compared to a control
fraction to determine
whether the candidate compound has phosphatase activity by assaying for the
amount of the
mammalian BAD, or said fragment, that is bound to Bcl-XL and/or Bcl-2 in the
reacted
fraction as compared to a control fraction. Preferably, treatment of the
control fraction is
essentially identical to that of the reacted fraction, except the control
fraction is not contacted
with the candidate compound.
Another embodiment of the present invention provides methods of assaying a
candidate compound for phosphatase activity capable of dephosphorylating a
mammalian
BAD, or fragment of a mammalian BAD, at a serine at a position in the amino
acid sequence
of the mammalian BAD, or said fragment, corresponding to position 118 of SEQ
ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of
the serine is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. The methods
comprise first
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contacting the candidate compound with the mammalian BAD, or said fragment, to
form a
reacted fraction, wherein the mammalian BAD, or said fragment, is de-
phosphorylated at the
specified serine. Second, the reacted fraction is compared to a control
fraction to determine
whether the candidate compound has phosphatase activity by assaying for an
amount of the
mammalian BAD, or said fragment, that is dephosphorylated at the serine in the
reacted
fraction as compared to the control fraction. Preferably, treatment of the
control fraction is
essentially identical to that of the reacted fraction, except the control
fraction is not contacted
with the candidate compound.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes cell survival. The methods comprise
first contacting
the candidate drug with a mammalian BAD, or fragment of a mammalian BAD, and,
optionally, a kinase, to form a reacted fraction. The mammalian BAD, or said
fragment, is
capable of being phosphorylated at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of the serine
is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:1, SEQ ID N0:2 and SEQ ID N0:3, respectively. Second, the reacted
fraction
is compared to a control fraction to determine whether the candidate drug has
activity that
promotes cell survival by assaying for the amount of the mammalian BAD, or
said fragment,
that is phosphorylated at the serine in the reacted fraction as compared to
the control fraction.
Preferably, treatment of the control fraction is essentially identical to that
of the reacted
fraction, except the control fraction is not contacted with the candidate
drug.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes cell survival. The methods comprise
first contacting
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the candidate drug with a mammalian BAD, or fragment of a mammalian BAD, and,
optionally, a kinase, to form a reacted fraction. The mammalian BAD, or said
fragment, is
capable of being phosphorylated at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of the serine
is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:1, SEQ ID N0:2 and SEQ ID N0:3, respectively. Second, the reacted
fraction
is contacted with Bcl-X~ and/or Bcl-2. Third, the reacted fraction is compared
to a control
fraction to determine whether the candidate drug has activity that promotes
cell survival by
assaying for the amount of the mammalian BAD bound to Bcl-XL and/or Bcl-2, or
the amount
of said fragment bound to Bcl-X~ and/or Bcl-2. Alternatively, the reacted
fraction is
compared to a control fraction to determine whether the candidate drug has
activity that
promotes cell survival by assaying for the amount of the mammalian BAD, or
said fragment,
that is phosphorylated at the specified serine in the reacted fraction as
compared to the control
fraction. Preferably, treatment of the control fraction is essentially
identical to that of the
reacted fraction, except that the control fraction is not contacted with the
candidate drug.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes cell survival. The methods comprise
first preparing
a cell culture containing a cell line expressing a mammalian BAD, or fragment
of a
mammalian BAD, wherein the cell line has activity that promotes apoptosis, or
is capable of
having activity that promotes apoptosis. The mammalian BAD, or said fragment,
comprises
an amino acid sequence containing a serine at a position corresponding to
position 118 of
SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The
position
of the serine is identified by alignment of the amino acid sequence of the
mammalian BAD,
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or fragment of the mammalian BAD, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3,
respectively. Second, the cell culture is contacted with the candidate drug to
form a reacted
fraction. Third, the cells in the reacted fraction are compared to cells of a
control culture to
determine whether the candidate drug has activity promoting cell survival by
monitoring the
S viability of the cells in the reacted fraction as compared to the cells of
the control culture.
Preferably, treatment of the control cell culture is essentially identical to
that of the cell
culture of the reacted fraction, except that the control cell culture is not
contacted with the
candidate drug. '
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes cell survival. The methods comprise
first preparing
a cell culture containing a cell line expressing a mammalian BAD, or fragment
of a
mammalian BAD, wherein the cell line has activity that promotes apoptosis, or
is capable of
having activity that promotes apoptosis. The mammalian BAD, or said fragment,
comprises
an amino acid sequence containing a serine at a position corresponding to
position 118 of
SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The
position
of the serine is identified by alignment of the amino acid sequence of the
mammalian BAD,
or fragment of the mammalian BAD, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3,
respectively. Second, the cell culture is contacted with the candidate drug to
forth a reacted
fraction. Third, cultured cells in the reacted fraction are compared to cells
of a control
culture, to determine whether the candidate drug has activity promoting cell
survival, by
monitoring the viability of the cells in the reacted fraction as compared to
the control cells.
Alternatively, the cells in the reacted fraction can be compared to the
control cells, in order to
determine whether the candidate drug has activity promoting cell survival, by
further
contacting the cells of both cultures with at least one antibody specific for:
1) the mammalian
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BAD, or said fragment, that is phosphorylated at the serine; or 2) the
mammalian BAD, or
said fragment, that is unphosphorylated at the serine; and then assaying for
the amount of the
antibody binding to the mammalian BAD, or said fragment. Preferably, treatment
of the
control cells is essentially identical to treatment of the cells in the
reacted fraction, except that
S the control cells are not contacted with the candidate drug.
Another embodiment of the present invention provides methods of inhibiting
apoptosis in a cell expressing a mammalian BAD, or fragment of a mammalian
BAD. The
methods comprise first preparing a cell culture containing a cell line
expressing a mammalian
BAD, or said fragment. The mammalian BAD, or said fragment, comprises an amino
acid
sequence containing a serine at a position corresponding to position 118 of
SEQ ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of
the serine is
identified by alignment of the amino acid sequence of the mammalian BAD, or
fragment of
the mammalian BAD, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
Second, the cultured cells are contacted with an extracellular agent, and/or
an intracellular
agent is induced, to form a reacted fraction and, thereby, a kinase in the
cells is activated,
wherein the kinase is capable of phosphorylating the mammalian BAD, or said
fragment, at
the specified serine. Third, the cells in the reacted fraction are compared to
a control cell line
to determine whether apoptosis is inhibited in the cells in the reacted
fraction, by 1) assaying
for the amount of the mammalian BAD, or said fragment, that is phosphorylated
at the
specified serine in the cells in the reacted fraction as compared to the
control cells; and/or 2)
monitoring indicia of apoptosis in the cells in the reacted fraction as
compared to the control
cells. Preferably, treatment of the control cells is essentially identical to
treatment of the cells
in the reacted fraction, except that the control cells do not express a
mammalian BAD, or said
fragment, that is capable of being phosphorylated by the kinase. Examples of a
serine kinase
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capable of phosphorylating the mammalian BAD, or said fragment, at the serine
include
PKA. Further examples of serine kinases capable of phosphorylating the
mammalian BAD,
or fragment of the mammalian BAD, include a heterologous kinase. Examples of a
mammalian BAD include a heterologous mammalian BAD, and examples of a fragment
of a
S mammalian BAD include a fragment of a heterologous mammalian BAD. Examples
of an
extracellular agent and/or said intracellular agent include a ligand of a G-
protein-coupled
receptor, such as L-epinephrine.
Another embodiment of the present invention provides methods of assaying a
candidate compound for a kinase activity capable of phosphorylating a
mammalian BAD, or
fragment of a mammalian BAD, at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1;
position
155 of SEQ ID N0:2; or position 113 of SEQ ID N0:3. The position of the serine
is
identified by alignment of the amino acid sequence of the mammalian BAD, or
fragment of
mammalian BAD, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. The
1 S methods comprise first contacting the candidate compound with the
mammalian BAD, or said
fragment, to form a reacted fraction. Second, to determine whether the
candidate compound
has kinase activity capable of phosphorylating the mammalian BAD, or said
fragment, the
reacted fraction is assayed for the amount of the mammalian BAD, or said
fragment, that is
phosphorylated at the specified serine. Alternatively, to determine whether
the candidate
compound has kinase activity capable of phosphorylating the mammalian BAD, or
said
fragment, at the specified serine, the reacted fraction is assayed, for
example, by detecting: 1)
the amount of radioactive label on the serine, wherein the cell is contacted
with radioactive
label and the radioactive label is attached to the serine when the serine is
phosphorylated; 2) a
difference in the electrophoretic mobility of the mammalian BAD, or said
fragment, that is
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phosphorylated at the serine as compared to the mammalian BAD, or said
fragment, that is
unphosphorylated at the serine; or 3) the amount of the mammalian BAD, or said
fragment,
bound to an antibody specific for the mammalian BAD, or said fragment, that is
phosphorylated at the serine. Examples of an antibody specific for the
mammalian BAD, or
said fragment, that is phosphorylated at the serine include a monoclonal
antibody.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes the phosphorylation of a mammalian
BAD, or
fragment of a mammalian BAD, at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of the serine
is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:l, SEQ ID N0:2, or SEQ ID N0:3, respectively. The methods
comprise first
contacting the candidate drug with a sample comprising Bcl-X~ and the
mammalian BAD, or
said fragment, to form a reacted fraction, wherein the mammalian BAD, or said
fragment is
capable of being phosphorylated at the serine. Second, the reacted fraction is
compared to a
control fraction to determine whether the candidate drug has activity that
promotes
phosphorylation,.by assaying for: 1) the amount of mammalian BAD, or said
fragment, that
is not bound to Bcl-X~ in the reacted fraction as compared to the control
fraction; and/or 2)
the amount of the mammalian BAD, or said fragment, that is phosphorylated at
the serine in
the reacted fraction as compared to the control fraction. Preferably,
treatment of the control
fraction is essentially identical to that of the reacted fraction, except the
mammalian BAD, or
fragment of the mammalian BAD, in the control fraction is not contacted with
the candidate
drug, andlor the control fraction contains a mammalian BAD, or said fragment,
that is not
capable of being phosphorylated at the specified serine.
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Another embodiment of the present invention provides methods of screening a
candidate drug for an activity that promotes the phosphorylation of a
mammalian BAD, or
fragment of a mammalian BAD, at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position of the serine
is
identified by alignment of the amino acid sequence of the mammalian BAD, or
said fragment,
to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. The methods
comprise first
contacting a candidate drug with a sample comprising the mammalian BAD, or
said fragment,
and a kinase, to form a reacted fraction, wherein the mammalian BAD, or said
fragment, is
capable of being phosphorylated by the kinase. Second, the reacted fraction is
compared to a
control fraction to determine whether the candidate drug has activity that
promotes
phosphorylation by the kinase by assaying for the amount of the mammalian BAD,
or said
fragment, that is phosphorylated at the specified serine in the reacted
fraction as compared to
the control fraction. Preferably, treatment of the control fraction is
essentially identical to that
of the reacted fraction, except the control fraction is not contacted with the
candidate drug
and/or the control fraction contains a mammalian BAD, or said fragment, that
is not capable
of being phosphorylated at the specified serine.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that promotes phosphorylation in a cell of a
mammalian BAD, or
fragment of a mammalian BAD, at a serine at a position in the amino acid
sequence of the
mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:1,
position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The position is identified
by
alignment of the amino acid sequence of the mammalian BAD, or said fragment,
to SEQ ID
NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively. The methods comprise first
preparing a
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cell culture containing a cell line expressing the mammalian BAD, or said
fragment, wherein
the cell line has activity that promotes apoptosis, or is capable of having
activity that
promotes apoptosis. Second, the cells are contacted with a candidate drug to
form a reacted
fraction. Third, the cells in the reacted fraction are compared to a control
cell line to
determine whether the candidate drug has activity that promotes
phosphorylation by assaying
for the amount of the mammalian BAD, or said fragment, that is phosphorylated
at the
specified serine, in the cells in the reacted fraction as compared to the
control cells, and/or
monitoring indicia of apoptosis in the cells in the reacted fraction as
compared to the control
cells. Alternatively, the cells in the reacted fraction are compared to
control cells to
determine whether the candidate drug has activity that promotes
phosphorylation by further
contacting cells of both fractions with at least one antibody, wherein the
antibody is selected
from a group consisting of at least one antibody specific for the mammalian
BAD, or said
fragment, that is phosphorylated at the serine, or at least one antibody
specific for the
mammalian BAD, or said fragment, that is unphosphorylated at the serine.
Preferably,
1 S treatment of the control cell line is essentially identical to that of the
cells in the reacted
fraction, except the control cells are not contacted with the candidate drug.
Another embodiment of the present invention provides methods of screening a
candidate drug for activity that modulates apoptosis promoting activity in a
cell. The methods
comprise first preparing a cell culture containing a cell line expressing a
mammalian BAD, or
fragment of a mammalian BAD. The mammalian BAD, or said fragment, comprises an
amino acid sequence containing a serine at a position corresponding to
position 118 of SEQ
ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. The
position of
the serine is identified by alignment of the amino acid sequence of the
mammalian BAD, or
said fragment, to SEQ ID NO:1, SEQ ID N0:2 or SEQ ID N0:3, respectively.
Second, the
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cells are contacted with an apoptosis promoting substance, wherein the cell
line has activity
that promotes apoptosis, or is capable of having activity that promotes
apoptosis. Third, the
cells are contacted with the candidate drug to form a reacted fraction.
Fourth, the cells in the
reacted fraction are compared to a control cell line to determine whether the
candidate drug
has activity that modulates apoptosis promoting activity by: 1) assaying for
the amount of the
mammalian BAD, or said fragment, that is phosphorylated or unphosphorylated at
the
specified serine in the cells in the reacted fraction as compared to the
control cells; or 2)
monitoring indicia of apoptosis in the cells in the reacted fraction as
compared to the control
cells. Preferably, treatment of the control cell line is essentially identical
to that of the cells in
the reacted fraction, except that the control cells are not contacted with the
candidate drug.
Another embodiment of the present invention provides antibodies that
specifically
bind to a mammalian BAD, or fragment of a mammalian BAD, phosphorylated at a
serine at
a position in the amino acid sequence of the mammalian BAD, or said fragment,
corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or
position
113 of SEQ ID N0:3. The position of the serine is identified by alignment of
the amino acid
sequence of the mammalian BAD, or said fragment, to SEQ ID NO:1, SEQ ID N0:2,
or SEQ
ID N0:3, respectively.
More particularly, an embodiment of the present invention provides monoclonal
antibodies that specifically bind to a mammalian BAD, or fragment of a
mammalian BAD,
phosphorylated at a serine at a position in the amino acid sequence of the
mammalian BAD,
or said fragment, corresponding to position 118 of SEQ ID NO:1, position 155
of SEQ ID
N0:2, or position 113 of SEQ ID N0:3. The position of the serine is identified
by alignment
of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID
NO:1, SEQ
ID N0:2, or SEQ ID N0:3, respectively.
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Also more particularly, an embodiment of the present invention provides
antibodies
that specifically bind to a mammalian BAD, or fragment of a mammalian BAD,
unphosphorylated at a serine at a position in the amino acid sequence of a
mammalian BAD,
or said fragment, corresponding to position 118 of SEQ ID NO:1, position 155
of SEQ ID
S N0:2, or position 113 of SEQ ID N0:3. The position of the serine is
identified by alignment
of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID
NO:1, SEQ
ID N0:2, or SEQ ID N0:3, respectively.
Even more particularly, an embodiment of the present invention provides
monoclonal
antibodies that specifically bind to a mammalian BAD, or fragment of a
mammalian BAD,
unphosphorylated at a serine at a position in the amino acid sequence of a
mammalian BAD,
or said fragment, corresponding to position 118 of SEQ ID NO:1, position 155
of SEQ ID
N0:2, or position 113 of SEQ ID N0:3. The position of the serine is identified
by alignment
of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID
NO:1, SEQ
ID N0:2, or SEQ ID N0:3, respectively.
Another embodiment of the present invention provides polynucleotides encoding
at
least one isolated or synthetic polypeptide comprising an amino acid sequence
of a mutant
BAD, or at least one fragment of an isolated or synthetic polypeptide
comprising a less than
full-length amino acid sequence of a mutant BAD, having cell death promoting
activity. The
amino acid sequence of the encoded isolated or synthetic polypeptides of a
mutant BAD, or
fragment of a mutant BAD, is: 1 ) derived from a naturally-occurring or wild-
type
mammalian BAD; 2) contains a domain that is substantially identical to a BH3
domain of a
naturally-occurring or wild-type mammalian BAD; and 3) does not contain a
serine at a
position corresponding to position 118 of SEQ ID NO:1, position 155 of SEQ ID
N0:2, or
position 113 of SEQ ID N0:3. The position corresponding to position 118 of SEQ
ID NO:1,
28
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position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is determined by
alignment
of the amino acid sequence of the isolated or synthetic polypeptides of the
mutant BAD, or
said fragments, to SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects of the present invention, the various features
thereof,
as well as the invention itself may be more fully understood from the
following description,
when read together with the accompanying drawings.
Fig. 1 is a western blot. Whole cell lysates were prepared from COS-7 cells
transiently expressing the double mutant HA-BAD S 112A/S 136A. Lysates were
treated
("+") with lambda phosphatase or a buffer control ("-") (lanes 1 and 2).
Separate aliquots of
the lysate were incubated with GST-Bcl-XL ("+") or a buffer control ("-") and
precipitated
with glutathione beads (lanes 3 and 4). Proteins were separated by SDS-PAGE.
Blots were
probed with an anti-HA antibody.
Fig. 2(A) is a western blot. Whole cell lysates were prepared from COS-7 cells
transiently expressing empty pcDNA3 vector ("mock") (lane 1), the double
mutant HA-BAD
S 112A1S 136A (lane 2), the triple mutant HA-BAD S 112A/S 134A/S 136A (lanes 3
and 4), or
the triple mutant HA-BAD S112A/S136A/S155A (lanes 5 and 6). Lysates were
treated ("+")
with lambda phosphatase or a buffer control ("-") and precipitated with an
anti-HA antibody.
Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA
antibody.
Fig. 2(B) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing empty pcDNA3 vector ("mock") (lane 1), shorter murine
HA-BAD
(SEQ ID N0:3) ("wildtype") (lane 2), HA-BAD S 112A (lane 3), HA-BAD S 136A
(lane 4),
29
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or HA-BAD S 155A (lane S). Proteins were separated by SDS-PAGE. Blots were
probed
with an anti-HA antibody.
Fig. 2(C) is a western blot. Whole cell lysates were prepared from serum-
starved
HeLa cells expressing HA-BAD ("wildtype") or HA-BAD S155A, pretreated with
epidermal
growth factor ("EGF"), fetal calf serum ("FCS") or buffer control. Proteins
were separated
by SDS-PAGE. Blots were probed with an anti-HA antibody.
Fig. 3(A) is an amino acid sequence alignment of BH3 domains of the Bcl-family
members, against the BH3 domain of BAD (SEQ ID NO:1). The open rectangle,
encompassing the closed rectangle, represents the amino acid sequence of the
longer murine
BAD (SEQ ID NO:1) protein. The closed rectangle represents the BH3 domain of
the longer
murine BAD (SEQ ID NO:1) protein. "S 112," S 136" and "S 155" indicate the
location of
serine residues at positions 112, 136 and 155 of the longer murine BAD (SEQ ID
NO:1)
protein, respectively. The amino acid sequences are those of BAD (SEQ ID
N0:4), BAK
(SEQ ID NO:S), BAX (SEQ ID N0:6), BIK (SEQ ID N0:7), BID (SEQ ID N0:8), HRK
(SEQ ID N0:9), BOK (SEQ ID NO:10), and BIM (SEQ ID NO:11). Residues surrounded
by
a black box are identical. Residues surrounded by a gray box are homologous
among BH3
domains. The code for the individual residues is A = alanine, C = cysteine, D
= aspartic acid,
E = glutamic acid, F = phenylalanine, G = glycine, H = histidine, I =
Isoleucine, K = lysine, L
= leucine, M = methionine, N = asparagine, P = proline, R = arginine, Q =
glutamine, S =
serine, T = threonine, V = valine, W = tryptophan, Y = tyrosine.
