Note: Descriptions are shown in the official language in which they were submitted.
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Title: A Caspase Activated Protein Kinase
FIELD OF THE INVENTION
The invention relates to apoptosis and particularly a novel protein, SMAK
that activates two distinct signalling pathways that are involved in mediating
apoptosis.
The invention includes nucleic acid molecules encoding the SMAK protein; the
SMAK
protein and truncations, analogs, and homologs of the protein; and uses of the
protein and
nucleic acid molecules in controlling cellular apoptosis. In addition, the
invention includes
identification of a myosin binding protein (mybp-c) that specifically binds
the smak kinase
domain and inactivates kinase activity.
BACKGROUND OF THE INVENTION
Programmed cell death or apoptosis is a genetically controlled process
triggered by various stimuli in different cells. External stimuli such as
cytokines, UV
irradiation, and numerous drugs induce an apoptotic response characterized by
a series of
morphological changes that include cytoplasmic shrinkage, membrane blebbing,
chromatin
condensation, DNA fragmentation, and the formation of apoptotic bodies (Kerr
et al., 1972;
Raff et al., 1993; Steller, 1995). Several studies have revealed the catalytic
activation of
several kinases during apoptosis. For example, the cJun-aminoterminal kinase
(JNK)
pathway is activated in response to apoptotic triggers such as TNF-a and Fas
ligand
(Verheij et al., 1996; Xia et al., 1995; Yang et al., 1997). In addition,
activation of other
kinases, such as ASKl, RIP, or ZIP kinases, stimulates apoptosis in cultured
cells (Ichijo et
al., 1997; Kawai et al., 1998; Stanger et al., 1995).
The induction of apoptosis involves a proteolytic activation of a cascade of a
family of cysteine proteases called caspases. Aggregation of death receptors
following
ligand binding activates initiator caspases, which in turn, activates
downstream effector
caspases through proteolytic processing (Thornberry and Lazebnik, 1998).
Caspases
contribute to cell death by direct inactivation of negative regulators of
apoptosis and by
promoting the disassembly of cellular structures such as focal adhesion
complexes
(Thornberry and Lazebnik, 1998). Although several caspase substrates such as
nuclear
lamins and poly-ADP ribose polymerase have been identified (Thornberry and
Lazebnik,
1998), little is known about the biological function of the cleavage products.
However,
recent studies have revealed that caspase-mediated cleavage of the
serine/threonine
kinase PAK2 (p65PAK) generates a catalytically active fragment involved in
regulating
some of the morphological changes associated with apoptosis (Lee et al., 1997;
Rudel and
Bokoch, 1997). Caspases similarly cleave the Ste20-related kinase MST1 to
release a
catalytically active kinase domain that activates SAPKs as well as MKK6 and
MKK7
(Graves et al., 1998; Lee et al., 1998).
Cell growth and differentiation are regulated by complex signaling processes
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involving a large number of protein kinases and phosphatases. The activation
or inhibition
of various pathways ultimately results in the expression of specific subsets
of genes directly
involved in proliferation or terminal differentiation (Davis, 1993; Fanger et
al., 1997).
Extracellular signals acting on growth factor receptors or G-protein coupled
receptors
transduce their signal through a kinase cascade resulting in the activation of
mitogen-activated kinases (MAPK) such as ERKl and ERK2 . Stress-inducing
agents as well
as apoptotic triggers signal through the stress-activated pathway leading to
the
activation of JNK, p38, and other members of the stress-activated protein
kinase (SAPK)
family (Fanger et al., 1997). Activation of these downstream kinases requires
the
activation of multiple kinases and transducing molecules such as small
GTPases, MAPK
kinase kinases (MEKK) and MAPK kinases (MEK) (Fanger et al., 1997).
In yeast, the Ste20 serine/threonine protein kinase regulates a
mitogen-activated protein kinase pathway consisting in Stel1 (MEK kinase),
Ste7 (MEK)
and Fus3/Kss1 (MAPK) protein kinases involved in the control of mating
response (Zhao et
al., 1995). Activation of Ste20 results from a factor pheromone binding to its
G-protein
coupled receptor and subsequent activation of Ste20 by the (3'y complex
released from the
heterotrimeric G protein. This interaction results in translocation of Ste20
to the
scaffolding protein Ste5, leading to the sequential activation of Stell, Ste7
and Fus3/Kssl
(Leberer et al., 1997). Ste20 also binds the small GTPase Cdc42, however the
Cdc42 binding
domain of Ste20 has been shown to be dispensable for pheromone signaling in
yeast (Leberer
et al., 1997).
The small GTPase proteins of the Rho subfamily mediate various cellular
processes such as growth and cytoskeleton reorganization through direct
binding of the
activated GTP-bound forms to downstream targets (Van Aelst and D'Souza-
Schorey, 1997).
RhoA is required for maintenance of actin stress fibers and focal adhesions in
cultured cells.
These activities have been shown to be mediated by several Rho-associated
protein kinases
such as ROKa, p160ROCK, MRCKa, PKN, and PRK2 (Amano et al., 1997; Amano et
al.,
1996; Ishizaki et al., 1997; Leung et al., 1996; Leung et al., 1998; Nakagawa
et al., 1996;
Watanabe et al., 1996). The Cdc42 GTPase promotes the formation of actin
microspikes
whereas Rac1 activation induces the formation of lamellipodia or membrane
ruffles. In
addition to playing an important role in cellular morphology, the Rho family
of GTPases
regulates transcription through the JNK and p38 pathways (Fanger et al.,
1997).
Mammalian targets of Cdc42 and Rac1 include the PAK family of protein kinases
(Van
Aelst and D'Souza-Schorey, 1997). Upon binding to activated Cdc42 or Racl, PAK
is
activated and translocated to focal adhesion sites (Manser et al., 1997).
Expression of
constitutively active aPAK causes the loss of focal adhesions and retraction
of actin stress
fibers to the periphery (Manser et al., 1997). In addition, PAK activation
leads to
stimulation of the SAPK and p38 kinase pathways (Fanger et al., 1997).
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SUMMARY OF THE INVENTION
The present inventors have identified and characterized a cDNA that
encodes a protein kinase that mediates apoptosis and actin stress fiber
dissolution through
caspase-3-cleavage and functions to activate the stress activated protein
kinases
(cJun-amino terminal kinase (JNK) signalling pathway).
The present invention therefore provides a purified and isolated nucleic acid
molecule comprising a sequence encoding a protein having about 70% homology in
the kinase
domain with LOK, about 65% homology with M-NAP; and about 60% homology with
ATI-46. The protein of the invention may be generally referred to as a Ste20-
like kinase
protein or SMAK.
In an embodiment of the invention, the purified and isolated nucleic acid
molecule comprises: (i) a nucleic acid sequence encoding a SMAK protein having
the amino
acid sequence as shown in Figure 1; and, (ii) nucleic acid sequences
complementary to (i).
In a preferred embodiment of the invention, the purified and isolated nucleic
acid molecule comprises:
(i) a nucleic acid sequence encoding a SMAK protein having the nucleic acid
sequence as shown in Figure 1, wherein T can also be U;
(ii) a nucleic acid sequence complementary to (i), preferably complementary
to the full length nucleic acid sequence shown in Figure 1;
(iii) a nucleic acid molecule differing from any of the nucleic acids of (i)
and
(ii) in codon sequences due to the degeneracy of the genetic code.
The invention also contemplates: (a) a nucleic acid molecule comprising a
sequence encoding a truncation of the SMAK protein, an analog or homolog of
the SMAK
protein or a truncation thereof; (b) a nucleic acid molecule comprising a
sequence which
hybridizes under high stringency conditions to the nucleic acid encoding a
SMAK protein
having the amino acid sequence as shown in Figure 2; and (c) a nucleic acid
molecule
comprising a sequence which hybridizes under high stringency conditions to the
nucleic acid
sequence as shown in Figure 1, wherein T can also be U, or complementary
sequences thereto.
The invention further contemplates a purified and isolated double stranded
nucleic acid molecule containing a nucleic acid molecule of the invention,
hydrogen bonded
to a complementary nucleic acid base sequence.
The nucleic acid molecules of the invention may be inserted into an
appropriate expression vector, i.e., a vector which contains the necessary
elements for the
transcription and translation of the inserted coding sequence. Accordingly,
recombinant
expression vectors adapted for transformation of a host cell may be
constructed which
comprise a nucleic acid molecule of the invention and one or more
transcription and
translation elements operatively linked to the nucleic acid molecule.
The recombinant expression vector can be used to prepare transformed host
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cells expressing a SMAK protein of the invention or a SMAK related protein.
The
expression vector can be used for gene therapy. The invention further provides
one or more
host cells containing a recombinant molecule of the invention. The invention
also
contemplates transgenic non-human mammals whose germ cells and somatic cells
contain a
recombinant molecule comprising a nucleic acid molecule of the invention which
encodes a
SMAK protein or an analog of a SMAK protein, i.e., the protein with an
insertion,
substitution or deletion mutation.
The invention further provides a method for preparing a SMAK protein, or a
SMAK related protein utilizing the purified and isolated nucleic acid
molecules of the
invention. In an embodiment a method for preparing a SMAK protein is provided
comprising: (a) transferring a recombinant expression vector of the invention
into a host cell;
(b) selecting one or more transformed host cells from untransformed host
cells; (c) culturing a
selected transformed host cell under conditions which allow expression of the
SMAK
protein; and (d) isolating the SMAK protein.
The invention further broadly contemplates a purified and isolated SMAK
protein that upon caspase-3 cleavage mediates apoptosis and actin stress fiber
dissolution.