Fig. 3(B) is a graphical representation of the results of an in vitro
competition binding
assay. Recombinant GST-Bcl-XL was incubated with a BAD BH3 peptide (residues
143-
168) phosphorylated on Ser155 ("BAD BH3-P"), a BAD BH3 peptide (residues 143-
168)
unphosphorylated on Ser155 ("BAD BH3"), or a BAK BH3 peptide (residues 71-89)
as a
positive control, at the indicated concentrations. The reaction mixtures were
then added to
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microtiter plates pre-coated with BAK BH3 peptide. The amount of bound GST-Bcl-
XL was
determined by ELISA using an anti-GST primary antibody and a horse-radish
peroxidase-
conjugated anti-mouse IgG secondary antibody with ABTS as substrate.
Fig. 3(C) is two autoradiographs. Full-length BAD ("wildtype") and mutant BAD
S155A ("S155A") were produced by in vitro translation and labeled with 35S-
methionine
therein. The labeled proteins were incubated with PKA ("+") or a buffer
control ("-"), and
then incubated with either GST or GST-Bcl-XL, followed by capture on
glutathione-agarose
beads. Proteins bound to the beads ("Bound") (lower panel) and samples of the
reactions
collected prior to incubation with the beads ("Total") (upper panel) were
analyzed by SDS-
PAGE followed by autoradiography.
Fig. 4 is a western blot and an autoradiograph. Whole cell lysates were
prepared from
COS-7 cells expressing GST-tagged shorter murine BAD (SEQ ID N0:3)
("wildtype") (lane
1 ), GST-BAD S 112A ("S 112A") (lane 2), GST-BAD S 136A ("S 136A") (lane 3),
GST-BAD
S155A ("S155A") (lane 4), GST-BAD S112A/S136A ("S112A/S136A") (lane S), and
GST-
BAD S 112A/S 136A/S 1 SSA ("S 112A/S 136A/S 1 SSA") (lane 6). Lysates were
precipitated
with glutathione beads, and the purified GST-tagged polypeptides were
incubated with PKA
in the presence of y-32P-ATP radiolabel. Proteins were separated by SDS-PAGE,
and
phosphorylation was judged by autoradiograph (upper panel), while equivalent
loading was
confirmed by probing with an anti-BAD antibody (lower panel) on the same
membrane.
Fig. 5(A) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing empty expression vector ("mock") (lane 1), HA-BAD S
112A/S 136A
(lanes 2, 3 and 4), and HA-BAD S 112A/S 136A/S 155A (lanes 5 and 6),
pretreated with
Forskolin ("+") or a buffer control ("-") and then lambda phosphatase ("+") or
a buffer
control ("-") prior to lysis. Proteins were separated by SDS-PAGE. Blots were
probed with
an anti-HA antibody.
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Fig. 5(B) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing empty expression vector ("mock") (lane 1), or HA-BAD
S 112A/S 136A (lanes 2-9), treated with Forskolin ("Fk") for the indicated
time periods prior
to lysis. Proteins were separated by SDS-PAGE. Blots were probed with an anti-
HA
antibody.
Fig. 5(C) is a series of western blots. Whole cell lysates were prepared from
HeLa
cells transiently expressing HA-BAD ("wildtype") (lanes 1 and 2), HA-BAD S155A
("S 15 SA") (lanes 3 and 4), HA-BAD S 112A/S 136A ("S 112A/S 136A") (lanes S
and 6), or
HA-BAD S 112A/S 136A/S 155A ("S 112A/S 136A/S 155A ") (lanes 7 and 8),
pretreated with
Forskolin ("+") or a buffer control ("-") prior to lysis. Proteins were
separated by SDS-
PAGE. Blots were probed with anti-phospho-Ser155 specific BAD antibody ("anti-
pS155
Ab probe") (upper panels), followed by stripping and reprobing with an anti-
BAD antibody
("anti-BAD Ab probe") (lower panels).
Fig. 6(A) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing empty expression vector ("-") (lane 1), or HA-BAD
S112A/S136A
(lanes 2-6), pretreated with Forskolin (lane 2), thyroid stimulating hormone
("TSH") (lane 3),
L-epinephrine ("L-epi") (lane 4), adrenocorticotropic hormone ("ACTH") (lane
5), or a
combination of thyroid stimulating hormone, L-epinephrine and
adrenocorticotropic hormone
("TSH L-epi ACTH") (lane 6) prior to lysis. Proteins were separated by SDS-
PAGE. Blots
were probed with an anti-HA antibody.
Fig. 6(B) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing HA-BAD ("wildtype") (lanes 1 and 2), HA-BAD S 112A ("S
112A")
(lanes 3 and 4), HA-BAD S 136A ("S 136A") (lanes 5 and 6) and HA-BAD S 155A
("S 1 SSA")
(lane 7 and 8), pretreated with L-epinephrine ("+") or a buffer control ("-")
prior to lysis.
Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA
antibody.
32
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Fig. 7(A) is a western blot. Whole cell lysates were prepared from HeLa cells
expressing endogenous wild-type BAD, and untreated (lane 1), pretreated with
Forskolin
(lanes 2-6) or L-epinephrine (lanes 7-9) for different lengths of time prior
to lysis. Proteins
were separated by SDS-PAGE. Blots were probed with an anti-BAD antibody.
Fig. 7(B) is a western blot. Whole cell lysates were prepared from HeLa cells
expressing endogenous wild-type BAD, and untreated (lane 1), pretreated with L-
epinephrine
alone (lane 2), L-epinephrine and lambda phosphatase (lane 3), L-epinephrine
and H89 (lane
4) or L-epinephrine and Wortmannin (lane 5) prior to lysis. Proteins were
separated by SDS-
PAGE. Blots were probed with an anti-BAD antibody.
Fig. 7(C) is two western blots. Whole cell lysates were prepared from Rat-1
cells
expressing endogenous wild-type BAD, untreated ("-") (lane 1, upper panel),
pretreated with
Forskolin for various times (lanes 2-6, upper panel) or treated with Forskolin
and H89 for 5
minutes (lane 7, upper panel) or 30 minutes (lane 8, upper panel) prior to
lysis. Proteins were
separated by SDS-PAGE. Blots were probed with an anti-BAD antibody.
In a separate comparison (lower panel), cells were untreated ("-") (lane 1,
lower
panel), treated with Forskolin (lane 2, lower panel) or treated with Forskolin
and lambda
phosphatase (lane 3, lower panel) prior to lysis. Proteins were separated by
SDS-PAGE.
Blots were probed with an anti-BAD antibody.
Fig. 8(A) is two western blots. HeLa cells transiently expressing HA-BAD
S 112A/S 136A and either an empty expression vector ("vector") (lanes 1 and 2)
or HA-PKI
("PKI") (lanes 3 and 4) were treated with L-epinephrine ("+") or a buffer
control ("-").
Whole cell lysates were prepared and proteins were separated by SDS-PAGE.
Blots were
probed with an anti-HA antibody. The upper panel displays proteins
corresponding, in size,
to HA-BAD S 112A/S 136A. The lower panel displays proteins corresponding, in
size, to HA-
PKI.
33
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Fig. 8(B) is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing a HA-BAD S 112A/S 136A and untreated ("-") (lane 1),
pretreated with
L-epinephrine (lane 2) or pretreated with L-epinephrine and H89 (lane 3) prior
to lysis.
Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA
antibody.
Fig. 9(A) is a western blot. Whole cell lysates were prepared from Rat-1 cells
transiently expressing HA-BAD S112A/S136A, untreated ("-") (lane 1) or
pretreated with
platelet-derived growth factor ("PDGF") (lane 2), epidermal growth factor
("EGF") (lanes 3
and 4), insulin-like growth factor I ("IGF-1") (lanes 5 and 6), N6-benzoyl-
adenosine 3',5'-
cyclic monophosphate ("cAMP") (lanes 7 and 8), or Forskolin ("Forskolin")
(lanes 9 and 10)
prior to lysis, for the time periods indicated. Proteins were separated by SDS-
PAGE. Blots
were probed with an anti-HA antibody.
Fig. 9(B) is two western blots. Rat-1 cells transiently expressing HA-BAD
S 112A/S 136A were treated with a buffer control, H89 (PKA inhibitor) or
Wortmannin (PI 3-
kinase inhibitor), prior to stimulation with Forskolin or platelet-derived
growth factor
("PDGF"). Whole cell lysates were then prepared and proteins were separated by
SDS-
PAGE. Blots were separately probed with an anti-phospho-S473 Akt antibody (the
kinase
that phosphorylates Ser136) (upper panel) and an anti-BAD antibody (lower
panel).
Fig. 9(C) is two western blots. Whole cell lysates were prepared from non-
transfected, serum-starved, Rat-1 cells pretreated with PDGF ("+") or a buffer
control ("-")
prior to lysis. Endogenous BAD was immunoprecipitated from the lysates and
separated by
SDS-PAGE ("lysate +"). As a negative control, the anti-BAD antibody was
incubated with
beads without cell lysates and the collected fraction was subjected to the
same anti-phospho-
5155 BAD antibody analysis ("lysate -"). Blots were probed with the anti-
phospho-Ser155
specific BAD antibody ("anti-pS155 Ab") (left panel) and then stripped and
reprobed with an
anti-BAD antibody ("anti-BAD Ab") (right panel).
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Fig. 10(A) is two western blots. Whole cell lysates were prepared from serum-
starved HeLa cells co-transfected with HA-BAD S 112A/S 136 and either the PKI
expression
vector ("PKI") (lanes 3 and 4) or empty expression vector ("vector") (lanes 1
and 2), and then
treated with EGF or a buffer control, prior to lysis. Proteins were separated
by SDS-PAGE.
Blots were probed with an anti-HA antibody (upper panel) or an anti-PKI
antibody (lower
panel).
Fig. 10(B) is a series of western blots. Serum-starved HeLa cells expressing
HA-
BAD S155A, were pretreated with a buffer control (lanes l and 2), AG1478
("AG") (lane 3),
Wortmannin ("Wm") (lane 4), or H89 (lane 5). The cell cultures were then
stimulated with a
buffer control (lane 1) or EGF (lanes 2-5). Whole cell lysates were made from
the cultures
and the proteins were separated by SDS-PAGE. Blots were probed with either an
anti-
activated EGF receptor antibody ("activated EGFR") (panel A), an anti-phospho-
S473 Akt
antibody ("pS473 Akt") (panel B), or an anti-phospho-5155 BAD antibody ("pS155
BAD")
(panel D). Reblots were probed with either an anti-Akt antibody ("Akt") (panel
C) or an anti-
HA BAD antibody ("HA-BAD") (panel E).
Fig. 10(C) is a western blot. Serum-starved Rat-1 cells were pretreated with a
buffer
control (lanes 1 and 2), H89 (lane 3) or Wortmannin ("Wm") (lane 4), followed
by
stimulation with PDGF. Whole cell lysates were prepared, endogenous BAD was
immunoprecipitated and the resulting proteins were separated by SDS-PAGE. The
blot was
probed with an anti-phospho-Ser155 BAD antibody.
Fig. 11(A) is a graphic representation of the results of an enzyme-linked
immunosorbent assay (ELISA). HeLa cells were co-transfected with (3-
galactosidase and one
of the following: empty vector ("mock") (lane 1), wild-type BAD ("WT") (lane
2), BAD
S112A ("S112A") (lane 3), BAD S136A ("S136A") (lane 4), BAD S155A ("S155A")
(lane
5), BAD S 112A/S 136A ("AA") (lane 6), or BAD S 112A/S 136A/S 1 SSA ("AAA")
(lane 7).
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Cells were treated with Forskolin four hours after co-transfection, and whole
cell lysates were
prepared 24 hours after co-transfection. The (3-galactosidase activity was
analyzed by ELISA
(absorbance = 410 nm).
Fig. 11(B) is a western blot. Whole cell lysates were prepared as described
above in
Fig. 11(a). Empty vector ("mock") (lane 1), wild-type BAD ("wild-type") (lane
2), BAD
S 112A ("S 112A") (lane 3), BAD S 136A ("S 136A") (lane 4), BAD S 15 SA ("S 1
SSA") (lane
5), BAD S 112A/S 136A ("S 112A/S 136A") (lane 6), or BAD S 112A/S 136A/S 155A
("S 112A/S 136A/S 155A") (lane 7). Proteins were separated by SDS-PAGE. Blots
were
probed with an anti-HA antibody.
Fig. 12(A) is a graphic representation of the results of an ELISA. HeLa cells
were co-
transfected with (3-galactosi.dase and one of the following: empty vector
("mock") (lanes 1
and 2), wild-type BAD ("WT") (lanes 3 and 4), BAD S 155A ("S 155A") (lanes 5
and 6),
BAD S 112A/S 136A ("AA") (lanes 7 and 8), or BAD S 112A/S 136A/S 1 SSA ("AAA")
(lanes
9 and 10). Cells were treated with Forskolin (open bars) or a buffer control
(closed bars) four
hours after co-transfection. Whole cell lysates were prepared 24 hours after
co-transfection.
The ~i-galactosidase activity was analyzed by ELISA (absorbance = 410 nm).
Fig. 12(B) is a graphic representation of the results of a (3-galactosidase
assay. HeLa
cells were co-transfected with the (3-galactosidase gene and either HA-BAD
("WT"), HA-
BAD S155A ("S155A"), HA-BAD S112A/S136A ("S112A/S136A") or HA-BAD
S112A/S136A/S155A ("AAA"). Transfected cells were then cultured in serum-free
medium
(SFM) or SFM supplemented with EGF (SFM+EGF) for 12 hours. Cell lysates were
prepared and BAD-induced cell death was measured by the loss of (3-
galactosidase activity in
a fluorescence-based assay. Fluorescence was measured at 465 nm. Results shown
are the
average and standard deviation of triplicate transfections (this experiment
was repeated two
more times with similar results). Expression levels of HA-tagged BAD and BAD
mutants in
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transfected cell lysates were detected by anti-HA western blot analysis: HA-
BAD ("WT"),
HA-BAD S 155A ("A"), HA-BAD S 112A/S 136A ("AA"), and HA-BAD
S 112A/S 136A/S 155A ("AAA")
Fig. 12(C) is a graphic representation of the results of a ~3-galactosidase
assay. HeLa
cells were co-transfected with the (3-galactosidase gene and either an empty
vector ("CTL"),
wild-type BAD ("WT"), the BAD S 155A mutant ("S 155A") or the BAD S 155D
mutant
("S 1 SSD"). Cell lysates were prepared and BAD-induced cell death was
measured by the
loss of ~3-galactosidase activity in a fluorescence-based assay. Fluorescence
was measured at
465 nm. Results shown are the average and standard deviation of triplicate
transfections (this
experiment was repeated two more times with similar results).
Fig. 13 is a western blot. Whole cell lysates were prepared from COS-7 cells
expressing empty vector ("mock") (lane l, upper panel), the 204 amino acid
form of murine
BAD (longer murine BAD) ("204 as") (lanes 2 and 3, upper panel) or the 162
amino acid
form of murine BAD (shorter murine BAD) ("162 as") (lanes 4 and 5, upper
panel). Proteins
were separated by SDS-PAGE. Blots were probed with an anti-BAD antibody.
Whole cell lysates were also prepared from FLS.i2 cells expressing endogenous
BAD,
pretreated with IGF-1 ("+") (lanes 2 and 3, lower panel), Wortmannin ("+")
(lane 3, lower
panel) or a buffer control ("-") (lane 1, lower panel) prior to lysis.
Proteins were separated by
SDS-PAGE. Blots were probed with an anti-BAD antibody.
Fig. 14 is a western blot. Whole cell lysates were prepared from HeLa cells
transiently expressing HA-BAD S 112A/S 136A, pretreated with Forskolin ("+")
or a buffer
control ("-") prior to lysis. Aliquots of the lysate were separated by SDS-
PAGE and probed
with pre-immune serum, crude rabbit anti-phospho-Ser155 BAD antibody or anti-
HA
antibody.
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DETAILED DESCRIPTION OF THE INVENTION
The positions of amino acid residues in the sequences of BAD, mutant BAD, and
fragments of a BAD or mutant BAD of the present invention are sometimes
described in
terms of their corresponding positions in the sequence of the human BAD of SEQ
ID NO:1,
murine BAD of SEQ ID N0:2 ("longer murine BAD"), and murine BAD of SEQ ID N0:3
("shorter murine BAD"). However, since much of the published research on BAD
has been
performed using the murine BAD of SEQ ID N0:2, the positions of the amino
acids in the
sequences of BAD, mutant BAD, and fragments of BAD or mutant BAD of the
present
invention are sometimes described in terms of their corresponding positions in
the sequence
of the murine BAD of SEQ ID N0:2, for ease in understanding and cross
comparing the
present invention and the published works.
The amino acid sequence of the human BAD of SEQ ID NO:1 and of the murine
BAD of SEQ ID N0:3 are aligned against the amino acid sequence of the murine
BAD of
SEQ ID N0:2 in Table 1, below, illustrating the corresponding positions of the
amino acids.
The serine phosphorylation sites at positions 112, 136, and 155 of SEQ ID N0:2
are denoted
with an asterisk, and the corresponding positions in the amino acid sequences
of SEQ ID
N0:3 and SEQ ID NO:1 are given below the serine at positions 112, 136, and 155
of SEQ ID
N0:2. The positions having an amino acid residue that is common between all
three BAD
amino acid sequences are denoted in bold.
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TABLE 1
SEQ ID N0:2 1 MGTPKQPSLAPAHALGLRKSDPGIRSLGSDAGGRRWRPAAQSMFQIPEF
SEQ ID N0:3 1 MFQIPEF
S SEQ ID N0:1 1 MFQIPEF
SEQ ID N0:2 50 EPSEQEDASATDRGLGPSLTEDQPGP YLAPGLLGSNIHQQGRAA
SEQ ID N0:3 8 EPSEQEDASATDRGLGPSLTEDQPGP YLAPGLLGSNIHQQGRAA
lO SEQ ID N0:1 8 EPSEQEDSSSAERGLGPSPAGDGPSGSGKHHRQAPGLLWDASHQQEQPT
SEQ ID N0:2 94 TNSHHGGAGAMETRSRHSSYPAGTEEDEGMEEELSPFRGRSRSAPPNLW
SEQ ID N0:3 52 TNSHHGGAGAMETRSRHSSYPAGTEEDEGMGEELSPFRGRSRSAPPNLW
15 SEQ ID NO:1 57 SSSHHGGAGAVEIRSRHSSYPAGTEDDEGMGEEPSPFRGRSRSAPPNLW
SEQ ID N0:2 143 AAQRYGRELRRMSDEFEGSFK GLPRPKSAGTATQMRQSAGWTRIIQSW
SEQ ID N0:3 101 AAQRYGRELRRMSDEFEGSFK GLPRPKSAGTATQMRQSAGWTRIIQSW
20 SEQ ID N0:1 106 AAQRYGRELRRMSDEFVDSFKKGLPRPKSAGTATQMRQSSSWTRVFQSW
SEQ ID N0:2 191 WDRNLGKGGSTPSQ
SEQ ID N0:3 149 WDRNLGKGGSTPSQ
2$ SEQ ID N0:1 155 WDRNLGRGSSAPSQ
* = serine phosphorylation site
The term "BAD" as used herein refers to a Bcl-XLBcI-2 Associated Cell Death
30 Regulator polypeptide, and includes any polypeptide of any origin that is a
cell death
promoter, that is substantially identical to and/or biologically equivalent to
BAD, and that
binds to Bcl-XL and/or Bcl-2 in competition with the cell death promoters BAX
and/or BAK
to inhibit the cell death repressor activity of Bcl-XL and/or Bcl-2.