SMAK protein also activates the JNK pathway and has protein interaction motifs
including several AU-rich motifs and an ATH domain. The SMAK protein has an
approximate molecular weight of 148KDa. In an embodiment of the invention, a
purified
SMAK protein is provided which has the amino acid sequence as shown in Figure
2. The
purified and isolated protein of the invention may be modified or activated,
i.e.,
phosphorylated. The invention also includes truncations of the protein and
analogs,
homologs, and isoforms of the protein and truncations thereof.
The SMAK proteins of the invention may be conjugated with other molecules,
such as proteins to prepare fusion proteins. This may be accomplished, for
example, by the
synthesis of N-terminal or C-terminal fusion proteins.
The invention further contemplates antibodies having specificity against one
or more epitopes of a SMAK protein of the invention. Antibodies may be
labelled with a
detectable substance and they may be used to detect the SMAK protein of the
invention in
tissues and cells.
The invention also permits the construction of nucleotide probes which are
unique to the nucleic acid molecules of the invention and accordingly to a
SMAK protein of
the invention. Thus, the invention also relates to a probe comprising a
sequence encoding a
SMAK protein or fragment thereof. The probe may be labelled, for example, with
a
detectable substance and it may be used to select from a mixture of nucleotide
sequences a
nucleotide sequence coding for a protein which displays one or more of the
properties of the
protein of the invention. The probes may also be used to detect a nucleic acid
encoding a
SMAK protein of the invention in tissues and cells.
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The SMAK protein is involved in cell apoptosis and actin stress fiber
dissolution. Accordingly, in one embodiment the present invention provides a
method of
modulating apoptosis comprising administering an effective amount of a SMAK
protein or a
nucleic acid encoding a SMAK protein to a cell or animal in need thereof.
JNK and caspases are associated with apoptosis. Stress inducing agents as
well as apoptotic triggers have been demonstrated to signal through stress-
activated
pathways leading to activation of JNK. The induction of apoptosis involves a
proteolytic
activation cascade of a family of cysteine proteases called caspases. The SMAK
protein
appears to be associated with apoptosis and accordingly may play a role in
preventing
neoplasia development, lymphoproliferative conditions, inflammation, and
autoimmune
disease. In such embodiments, it may not be desirable to inhibit the
expression of the
SMAK protein. Accordingly, the present invention provides a method of
inhibiting or
reducing cell proliferation comprising administering to an undesirable cell or
to an animal in
need thereof, an effective amount of an agent that promotes the expression or
activity of the
SMAK protein. Agents include those that may promote the SMAK protein promoter
sequences to upregulate a portion of a nucleic acid sequence encoding the SMAK
protein.
Under conditions where apoptotic responses are detrimental, such as
ischemia and stroke, and where SMAK activity would be increased, it may be
desirable to
inhibit SMAK activity or expression. Accordingly, the present invention
provides a
method of inhibiting or reducing SMAK activity comprising administering to a
cell or
animal in need thereof an effective amount of an agent that inhibits the
expression or
activity of the SMAK protein. Agents include those that may inhibit the SMAK
protein
promoter sequences to downregulate a portion of a nucleic acid sequence
encoding the SMAK
protein.
The invention still further provides a method for identifying a substance
which is capable of binding to a SMAK protein or an activated form thereof,
comprising
reacting the SMAK protein, or an activated form thereof, with at least one
substance which
can potentially bind with the SMAK protein or an activated form thereof, under
conditions
which permit the formation of complexes between the substance and the SMAK
protein or
an activated form thereof, and assaying for complexes, for free substance, for
non-complexed
SMAK protein or an activated form thereof, or for activation of SMAK.
Specifically, a
yeast two hybrid assay system may be utilized as a method for identifying
proteins which
interact with the protein (Fields, S. and Song, O. 1989, Nature, 34:245-247).
Still further, the invention provides a method for assaying a medium for the
presence of an agonist or antagonist of the interaction of a SMAK protein or
an activated
form thereof, and a substance which binds to the SMAK protein or an activated
form
thereof. In an embodiment, the method comprises providing a known
concentration of a
SMAK protein, with a substance which is capable of binding to the SMAK protein
and a
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suspected agonist or antagonist substance under conditions which permit the
formation of
complexes between the substance and SMAK protein, and assaying for complexes,
for free
substance, for non-complexed SMAK protein, or for activation of SMAK protein.
Substances which affect the SMAK protein may also be identified using the
methods of the invention by comparing the pattern and level of expression of
the SMAK
protein of the invention in tissues and cells in the presence, and in the
absence of the
substance.
Further, the present invention provides a method of identifying agents for
treatment of neoplasia, lymphoproliferative conditions, arthritis,
inflammation,
autoimmune diseases, apoptosis, muscle atrophy, cardiomyopathy, and the like,
that are
related to SMAK signal transduction and actin reorganizing pathways.
The antibodies to the SMAK protein or antisense oligonucleotides
complimentary to a nucleic acid encoding the SMAK protein as well as
substances identified
using the method of the invention may be used in the treatment of conditions
involving
apoptosis and actin stress fiber dissolution, preferably arthritis, apoptosis,
muscle atrophy,
and cardiomyopathy. Accordingly, the substances may be formulated into
pharmaceutical
compositions for adminstration to individuals suffering from such afflictions.
Other features and advantages of the present invention will become apparent
from the following detailed description. It should be understood, however,
that the
detailed description and the specific examples while indicating preferred
embodiments of
the invention are given by way of illustration only, since various changes and
modifications
within the spirit and scope of the invention will become apparent to those
skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the cDNA sequence of SMAK.
Figure 2 shows the deduced amino acid sequence of SMAK.
Figure 3A shows the sequence alignment of SMAK with that of the
Ste20-related kinases human SLK (hSLK), LOK and MST.
Figure 3B shows the sequence alignment of SMAK with Rat SLK, LOK,
M-NAP and AT1-46.
Figure 3C shows the schematic representation of SMAK and similarity
indices (%) to various other polypeptide. The catalytic domain (black) is most
similar to
LOK and MSTl/2, two Ste20-like kinases. SMAK is highly homologous to
microtubule and
nuclear associated protein (M-NAP; grey) and to ATl-46 (white) in the carboxy
terminal
region. Numbers in brackets represent SMAK1 amino acid residues.
Figure 4A shows the Northern blot analysis of various tissue analyzed for
SMAK expression and normalized for PGK-1 mRNA levels.
Figure 4B shows the Northern blot analysis of various tissue analyzed for
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SMAK expression and normalized for b-actin mRNA levels.
Figure 4C shows the Northern blot analysis of SMAK mRNA expression in
murine cell lines.
Figure 4D shows the Northern blot analysis of SMAK expression in various
human cell lines.
Figure 5A shows expression of SMAK during C2C12 cellular differentiation.
Figure 5B shows expression of SMAK during serum stimulation of NIH3T3
fibroblasts.
Figure 6A shows an immunoblot of in vitro kinase assay of purified or
immunoprecipitated SMAK 1.
Figure 6B shows inactivation of SMAK by calf intestinal alkaline
phosphatase (CIAP) treatment.
Figure 7A shows immunoprecipitation and in vitro kinase assay of
transfected Myc-SMAK.
Figure 7B shows immunoblot analysis illustrating that the cultures of Figure
7A expressed equivalent amounts of transfected SMAK protein.
Figure 7C shows the activation of JNK1 by SMAK in 293 cells.
Figure 8A shows immunolocalization of SMAK in C2C12 myoblasts.
Figure 8B shows the photomicrographs of Figure 8A at 400X.
Figure 9A shows induction of apoptosis in SMAK Transfected C2C12
Myoblasts.
Figure 9B shows induction of apoptosis in SMAK Transfected C2C12
Myoblasts.
Figures 9C and 9D shows double immunofluorescent staining of Myc-SMAK
and annexin V-FITC in transfected C2C12 cells. Arrows denote identical cells
in Figures 9C
and 9D.
Figures 9E and 9F show immunodetection of the Myc-SMAKK63R lacking
functional kinase activity in transfected C2C12 cells and co-localization of
annexin V-FITC.
Arrows denote identical cells in Figures 9E and 9F.
Figures 9G and 9H shows immunodetection of Myc-SMAK expression in cells
undergoing DNA fragmentation as detected by TUNEL labeling. Arrows denote
identical
cells in Figures 9G and 9H.
Figure 10 shows Gal4-Luciferase reporter and Gal4-Elkl, Gal4-Jun and
Gal4-CREB fusions with or without SMAK transiently transfected into 293 cells.
Figures 11A and 11B shows expression of Myc-SMAK in transiently
transfected C2C12 cells inducing loss of actin stress fibers as detected by
phalloidin
staining. Arrow denotes the identical cell in Figures 10A and 10B.
Figures 11C and 11D show expression of Myc-RacVl2 in C2C12 cells
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illustrating similarly induced loss of stress fibers as evidenced by
phalloidin staining.
Arrow denotes the identical cell in Figures 11C and 11D.
Figures 11E and 11F show expression of transfected Myc-p65PAK illustrating
induced loss of actin stress fibers. Arrow denotes the identical cell in
Figures 11E and 11F.
Figure 12 shows the colocalization of SMAK, PAK, and RhoA in transfected
C2C12 myoblasts.
Figures 12A, 12B, 12C, and 12D show immunodetection of transfected
Myc-tagged SMAK, PAK, RhoANl9 and Rac1V12, respectively with antibody 9E10.
Figure 12E shows induction of cell death by activated RhoA in C2C12 cells.
RhoAVl4 expression was detected using 9E10 16 hours following transfection.
Figure 12F shows a phase contrast photomicrograph of Figure 12E.
Figures 12G and 12H show double immunodetection of transfected HA-tagged
SMAK with a rabbit anti-HA antibody and of transfected Myc-RhoANl9 expressing
cells
using 9E10.