The term "mutant BAD" as used herein refers to a BAD having at least an amino
acid
35 sequence in which the serine at a position corresponding to position 118 of
SEQ ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3, has been replaced
with an
amino acid other than serine. The position of the serine, or other amino acid,
corresponding
to position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113
of SEQ ID
N0:3, is identified by alignment of the amino acid sequence of the mutant BAD
to SEQ ID
40 NO:1, SEQ ID N0:2, or SEQ ID N0:3, respectively.
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The term "BAD mutants" as used herein refers to the plural of a "mutant BAD."
The term "fragment" as used herein refers to a polypeptide comprising an amino
acid
sequence of a less than full-length polypeptide. A fragment contains at least
10 amino acids,
more preferably at least 25 amino acids, and can approach the number of amino
acids in the
full-length polypeptide. Further, a fragment of the present invention is
characterized as
having biological activity such as, e.g., cell death promoting activity and/or
the ability to bind
to Bcl-X~ and/or Bcl-2.
Examples of a fragment are a fragment of a BAD and a fragment of a mutant BAD.
A fragment of a BAD comprises an amino acid sequence of a less than full-
length BAD.
Preferably, a fragment of a BAD contains a domain substantially identical to a
BH3 domain
of a naturally-occurring or wild-type mammalian BAD, wherein the amino acid
sequence of
the fragment has a serine at a position corresponding to position 118 of SEQ
ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. A fragment of a
mutant
BAD comprises an amino acid sequence of a less than full-length mutant BAD.
Preferably, a
fragment of a mutant BAD contains a domain substantially identical to a BH3
domain of a
naturally-occurring or wild-type mammalian BAD, wherein the amino acid
sequence of the
fragment of a mutant BAD has an amino acid other than serine at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3. The position of the amino acid corresponding to position 118 of SEQ ID
NO:l,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3, is identified by
alignment of
the amino acid sequence of the fragment to SEQ ID NO:l, SEQ ID N0:2, or SEQ ID
N0:3,
respectively.
The terms "naturally-occurnng" or "wild-type" as used herein refer to a
polynucleotide or polypeptide that can be found in nature and is present in an
organism
(including viruses) although not necessarily in a discrete or isolated form,
which can be
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isolated from a source in nature and which has not been intentionally modified
by man in the
laboratory.
The term "BH3 domain" as used herein refers to the amino acids comprising from
approximately residue 143 to residue 168 of the murine BAD of SEQ ID N0:2, or
any
portion thereof, or any sequence of amino acids that corresponds to this
sequence when
aligned with the murine BAD of SEQ ID N0:2, or any portion thereof.
The BAD, mutant BAD, and fragments of a BAD or mutant BAD of the present
invention in one embodiment are provided in an isolated form. The term
"isolated" as used
herein refers to the fact that the object species such as a BAD, mutant BAD,
or fragment of a
BAD or mutant BAD, is substantially free of other substances which are not the
object
species, i.e. not in a natural environment. Generally, a substantially pure
composition will
comprise more than about 80 to 90 percent of all macromolecular species
present in the
composition. Most preferably, the object species is purified to essential
homogeneity such
that contaminant species cannot be detected in the composition by standard
methods of
detection and wherein the composition consists essentially of a single
macromolecular
species. Solvent species, small molecules (<500 Daltons), and elemental ion
species are not
considered macromolecular species.
The BAD, mutant BAD, and fragments of BAD or mutant BAD of the present
invention may be derived from any naturally-occurring or wild-type BAD native
to any tissue
or species. Similarly, the biological activity, e.g., cell death promoting
activity and/or
binding to Bcl-XL and/or Bcl-2, of such BAD, mutant BAD, and fragments of BAD
or
mutant BAD, can be characterized using any number of biological assay systems
known to
those skilled in the art.
The terms "activity that promotes apoptosis" and "cell death promoting
activity" as
used herein mean functional activity of a protein, such as BAD, mutant BAD,
and fragments
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of BAD or mutant BAD of the present invention, that induces, instigates,
causes or triggers
apoptosis or cell death, or the signal transduction pathway that leads to
apoptosis or cell
death.
The term "monitoring indicia of apoptosis" as used herein means examining
culture
cells, whether by assay or visual inspection, for one or more of loss of
cellular junctions and
microvilli, cytoplasmic condensation and nuclear chromatin margination,
nuclear
fragmentation, cytoplasmic contraction, mitochondria) and ribosomal
compaction,
endoplasmic reticulum dilation and fusion with the plasma membrane, and
cellular break up
into membrane-bound apoptotic bodies (Wyllie, 1980).
The term "biologically equivalent" as used herein means that the BAD, mutant
BAD,
or fragments of a BAD or mutant BAD, of the present invention are capable of
demonstrating
some or all of the same biological activity of a naturally-occurring or wild-
type BAD, e.g.,
cell death promoting activity, and/or binding to Bcl-X,_, andlor Bcl-2,
although not necessarily
to the same degree as the naturally-occurnng or wild-type BAD. The biological
activity of a
BAD, mutant BAD, or fragment of a BAD or mutant BAD, of the present invention
can be
determined using assays known to those skilled in the art. For example, the
percent viability
of cells expressing a BAD, mutant BAD, or fragment of a BAD or mutant BAD, of
the
present invention can be monitored as an indication of the cell death
promoting activity of the
expressed polypeptide. A mutant BAD, or fragment of the mutant BAD, can show
increased
or decreased cell death promoting activity compared to, for example, the
naturally-occurring
or wild-type BAD from which the mutant BAD, or fragment of the mutant BAD, was
derived, depending upon the particular mutant.
The terms "percent sequence identity," "percent identity," "% sequence
identity," or
"% identity" as used herein are intended to mean the percentage of the same
residues or
nucleotides between two or more amino acid sequences or nucleic acid
sequences,
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respectively (Higgins et al., 1992). The percentage of the same residues or
nucleotides
between multiple amino acid or nucleic acid sequences can be determined by
aligning the
amino acid or nucleic acid sequences, respectively, using sequence analysis
software such as,
for example, the Lasergene biocomputing software (DNASTAR, Inc., Madison,
Wis.). The
amino acid residue weight table used for the Lasergene alignment program is
PAM250
(Dayhoff et al., 1978). Sequence alignment allows identification of regions of
sequence
homology, such as, for example, the BH3 domain and, in particular, it allows
identification of
the serine, or other amino acid, at a position corresponding to the serine at
position 118 of
SEQ ID NO:1, position 155 of SEQ ID N0:2, and position 113 of SEQ ID N0:3.
The term "substantially identical" as used herein is intended to mean that
there is at
least 75%, preferably 85%, and more preferably 90 to 95% identity between two
or more
amino acid sequences or between two or more nucleotide sequences, and
preferably the
amino acid sequence includes a BH3 domain, or the nucleotide sequence encodes
a BH3
domain.
Reference to a mutant BAD herein preferably includes a mutant BAD having an
amino acid sequence that is substantially identical to at least one of SEQ ID
NO:1, SEQ ID
N0:2, or SEQ ID N0:3.
Preferably, reference to a mutant BAD herein includes a mutant BAD having an
amino acid sequence with at least 85 percent sequence identity with at least
one of SEQ ID
NO:1, SEQ ID N0:2, or SEQ ID N0:3.
More preferably, reference to a mutant BAD herein includes a mutant BAD having
an
amino acid sequence with at least 90-95 percent sequence identity with at
least one of SEQ
ID NO:1, SEQ ID N0:2, or SEQ ID N0:3.
The BAD, mutant BAD, and fragments of BAD and mutant BAD, of the present
invention can also include synthetic, derivative, heterologous, hybrid,
fusion, and modified
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forms of such polypeptides. The different forms of such polypeptides include
forms in which
certain amino acids have been deleted, added, replaced, or substituted; one or
more amino
acids has/have been changed to an amino acid analogue; and/or there are
glycosylations of
such polypeptides. Such polypeptides are characterized as having biological
activity, e.g.,
cell death promoting activity and/or the ability to bind to Bcl-X,_, and/or
Bcl-2. Furthermore,
in the case of a BAD, mutant BAD, or fragment of a BAD or mutant BAD, having
an amino
acid substitution at a position corresponding to the serine at position 118 of
SEQ ID NO:1,
155 of SEQ ID N0:2, or 113 of SEQ ID N0:3, such biological activity may be
blocked by
the phosphorylation of the amino acid at such a corresponding position.
The polynucleotides of the present invention encoding BAD, mutant BAD, or a
fragment of BAD or mutant BAD include, for example, isolated or synthetic DNA,
genomic
DNA, mRNA, and cDNA. The isolated polynucleotides may be isolated, for
example,
through hybridization with the complementary sequence of genomic, subgenomic
DNA,
cDNA, or mRNA encoding BAD, mutant BAD, or fragments of BAD or mutant BAD. The
polynucleotides may encode BAD, mutant BAD, or fragments of BAD or mutant BAD,
having substituted serine residues and/or phosphorylated serine residues. It
will also be
appreciated by one skilled in the art that degenerate DNA sequences can encode
BAD,
mutant BAD, or fragments of BAD or mutant BAD having serine substitutions or
having
serines which can be phosphorylated. Also intended to be included within the
present
invention are those polynucleotides encoding allelic variants of BAD and
serine substituted
and/or serine phosphorylated derivatives of such BAD.
Polynucleotides of the present invention encoding a BAD, mutant BAD, or
fragment
of a BAD or mutant BAD, may include sequences that facilitate RNA
transcription
(expression sequences) and protein translation of the coding sequences, such
that the encoded
polypeptide product is produced. Methods for construction of such
polynucleotides are
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known to those skilled in the art and are described, for example, in Sambrook
et al.,
Molecular Cloning, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. For
example, such
polynucleotides may include a promoter, a transcription termination site,
polyadenylation
site, ribosome binding site, an enhancer for the use in eukaryotic expression
hosts, and/or,
optionally, sequences necessary for replication of a vector. A typical
eukaryotic expression
vector may include a polynucleotide sequence encoding a BAD, mutant BAD, or
fragment of
a BAD or mutant BAD, inserted in-frame and downstream of a promoter such as,
e.g., the
herpes simplex virus thymidine kinase ("HSV-tk") promoter or the
phosphoglycerate kinase
("pgk") promoter, optionally linked in-frame to an enhancer and a downstream
polyadenylation site (e.g., the SV40 large T polyadenylation site).
The degeneracy of the genetic code gives a finite set of polynucleotide
sequences
encoding these amino acid sequences of the BAD, mutant BAD, and fragments of
the BAD
or mutant BAD of the present invention. This set of degenerate sequences may
be readily
generated by one skilled in the art, by hand or by computer using commercially
available
software. Isolated polynucleotides encoding a BAD, mutant BAD, or fragment of
a BAD or
mutant BAD, are typically less than approximately 10,000 nucleotides, more
preferably less
than approximately 3,000 nucleotides, still more preferably less than
approximately 1,500
nucleotides, and most preferably approximately 600 nucleotides.
Preferred polynucleotides are those polynucleotides encoding a mutant BAD, or
fragment of a mutant BAD, having an amino acid sequence that is substantially
identical to
SEQ ID NO:I, SEQ ID N0:2, or SEQ ID N0:3.
Preferred polynucleotides~ also include those polynucleotides capable of
hybridizing,
under stringent conditions, with a polynucleotide encoding a naturally-
occurring or wild-type
mammalian BAD. Such stringent conditions are known to, and/or can be
determined by
standard methods, by those skilled in the art. Examples of such stringent
conditions are
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provided in Sambrook et al., 1989. Such polynucleotides may be further
screened, under the
stringent conditions described above, to select for polynucleotides that do
not hybridize to
polynucleotides encoding any other members of the Bcl-2 family. Such selective
hybridization can be performed using standard cross-hybridization tests known
to those
skilled in the art.
Polynucleotides encoding a fragment of a BAD or mutant BAD of the present
invention may be short oligonucleotides, for example oligonucleotides that are
20-100
nucleotides in length, wherein such oligonucleotides have biological activity,
for example,
have cell death promoting activity and/or the ability to bind to Bcl-XL and/or
Bcl-2.
Polynucleotides encoding a BAD, mutant BAD, or fragment of a BAD or mutant
BAD of the present invention may also comprise part of a larger
polynucleotide, such as, for
example, a cloning vector. Also, such polynucleotides encoding a BAD, mutant
BAD, or
fragment of a BAD or mutant BAD, of the present invention may be fused in-
frame, by
polynucleotide linkage, to another polynucleotide sequence encoding a
different polypeptide
such as, for example, a polynucleotide encoding a heterologous polypeptide
such as the Tat
polypeptide (YGRI~KRRQRRRG) (SEQ ID N0:20) which facilitates intracellular
delivery of
the BAD, mutant BAD, or fragment of the BAD or mutant BAD. Similarly, the
encoded
polypeptide can be a fusion polypeptide, such as, for example, a BAD, mutant
BAD, or
fragment of a BAD or mutant BAD, fused to a heterologous polypeptide such as,
for
example, the Tat polypeptide (YGRKKRRQRRRG) (SEQ ID N0:20).
Typically, the polynucleotides encoding a mutant BAD, or fragment of a mutant
BAD, comprise at least 25 consecutive nucleotides which are substantially
identical to the
polynucleotide sequence encoding a naturally-occurnng or wild-type mammalian
BAD and
encoding a codon for an amino acid substitution of the serine at a position
corresponding to
position 118 of SEQ ID NO:1, 155 of SEQ ID N0:2, or 113 of SEQ ID N0:3. More
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preferably the polynucleotides encoding a mutant BAD, or fragment of a mutant
BAD,
comprise at least SO to 100 consecutive nucleotides, and still more preferably
at least 500 to
550 consecutive nucleotides, which are substantially identical to the
polynucleotide sequence
encoding a naturally-occurring or wild-type mammalian BAD and encoding an
amino acid
substitution of the serine at a position corresponding to position 118 of SEQ
ID NO:1, 155 of
SEQ ID N0:2, or 113 of SEQ ID N0:3.
Additionally, a polynucleotide encoding a mutant BAD, or fragment of a mutant
BAD, can be used to construct transgenes for expressing such polypeptides at
high levels,
and/or under the transcriptional control of transcription control sequences
which do not
naturally occur adjacent to a naturally-occurnng or wild-type Bad gene. For
example, a
constitutive promoter (e.g., a HSV-tk or pgk promoter) or a cell-lineage
specific
transcriptional regulatory sequence (e.g., a CD4 or CD8 gene
promoter/enhancer) or tissue-
specific transcriptional regulatory sequence may be operably linked to a
polynucleotide
encoding a mutant BAD, or fragment of a mutant BAD, to form a transgene
(typically in
combination with a selectable marker such as, e.g., a neo gene expression
cassette). Such
transgenes can be introduced into cells, such as hematopoietic stem cells and
transgenic cells,
and transgenic nonhuman animals can be obtained according to standard methods
known to
those skilled in the art. Transgenic cells and/or transgenic nonhuman animals
may be used to
generate models of diseases involving overexpression or inappropriate
expression of BAD
and to screen for agents to treat such diseases as, for example,
immunodeficiency diseases,
including AIDS, senescence, neurodegenerative disease, ischemic and
reperfusion cell death,
infertility, wound-healing, and the like. Polynucleotides encoding a BAD, or
fragment of a
BAD, can also be used to construct, express, and use a transgene in the same
manner as
described above for a mutant BAD, or fragment of a mutant BAD.
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In another embodiment the present invention provides a method for producing a
mutant BAD, or fragment of a mutant BAD, with increased cell death promoting
activity,
relative to a corresponding wild-type BAD or mutant BAD (i.e. one having the
same
sequence except for the particular amino acid changes) or with the ability to
modulate cell
death promoting activity. The method comprises preparation of a mutant BAD, or
fragment
of a mutant BAD, having an amino acid substitution at a serine at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3. The mutant BAD, or fragment of a mutant BAD, can be made based upon the
complete or partial sequence of a BAD from any source, for example a mammalian
BAD
such as the BAD of SEQ ID NO:1, SEQ ID N0:2, or SEQ ID N0:3. The position of
the
serine, or other amino acid, at a position corresponding to position 118 of
SEQ ID NO:1,
position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3 is identified by
alignment of
the sequence of the mutant BAD, or fragment of a mutant BAD, with SEQ ID NO:1,
SEQ ID
N0:2, or SEQ ID N0:3, respectively. Such a mutant BAD, or fragment of a mutant
BAD,
can be prepared using standard methods known to those skilled in the art.
For example, a mutant BAD, or fragment of a mutant BAD, may be made by
expression of the DNA sequence encoding the mutant BAD, or fragment of a
mutant BAD, in
a suitable transformed host cell. Using methods well known in the art, the DNA
encoding the
mutant BAD, or fragment of a mutant BAD, can be prepared and inserted into an
expression
vector, transformed into a host cell, and suitable conditions established for
expression of the
mutant BAD, or fragment of the mutant BAD, in the transformed cell. A BAD, or
fragment
of a BAD, can also be produced and expressed in the same manner described
above for the
mutant BAD, and fragment of a mutant BAD.
Any suitable expression vector may be employed to produce a recombinant BAD,
mutant BAD, or fragment of a BAD or mutant BAD, such as, for example, the
mammalian
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expression vector pCB6 (Brewer, 1994) or the E. coli pET expression vectors,
for example,
pET-30a (Studier et al., 1990). Other suitable expression vectors for
expression in
mammalian and bacterial cells that are known in the art are, for example,
expression vectors
for use in yeast or insect cells. For example, Baculovirus vectors and
expression systems can
be employed. A BAD, mutant BAD, or fragments of a BAD or mutant BAD, can also
be
prepared by chemical synthesis, by expression in in vitro translation systems
using a
polynucleotide template, or by isolation from biological samples. Chemical
synthesis of a
polypeptide can be performed, for example, by the classical Merrifeld method
of solid phase
peptide synthesis (Merrifeld, 1963), or by the FMOC strategy on a Rapid
Automated
Multiple Peptide Synthesis System (DuPont Company, Wilmington, Del.) (Caprino
and Han,
1972). Fragments, analogs, and modified forms of a BAD or mutant BAD can also
be
constructed or synthesized using methods well known in the art.
The terms "analog," "mutein," or "mutant" as used herein refer to polypeptides
which
are comprised of a segment of at least l0.amino acids that have substantial
identity to a
portion of the sequence of a naturally-occurring or wild-type polypeptide, for
example, a
naturally-occurnng or wild-type BAD. Typically, analog polypeptides comprise a
conservative amino acid substitution (or addition or deletion) with respect to
a naturally-
occurring or wild-type polypeptide sequence, or a mutant polypeptide, for
example, the
serine-substituted mutant BAD or fragment of a mutant BAD of the present
invention.
Analogs typically are at least 20 amino acids long, preferably at least 50
amino acids long or
longer up to the length of a full-length naturally-occurring or wild-type
polypeptide.
The discovery of the inhibitory effect of the phosphorylation of BAD on the
binding
of BAD to Bcl-X~ and/or Bcl-2 provides a new site for intervention in the
modulation of
apoptosis or programmed cell death. Such intervention can involve the
administration of, for
example, a mutant BAD, fragment of a mutant BAD, analog of a BAD, or fusion
thereof,
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having an amino acid other than serine at a position corresponding to position
118 of SEQ ID
NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3, and
preferably,
having an amino acid sequence that is substantially identical to SEQ ID NO:1,
SEQ ID N0:2,
or SEQ ID N0:3, respectively.
Modulation of the phosphorylation of BAD within a cell can be accomplished by
altering the intracellular phosphorylation state of BAD. The phosphorylation
state of many
polypeptides is dynamically controlled by both protein kinases and protein
phosphatases
(Cohen, 1989). The present work shows that both protein kinases and protein
phosphatases
can alter the phosphorylation state of BAD.