Figure 12I illustrates a superposition of Figures 12G and 12H showing
colocalization of SMAK and RhoA.
Figures 12J, 12K, and 12L illustrate double immunodetection of transfected
HA-tagged SMAK with a rabbit anti-HA antibody and of transfected Myc-PAK
expressing
cells using 9E10.
Figure 12L shows a superposition of Figures 12J and 12L showing
colocalization of SMAK and PAK.
Figure 13 shows the schematic representation of the plasmid expression
vectors used in this study. The Ste20 kinase domain (black), M-NAP (grey) and
ATH
(white) domains are indicated. SMAK amino acid residues are indicated at the N-
and
C-termini.
Figures 14A and 14B show immunodetection and phalloidin staining of
Myc-SMAIC~C-expressing C2C12 cells 16 hours post-transfection.
Figures 14C and 14D show that overexpression of the Myc-SMAKOCK63R
mutant did not result in any morphological changes in C2C12 cells.
Figures 14E and 14F show forced expression of Myc-SMAK~N induced stress
fiber dissolution in overexpressing cells as shown by phalloidin staining.
Figures 14G and 14H show transfection of the ATl-46 domain (pXh2973; H
and I) resulted in a loss of strongly staining actin stress fibers.
Photomicrographs are shown
at 400X.
Figure 14I shows enhanced apoptotic response by SMAKOC overexpression.
Figure 14J shows the indicated expression vectors were transfected into 293T
cells, immunoprecipitated using 9E10 antibodies and assayed for kinase
activity on myelin
basic protein MBP arrow. Equivalent aliquots of protein were also subjected to
Western blot
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analysis for normalization (upper panel).
Figure 15A shows caspase-3 cleavage of in vitro translated wildtype (WT)
and caspase-3 cleavage site mutant (D436N) SMAK proteins.
Figure 15B shows apoptosis-induced cleavage of endogenous SMAK protein in
stimulated Rat1-Myc/ER cells.
Figure 15C shows N-terminal-specific antibodies identifying the 60 kDa
fragment as the kinase domain in induced Rat1-Myc/ER cells and NIH3T3 cells
exposed to
apoptotic triggers.
Figure 15D shows in vitro caspase-3/kinase assay on immunoprecipitated
Myc-SMAK proteins.
Figure 15E shows anti-Myc tag western blot analysis showing expression of
all Myc-SMAK proteins in transfected 293 cells.
DETAILED DESCRIPTION OF THE INVENTION
The following standard abbreviations for the amino acid residues are used
throughout the specification: A, Ala - alanine; C, Cys - cysteine; D, Asp-
aspartic acid; E,
Glu - glutamic acid; F, Phe - phenylalanine; G, Gly - glycine; H, His -
histidine; I, Ile -
isoleucine; K, Lys - lysine; L, Leu - leucine; M, Met - methionine; N, Asn -
asparagine; P, Pro
- proline; Q, Gln - glutamine; R, Arg - arginine; S, Ser - serine; T, Thr -
threonine; V, Val
valine; W, Trp- tryptophan; Y, Tyr - tyrosine; and p.Y., P.Tyr -
phosphotyrosine, P, Ser -
phosphoserine.
I. Nucleic Acid Molecules Encoding SMAK
As hereinbefore mentioned, the invention provides an isolated and purified
nucleic acid molecule having a sequence encoding a SMAK protein. The term
"isolated and
purified" refers to a nucleic acid substantially free of cellular material or
culture medium
when produced by recombinant DNA techniques, or chemical precursors, or other
chemicals
when chemically synthesized. The term "nucleic acid" is intended to include
DNA and
RNA and can be either double stranded or single stranded.
The nucleic acid sequence of the cDNA encoding the SMAK protein is shown
in Figure 1.
It will be appreciated that the invention includes nucleic acid molecules
encoding truncations of SMAK protein, and analogs and homologs of SMAK and
truncations
thereof, as described herein. It will further be appreciated that variant
forms of the
nucleic acid molecules of the invention which arise by alternative splicing of
an mRNA
corresponding to a cDNA of the invention are encompassed by the invention.
Another aspect of the invention provides a nucleic acid molecule which
hybridizes under high stringency conditions to a nucleic acid molecule which
comprises a
sequence which encodes SMAK protein having the amino acid sequence shown in
Figure 2.
Appropriate stringency conditions which promote DNA hybridization are known to
those
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skilled in the art, or can be found in Current Protocols in Molecular Biology,
John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0 x sodium chloride/sodium
citrate (SSC) at
about 45°C, followed by a wash of 2.0 x SSC at 50°C may be
employed. The stringency may
be selected based on the conditions used in the wash step. By way of example,
the salt
concentration in the wash step can be selected from a high stringency of about
0.2 x SSC at
50°C. In addition, the temperature in the wash step can be at high
stringency conditions, at
about 65°C.
Isolated and purified nucleic acid molecules encoding a protein having the
activity of SMAK protein as described herein, and having a sequence which
differs from
the nucleic acid sequence shown in Figure 1, due to degeneracy in the genetic
code are also
within the scope of the invention.
In addition, DNA sequence polymorphisms within the nucleotide sequence of
the SMAK protein (especially those within the third base of a codon) may
result in "silent"
mutations in the DNA which do not affect the amino acid encoded. However, DNA
sequence polymorphisms may lead to changes in the amino acid sequences of SMAK
protein
within a population. It will be appreciated by one skilled in the art that
these variations
in one or more nucleotides of the nucleic acids encoding proteins having the
activity of the
SMAK protein may exist among individuals within a population due to natural
allelic
variation. Any and all such nucleotide variations and resulting amino acid
polymorphisms
are within the scope of the invention.
An isolated and purified nucleic acid molecule of the invention which
comprises DNA can be isolated by preparing a labelled nucleic acid probe based
on all or
part of the nucleic acid sequence shown in Figure 1 and using this labelled
nucleic acid probe
to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library). For
instance,
a cDNA library made from human cells such as B cells can be used to isolate a
cDNA
encoding a protein having SMAK activity by screening the library with the
labelled probe
using standard techniques. Alternatively, a genomic DNA library can be
similarly screened
to isolate a genomic clone encompassing a gene encoding a SMAK protein.
Nucleic acids
isolated by screening of a cDNA or genomic DNA library can be sequenced by
standard
techniques.
An isolated and purified nucleic acid molecule of the invention which is
DNA can also be isolated by selectively amplifying a nucleic acid encoding a
SMAK protein
using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It
is
possible to design synthetic oligonucleotide primers from the nucleotide
sequence shown in
Figure 1. A nucleic acid can be amplified from cDNA or genomic DNA using these
oligonucleotide primers and standard PCR amplification techniques. The nucleic
acid so
amplified can be cloned into an appropriate vector and characterized by DNA
sequence
analysis. It will be appreciated that cDNA may be prepared from mRNA, by
isolating
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total cellular mRNA by a variety of techniques, for example, by using the
guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry,
18,
5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse
transcriptase
(for example, Moloney MLV reverse transcriptase available from Gibco/BRL,
Bethesda,
MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St.
Petersburg,
FL).
An isolated and purified nucleic acid molecule of the invention which is
RNA can be isolated by cloning a cDNA encoding a SMAK protein into an
appropriate
vector which allows for transcription of the cDNA to produce an RNA molecule
which
encodes a protein which exhibits SMAK protein activity. For example, a cDNA
can be
cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a
vector, cDNA can
be transcribed in vitro with T7 polymerase, and the resultant RNA can be
isolated by
standard techniques.
A nucleic acid molecule of the invention may also be chemically synthesized
using standard techniques. Various methods of chemically synthesizing
polydeoxynucleotides are known, including solid-phase synthesis which, like
peptide
synthesis, has been fully automated in commercially available DNA synthesizers
(See e.g.,
Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No.
4,458,066; and
Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
Determination of whether a particular nucleic acid molecule encodes a
protein having SMAK protein activity can be accomplished by expressing the
cDNA in an
appropriate host cell by standard techniques, and testing the ability of the
expressed
protein to undergo caspase-3 cleavage and mediate apoptosis and actin stress
fibre
dissolution. A cDNA having the biological activity of a SMAK protein so
isolated can be
sequenced by standard techniques, such as dideoxynucleotide chain termination
or
Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and
the
predicted amino acid sequence of the encoded protein.
The sequence of a nucleic acid molecule of the invention may be inverted
relative to its normal presentation for transcription to produce an antisense
nucleic acid
molecule. An antisense nucleic acid molecule may be constructed using chemical
synthesis
and enzymatic ligation reactions using procedures known in the art.
II. SMAK Proteins
As hereinbefore mentioned, the present inventors have isolated and
characterized a novel caspase-3 activated protein kinase, designated SMAK.
SMAK is
highly related to LOK, an Ste20-related protein kinase preferentially
expressed in
lymphocytes in that it shares about a 70% homology in the kinase domain. SMAK
also
shares extensive homology to microtubule and nuclear associated protein (M-
NAP)
amounting to about 65% homology and to ATl-46 with a homology of about 60%.
The SMAK
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cDNA encodes a protein with a predicted molecular weight of 148 kDa. The amino
acid
sequence of the SMAK protein is shown in Figure 2.
The inventors have shown that the SMAK protein contains an SH3-binding
motif and a coiled-coil structure in the C-terminal region and an N-terminal
serine/threonine kinase catalytic domain with the signature sequence Gly-X-Gly-
X-X-Gly.
SMAK mRNA is widely expressed in a variety of tissues including skeletal
muscle, heart, thymus, brain, colon, spleen, lung, kidney, testes, uterus, and
liver. The
SMAK protein is not expressed in undifferentiated P19 embryo carinoma cells
which
suggests that SMAK has more of a role in differentiated cell types.