Phosphatases that dephosphorylate serine residues in polypeptides have been
extensively studied and both inhibitors and activators have been reported (for
reviews, see
Wera and Hemmings, 1995; Shenolikar, 1995). Thus, either inhibitors or
activators can be
used to modulate, increase, or decrease, including diminish, the ability of
intracellular
phosphatase to remove the phosphate from the serine at a position
corresponding to position
118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of SEQ ID
N0:3. Many
phosphatase inhibitors and activators are known and these can be readily
screened by one
skilled in the art for activity, for example, by determining the effect of a
test agent on the in
vitro or in vivo cleavage, by phosphatase activity, of a radiolabeled
phosphate group from the
serine at position corresponding to position 118 of SEQ ID NO:1, position 155
of SEQ ID
N0:2, or position 113 of SEQ ID N0:3 (see, e.g., Matthews, 1995; Shenolikar,
1995). It is
preferred that the inhibitors and activators have selective actions on the
phosphatase(s) acting
upon the BAD, or fragments of BAD, of the present invention.
Similarly, inhibitors and activators of protein kinases are known and can be
used as
therapeutic agents (see, e.g., Levitski, 1994). Thus, either kinase inhibitors
or activators can
act to modulate, increase, or decrease, including diminish, the action of
intracellular kinases
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which phosphorylate a BAD, or fragment of a BAD, at a serine at a position
corresponding to
position 118 of SEQ ID NO:1, position 155 of SEQ ID N0:2, or position 113 of
SEQ ID
N0:3. It is preferred that the inhibitors and activators have selective
actions on the kinase(s)
acting upon the BAD, or fragment of BAD, of the present invention. It has been
demonstrated that kinases act selectively and that neither phosphokiriase C
(PKC) nor RAF1
could phosphorylate BAD, in vitro, at the serine at position 118 of SEQ ID
NO:1, position
155 of SEQ ID N0:2, and position 113 of SEQ ID N0:3. However, heart muscle
kinase
(HMK), a form of phosphokinase A, could phosphorylate BAD, in vitro, at the
serine at
position 118 of SEQ ID NO:I, position 155 of SEQ ID N0:2, and position 113 of
SEQ ID
N0:3. Thus, inhibitors and activators effective in modulating, increasing, or
decreasing,
including diminishing, the phosphorylation state of BAD could be tested for
their effect on
BAD phosphorylation in vitro using HMK, for example, as a selective serine
kinase that
phosphorylates a serine at a position corresponding to position 118 of SEQ ID
NO:l, position
155 of SEQ ID N0:2, or position 113 of SEQ ID N0:3. Alternatively, in vivo
testing could
be done using a number of experimental approaches, and one example, as
reported herein,
utilizes the endogenous phosphorylation of BAD upon re-addition of IL-3 after
withdrawal
for two hours. Such standard testing systems could be used to test candidate
compounds as
inhibitors or activators of BAD phosphorylation. PMA promotes BAD
phosphorylation,
whereas staurosporin inhibits BAD phosphorylation.
It may be desirable to modulate or decrease, including diminish, the amount of
BAD
that is able to bind to Bcl-XL and/or Bcl-2 in the cells. Such as in the
treatment of diseases
involving overexpression or inappropriate expression of BAD, or the active
form of BAD, at
any level for which decreasing the amount of BAD can interfere with apoptosis
and promote
cell survival. In such disease conditions, treatments to modulate or decrease,
including
diminish, non-phosphorylated BAD can be used. Such treatments can involve
administration
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of BAD serine phosphatase inhibitors or selective serine kinase activators
that phosphorylate
the serine at a position corresponding to position 118 of SEQ ID NO:1, 155 of
SEQ ID N0:2,
and/or 113 of SEQ ID N0:3 to decrease the ability of BAD to bind to Bcl-X~
and/or Bcl-2.
Such treatment would be useful in diseases such as, for example,
immunodeficiency diseases,
senescence, neurodegenerative disease, ischemic cell death, reperfusion cell
death, infertility
and wound-healing.
Conversely, in the treatment of diseases involving underexpression or
inappropriately
low levels of active BAD, it may be desirable to modulate and increase the
amount of BAD
that is able to bind to Bcl-XL and/or Bcl-2 in the cells. In such disease
conditions, treatments
to modulate or increase the non-phosphorylated, active BAD can be used. Such
treatments
can involve administration of BAD serine phosphatase activators or inhibitors
of kinase that
phosphorylate the serine at a position corresponding to position 118 of SEQ ID
NO:1,
position 155 of SEQ ID N0:2, andlor position 113 of SEQ ID N0:3 to increase
the ability of
BAD to bind to Bcl-X~ and/or Bcl-2. Such treatments would be useful in
diseases such as,
for example, cancer, viral infections, lymphoproliferative conditions,
arthritis, infertility,
inflammation and autoimmune diseases.
The BAD, mutant BAD, and fragments of BAD or mutant BAD, of the present
invention can be prepared by chemical synthesis; in recombinant cells
transformed with a
polynucleotide encoding at least one of such polypeptides; by expression in in
vitro
translation systems using a polynucleotide template encoding at least one of
such
polypeptides; or by isolation of such polypeptides from biological samples.
Phosphorylation
of one or more amino acid residues, such as to produce the phosphorylated
serine-containing
polypeptides of the present invention, can be accomplished by well known
methods in the art.
For example, the amino acid residues) can be phosphorylated prior to
polypeptide synthesis.
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In other methods the amino acids) is phosphorylated after synthesis of the
polypeptide, such
as in the case when a kinase such as HMK is used.
Derivatives of the BAD, mutant BAD, fragments of BAD or mutant BAD, and/or
phosphorylated forms of such polypeptides, of the present invention can
include non-peptide
substances possessing the biological properties of the BAD, mutant BAD,
fragments of BAD
or mutant BAD, and/or phosphorylated forms of such polypeptides, in inducing
and/or
promoting an apoptotic state and/or binding to Bcl-XL or Bcl-2. The techniques
for making
peptide mimetics are well known to those skilled in the art (see, e.g., Navia
and Peattie, 1993;
Olson et al., 1993). Typically, making peptide mimetics involves
identification and
characterization of the polypeptide target site as well as the polypeptide
ligand using X-ray
crystallography and nuclear magnetic resonance. For example, the amino acid
sequence of
two marine BAD (SEQ ID N0:2 and SEQ ID N0:3) and a human BAD (SEQ ID NO:1),
containing a BH3 domain and containing a phosphorylation site at the serine at
position 155
of SEQ ID NO:1, position 118 of SEQ N0:2, and position 113 of SEQ ID N0:3,
respectively, has been identified. Using this information along with
computerized molecular
modeling, a pharmacophore hypothesis can be developed and compounds can be
made and
tested in an assay system as described herein or known in the art.
The BAD, mutant BAD, and fragment of BAD or mutant BAD, of the present
invention can also be used to detect new polypeptides as well as non-peptide
compositions
capable of associating with or binding to Bcl-XL and/or Bcl-2 and thereby
acting as inhibitors
to the binding of BAD to Bcl-X,_, and/or Bcl-2, for example, by using a
standard radioligand
assay system (see, e.g., Bylund and Toews, 1993). Such inhibitors could serve
to remove any
apoptotic inducing or modulating effect that the binding of BAD to Bcl-X~
and/or Bcl-2
might have. In one embodiment, the inhibitors can be polypeptides that also
contain the
conserved serine residues as described above, including BAD, or a fragment of
BAD. It is
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also possible to utilize as inhibitory polypeptides having a modified or
substituted amino acid
or non-amino acid residue at a position corresponding to position 118 of SEQ
ID NO:1,
position 155 of SEQ ID N0:2, and/or position 113 of SEQ ID N0:3, for example,
in place of
a serine residue, such that the polypeptide containing the modified amino acid
or non-amino
acid residue functions in the same way as dephosphorylated BAD, in that it
binds to Bcl-X,,
and/or Bcl-2, and thereby displaces BAD.
The radioligand assays useful in screening for inhibitors of the binding of
BAD to
Bcl-XL and/or Bcl-2 can involve preparation of a radiolabeled form of BAD,
mutant BAD, or
fragment of BAD or mutant BAD, capable of binding to Bcl-X~ and/or Bcl-2
using, for
example, either a 3H or'25I according to standard methods. For example, the
Bolton Hunter
Reagent can be used (ICN Chemicals, Radioisotope Division, Irvine, Calif.).
The
radiolabeled BAD ligand binds to the Bcl-XL and/or Bcl-2 immobilized to a
substrate such as
in a standard ELISA-style plate assay. The amount of bound and/or free
radiolabeled ligand
is then measured (see, e.g., Slack et al., 1989; Dower et al., 1989).
Alternatively, the Bcl-XL
and/or Bcl-2 can be radiolabeled and the BAD, mutant BAD, or fragment of BAD
or mutant
BAD, immobilized to a substrate. In a variation of this approach, the binding
assay is
performed with soluble, non-immobilized BAD, mutant BAD, or fragments of BAD
or
mutant BAD, and Bcl-XL and/or Bcl-2. Competitive inhibition of the binding of
the
radiolabeled BAD ligand to Bcl-XL and/or Bcl-2 on addition of a test compound
can be
evaluated by standard methods of analysis (see, e.g., Rovati, 1993).
The present invention also includes therapeutic or pharmaceutical compositions
comprising an active agent which is: a phosphatase inhibitor or activator; a
kinase inhibitor
or activator; or a BAD, mutant BAD, fragment of a BAD or mutant BAD; or a
phosphorylated BAD, mutant BAD, or fragment of a BAD; for treating diseases or
disease
conditions in which the propensity for cell death can be advantageously
modulated, and
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methods of making and using such compositions. These compositions and methods
are
useful for treating a number of diseases such as, for example, neoplasia,
certain viral
infections (e.g., Epstein-Barr virus), lymphoproliferative conditions,
arthritis, inflammation,
autoimmune diseases and the like resulting from an inappropriate decrease in
cell death as
well as diseases such as, for example, immunodeficiency diseases, senescence,
neurodegenerative disease, ischemic cell death, reperfusion cell death,
infertility, wound-
healing and the like resulting from an inappropriate increase in cell death.
Treatment can also
involve administration to affected cells ex vivo.
The therapeutic or pharmaceutical compositions of the present invention can be
administered by any suitable route known in the art including for example
intravenous,
subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or
administration to
cells in ex vivo treatment protocols. Administration can be either rapid as by
injection or over
a period of time as by slow infusion or administration of a slow release
formulation. For
treating tissues in the central nervous system, administration can be by
injection or infusion
into the cerebrospinal fluid (CSF). When it is intended that the active agent
be administered
to cells in the central nervous system, administration can be with one or more
agents capable
of promoting penetration of the active agent across the blood-brain barrier
(see, e.g., Friden et
al., 1993). Furthermore, BAD, mutant BAD, fragments of BAD or mutant BAD, and
serine-
phosphorylated forms of such polypeptides, can be stably linked to a polymer
such as
polyethylene glycol to obtain desirable properties of solubility, stability,
half life and other
pharmaceutically advantageous properties (see, e.g., Davis et al., 1978;
Burnham, 1994).
Furthermore, the active agent can be in a composition which aids in delivery
into the
cytosol of a cell. For example, the peptide may be conjugated with a Garner
moiety such as a
liposome that is capable of delivering the peptide into the cytosol of a cell.
Such methods are
well known in the art (see, e.g., Amselem et al., 1993). Alternatively, the
active agent can be
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modified to include specific transit peptides or fused to such transit
peptides which are
capable of delivering the BAD, mutant BAD, or fragment of BAD or mutant BAD,
of the
present invention into a cell. In addition, such polypeptides can be delivered
directly into a
cell by microinjection.
The phosphatase inhibitors and activators, and kinase inhibitors and
activators, can
also be linked or conjugated with agents that provide desirable pharmaceutical
or
pharmacodynamic properties as described above, such as the coupling of the
active substance
to a compound which promotes penetration or transport across the blood-brain
barrier or
stably linking the active substance to a polymer to obtain desirable
properties of solubility,
stability, half life and the like.
The compositions are usually employed in the form of pharmaceutical
preparations.
Such preparations are made in a manner well known in the pharmaceutical art.
One preferred
preparation utilizes a vehicle of physiological saline solution, but it is
contemplated that other
pharmaceutically acceptable carriers such as physiological concentrations of
other non-toxic
salts, five percent aqueous glucose solution, sterile water or the like may
also be used. It may
also be desirable that a suitable buffer be present in the composition. Such
solutions can, if
desired, be lyophilized and stored in a sterile ampoule ready for
reconstitution by the addition
of sterile water for ready injection. The primary solvent can be aqueous or
alternatively non-
aqueous. The active agent can also be incorporated into a solid or semi-solid
biologically
compatible matrix which can be implanted into tissues requiring treatment.
The carrier can also contain other pharmaceutically-acceptable excipients for
modifying or maintaining the pH, osmolarity, viscosity, clarity, color,
sterility, stability, rate
of dissolution, or odor of the formulation. Similarly, the carrier may contain
still other
pharmaceutically-acceptable excipients for modifying or maintaining release or
absorption or
penetration across the blood-brain barrier. Such excipients are those
substances usually and
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customarily employed to formulate dosages for parenteral administration in
either unit dosage
or multi-dose form or for direct infusion by continuous or periodic infusion.
Dose administration can be repeated depending upon the pharmacokinetic
parameters
of the dosage formulation and the route of administration used.
It is also contemplated that certain formulations containing the active agent
may be
administered orally. Such formulations are preferably encapsulated and
formulated with
suitable carriers in solid dosage forms. Some examples of suitable carriers,
excipients, and
diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium
phosphate, alginates, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone,
cellulose, gelatin, syrup, methyl cellulose, methyl- and
propylhydroxybenzoates, talc,
magnesium, stearate, water, mineral oil, and the like. The formulations can
additionally
include lubricating agents, wetting agents, emulsifying and suspending agents,
preserving
agents, sweetening agents or flavoring agents. The compositions may be
formulated so as to
provide rapid, sustained, or delayed release of the active ingredients after
administration to
the patient by employing procedures well known in the art. The formulations
can also
contain substances that diminish proteolytic degradation and/or substances
which promote
absorption such as, for example, surface active agents.
The specific dose is calculated according to the approximate body weight or
body
surface area of the patient or the volume of body space to be occupied. The
dose will also be
calculated dependent upon the particular route of administration selected.
Further refinement
of the calculations necessary to determine the appropriate dosage for
treatment is routinely
made by those of ordinary skill in the art. Such calculations can be made
without undue
experimentation by one skilled in the art in light of the activity disclosed
herein in assay
preparations of target cells. Exact dosages are determined in conjunction with
standard dose-
response studies. It will be understood that the amount of the composition
actually
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administered will be determined by a practitioner, in light of the relevant
circumstances
including the condition or conditions to be treated, the choice of composition
to be
administered, the age, weight, and response of the individual patient, the
severity of the
patient's symptoms, and the chosen route of administration. Whereas typically
the patient as
referenced herein is human, nevertheless, the formulations and methods herein
can be
suitably prepared and used for veterinary applications in addition to human
applications and
the term "patient" as used herein is intended to include human and veterinary
patients.
In a number of circumstances it would be desirable to determine the level of a
phosphorylated BAD with respect to the non-phosphorylated BAD in a cell. This
would
provide an assessment of the apoptotic status of the cell and allow the design
of a rational
treatment program designed to change the level and/or ratio of phosphorylated
to non-
phosphorylated BAD. A high level of non-phosphorylated BAD, or a high ratio of
non-
phosphorylated BAD to phosphorylated BAD, might indicate an increase in
apoptotic activity
in the cell, or progression of the state of apoptosis in the cell, and could
indicate the need for
treatment to decrease the non-phosphorylated BAD, or the ratio of non-
phosphorylated BAD
to phosphorylated BAD. Conversely, low levels of non-phosphorylated BAD or a
low ratio
of non-phosphorylated BAD to phosphorylated BAD could indicate the need to
increase
either the non-phosphorylated BAD or the ratio of non-phosphorylated BAD to
phosphorylated BAD.
Furthermore, in the treatment of disease conditions, compositions containing
BAD
can be administered exogenously and it would likely be desirable to achieve
certain target
levels of BAD, as well as a ratio of non-phosphorylated to phosphorylated BAD
in sera, in
any desired tissue compartment or in the affected cells or tissue. It would,
therefore, be
advantageous to be able to monitor the levels of non-phosphorylated and
phosphorylated
BAD in a patient or in a biological sample, including a tissue biopsy sample
obtained from a
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patient and, in some cases, it might also be desirable to monitor the level of
other members of
the Bcl-2 family, including Bcl-X,, and/or Bcl-2. Accordingly, the present
invention also
provides methods for detecting the presence of BAD, and the ratio of non-
phosphorylated to
phosphorylated BAD, in a cell or a population of cells or in a sample from a
patient.
The term "detection" as used herein, in the context of detecting the presence
of non-
phosphorylated and phosphorylated BAD in a patient, is intended to include the
ability to
determine the amount of, or distinguish, non-phosphorylated and phosphorylated
BAD; to
determine the amount of, or distinguish from other polypeptides, expressed
and/or post-
translationally modified BAD; distinguish non-phosphorylated and
phosphorylated forms of
BAD from each other, and from other members of the Bcl-2 family; estimate the
probable
outcome of a disease involving non-phosphorylated and phosphorylated BAD;
estimate the
prospect for recovery; determine the level of non-phosphorylated and
phosphorylated BAD
over a period of time as a measure of the status of a disease or condition;
and/or monitor the
phosphorylated and non-phosphorylated level of BAD for determining a preferred
therapeutic
regimen for the patient.
To detect the presence and level of non-phosphorylated and phosphorylated BAD
in a
cell or population of cells or patient, a sample is obtained from the
population of cells or from
the patient. The sample can be a population of cells, a tissue biopsy sample,
a sample of
blood, or a cell fraction from blood, plasma or the like. When the sample is
from a patient
any of a variety of tissues known to express BAD can serve as the source of
cells for testing
as can a sample or biopsy from a diseased tissue such as a neoplasia. When
assessing
peripheral levels of BAD, the sample can be a sample of cells obtained from
blood or a cell-
free sample such as plasma or serum.
The present invention further provides for methods to detect the presence of
the non-
phosphorylated and phosphorylated forms of BAD in a sample obtained from a
patient. Any
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method known in the art for detecting proteins can be used. Such. methods
include, but are
not limited to immunodiffusion, immunoelectrophoresis, immunochemical methods,
binder-
ligand assays, immunohistochemical techniques, agglutination and complement
assays (for
example see Sites and Terr, eds., 1991). Preferred are binder-ligand
immunoassay methods,
including reacting antibodies with an epitope or epitopes of BAD and
competitively
displacing a labeled BAD or derivative thereof. The measurement of levels of
phosphorylated BAD can be by any of a variety of methods. A particularly
useful method
based on the work described herein would involve the determination of the
amount of
phosphorylated BAD bound to Bcl-XL and/or Bcl-2. This can be done by
immunoprecipitation and western blot analysis using, for example, anti-BAD,
anti-serine-
phosphorylated-BAD, anti-Bcl-XL, and/or anti-Bcl-2 antibodies, all of which
can be prepared
by known methods. In addition, recombinant BAD tagged with the hemagglutinin
("HA")
epitope can be immunoprecipitated or probed on a western blot using anti-HA
antibody.
Also, recombinant BAD tagged with a glutathione S-transferase ("GST") moiety
can be
precipitated using glutathione beads. Alternatively, serine-phosphorylated and
-
unphosphorylated BAD can be determined by using anti-BAD and anti-serine-
phosphorylated-BAD antibodies in an immunoassay method as described below.
Such methods of detection described above can also be used to detect the
phosphorylation state and/or the presence of a mutant BAD, or fragment of a
BAD or mutant
BAD, including a recombinant form thereof.
Polyclonal antibodies can be prepared by immunizing rabbits or other animals
by
injecting antigen followed by subsequent boosts at appropriate intervals. The
animals are
bled and sera assayed against the purified BAD, mutant BAD, or fragments of
the BAD or
mutant BAD of the present invention, usually by enzyme-linked immunosorbent
assay
(ELISA) or by bioassay based upon the ability to accelerate apoptosis in
cells. Monoclonal
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antibodies can be prepared by methods known in the art, such as by the method
of Milstein
and Kohler by fusing splenocytes from immunized mice with continuously
replicating tumor
cells such as myeloma or lymphoma cells (Milstein and Kohler, 1975; Gulfre and
Milstein,
1981). The hybridoma cells formed in this manner are then cloned by limiting
dilution
methods and the supernatants assayed for antibody production ELISA,
radioimmunoassay
(RIA), or bioassay.
Numerous competitive and non-competitive polypeptide binding immunoassays are
well known in the art. Antibodies employed in such assays may be unlabeled,
for example as
used in agglutination tests, or labeled for use in a wide variety of assay
methods. Labels that
can be used include, for example, radionucleides, enzymes, fluorescers,
chemiluminescers,
enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the
like for use in
RIA, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays and the like.
Polyclonal or monoclonal antibodies directed against a BAD, mutant BAD, a
fragment of a
BAD or mutant BAD, or an epitope thereof, can be made for use in immunoassays
by any of
1 S a number of methods known in the art.
Immunoprecipitating BAD heterodimers from cells expressing BAD and other
polypeptides that bind to BAD, for example, members of the Bcl-2 family,
including Bcl-XL,
and Bcl-2, can be accomplished by methods well known in the art. For example,
a
polynucleotide encoding a BAD can be inserted into a plasmid expression vector
encoding
the GST moiety and the amino acid sequence of the heart muscle kinase (HMK)
phosphorylation target sequence, so that the encoded BAD is operably linked,
in-frame, to the
GST moiety and HMK target sequence. Cells, for example E. coli, can then be
transformed
with such a plasmid vector and produce a BAD which is fused to a GST moiety
and the HMK
target sequence. The resulting polypeptide can be purified after
overexpression in E. coli by
standard methods, for example, using GST agarose beads. The purified GST-BAD
can then
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be labeled in vitro, for example, with y-32P-ATP and heart muscle kinase to
produce
radiolabeled, phosphorylated GST-BAD.
Such methods for immunoprecipitation as described above can also be used to
immunoprecipitate heterodimers in cells expressing a mutant BAD, or fragments
of a BAD or
mutant BAD, and polypeptides that bind to such a mutant BAD, or fragments of a
BAD or
mutant BAD, and forming heterodimers.
An expression library, such as a cDNA expression library, can then be screened
with
the radiolabeled, phosphorylated GST-BAD for clones that produce a polypeptide
which
binds to the GST-BAD (see Neilsen, 1991). The identified clone can then be
isolated and the
DNA sequence of the clone determined by standard methods known in the art. One
skilled in
the art would know how to predict the amino acid sequence of a potential BAD
from the
DNA sequence of the clone (Muslin et al., 1996).
Such methods for screening, cloning, and sequencing as described above can
also be
applied to phosphorylate and screen with a radiolabeled mutant BAD, or
fragment of a BAD
or mutant BAD.
For metabolic labeling, cells can be labeled, for example, either in phosphate-
free
media (e.g., RPMI 1640 media) with 32P-orthophosphate (e.g., 1 mCi/106 cells)
or in
methionine-free media (e.g., RPMI 1640 media) with 35S-methionine (e.g., 200
mCi/106
cells). The cells can then be lysed (e.g., in either in 137 mM NaCI, 20 mM
Tris (pH 8.0), 1.5
mM MgCl2, 1 mM EDTA, 50 mM NaF, 0.2%-0.5% NP40 containing aprotinin (0.15
U/ml),
20 mM leupeptin, and 1 mM phenylmethysulfonyl fluoride) for co-
immunoprecipitation, or
for direct immunoprecipitation (in RIPA buffer). The lysates are first
cleared, for example,
with protein A beads (e.g., 30 min), followed by incubation with an antibody
(e.g., 1.5 hour,
on ice). The antibody complexes are then captured with protein A beads (e.g.,
1 hour). The
immunoprecipitate is then washed (e.g., with 0.2% NP-40 lysis buffer),
resuspended in
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loading buffer, and separated by SDS-PAGE. The gel is then treated, for
example, with
fluorography and visualized by autoradiography, or transferred to a
nitrocellulose membrane
for further immunoblot analysis. For western blots, lysates are separated by
SDS-PAGE, and
transferred, typically, to a nitrocellulose membrane. The membrane is first
blocked with a
S milk solution (e.g., 3% milk solution, 1 hour), followed by incubation with
primary and
secondary antibodies (e.g., for one hour each), and finally developed by
enhanced
chemiluminescence using, for example, a commercially available kit (e.g., a
kit supplied by
Amersham Pharmacia Biotech, Piscataway, NJ).
Plasmids capable of expressing a fusion polypeptide comprising a naturally-
occurring
or wild-type BAD, mutant BAD, or fragment of a BAD or mutant BAD; or a fusion
polypeptide capable of binding to a BAD, mutant BAD, or fragment of a BAD or
mutant
BAD, for example, Bcl-XL or Bcl-2, can be constructed by inserting a
polynucleotide
encoding the amino acid sequence of at least one of such polypeptides into an
expression
vector. A fusion polypeptide can be constructed, for example, by inserting a
polynucleotide
encoding the amino acid sequence of a first polypeptide into an expression
vector containing
a polynucleotide encoding the amino acid sequence of a second polypeptide,
such that the
amino acid sequence of the first polypeptide is in-frame with, and operably
linked to the
amino acid sequence of the second polypeptide. For example, the amino acid
sequence of a
BAD, mutant BAD, or fragment of a BAD or mutant BAD, can be operably linked to
the
amino acid sequence of the HA epitope. In this manner a plasmid is constructed
that is
capable of expressing a fusion polypeptide that comprises a BAD, mutant BAD,
or fragment
of a BAD or mutant BAD, that is tagged with the HA epitope. Using the same
approach,
plasmids capable of expressing a fusion polypeptide that comprises a
polypeptide that can
bind to a BAD, mutant BAD, or fragment of a BAD or mutant BAD, and is operably
linked
with the HA epitope or other polypeptide, can be constructed. Examples of
polypeptides that
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bind to BAD are Bcl-X~ and Bcl-2. Also, plasmids capable of expressing a
naturally-
occurring or wild-type BAD, or polypeptide that binds to a naturally-occurring
or wild-type
BAD, can be constructed by inserting a polynucleotide encoding the amino acid
sequence of
at least one of such polypeptides into an expression vector. The methods and
vectors for
constructing such plasmids are well known to those skilled in the art.
Using standard methods known in the art, cells (e.g., FLs.iz cells) can be
transformed
(e.g., using electroporation) with the expression plasmid containing the
fusion polypeptide.
More particularly, cells can be co-transformed with a plasmid capable of
expressing a BAD,
mutant BAD, or fragment of a BAD or mutant BAD, and another plasmid capable of
expressing a polypeptide capable of binding to a BAD, mutant BAD, or fragment
of a BAD
or mutant BAD. Thereafter, (e.g., forty-eight hours after transformation) a
limited dilution of
the cells can be performed in selection medium (e.g., in medium containing
G418). Single-
cell clones, expressing the polypeptide encoded by the polynucleotide
contained in the
plasmid used to transform the cells and detected by western blot analysis, can
be selected
several days later (e.g., 7-10 days later). Lysates from cells expressing the
fusion polypeptide
can be analyzed by immunoprecipitation and/or western blot analysis using, for
example,
anti-BAD antibody, anti-HA antibody, anti-Bcl-XL, and/or anti-Bcl-2 antibody.
The phosphorylation of a BAD, mutant BAD, or fragment of a BAD or mutant BAD,
can be detected in vivo, for example, by radiolabeling the BAD, mutant BAD, or
fragment of
a BAD or mutant BAD expressed in cells. The cells are first transformed with a
plasmid
capable of expressing BAD, mutant BAD, or fragment of a BAD or mutant BAD, and
then,
using standard methods, the polypeptides expressed in the transformed cells
are labeled, for
example, with 32P-orthophosphate. Thereafter, the cells are lysed and the BAD,
mutant BAD,
or fragment of a BAD or mutant BAD, expressed in the transformed cells can be
immunoprecipitated with, for example, anti-BAD antibody, resolved on an SDS-
PAGE gel,
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and analyzed, for example, by fluorography. Alternatively, a sample of the
labeled cell lysate
can be analyzed by western blot with an anti-BAD antibody. In particular, the
serine
phosphorylation of a BAD, mutant BAD, or fragment of a BAD or mutant BAD can
be
detected, in vivo, using the methods described above.
32P-radiolabeled BAD, mutant BAD, or fragment of a BAD or mutant BAD, bound to
another polypeptide, for example, Bcl-XL or Bcl-2, can be immunoprecipitated
from cells
expressing BAD, mutant BAD, or a fragment of a BAD or mutant BAD, and a
polypeptide
that binds to BAD, mutant BAD, or fragment of a BAD or mutant BAD as described
above.
Using standard protocols known to those skilled in the art, the
immunoprecipitated BAD,
mutant BAD, or fragment of a BAD or mutant BAD, can then be treated with
phosphatase.
For example, protein A-BAD complexes can be suspended in an appropriate buffer
(e.g., 500 ml of 40 mM PIPES piperazine-N,N'-bis(2-thansulfonic acid) buffer
(pH 6.0)
containing 1 mM DTT, aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM
phenylmethysulfonyl fluoride) and treated with a commercially available
phosphatase (e.g.,
potato acid phosphatase, Sigma). Samples containing phosphatase inhibitors can
be
supplemented with, for example, 50 mM sodium fluoride, 5 mM sodium phosphate,
10 mM
sodium pyrophosphate, 10 mM ammonium molybdate, 5 mM EDTA and 5 mM EGTA. The
protein A beads can then be pelleted by centrifugation, washed (e.g., with NP-
40 lysis
buffer), resuspended in a gel loading buffer, and the bound proteins can be
examined by
western blot analysis. BAD can resolve on a gel as a doublet; phosphorylated
BAD resolves
as a higher molecular weight band relative to unphosphorylated or
dephosphorylated BAD,
which migrates as a lower molecular weight relative to phosphorylated BAD.
Treatment of
immunoprecipitated BAD with potato acid phosphatase (PAP) can eliminate the
higher
molecular weight band by converting the higher molecular weight phosphorylated
BAD to
the lower molecular weight dephosphorylated BAD.
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The immunoprecipitated 32P-radiolabeled BAD, mutant BAD, or fragment of a BAD
or mutant BAD can be separated by SDS-PAGE and transferred to a nitrocellulose
membrane. Membrane slices containing either the phosphorylated,
dephosphorylated or
unphosphorylated BAD, mutant BAD, or fragment of a BAD or mutant BAD, can be
isolated
S from the membrane, and digested with trypsin under appropriate conditions.
Standard
methods known to those skilled in the art can be used to isolate the
nitrocellulose membrane-
bound BAD, mutant BAD, or fragment of a BAD or mutant BAD, and digest it with
commercially available trypsin (e.g., Worthington Biochemicals). The peptides
resulting
from the trypsin digest can then be dried, washed, hydrolyzed, and resolved by
TLC, using
standard methods known to those skilled in the art (see Boyle et al., 1991).
The location of
phosphoamino acids can then be determined, for example, by ninhydrin staining
and
autoradiography. Phosphoamino acid analysis of the BAD doublet revealed that
BAD was
exclusively phosphorylated at serine residues.
Using methods well known in the art, for example, using two-dimensional
tryptic
peptide mapping, it is possible to identify the specific amino acids and their
positions in the
amino acid sequence of a BAD, mutant BAD, or fragment of a BAD or mutant BAD,
that
were phosphorylated in vivo. For example, by generating two dimensional
tryptic peptidic
maps of a BAD the precise sites of serine phosphorylation can be identified.
After the BAD
is immunoprecipitated from 32P-orthophosphate labeled cells, separated by SDS-
PAGE,
transferred to a nitrocellulose membrane, and enzymatically digested with
trypsin, the tryptic .
peptides can be separated, horizontally, in the first dimension (e.g., in pH
8.9 buffer; see
Boyle et al., 1991) by TLC (e.g., utilizing a HTLE-7000 apparatus and
electrophoresing for
minutes at 1000 V at 4°C). Separation in the second dimension can
performed by
ascending chromatography (e.g., in 37.5% n-butanol, 25% pyridine and 7.5%
acetic acid for
25 10 hours). The 32P-phosphopeptides can then be visualized by
autoradiography and the non-
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radiolabeled phosphopeptides can be visualized, for example, with ninhydrin
staining. The
labeled peptides can then be eluted (e.g., in pH 1.9 buffer) from the TLC
plates, conjugated to
membrane (e.g., Sequelon-AA membrane (Perspective Biosystem, Framingham,
Mass.),
using methanol:waterariethylamine:phenylisothiocyanate, in a 7:1:1:1 ratio, at
55°C). The
eluted peptides can then be either directly applied onto a TLC plate or
treated with performic
acid to uniformly oxidize the peptides before application to the TLC plate.
The peptides are
then separated according to charge in the first dimension, followed by
hydrophobicity in the
second dimension, as above. The 2D maps of the upper and lower molecular
bands,
representing phosphorylated and unphosphorylated or dephosphorylated BAD
species can
then be resolved. The resolved 3zP-labeled tryptic peptides can then be
subjected to manual
Edman degradation performed as previously described (Boyle et al., 1991; Luo,
et al., 1991)
and the identity and position of the specific amino acids in the BAD amino
acid sequence
determined.
EXAMPLES
Preferred embodiments of the invention are described in the following
examples. The
Examples provide exemplary guidance for practicing the various aspects of the
invention
described above. Other embodiments within the scope of the claims herein will
be apparent
to one skilled in the art from consideration of the specification or practice
of the invention as
disclosed herein. It is intended that the specification, together with the
examples, be
considered exemplary only, with the scope and spirit of the invention being
indicated by the
claims that follow the examples. Unless otherwise indicated, all parts,
percents and ratios are
by volume.
Additional details for methods employed herein and normally used in the art
can be
found, for example, in the cited references. The inventions described herein
are useful for
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investigating and controlling apoptosis events, including situations where
apoptosis is
desirably enhanced and situations where apoptosis is desirably inhibited.
Specific utilities
and applications are apparent to the ordinary skilled artisan.
Example 1
Phosphorylation of BAD at a novel site renders BAD unable to associate with
Bcl-XL
In order to study the role of BAD phosphorylation in the regulation of tumor
cell
survival, mammalian expression vectors encoding the shorter murine BAD of SEQ
ID N0:3
or a mutant BAD derived from the shorter murine BAD of SEQ ID N0:3 were
constructed,
wherein the amino acid sequence of the mutant BAD contained an amino acid
substitution at
the serine at a position corresponding to the serine at position 112 ("Serl
12") and/or position
136 ("Ser136") of SEQ ID N0:2. The mutant BAD S 112A has an amino acid
sequence
wherein the serine at a position corresponding to Serl 12 is substituted with
alanine; the
mutant BAD S 136A has an amino acid sequence wherein the serine at a position
corresponding to Ser136 is substituted with alanine; and the mutant BAD S
112A/S 136A has
an amino acid sequence wherein the serines at positions corresponding to both
Serl 12 and
Ser136 are substituted with alanine.
Mouse FLS.i2 cells were then transfected with the expression vector encoding
the
shorter murine BAD of SEQ ID N0:3 or one of the three BAD mutants. Total mRNA
was
isolated from the transfected FLs.iz cells, and reverse transcribed into cDNA.
The cDNA was
then subcloned into the expression vector pcDNA3 so that the amino acid
sequence of the
BAD or mutant BAD, encoded by the subcloned cDNA, was in-frame and operably
linked to
an N-terminal hemagglutinin ("HA") epitope encoded by the expression vector.
The
expression vectors encoding the HA-tagged shorter murine BAD and mutant BAD
were
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expressed in COS-7 cells. Whole cell lysates ("WCL") were then prepared from
the COS-7
cells expressing the polypeptides. The phosphorylation of the shorter murine
BAD of SEQ
ID N0:3 and mutant BAD were detected by a western blot of the WCL, probed with
an anti-
HA antibody ("anti-HA antibody"). Consistent with previous observations of
phosphorylated
BAD (Zha et al., 1996), the phosphorylation of the shorter murine BAD resulted
in a shift of
a lower molecular weight band, representing unphosphorylated shorter murine
BAD, to a
higher molecular weight band, representing phosphorylated shorter murine BAD.
Of note,
each of the BAD mutants resolved as a doublet on a 16% SDS-PAGE, even though
in the
amino acid sequence of each of the BAD mutants one or both of the serines
corresponding to
Serl 12 and Ser136 were mutated, i.e., substituted with alanine. The higher
molecular weight
band of the doublet comigrated on the SDS-PAGE with the band representing
phosphorylated
shorter murine BAD, and the lower molecular weight band of the doublet
comigrated on the
SDS-PAGE with the band representing unphosphorylated shorter murine BAD
(results not
shown). To verify that the higher molecular band in the doublet was a result
of
phosphorylation, COS-7 cells transfected with the HA-BAD S 112A/S 136A double
mutant
were lysed and treated with lambda phosphatase or a buffer control. The
proteins were
separated via SDS-PAGE, transfer to nitrocellulose and probed with an anti-HA
antibody.
The results demonstrated that the phosphatase treatment completely eliminated
the higher
molecular weight band of the doublet, suggesting the existence of a
phosphorylation site on
BAD that is distinct from either of the serines at positions corresponding to
Ser112 and
Ser136 (Fig. l, compare lanes 1 and 2).
In order to test whether the additional and novel phosphorylation site on BAD
has
functional significance, the effect on BAD binding to Bcl-X~ was examined.
Recombinant
Bcl-XL tagged with the glutathione S-transferase ("GST") moiety ("GST-Bcl-X~")
was
incubated with WCL prepared from COS-7 cells expressing the double mutant HA-
BAD
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S 112A/S 136A, and the HA-BAD S 112A/S 136A that bound to the GST-Bcl-XL was
precipitated with GST beads. In a western blot of the WCL incubated with GST-
Bcl-XL and
probed with anti-HA antibody, only the lower molecular weight band of the
doublet was
detected in the WCL where HA-BAD S 112A/S 136A was bound to the GST-Bcl-XL and
S precipitated with GST beads (Fig. 1, lane 3), suggesting that the
phosphorylation of BAD at
the additional and novel phosphorylation site prevents BAD heterodimer
formation with Bcl-
X~. No HA-BAD S 112A/S 136A was precipitated with GST beads from the WCL that
were
not incubated with GST-Bcl-XL (Fig. 1, lane 4).
Example 2
Localization and identification of the novel BAD phosphorylation site, Ser155
In order to localize the novel phosphorylation site, selected serine residues
in the
amino acid sequence of the shorter murine BAD of SEQ ID N0:3 were substituted
with
alanines, in addition to the serines at positions corresponding to Ser112 and
Ser136. Two
HA-BAD triple mutants were constructed. The triple mutant HA-BAD
S 112A/S 134A/S 136A has an amino acid sequence, wherein the serines at
positions
corresponding to Ser112 and Ser136, and the serine at position 134 ("Ser134"),
of SEQ ID
N0:2, are substituted with alanine. The triple mutant HA-BAD S 112A/S 136A/S
155A has an
amino acid sequence, wherein the serines at positions corresponding to Ser112
and Ser136, as
well as the serine at position 155 ("Ser155") of SEQ ID N0:2, are substituted
with alanine.
The correct DNA sequence encoding BAD or a mutant BAD was confirmed by DNA
sequencing. The DNA encoding BAD or a mutant BAD was then inserted into the
vector
pcDNA3, between the BamHI and EcoRI restriction enzyme sites, so that the
encoded amino
acid sequence of the BAD or mutant BAD was in-frame with and operably linked
to an HA
epitope at the amino terminus of the encoded BAD or mutant BAD. The DNA was
then used
to transform COS-7 cells.
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COS-7 cells were transfected with the expression vector encoding the BAD or a
mutant BAD. A western blot was performed on WCL prepared from the transfected
COS-7
cells expressing HA-BAD S 112A/S 136A, HA-BAD S 112A/S 134A/S 136A, or HA-BAD
S 112A/S 136A/S 1 SSA and treated with phosphatase or a buffer control. The
western blot was
S probed with anti-HA antibody. The results of the western blot demonstrated
that mutation of
the serine at a position in the amino acid sequence of the shorter murine BAD
(SEQ ID
N0:3) corresponding to Serl SS, eliminates the phosphorylation of BAD (Fig.