The proteins of the present invention include truncations of the SMAK
protein, and analogs, and homologs and truncations thereof as described
herein.
The truncated proteins may have an amino group (-NH2), a hydrophobic
group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl
group, a
9-fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not
limited
to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the
amino terminal
end. The truncated proteins may have a carboxyl group, an amido group, a
T-butyloxycarbonyl group, or a macromolecule including but not limited to
lipid-fatty acid
conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.
The proteins of the invention may also include analogs of the SMAK protein
as shown in Figure 2 or truncations thereof as described herein, which may
include, but are
not limited to one or more amino acid substitutions, insertions, and/or
deletions. Amino acid
substitutions may be of a conserved or non-conserved nature. Conserved amino
acid
substitutions involve replacing one or more amino acids of the SMAK amino acid
sequence
with amino acids of similar charge, size, and/or hydrophobicity
characteristics. When
only conserved substitutions are made the resulting analog should be
functionally
equivalent to the SMAK protein. Non-conserved substitutions involve replacing
one or more
amino acids of the SMAK amino acid sequence with one or more amino acids which
possess
dissimilar charge, size, and/or hydrophobicity characteristics.
One or more amino acid insertions may be introduced into the SMAK protein.
Amino acid insertions may consist of single amino acid residues or sequential
amino acids
ranging from 2 to 15 amino acids in length.
The proteins of the invention also include homologs of the SMAK protein
(Figure 2) and/or truncations thereof as described herein. Such homologs are
proteins whose
amino acid sequences are comprised of the amino acid sequences of SMAK protein
regions
from other species that hybridize under stringent hybridization conditions
with a probe
used to obtain the SMAK protein.
The invention also contemplates isoforms of the protein of the invention. An
isoform contains the same number and kinds of amino acids as the protein of
the invention or
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subtle spliced differences which alter the overall number of amino acids
resulting in a
slight shift in molecular weight. The isoforms contemplated by the present
invention are
those having the same properties as the protein of the invention as described
herein. The
inventors have demonstrated that the SMAK mRNA is expressed and exists in at
least
three distinct isoforms, all of which are expressed at similar levels in all
tissues tested
with the exception of the testes and colon tissues.
The present invention also includes SMAK protein conjugated with a selected
protein, or a selectable marker protein (see below) to produce fusion
proteins. Further, the
present invention also includes activated or phosphorylated SMAK proteins of
the
invention. Additionally, immunogenic portions of SMAK proteins are within the
scope of
the invention.
SMAK proteins of the invention may be prepared using recombinant DNA
methods. Accordingly, the nucleic acid molecules of the present invention
having a sequence
which encodes a SMAK protein of the invention may be incorporated in a known
manner into
an appropriate expression vector which ensures good expression of the protein.
Possible
expression vectors include but are not limited to cosmids, plasmids, or
modified viruses (e.g.
replication defective retroviruses, adenoviruses and adeno-associated
viruses), so long as
the vector is compatible with the host cell used. The expression vectors are
"suitable for
transformation of a host cell", means that the expression vectors contain a
nucleic acid
molecule of the invention and regulatory sequences selected on the basis of
the host cells to
be used for expression, which is operatively linked to the nucleic acid
molecule.
Operatively linked is intended to mean that the nucleic acid is linked to
regulatory
sequences in a manner which allows expression of the nucleic acid.
The invention therefore contemplates a recombinant expression vector of the
invention containing a nucleic acid molecule of the invention, or a fragment
thereof, and the
necessary regulatory sequences for the transcription and translation of the
inserted
protein-sequence. Suitable regulatory sequences may be derived from a variety
of sources,
including mammalian, bacterial, fungal, viral or insect genes (For example,
see the
regulatory sequences described in Goeddel, Gene Expression Technology: Methods
in
Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate
regulatory sequences is dependent on the host cell chosen as discussed below,
and may be
readily accomplished by one of ordinary skill in the art. Examples of such
regulatory
sequences include: a transcriptional promoter and enhancer or RNA polymerase
binding
sequence, a ribosomal binding sequence, including a translation initiation
signal.
Additionally, depending on the host cell chosen and the vector employed, other
sequences,
such as an origin of replication, additional DNA restriction sites, enhancers,
and sequences
conferring inducibility of transcription may be incorporated into the
expression vector. It
will also be appreciated that the necessary regulatory sequences may be
supplied by the
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native SMAK gene and/or its flanking regions.
The invention further provides a recombinant expression vector comprising a
DNA nucleic acid molecule of the invention cloned into the expression vector
in an antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in a
manner which allows for expression, by transcription of the DNA molecule, of
an RNA
molecule which is antisense to the nucleotide sequence shown in Figure 1.
Regulatory
sequences operatively linked to the antisense nucleic acid can be chosen which
direct the
continuous expression of the antisense RNA molecule in a variety of cell
types, for instance a
viral promoter and/or enhancer, or regulatory sequences can be chosen which
direct tissue or
cell type specific expression of antisense RNA.