2(a), lanes S
and 6). However, mutation of the serine at a position in the amino acid
sequence of the
shorter murine BAD (SEQ ID N0:3) corresponding to Ser134 does not eliminate
the
phosphorylation of BAD (Fig. 2(a), lanes 3 and 4). Moreover, this experiment
confirmed that
mutation of the serines at positions in the amino acid sequence of the shorter
murine BAD
(SEQ ID N0:3) corresponding to Serl 12 and Serl36 do not eliminate the
phosphorylation of
BAD (Fig. 2(a), lane 2). These results indicate that the serine corresponding
to SerlSS is a
third and novel BAD phosphorylation site.
1 S A mammalian expression vector encoding mutant HA-BAD S 1 SSA, derived from
the
shorter murine BAD of SEQ ID N0:3, was constructed. HA-BAD S1SSA has an amino
acid
sequence wherein the serine at a position corresponding to SerISS is
substituted with alanine.
A western blot was performed on WCL prepared from cells expressing the shorter
murine
BAD (SEQ ID N0:3), HA-BAD S112A, HA-BAD S136A, or HA-BAD S1SSA, and probed
with anti-HA antibody. The results of the western blot analysis demonstrated
that a single
mutation of the amino acid sequence of the shorter murine BAD (SEQ ID N0:3),.
at the
serine at a position corresponding to SerlSS, dramatically reduced the
phosphorylation of the
murine BAD in HeLa cells (Fig. 2(b), lane S), as compared to a single mutation
of the serine
at a position corresponding to Ser112 or Ser136 (Fig. 2(b), lanes 3 and 4,
respectively).
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These results indicate that phosphorylation of the serine at a position
corresponding to Serl55
contributed significantly to the overall phosphorylation of BAD.
A further experiment demonstrated that growth factor-induced phosphorylation
of
BAD was also eliminated in the S 155A mutant. HeLa cells transiently
expressing HA-BAD
or mutant HA-BAD S 155A were cultured in the presence of epidermal growth
factor (EGF),
fetal calf serum (FCS), or a buffer control. A western blot was performed on
whole cell
lysates prepared from the cultures, and probed with anti-HA antibody. The
results of the
western blot analysis demonstrated that a single mutation of the amino acid
sequence of the
shorter murine BAD (SEQ ID N0:3), at the serine at a position corresponding to
Ser155,
dramatically reduced the growth factor-induced phosphorylation of the murine
BAD in HeLa
cells (Fig. 2(c), compare lanes 2 and 5), as well as the serum-induced
phosphorylation of the
murine BAD in HeLa cells (Fig. 2(c), compare lanes 3 and 6). These results
further indicate
that phosphorylation of the serine at a position corresponding to Ser155
contributed
significantly to the overall phosphorylation of BAD.
Notably, Ser155 is located at the center of the BH3 domain of the longer
murine BAD
(SEQ ID N0:2) (Fig. 3(a)), i. e., the domain that is involved in the
association of BAD with
Bcl-X,, (Ottilie et al., 1997; Zha et al., 1997). In contrast to BAD, other
members of the Bcl-
2 family have a glycine at a position corresponding to Ser155. Ser155 is also
within a PKA
consensus site.
NMR studies have revealed that BH3 forms an alpha helical structure, which
binds to
a hydrophobic cleft on the surface of Bcl-XL (Sattler et al., 1997). Whether
the addition of a
phosphate group on BAD Ser155 interferes with this interaction was tested by
measuring the
affinity of BAD BH3 peptides for Bcl-X~ in an in vitro competition binding
assay. Synthetic
BAD BH3 peptide, encompassing BAD residues 143 to 168, was incubated with
recombinant
GST-Bcl-XL at the indicated concentrations (Fig. 3(b)). The affinity of the
BAD BH3
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peptide for GST-Bcl-X~ was measured as the ability of the peptide to block
subsequent
binding of GST-Bcl-X,, to a BAK BH3 peptide when the reacted mixture was added
to a
microtiter plate pre-immobilized with a BAK BH3 peptide, as detected by ELISA
(triplicate
samples, +/- standard deviation). Both phosphorylated (BAD BH3-P) and
unphosphorylated
S (BAD BH3) BAD were assayed, as was a BAK BH3 peptide (residues 71-89) as a
positive
control. The unphosphorylated BAD BH3 peptide bound to Bcl-XL with an affinity
similar to
the BAK BH3 control peptide, however, the affinity of BAD phosphorylated on
Ser155 for
Bcl-XL was reduced by greater than 100-fold (Fig. 3(b)).
The ability of full-length BAD and mutant BAD S155A to bind Bcl-XL was also
determined (Fig. 3(c)). Full-length BAD ("WT") and mutant BAD S155A ("S155A")
were
produced by in vitro translation and labeled with 35S-methionine therein. The
labeled
proteins were then incubated with PKA ("+") in the presence of unlabeled ATP
or a buffer
control ("-"). Following the kinase reactions, aliquots of each sample of the
four samples
were incubated with either GST or GST-Bcl-XL. The protein complex were
captured on
glutathione-agarose beads. Proteins bound to the beads ("Bound") (lower panel)
and
samples of the reactions collected prior to incubation with the beads
("Total") (upper panel)
were analyzed by SDS-PAGE followed by autoradiography. The results demonstrate
that
phosphorylation by PKA induced a gel-mobility shift of wild-type BAD (Fig.
3(c), compare
lanes 1 and 2, and also lanes S and 6, upper panel), but not mutant S 155A BAD
(Fig. 3(c),
compare lanes 3 and 4, and also lanes 7 and 8, upper panel), and blocked the
ability of BAD
to bind to Bcl-XL (Fig. 3(c), lower panel). These results demonstrate that
phosphorylation on
Ser155 is sufficient to directly inactivate the heterodimerization function of
BH3 and provide
a biochemical mechanism for how the pro-apoptotic function of BAD is
suppressed by
Ser155 phosphorylation.
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EXAMPLE 3
Ser155 of BAD is phosphorylated by PKA in vitro
The amino acid sequence surrounding Serl SS of the longer murine BAD (SEQ ID
N0:2) was examined for the presence of serine-threonine kinase recognition
motifs. The
amino acid sequence LRRMSD (SEQ ID NO: 19) matched well with the consensus
recognition sequence of mammalian PKA, which is XRRXSX (Kemp and Pearson,
1990).
Based on this finding, PKA was tested to determine whether it could
phosphorylate BAD, in
vitro, at the serine at a position corresponding to Ser155.
Expression vectors encoding fusion polypeptides of the shorter murine BAD (SEQ
ID
N0:3) and BAD mutants derived from the shorter murine BAD, were constructed
such that
the encoded amino acid sequence of the BAD or mutant BAD was fused in-frame
and
operably linked to the GST moiety. The amino acid sequence of GST-BAD encodes
a GST-
tagged shorter murine BAD. The amino acid sequence of GST-BAD S 112A encodes a
GST-
tagged mutant BAD, wherein the serine at a position corresponding to Serl 12
is substituted
with alanine. The amino acid sequence of GST-BAD S136A encodes a GST-tagged
mutant
BAD, wherein the serine at a position corresponding to Ser136 is substituted
with alanine.
The amino acid sequence of GST-BAD S155A encodes a GST-tagged mutant BAD,
wherein
the serine at a position corresponding to Ser155 is substituted with alanine.
The amino acid
sequence of GST-BAD S 112A/S 136A encodes a GST-tagged mutant BAD, wherein the
serines at positions corresponding to Ser112 and Ser136 are substituted with
alanine. The
amino acid sequence of GST-BAD S112A/S136A/S155A encodes a GST-tagged mutant
BAD, wherein the serines at positions corresponding to position 112, position
136, and
position 155 of SEQ ID N0:2 are substituted with alanine.
The GST-tagged BAD and BAD mutants were then expressed individually in cells
and the purified GST-tagged polypeptides incubated with purified PKA, in the
presence of
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3ZP radiolabel. A western blot, probed with anti-BAD antibody, was performed
on the
incubated GST-tagged BAD and BAD mutants to determine whether PKA could
phosphorylate the BAD and/or mutant BAD in vitro. The GST-tagged BAD and BAD
mutants phosphorylated by PKA could be detected directly by autoradiography
due to the
incorporation of 3zP radiolabel when the polypeptides were phosphorylated
(Fig. 4, upper
panel), whereas the total amount of BAD and mutant BAD present in the in vitro
reaction
was detected by the anti-BAD antibody probe (Fig. 4, lower panel).
The results of the western blot demonstrate that the shorter murine BAD, and
BAD
mutants containing mutations at the serine at a position corresponding to Serl
12 and/or
Ser136, were good in vitro substrates for PKA (Fig. 4, lanes 1-3 and 5).
However, in
contrast, the BAD mutants containing a mutation at the serine at a position
corresponding
Ser155 were poor in vitro substrates for PKA. The mutation at the serine at a
position
corresponding to Ser155 dramatically reduced the phosphorylation of the single
mutant GST-
BAD S 1 SSA and abolished phosphorylation of the triple mutant GST-BAD
S 112A/S 135A/S 1 SSA (Fig. 4, lanes 4 and 6, respectively). Thus, the serine
at a position in
the amino acid sequence of the shorter murine BAD, corresponding to Ser155 is
a major site
of PKA ~phosphorylation.
The same western blot was then probed with anti-BAD antibody to confirm that a
similar amount of BAD and mutant BAD was added to each kinase reaction (Fig.
4, lower
panel).
Moreover, the in vitro phosphorylation of BAD by PKA was completely inhibited
by
PKI (data not shown), a PKA-specific inhibitor (Chijiwa et al., 1990). These
results indicate
that Ser155, of the longer murine BAD, is a major phosphorylation site of BAD
by PKA in
vitro.
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Example 4
Forskolin induces phosphorylation of BAD Ser155
The fact that PKA preferentially phosphorylated BAD at the serine at a
position
corresponding to Ser155 in vitro suggested that PKA may be the cellular kinase
responsible
for the phosphorylation of BAD at this phosphorylation site as well. To test
this possibility,
HA-BAD and mutants HA-BAD S 112A/S 136A and HA-BAD S 112A/S 136A/S 1 SSA were
transiently expressed in HeLa cells, and samples of the transfected cells were
treated with
Forskolin, an activator of adenylate cyclase (and, ultimately, PKA) (Fischer
et al., 1981).
A western blot analysis, probed with anti-HA antibody, was performed on
lysates
produced from the Forskolin treated and untreated (control) cells to determine
whether
Forskolin could induce the phosphorylation BAD or mutant BAD. The results of
the western
blot demonstrated that treatment with Forskolin induced a significant band-
shift' of HA-BAD
S 112A/S 136A (Fig. 5(a), lanes 2 and 3) that was rapid and sustained (Fig.
5(b)). Because
Forskolin is a known activator of the pathway leading to PKA activation, these
results
indicate that cellular PKA is responsible for phosphorylation of Ser155 of
BAD. Consistent
with this observation, treatment of the cells with the cell permeant cAMP
analog, 6-Bnz-
cAMP, in place of Forskolin; also led to Ser155 phosphorylation of BAD (see
Fig. 9(a)).
In contrast, the BAD triple mutant HA-BAD S 112A/S 136A/S 155A failed to show
such a band-shift, again indicating that Serl SS is the likely site of PKA
phosphorylation of
BAD in cells (Fig. 5(a), lanes 5 and 6).
An anti-phospho-Ser155 specific BAD antibody (see Materials and Methods) was
used to provide direct evidence for BAD phosphorylation on Ser155. HeLa cells
transiently
expressing HA-BAD, HA-BAD S155A, HA-BAD S112A/S136A, or HA-BAD
S112A/S136A/S155A were treated with Forskolin or a buffer control. A western
blot
analysis was performed on lysates prepared from these cells and probed with
either anti-
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phospho-Ser155 specific BAD antibody (Fig. 5(c), upper panels) or an anti-BAD
antibody
(Fig. S(c), lower panels). The failure of the anti-phospho-Ser155 specific BAD
antibody to
react with BAD S 155A and the BAD triple mutant, although the expression of
BAD and
BAD mutants were at similar levels (Fig. Sc, lower panels), provides direct
evidence for
S BAD phosphorylation on Ser155.
Example 5
G-protein coupled receptor ligand induces phosphorylation of BAD Ser155
Additional experiments were conducted to further identify extracellular
signaling
molecules that promote BAD phosphorylation on Ser155. Because adenylate
cyclase is
known to be a target of G protein-coupled receptors (GPCRs) (Linder and
Gilman, 1992),
which in turn catalyzes the formation of CAMP and eventual activation of PKA,
three GPCR
ligands were tested for their effect on BAD Ser155 phosphorylation: thyroid
stimulating
hormone (TSH), L-epinephrine (L-epi), and adrenocorticotropic hormone (ACTH).
The HA-BAD S 112A/S 136A double mutant was transiently expressed in HeLa
cells,
and transfected cell cultures were treated with each of the GPCR ligands,
individually and as
a group. A western blot analysis, probed with anti-HA antibody, was performed
on lysates
produced from the treated and untreated (negative control) cells. The results
of the western
blot demonstrated that treatment with L-epinephrine, either alone, or in
combination with TK
and ACTH, induced a significant band-shift of HA-BAD S112A/S136A (Fig. 6(a)
lanes 4
and 6). In contrast, neither TK (Fig. 6(a), lane 3) nor ACTH (Fig. 6(b), lane
5) alone induces
a similar band-shift of the BAD double mutant, an indication that the
activation of adenylate
cyclase by GPCR is specific to particular GPCR ligands (Fig. 5(a) lanes 5 and
6). Forskolin
(FK) and no treatment (-) were positive and negative controls, respectively
(Fig. 6(a), lanes
and 1, respectively). These results indicate that L-epinephrine is an
effective inducer of BAD
phosphorylation.
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To determine whether the effects of L-epinephrine were due to non-specific
phosphorylation of the BAD double mutant, four HA-BAD constructs (HA-BAD, HA-
BAD
S 112A, HA-BAD S 136A, and HA-BAD S 155A) were transiently expressed in HeLa
cells,
and samples of the transfected cells were treated with L-epinephrine.
Untreated controls
received no L-epinephrine. A western blot analysis, probed with anti-HA
antibody, was
performed on lysates produced from the treated and untreated (control) cells.
The results of
the western blot demonstrated that while HA-BAD, HA-BAD S 112A, and HA-BAD S
136A
all showed a significant band-shift in response to L-epinephrine stimulation,
the S 155A
single mutant only had a limited response (Fig. 6(b)), suggesting that Ser155,
and not Ser112
or Ser136, is the major phosphorylation site in cells in response to L-
epinephrine. This result
was consistent with the in vitro phosphorylation of BAD at Serl SS by PKA and
with the
induction of Ser155 phosphorylation by Forskolin, which together suggest the
cellular BAD
Ser155 kinase is a cAMP-dependent protein kinase that may be activated by G
protein-
coupled receptors. For HeLa cells as a particular example, the Ser155 kinase
could be
activated, sequentially, by the L-epinephrine receptor, i. e. the (3-
adrenergic receptor (Linder
and Gilman, 1992), stimulatory G proteins, adenylate cyclase and elevated
intracellular
cAMP.
Example 6
Forskolin and L-epinephrine induces phosphorylation of endogenous BAD
To rule out the possibility that the evidence for Forskolin- or L-epinephrine-
induced
BAD Ser155 phosphorylation was an artifact of BAD overexpression, the effects
of these
agents on endogenous BAD were examined. Non-transfected HeLa cells were
treated with
Forskolin or L-epinephrine, followed by western blot analysis of cell lysates
using anti-BAD
antibodies. In untreated HeLa cells, a portion of BAD exhibited retarded
migration,
suggesting that there was some basal BAD phosphorylation in quiescent cells
(Fig. 7(a), lane
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1 ). However, treatment with Forskolin or L-epinephrine resulted in a new band-
shift, seen as
a third band, as shown in the time course in Fig. 7(a).
The results shown in Fig. 7(b) demonstrate that the band-shift was sensitive
to the
PKA inhibitor, H89 (Fig. 7(b), lane.4), but not to Wortmannin, a PI 3-kinase
inhibitor (Fig.
7(b) lane 5). Furthermore, both upper bands were sensitive to phosphatase,
indicating that
they represent phosphorylated BAD (Fig. 7(b), lane 3).
Forskolin treatment also resulted in a BAD band-shift in Rat-1 cells that was
sensitive
to the PKA inhibitor H89 (Fig. 7(c) lanes 7 and 8), but not to Wortmannin (not
shown). The
upper band induced by Forskolin was sensitive to phosphate (Fig. 7(c), lane 3,
lower panel),
again indicating that it represents phosphorylated BAD. In the absence of a
phospho-Serl SS
antibody, it was difficult to conclude that this band-shift was solely the
result of the
phosphorylation of Ser155. However, it appeared that this response was at
least primarily
due to the phosphorylation at this site. This was based on the combined
evidence, both in
vitro and in cells, as described above (Figs. 1, 2 and 4-7) and the fact that
Ser155 is the only
site of BAD homologous to the substrate consensus sequence of PKA. These
results also
suggest that cAMP-dependent induction of BAD phosphorylation does not require
overexpression of BAD in cells, nor does it require mutations of Serl 12 or
Ser136.
Example 6
PHI inhibition of L-epinephrine induces phosphorylation of the BAD double
mutant
To further verify that the cAMP-dependent BAD Ser155 kinase is PKA, cDNA
encoding PKI, the highly specific endogenous PKA inhibitor (Day et al., 1989),
was derived
from HeLa cells, and HeLa cells were co-transfected with HA-PKI and HA-BAD
S 112A/S 136A. Transformants were treated with L-epinephrine, then lysed and
subjected to
anti-HA western blot analysis. Overexpressed PKI blocked L-epinephrine
induction of
Ser155 phosphorylation (Fig. 8(a)). This result is consistent with the
inhibitory effect of H89
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(Fig. 8(b)), and together indicate that PKA is a BAD Ser155 kinase that is
activated by
elevated intracellular cAMP.
Example 7
Growth factor induction of BAD phosphorylation in fibroblasts
Growth factors also have been shown to confer protection from apoptosis.
Whether
the BAD Ser155 kinase, PKA, is also activated by growth factors, and if so,
whether such
activation is PI 3-kinase dependent, was investigated. Rat-1 cells transiently
expressing HA-
BAD S 112A/S 136A were treated with platelet-derived growth factor (PDGF),
epidermal
growth factor (EGF), insulin-like growth factor 1 (IGF-1), N6-Benzoyl-
Adenosine 3',5'-
cyclic monophosphate (6-Bnz-cAMP) and Forskolin (Fig. 9(a)). Cell lysates were
analyzed
via western blot analysis, probed using anti-HA antibody. IGF-1, EGF and PDGF
induced a
band-shift in the BAD double mutant in Rat-1 fibroblasts. However, the
magnitude of
phosphorylation was weaker and may be more transient, than the effect of
Forskolin (Fig.
9(a)). Of note, Ser155 phosphorylation following IGF-1, EGF, or PDGF
stimulation was not
detected in HeLa cells (results not shown).
It has been documented that Akt kinase activity, the serine kinase shown to
phosphorylate BAD Ser136, is induced by growth factors and is dependent on PI
3-kinase
activation (Datta et al., 1997; del Peso et al., 1997). In related
experiments, Rat-1 cells were
pretreated with either the PKA inhibitor H89, or the PI 3-kinase inhibitor
Wortmannin
(available, e.g., from Sigma, St. Louis, MO), followed by stimulation with
Forskolin (also
available from Sigma) or PDGF. Resulting western blot analyses of cell lysates
demonstrated
that a PDGF-induced BAD band-shift was significantly reduced by H89
pretreatment. This
indicated the shift was PKA dependent (Fig. 9(b), lower panel, compare lanes 5
and 6).
However, this PKA inhibitor had no effect on PDGF-induced Akt activation,
indicating Akt
activation was not PKA dependent (Fig. 9(b), upper panel, compare lanes 5 and
6). It
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therefore presumably had no effect on PDGF-induced activation of PI 3-kinase.
This result
suggested that PKA is activated by PDGF in Rat-1 cells, and this activation
does not appear
to depend on activation of PI 3-kinase and Akt.
In contrast, pretreatment of cells with Wortmannin completely blocked PDGF-
induced Akt activation (as expected) (Fig. 9(b), upper panel, compare lanes S
and 7), yet it
only partially inhibited PDGF-induced BAD phosphorylation (Fig. 9(b), lower
panel,
compare lanes S and 7). This suggested that part of the phosphorylation of BAD
in response
to PDGF is PI 3-kinase and Akt independent.
To examine whether endogenous BAD is phosphorylated on Ser155 in response to
growth factors, serum-starved Rat-1 fibroblasts were stimulated with PDGF.
Whole cell
lysates were prepared and endogenous BAD was immunoprecipitated. Proteins were
0
separated by SDS-PAGE, and Ser155 phosphorylation was analyzed by Western blot
using
the phospho-S 155 antibody and an anti-BAD antibody. As demonstrated in Fig.
9(c), PDGF
induced the phosphorylation of endogenous BAD on Ser155 (Fig. 9(c)).
To determine whether growth factor-induced Ser155 phosphorylation of BAD is
sensitive to the PKA inhibitor protein PKI, HeLa cells were co-transfected
with HA-BAD
S 112A/S 136 and either the PKI expression vector or empty expression vector,
and then
treated with EGF. Western blots probed with anti-HA antibody demonstrate that
while EGF
stimulated the phosphorylation of BAD on Ser155, this phosphorylation was
blocked by co-
expression of PKI (Fig. 10(a)).
To further test whether phosphorylation of Ser155 is dependent on the PI 3-
kinase/Akt pathway, HeLa cells were transfected with the mutant HA-BAD S155A
and
pretreated with Wortmannin, an inhibitor of PI 3-kinase. These cells were then
stimulated
with epidermal growth factor (EGF), lysed and analyzed by western blot
analysis.
Wortmannin treatment prevented the activation of endogenous Akt by PI-3 kinase
following
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EGF stimulation (Fig. 10(b), compare lanes 2 and 4, panel B), but did not
impair EGF-
induced phosphorylation of BAD on Serl55 (Fig. 10(b), compare lanes 2 and 4,
panel D). In
parallel assays, BAD Serl 55 phosphorylation was prevented by the addition of
the PKA
inhibitor H89 (Fig. 10(b), compare lanes 2 and 5, panel D) without affecting
the
phosphorylation of Akt (Fig. 10(b), compare lanes 2 and 5, panel B). The EGF
receptor
(EGFR) kinase inhibitor AG1478 (AG) blocked the phosphorylation of both Akt
(Fig. 10(b),
compare lanes 2 and 3, panel B) and BAD Ser155 (Fig. 10(b), compare lanes 2
and 3, panel
D). These results demonstrate that EGF stimulates the phosphorylation of BAD
Serl55
through a PKA-dependent mechanism that is distinct from the PI-3 kinase/Akt
pathway.
PDGF-induced Ser155 phosphorylation of endogenous BAD was also significantly
reduced by pretreatment of cells with the PKA inhibitor H89, but not the PI 3-
kinase inhibitor
Wortmannin (Fig. 10(c)).
Taken together, these results suggest that Akt, the Ser136 BAD kinase, and
PKA, the
putative Ser155 BAD kinase, are both downstream of activated growth factor
receptors, and
both contribute to the overall phosphorylation of BAD following growth factor
stimulation,
and the former, but not the latter, is dependent on PI 3-kinase.
Example 8
Mutation of BAD Ser155 to a non-phosphorylatable residue promotes cell death
Because BAD Ser155 phosphorylation, like Ser136 phosphorylation, prevents BAD
from binding to Bcl-X~ (Fig. 1), it was possible that by analogy, Ser155
dephosphorylation
promotes cell death. To test this idea, HeLa cells were co-transfected with
BAD or various
BAD serine mutants and a (3-gal reporter plasmid. Twenty-four hours post-
transfection, cell
viability was analyzed by (3-gal ELISA.
Of the three single serine mutations, the mutation of Ser155 (BAD S 155A) had
the
greatest effect on cellular survival, with a significant increase in cell
death compared to the
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control (Fig. 11(a)). Mutation of Ser136 (BAD S136A) had the next greatest
effect, followed
by mutation of Serl 12 (BAD S 112A). The BAD triple.mutant (BAD S 112A/S
136A/S 1 SSA)
had a greater effect than the BAD double mutant (BAD S 112A/S 136A),
suggesting that these
phosphorylation sites may not be functionally redundant. To ensure that the
greater pro-
apoptotic activity of the Ser155 mutants was not due to higher expression
levels of these
mutant BADs, cells transfected in parallel were lysed 24 hours after
transfection and BAD
expression levels probed on a western blot with anti-HA antibody (Fig. 11(b)).
Although the
S155A mutations enhanced apoptotic activity of mutants BAD S155A and BAD
S112A/S136A/S155A, the expression levels of these mutants were actually lower
than their
counterparts without the S 1 SSA mutation (compare Fig. 11 (b), lanes 3 and 4
to lane S, and
lane 6 to lane 7), a result that appeared to be consistent with the apoptosis
data.
Example 9
Phosphorylation of BAD Ser155 promotes cell survival
Based on the observation that substitution of BAD Ser155 with a
nonphosphorylatable residue enhances the pro-apoptotic activity of BAD, it was
thought that
induced phosphorylation of Ser155 might promote cell survival.
To test this idea, HeLa cells were again co-transfected with BAD or various
BAD
serine mutants and a (3-gal reporter plasmid. Forskolin was then added to half
of the cultures
while the other half was left untreated. Beta-gal ELISAs were performed 24
hours post
transfection. As shown in Fig. 12(a), Forskolin was able to reduce apoptosis
in wild-type
BAD and the BAD S 112A/S 136A double mutant, but not in the BAD S 1 SSA mutant
or the
BAD S112A/S136A/S155A triple mutant transfected cells. Thus, phosphorylation
of Ser155
rescues cells from BAD-induced apoptosis, while substitution of Ser155 with a
nonphosphorylatable residue results in increased cell death and the failure of
cells to be
protected from induced PKA activity.
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Like BAD phosphorylated on Serl 12 or Ser136, Ser155-phosphorylated BAD is
deficient in binding to Bcl-X,_,. When introduced into HeLa cells, the BAD
S155A mutant
showed enhanced apoptotic activity compared with the wild-type mammalian BAD,
suggesting that phosphorylation on Ser155 is anti-apoptotic (Figs. 11(a) and
12(a)). In
addition to and consistent with this result, the BAD S 112A/S 136A/S 155A
triple mutant was
more toxic to transfected HeLa cells than the S 112A/S 136A double mutant,
suggesting the
apoptotic effect of Ser155 dephosphorylation is additive to those of Ser112
and Ser136 (Fig.
11 (a)).
EGF stimulation also suppressed the pro-apoptotic function of BAD in
transfected
HeLa cells. HeLa cells were co-transfected with the ~i-galactosidase gene and
either wild-
type BAD, BAD S 155A, BAD S 112A/S 136A or BAD S 112A/S 136A/S.155A.
Transfected
cells were then cultured in serum-free medium (SFM) or SFM supplemented with
EGF for 12
hours. Cell lysates were prepared and BAD-induced cell death was measured by
the loss of
(3-galactosidase activity in a fluorescence-based assay. The results
demonstrate that mutation
of Ser155 to alanine alone does not eliminate the protective effect of EGF,
consistent with the
ability of EGF to inactivate BAD through the phosphorylation of Serl 12 and/or
Ser136 (Fig.
12(b)). However, the pro-apoptotic activity of a BAD S 112A/S 136A double
mutant was also
suppressed by EGF stimulation, revealing the ability of EGF to inactivate BAD
through a
distinct mechanism. This mechanism involves Ser155, since mutation of Ser155
to alanine in
the context of S112A/S136A completely inhibited the anti-apoptotic effect of
EGF
stimulation (S 112A/S 136A/S 155A triple mutant) (Fig. 12b, "AAA"). The (3-gal
reporter
assay was valid as a measurement of apoptosis, since BAD-induced reduction of
(3-gal
activity was reversed by the treatment of cells with z-VAD, a broad-spectrum
caspase
inhibitor (results not shown). These results indicate that the anti-apoptotic
effects of EGF can
be mediated through Ser155 phosphorylation, independently of Ser112/Ser136
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phosphorylation, and are consistent with the distinct EGF-activated signaling
pathways that
lead to the phosphorylation of BAD on Ser136 and on Ser155.
To further evaluate the impact of Ser155 phosphorylation on the anti-apoptotic
activity of BAD, Ser155 was replaced with aspartic acid (S 155D) to mimic the
negatively
charged phospho-Ser155 residue. HeLa cells were co-transfected with (3-gal and
either an
empty vector, wild-type BAD, the BAD S 155A mutant or the BAD S 155D mutant.
The
results of the assay demonstrated that BAD S 155D showed no pro-apoptotic
activity
compared to wild-type BAD (Fig. 12(c)). These results further supported the
notion that
phosphorylation of Ser155 leads to inactivation of BAD in cells.
In summary, Ser155 is the major site of phosphorylation by PKA in vitro. A
single
S155A mutation reduced PKA catalyzed 32P-ATP incorporation by more than 90%,
compared to the wild-type mammalian BAD substrate (Fig. 4). Because mutations
of Serl 12
or Ser136 did not result in reduced phosphorylation, and the BAD
S112A/S136A/S155A
triple mutant failed to show detectable phosphorylation by PKA (Fig. 4),
Ser155 is likely to
be the only major in vitro PKA phosphorylation site. The low, but detectable
level of BAD
S 1 SSA mutant phosphorylation, compared with the almost undetectable level of
BAD triple
mutant phosphorylation suggested that Ser112 and/or Ser136 might be minor in
vitro PKA
phosphorylation site(s).
Activation of PKA in vitro also resulted in phosphorylation of BAD on Ser155.
Treatment of BAD S 112A/S 136A transfected HeLa cells with the membrane-
permeable
cAMP analog 6-Bnz-cAMP, a PKA activator, led to marked Ser155 phosphorylation
(Fig.
9(a)). Stimulation of the BAD double mutant transfected cells with Forskolin,
an adenylate
cyclase activator that induces cAMP production, also led to a rapid and
sustained
phosphorylation of BAD on Serl SS (Fig. 5). Consistent with this result,
treatment of cells
with L-epinephrine, the ligand for a G protein-coupled receptor ((3-adrenergic
receptor) that
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sequentially activates the receptor, stimulatory G proteins and adenylate
cyclase (Birnbaumer
et al., 1990), activated the BAD Ser155 kinase and resulted in the
phosphorylation of this site
(Fig. 6). This is thought to be the first connection found between G protein-
coupled receptors
and the regulation of a Bcl-2 family member.
Similar to the results obtained in vitro, Ser155 appeared to be the only major
in vivo
phosphorylation site of BAD in response to treatment with Forskolin or L-
epinephrine. The
S155A single mutation significantly reduced L-epinephrine-induced BAD
phosphorylation
(measured by phosphatase-sensitive band-shift), whereas mutations on Serl 12
or Serl36 did
not (Fig. 6(b)). Serl SS phosphorylation was not triggered by transfection,
since MCF-7 cells
stably expressing BAD S 112A/S 136A also showed a similar response to
Forskolin
stimulation (data not shown). The phosphorylation of BAD following elevation
of
intracellular cAMP was not an artifact of overexpression of the BAD in cells
since
endogenous BAD responded similarly to such treatment and the phosphorylation
was
sensitive to the PKA inhibitor H89 (Fig. 7).
Overexpression of PKI, an endogenous PKA inhibitor, completely blocked L-
epinephrine-induced Ser155 phosphorylation in transfected HeLa cells (Fig.
8(a)). H89 also
had a similar effect (Fig. 8(b)). Together these results indicate that the BAD
Ser155
phosphorylation following elevated intracellular cAMP is mediated by PKA.
Importantly,
treatment of HeLa cells with H89 for one hour, not only blocked L-epinephrine-
induced BAD
phosphorylation, but also reduced basal phosphorylation of endogenous BAD
(Fig. 7(a)).
This suggests that PKA is also a BAD kinase in quiescent HeLa cells.
Consistent with the failure of Ser155-phosphorylated BAD to bind to Bcl-XL
(Fig. 1)
and the enhanced apoptotic activity of BAD that was mutated at Ser155 (Fig.
11(a)), induced
phosphorylation of BAD on Ser155 seemed to promote cell survival (Fig. 12(a)).
Approximately 50% more cells survived the transient expression of wild-type
mammalian
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BAD, than the BAD S 155A mutant, in the presence of Forskolin after 24 hours.
A similar
observation was made with the BAD double mutant- and triple mutant-transfected
cells:
additional mutations on BAD abolished the protective function of Forskolin. In
fact,
Forskolin also protected control (pcDNA3-GST) transfected cells from the
toxicity of lipid-
mediated transfection, presumably by phosphorylating Serl SS of endogenous BAD
(also see
Fig. 7(a)). Using ~3-gal activity as the measure of cell survival, the effect
of endogenous
BAD should be minimal in untransfected or BAD mutant-transfected cells; BAD
and the
reporter (3-gal were introduced into cells at a ratio of 5:1, therefore the
exogenous BAD or
BAD mutants were expected to be dominant over endogenous BAD. The transient
expression of BAD-induced apoptosis appeared to depend on activation of
caspases,
treatment of the transfected cells with a caspase inhibitor z-VAD, efficiently
protected the
cells (data not shown).
The protection of cells from apoptosis via PI 3-kinase/Akt-mediated BAD
phosphorylation has been documented (Datta et al., 1997; del Peso et al.,
1997). An
additional BAD phosphorylation site, Ser155, that suppresses BAD-induced
apoptosis in a PI
3-kinase/Akt-independent manner was demonstrated herein. The PKA inhibitor
H89, but not
the PI 3-kinase inhibitor Wortmannin, blocked growth factor-induced BAD
phosphorylation
in HeLa cells (Fig. 10(b)) and in Rat-1 cells (Fig. 10(c)), and Forskolin-
induced BAD
phosphorylation in Rat-1 cells (Fig. 7(b)). In Rat-1 cells, PDGF appears to
activate both
PKA- and Akt-mediated phosphorylation of BAD. The PKA inhibitor blocked a part
of the
growth factor-induced phosphorylation without interrupting the activation of
PI 3-kinase and
Akt; the PKA activator Forskolin induced BAD phosphorylation without
activating Akt; and
the PI 3-kinase inhibitor only partially blocked PDGF-induced BAD
phosphorylation (Fig.
9(b)). Together, these results suggest that in Rat-1 cells, the BAD kinases
Akt and PKA are
both activated by PDGF, but are likely to function independently.
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Kulik and Weber (1998) suggested an IGF-1-induced, Akt-independent survival
signaling pathway was present in Rat-1 cells overexpressing the IGF-1
receptor, although the
components of this pathway were not known. Results shown herein now suggest
that in
fibroblasts, the Akt-independent survival pathway activated by growth factor
stimulation
includes activated PKA. Exactly how growth factors activate PKA in Rat-1 cells
is not clear,
although there have been reports showing transactivation of G proteins by
growth factors
(Malbon and Karoor, 1998) and MAP kinase-dependent activation of PKA by PDGF
(Graves
et al., 1996).
Mutational studies, as taught herein, have provided strong evidence that BAD
promotes apoptosis by dimerizing with Bcl-XL and related proteins, emphasizing
the
importance of understanding how phosphorylation regulates this interaction.
The findings
presented here suggest that phosphorylation on different sites within BAD have
mechanistically distinct consequences: Ser155 phosphorylation directly
prevents
heterodimerization by abolishing the affinity of BAD BH3 for Bcl-XL, whereas
phosphorylation on Ser112 or Ser136, located outside the BH3 domain, may
inhibit
dimerization with Bcl-X,_, indirectly. In particular, phosphorylation on Serl
12 or Ser136, but
not Ser155, generates a consensus binding site for the cytosolic protein, 14-3-
3, which may
bind and alter the sub-cellular distribution of BAD to prevent interaction
with Bcl-XL at
mitochondrial membranes.
The discovery of the novel phosphorylation site, Serl S5, of BAD, and the
possible
cellular regulatory mechanisms that lead to its phosphorylation, indicate that
cells have the
ability to protect themselves via multiple survival pathways. Which pathway is
used could be
dependent on the intrinsic properties of the cells, such as the distribution
of cell membrane
receptors, as well as the extracellular environment.
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MATERIAL AND METHODS
Isolation of BAD cDNA, plasmid construction and mutagenesis
The cDNA encoding the murine BAD of SEQ ID N0:2 ("longer murine BAD") was
obtained by the Reverse Transcription Polymerase Chain Reaction ("RT-PCR") of
mRNA
S isolated from FLS.iz cells using the primers:
S' - GCCTCCAGGATCCAAGATGGGAACC - 3' (SEQ ID N0:12);
and
5' - GGAGCGGGTAGAATTCCGGGATG - 3' (SEQ ID N0:13).
The open-reading frame of the RT-PCR product was predicted to encode a protein
of
204 amino acids (aa) (Yang et al., 1995). The cDNA encoding the longer murine
BAD was
cloned into the pcDNA3 vector (Invitrogen), expressed in FLs.iz cells, and the
expressed
longer murine BAD detected with an anti-BAD antibody (C-20, Santa Cruz). The
expressed
longer murine BAD was significantly larger in molecular weight than the
endogenous BAD
(approximately 30 kD and 23 kD, respectively) (Fig. 13, compare lane 1 against
lanes 2 and
3, left panel). It was discovered that there is a methionine residue at
position 43 of the amino
acid sequence of the longer murine BAD that, when aligned with the amino acid
sequence of
the human BAD, corresponds to the first residue (a methionine) in the human
BAD amino
acid sequence of SEQ ID NO:1.
Therefore, a second murine BAD cDNA construct was produced, the murine BAD of
SEQ ID N0:3 ("shorter murine BAD"). A PCR primer having a sequence
complementary to
and upstream of the sequence encoding residue 43 of the murine BAD sequence of
SEQ ID
N0:2
S'- TGGAGACCAGGATCCCAGAGTAGCT - 3' (SEQ ID N0:14)
and the same downstream primer as above (i.e., SEQ ID N0:13) were used to
generate the
cDNA of SEQ ID N0:3, which was then cloned into the pcDNA3 vector. This
construct,
when expressed in FLs.iz cells, co-migrated with the endogenous BAD (Fig. 13,
compare
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lanes 4 and S, left panel, with lane 1, right panel), suggesting that the
shorter murine BAD,
which is 162 as in length, is likely to be the major translation product of
BAD in FLS,,z cells.
BAD mutants have been produced where one or more serine residues were changed
to
alanine residues using PCR-mediated mutagenesis (Ge and Rudolph, 1997).
Herein, BAD
mutants were also produced, where one or more of the serine residues at
positions 112, 134,
136, and 155, corresponding to the amino acid positions of the longer murine
BAD of SEQ
ID N0:2, were changed to alanine or aspartic acid. These positions correspond
to serine
residues in positions 75, 99, 101 and 118 in SEQ ID NO:1 and positions 70, 94,
96 and 113 in
SEQ ID NO: 3. One of ordinary skill in the art would understand how to use PCR-
mediated
mutagenesis to make such changes. Correct DNA sequences encoding for BAD and
its
mutants were confirmed by DNA sequencing. Resulting BAD mutant cDNAs were then
inserted into the pcDNA3 expression vector. Epitope-tagged BAD and BAD mutants
were
produced by subcloning each of the cDNAs in-frame into a second pcDNA3 vector,
between
BamHI and EcoRI sites, that already contained the HA epitope sequence upstream
to the
insertion site.
The cDNA encoding human protein kinase inhibitor (PKI) was isolated from total
mRNA of HeLa cells by RT-PCR. The primers used for the reaction were:
5' - CTATGTGGATCCTTGGTAGCAATG (SEQ ID NO:15);
and
5' - CCTCATAGACCTTAAGTAAACAAA (SEQ ID N0:16).
The product of the RT-PCR was then digested with the restriction enzymes BamHI
and EcoRI, and the sequence encoding the PKI was inserted into the polylinker
region of the
pcDNA3 vector, so that the nucleotide sequence encoding PKI was in-frame with
and
operably linked to an HA epitope at the amino ("N") terminus of the encoded
PKI. The
correct DNA sequence encoding the HA-tagged PKI was confirmed by DNA
sequencing.
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Expression and purification of glutathione s-transferase (GST) fusions of wild-
type and
mutant BAD
DNA encoding BAD and various mutant BAD were excised from pcDNA3 as a
BamHI-EcoRI fragment and inserted into BamHI-EcoRI cloning sites of pGEX-2T
S (Pharmacia), downstream of and in-frame with the GST coding region. GST-BAD
constructs
were then transfected into E. coli strain DHSa. Cells (500 ml) were grown to
an ODs9s of
0.7-0.9 at 37°C and induced with 2 mM isopropyl (3-D-
thiogalactopyranoside (Bachem) at
30°C overnight. Cells were collected by centrifugation and resuspension
in 20 ml of HEPES-
buffered saline (HBS) (10 mM HEPES, pH 7.5, 3.4 mM EDTA, 150 mM NaCI) plus 1%
(vol/vol) Triton X-100 and 10 mM (3-mercaptoethanol (BME). Cells were then
lysed by two
passages through a Microfluidizer M110S (Microfluidics) and cellular debris
was removed
by centrifugation for 30 minutes at 20,000 x g. A 20% ammonium sulfate
precipitation was
performed on the cell lysate to remove aggregated polypeptide, and GST-BAD was
purified
from the supernatant by glutathione agarose chromatography (Smith and Johnson,
1988).
The fractions containing GST-BAD were pooled and concentrated using a
Centriprep 3
(Amicon), and the polypeptide concentration determined by Bradford assay (Bio-
Rad).
Polypeptide was stored on ice for immediate use or frozen, in liquid nitrogen
and stored at -80
°C.
Cell culture and transfections
The Rat-1, COS-7, HeLa and MCF-7 cell lines were cultured in glutamine-free
DMEM supplemented with 10% fetal calf serum (FCS), 1% glutamine and 1%
penicillin/streptomycin (GIBCO). FLs.~2 cells were cultured in glutamine-free
IMEM
supplemented with 10% FCS, 10% WEHI-conditioned media (collected from WEHI
cell
cultures), 1 % glutamine and 1 % penicillin/streptomycin. Transfections were
carned out by
using the Superfection Kit from Qiagen or the GenePROTOR transfection reagent
from Gene
Therapy Systems. MCF-7 cells stably expressing BAD or mutant BAD were isolated
by
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6418 selection at 1 mg/ml; 6418-resistant clones were then pooled and used for
further
assays.
Antibodies, immunoprecipitation and western blotting analyses
Anti-BAD antibody (C-20) was from Santa Cruz Biotechnology, Inc. and anti-HA
S antibody (3F10) was from Boehringer Mannheim.
An antibody specific against phospho-Ser155 BAD was generated using an 18 as
phosphopeptide from the BAD BH3 domain region (aa 108-125) by Bio-Synthesis
Incorporated (Lewisville, Texas). The sequence of this region is:
QRYGRELRRMSDESVDSF (SEQ ID N0:17).
A phosphopeptide of the sequence
NHZ - (GC)QRYGRELRRMpSDESVDSF - COOH (SEQ ID N0:18)
was synthesized at >70% purity and conjugated to keyhole limpet hemocyanin.
This antigen
was injected into two rabbits, from which serum was collected. The serum
included pre-
bleed, 6th, 8'h and 10th week bleeds. The GC in parenthesis represent the
linker amino acids.
When used at 1:500 dilution, the 10'h week bleed from one of the animals was
reactive with
Ser155-phosphorylated BAD in western blot analysis. The pre-bleed was not
reactive. Fig.
14 shows the results of a western blot probe with the polyclonal antibody. The
anti-serum
was found to recognize both forms of phosphorylated murine BAD and human BAD,
as well
as fragments of all three proteins and mutant BAD where the serine residue
corresponding to
Ser155 was not mutated to alanine. In a separate experiment, HeLa cells
transiently
expressing HA-BAD S 112A/S 136A were kept in culture for two days in the
absence or
presence of Forskolin. Lysates were then prepared. Following SDS-PAGE
separation, lysate
samples were probed with either the anti-HA antibody or the rabbit anti-serum.
Lysates from
cells prepared in the presence and absence of Forskolin show a band reactive
with anti-HA
antibody (Mr ~ 30 kD). In contrast, for lysates probed with the rabbit anti-
human BAD
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phospho-Ser155 anti-serum, only the Forskolin (a PKA activator) treated cells
reacted to
display a band at Mr ~ 30 kD, demonstrating that the rabbit anti-human BAD
phospho-Ser155
antibody is specific to phosphorylated BAD.
For the generation of cell lysates, cells were washed with ice-cold PBS and
lysed in
RIPA buffer (50 mM HEPES, pH 7.5, 1% deoxycholate, 1% NP-40, 0.1% SDS, 150 mM
NaCI, 1 mM Na3(VO)4, 1 mM Na03P~, 1 mM PMSF, 10 pg/ml leupeptin and 20 p,g/ml
aprotinin). Lysates were centrifuged at 16,000 x g for 1 S min at 4°C
and the cleared
supernatant was transferred to new tubes.
For immunoprecipitation of all HA-BAD species, 1 p,g of the anti-HA antibody
was
added to the cell lysates, and incubated at 4°C for one hour. Protein G
agarose (Pierce) was
then added (50 p.l/fraction of 50% slurry) and followed by a one hour
incubation at 4°C.
Beads were washed with RIPA buffer, boiled in loading buffer (2% SDS, 10%
glycerol, 5%
~3-ME, 60 mM Tris, pH 6.8, 0.002% Bromophenol Blue) and loaded onto
polyacrylamide
gels. After electrophoresis, proteins were transferred to nitrocellulose
membranes, and blots
blocked in western wash buffer (40 mM Tris, pH 8.0, 150 mM NaCI, 0.2% NP-40)
with 5%
BSA for one hour at room temperature. Blots were incubated with primary
antibody diluted
in western wash buffer with 3% BSA at room temperature for 1-2 hours, washed
with
western wash buffer and incubated with secondary HItP-coupled antibody diluted
in western
wash buffer with 1.5% BSA, and washed extensively. Polypeptides were detected
with the
ECL chemiluminescence according to the manufacturers instructions (Amersham).
In vitro polypeptide binding, phosphatase treatment, and in vitro kinase
assays
Phosphoserine BADs can be used in assays to screen for inhibitors or
activators of
serine-phosphatase agents in which a test agent converts the serine-
phosphorylated BAD to
the non-phosphorylated BAD death promoter. The polypeptides can also be used
to screen
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for and identify serine phosphatase agents that are capable of participating
in the control of
apoptosis.
For Bcl-X~ binding studies, GST-Bcl-XL (1 mg/ml final concentration) was added
to
cell lysates derived from BAD expressing cells, and incubated with rotation
for two hours at
4°C. GST-Bcl-X,_, and any associated BAD were then isolated using
glutathione beads
(Pharmacia) that were added to lysate/GST- Bcl-XL mixture, and incubated for
an hour. The
beads were then washed with 1RIPA buffer, the proteins were separated by SDS-
PAGE and
associated BAD detected by probing the blot with anti-HA antibody.
For phosphatase treatments, cells were lysed in RIPA buffer without
phosphatase
inhibitors. Lambda protein phosphatase (New England Biolabs) was added to the
lysate at 4
U/ml, supplemented with MnClz and phosphatase reaction buffer (50 mM Tris-HCI,
pH 7.8,
containing 5 mM dithiothreitol). The reaction proceeded for 30 min. at
30°C.
In vitro kinase assays were carried out in 30 pl volumes containing 10 mM
HEPES
buffer, pH 7.5, 100 mM NaCI, 12 mM MgClz, 1 mM dithiothreitol, 15 mCI Y3zP-ATP
(6000
Ci/mol, NEN), 11 units PKA (catalytic subunit from bovine heart, Calbiochem),
and 1 ~g
purified GST-BAD. Reactions were incubated for 30 min at 30°C and
terminated by the
addition of SDS-PAGE sample buffer. Samples were run on a 4-20% SDS-PAGE gel
(Novex), transferred to a nitrocellulose membrane and phosphorylated GST-BAD
was
visualized by autoradiography. To verify that equal amounts of GST-BAD
substrates were
used, the nitrocellulose was subjected to western blot analysis with 133 ng/ml
anti-BAD
antibody (C-20).
Apoptosis assays
Beta-gal ELISA assays were performed using the (3-gal ELISA system from
Boehringer Mannheim, according to manufacture's instructions. Cells were
transfected with
BAD (or BAD mutant) and (3-gal in a ratio of 5:1. The transfected cells were
trypsinized and
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distributed into 96-well tissue culture plates. Twenty-four hours after
transfection, cell
extracts were obtained by lysing the cells in lysis buffer. Cell extracts (200
~l) were then
added to anti-(3-gal-coated microtiter plates (MTP). The MTPs were foil-
covered and
incubated for one hour at 37°C. Solutions were then decanted and the
MTPs were washed,
followed by the addition of 200 pl of anti-~3-gal-DIG working solution, and
incubation for an
additional hour as described above. Solutions were again decanted, the MTPs
washed and
200 p.l of DIG-POD working solution were added. The MTPs were incubated for
another
hour at 37°C, the solution decanted, and the wells washed. 200 pl of
substrate with enhancer
were added to each well and incubated at room temperature until color
development. The (3-
gal activity was measured by the absorbance of the sample at 410 nM. Data were
based on
triplicate wells with standard deviations calculated.
To measure ~3-gal activity, HeLa cells were plated in 12-well plates at 5 x
104
cells/well one day prior to transfection. Cells were transfected with BAD (or
BAD mutants)
and (3-gal expression plasmids in a ratio of 4:1 (0.6 pg:0.15 pg) using the
Superfect
1 S transfection reagent (Qiagen). Twenty-four hours after transfection, cells
were cultured in
serum-free medium (SFM) or in SFM plus EGF, for additional 12 hours. Beta-gal
activity
was measured in extracts using a fluorogenic substrate (MUG, BIO-RAD
FluorAceTM (3-gal
Reporter Assay, #170-3150). Loss of (3-gal activity in these assays reflects
apoptosis and
elimination of the transfected cells, and the (3-gal reductions were reversed
by the addition of
a broad spectrum caspase inhibitor, z-VAD-fmk.
Competition binding assay
Immunlon 2 (Dynatech) microtiter plates were coated with 5 ~g/ml neutravidin
(50
pl/well; Pierce) in sodium bicarbonate buffer, pH 9.0, overnight at 4~C. All
remaining steps
were conducted at room temperature. Plates were washed twice with PBS
containing 0.1%
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Tween 20 (wash buffer) and blocked for 1 hour with 0.2 ml/well of 1% normal
goat serum in
PBS. Following two additional washes, 1.25 ~g/ml of a BAK BH3 peptide 19-mer
(residues
71-89) biotinylated at the amino terminus was added to the wells in 50 p.l of
10 mM HEPES
buffer, pH 7.2 containing 150 mM KCI, 5 mM MgClz , 1 mM EGTA, and 0.2% NP-40
(NP-
40 buffer). After 30 min, the wells were washed twice with wash buffer and GST-
Bcl-XL
(0.25 ~M in 50 ~l of NP-40 buffer) was added in the absence or presence of BAD
or BAK
BH3 peptides. Following a one hour incubation, the plates were washed twice
with wash
buffer and the amount of bound GST-Bcl-XL was determined by ELISA using an
anti-GST
primary antibody and an HRP-conjugated anti-mouse IgG secondary antibody
(Jackson) with
ABTS (Zymed) as substrate. Five washes were conducted following each one hour
antibody
incubation. GST-Bcl-XL fusion protein was produced in E. coli by a similar
procedure
described for the production of GST-BAD (see above).
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REFERENCE LIST
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99
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SEQUENCE LISTING
<110> APOPTOSIS TECHNOLOGY, INC.
<120> COMPOUNDS AND METHODS FOR REGU1~ATING APOPTOSIS,
AND METHODS OF MAKING AND SCREENING FOR COMPOUNDS
THAT REGULATE APOPTOSIS
<130> F137122
<140>
<141>
<160> 20
<170> PatentIn Ver. 2.1
<210> 1
<211> 168
<212> PRT
<213> Homo sapiens
<400> 1
Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp Ser Ser
1 5 10 15
Ser Ala Glu Arg Gly Leu Gly Pro Ser Pro Ala Gly Asp Gly Pro Ser
20 25 30
Gly Ser Gly Lys His His Arg Gln Ala Pro Gly Leu Leu Trp Asp Ala
35 40 45
Ser His Gln Gln Glu Gln Pro Thr Ser Ser Ser His His Gly Gly Ala
50 55 60
Gly Ala Val Glu Ile Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr
65 70 75 80
Glu Asp Asp Glu Gly Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg
85 90 95
Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg
100 105 110
Glu Leu Arg Arg Met Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly
115 120 125
Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Thr Gln Met Arg Gln Ser
130 135 140
Ser Ser Trp Thr Arg Val Phe Gln Ser Trp Trp Asp Arg Asn Leu Gly
145 150 155 160
Arg Gly Ser Ser Ala Pro Ser Gln
165
SUBSTITUTE SHEET (RULE 26)
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<210> 2
<211> 204
<212> PRT
<213> Mus musculus
<400> 2
Met Gly Thr Pro Lys Gln Pro Ser Leu Ala Pro Ala His Ala Leu Gly
1 5 10 15
Leu Arg Lys Ser Asp Pro Gly Ile Arg Ser Leu Gly Ser Asp Ala Gly
20 25 30
Gly Arg Arg Trp Arg Pro Ala Ala Gln Ser Met Phe Gln Ile Pro Glu
35 40 45
Phe Glu Pro Ser Glu Gln Glu Asp Ala Ser Ala Thr Asp Arg Gly Leu
50 55 60
Gly Pro Ser Leu Thr Glu Asp Gln Pro Gly Pro Tyr Leu Ala Pro Gly
65 70 75 80
Leu Leu Gly Ser Asn Ile His Gln Gln Gly Arg Ala Ala Thr Asn Ser
g5 90 95
His His Gly Gly Ala Gly Ala Met Glu Thr Arg Ser Arg His Ser Ser
100 105 110
Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly Met Glu Glu Glu Leu Ser
115 120 125
Pro Phe Arg Gly Arg Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala
130 135 140
Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser Asp Glu Phe Glu Gly
145 150 155 160
Ser Phe Lys Gly Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Thr Gln
165 170 175
Met Arg Gln Ser Ala Gly Trp Thr Arg Ile Ile Gln Ser Trp Trp Asp
180 185 190
Arg Asn Leu Gly Lys Gly Gly Ser Thr Pro Ser Gln
195 200
<210> 3
<211> 162
<212> PRT
<213> Mus musculus
<400> 3
Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp Ala Ser
SUBSTITUTE SHEET (RULE 26)
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1 5 10 15
Ala Thr Asp Arg Gly Leu Gly Pro Ser Leu Thr Glu Asp Gln Pro Gly
20 25 30
Pro Tyr Leu Ala Pro Gly Leu Leu Gly Ser Asn Ile His Gln Gln Gly
35 40 45
Arg Ala Ala Thr Asn Ser His His Gly Gly Ala Gly Ala Met Glu Thr
50 55 60
Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly
65 70 75 80
Met Glu Glu Glu Leu Ser Pro Phe Arg Gly Arg Ser Arg Ser Ala Pro
85 90 95
Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met
100 105 110
Ser Asp Glu Phe Glu Gly Ser Phe Lys Gly Leu Pro Arg Pro Lys Ser
115 120 125
Ala Gly Thr Ala Thr Gln Met Arg Gln Ser Ala Gly Trp Thr Arg Ile
130 135 140
Ile Gln Ser Trp Trp Asp Arg Asn Leu Gly Lys Gly Gly Ser Thr Pro
145 150 155 160
Ser Gln
<210> 4
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BAD BH3
consensus sequence
<400> 4
Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser Asp Glu Phe
1 5 10 15
Val Asp Ser Phe Lys Lys Gly Leu Pro Arg
20 25
<210> 5
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
SUBSTITUTE SHEET (RULE 26)
CA 02373814 2001-11-09
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4/8
<223> Description of Artificial Sequence: BAK BH3
consensus sequence
<400> 5
Thr Met Gly Gln Val Gly Arg Gln Leu Ala Ile Ile Gly Asp Asp Ile
1 5 10 15
Asn Arg Arg Tyr Asp Ser Glu Phe Gln Thr
20 25
<210> 6
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BAX BH3
consensus sequence
<400> 6
Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys Arg Ile Gly Asp Glu Leu
1 5 10 15
Asp Ser Asn Met Glu Leu Gln Arg Met Ile
20 25
<210> 7
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BIK BH3
consensus sequence
<400> 7
Gly Ser Asp Ala Leu Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met
1 5 10 15
Asp Val Ser Leu Arg Ala Pro Arg Leu Ala
20 25
<210> 8
<211> 26
<212> PRT
<213> Artificial Sequence
<220>'
<223> Description of Artificial Sequence: BID BH3
consensus sequence
<400> 8
SUBSTITUTE SHEET (RULE 26)
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5/8
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Gln Val Gly Asp Ser Met
1 5 10 15
Asp Arg Ser Ile Pro Pro Gly Leu Val Asn
20 25
<210> 9
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: HRK BH3
consensus sequence
<400> 9
Ala Ala Gln Leu Thr Ala Ala Arg Leu Lys Ala Leu Gly Asp Glu Leu
1 5 10 15
His Gln Arg Thr Met Trp Arg Arg Arg Ala
20 25
<210> 10
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BOK BH3
consensus sequence
<400> 10
Arg Leu Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Asp Glu Leu
1 5 10 15
Glu Gln Ile Arg Pro Ser Val Tyr Arg Asn
20 25
<210> 11
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BIM BH3
consensus sequence
<400> 11
Pro Glu Ile Trp Ile Ala Gln Glu Leu Arg Arg Ile Gly Asp Glu Phe
1 5 10 15
Asn Ala Tyr Tyr Ala Arg Arg Val Phe Leu
SUBSTITUTE SHEET (RULE 26)
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20 25
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BAD primer
(murine)
<400> 12
gcctccagga tccaagatgg gaacc 25
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BAD primer
(murine)
<400> 13
ggagcgggta gaattccggg atg 23
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: BAD primer
(murine short)
<400> 14
tggagaccag gatcccagag tagct 25
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Human PKI
primer
<400> 15
ctatgtggat ccttggtagc aatg 24
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
SUBSTITUTE SHEET (RULE 26)
CA 02373814 2001-11-09
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7/8 _
<220>
<223> Description of Artificial Sequence: Human PKI
primer
<400> 16
cctcatagac cttaagtaaa caaa 24
<210> 17
<211> 18
<212> PRT
<213> Homo Sapiens
<400> 17
Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser Asp Glu Ser Val Asp
1 5 10 15
Ser Phe
<210> 18
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: antibody
generating phosphopeptide
<400> 18
Gly Cys Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser Asp Glu Ser
1 5 10 15
Val Asp Ser Phe
<210> 19
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: ST-kinase
recognition motif
<400> 19
Leu Arg Arg Met Ser Asp
1 5
<210> 20
<211> 12
<212> PRT
<213> Human immunodeficiency virus
SUBSTITUTE SHEET (RULE 26)
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<220>
<223> Description of Artificial Sequence: Tat polypeptide
<400> 20
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly
1 5 10
SUBSTITUTE SHEET (RULE 26)