The recombinant expression vectors of the invention may also contain a
selectable marker gene which facilitates the selection of host cells
transformed or
transfected with a recombinant molecule of the invention. Examples of
selectable marker
genes are genes encoding a protein such as 6418 and hygromycin which confer
resistance to
certain drugs, b-galactosidase, chloramphenicol acetyltransferase, firefly
luciferase, green
fluorescent protein, alkaline phosphatase, yellow fluorescent protein, blue
fluorescent
protein or an immunoglobulin or portion thereof such as the Fc portion of an
immunoglobulin
preferably IgG. Transcription of the selectable marker gene is monitored by
changes in the
concentration of the selectable marker protein such as b-galactosidase,
chloramphenicol
acetyltransferase, or firefly luciferase. If the selectable marker gene
encodes a protein
conferring antibiotic resistance such as neomycin resistance transformant
cells can be
selected with 6418. Cells that have incorporated the selectable marker gene
will survive,
while the other cells die. This makes it possible to visualize and assay for
expression of
recombinant expression vectors of the invention and in particular to determine
the effect of a
mutation on expression and phenotype. It will be appreciated that selectable
markers can
be introduced on a separate vector from the nucleic acid of interest.
The recombinant expression vectors may also contain genes which encode a
fusion moiety which provides increased expression of the recombinant protein;
increased
solubility of the recombinant protein; and aid in the purification of the
target recombinant
protein by acting as a ligand in affinity purification. For example, a
proteolytic cleavage
site may be added to the target recombinant protein to allow separation of the
recombinant
protein from the fusion moiety subsequent to purification of the fusion
protein. Typical
fusion expression vectors include pGEX (Pharmacia), pMAL (New England Biolabs,
Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A, respectively, to
the
recombinant protein.
Recombinant expression vectors can be introduced into host cells to produce a
transformant host cell. The term "transformant host cell" is intended to
include
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prokaryotic and eukaryotic cells which have been transformed or transfected
with a
recombinant expression vector of the invention. The terms "transformed with",
"transfected
with", "transformation" and "transfection" are intended to encompass
introduction of
nucleic acid (e.g. a vector) into a cell by one of many possible techniques
known in the art.
Prokaryotic cells can be transformed with nucleic acid by, for example,
electroporation or
calcium-chloride mediated transformation. Nucleic acid can be introduced into
mammalian
cells via conventional techniques such as calcium phosphate or calcium
chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofectin,
electroporation or
microinjection. Suitable methods for transforming and transfecting host cells
can be found in
Sarnbrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor
Laboratory press (1989)), and other laboratory textbooks.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host
cells. For example, the SMAK proteins of the invention may be expressed in
mammalian
cells, bacterial cells such as E. coli, insect cells (using baculovirus) or
yeast cells. Other
suitable host cells can be found in Goeddel, Gene Expression Technology:
Methods in
Enzymology 185, Academic Press, San Diego, CA (1991).
Mammalian cells suitable for carrying out the present invention include,
among others: Cos-7 (green monkey kidney), Saos-2 (human osteosarcoma), PC12,
NIH-3T3,
MONC, SYSY, P19, 6361 (human melanoma), A549 (human lung carcinoma), SW480
(human
colorectal adenocarcinoma), Raji (human Burkitt's lymphoma), MOLT-4 (human
lymphoblastic leukemia), K562 (human chronic myelogenous leukemia), S3 (Hela
cells),
HL-60 (human promyelocytic leukemia, and breast neoplasia cell lines; T47Ds,
MCF-7s and
C127s, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281),
CHO
(ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1
cells.
Suitable expression vectors for directing expression in mammalian cells
generally include a
promoter (e.g., derived from viral material such as polyoma, Adenovirus 2,
cytomegalovirus, retrovirus (pBabe and LXHSD), and Simian Virus 40), as well
as other
transcriptional and translational control sequences. Examples of mammalian
expression
vectors include pCDMB (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et
al.
(1987), EMBOJ. 6:187-195).
Bacterial host cells suitable for carrying out the present invention include
E.
coli, B. subtilis, Salmonella typhimurium, and various species within the
genus'
Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial
species
well known to one of ordinary skill in the art. Suitable bacterial expression
vectors
preferably comprise a promoter which functions in the host cell, one or more
selectable
phenotypic markers, and a bacterial origin of replication. Representative
promoters
include the b-lactamase (penicillinase) and lactose promoter system (see Chang
et al.,
Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth in
Enzymology
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101:155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982).
Representative
selectable markers include various antibiotic resistance markers such as the
kanamycin or
ampicillin resistance genes. Suitable expression vectors include but are not
limited to
bacteriophages such as lambda derivatives or plasmids such as pBR322 (see
Bolivar et al.,
Gene 2:9S, 1977), the pUC plasmids pUCl8, pUCl9, pUC118, pUC119 (see Messing,
Meth in
Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982), and
pNHBA,
pNHl6a, pNHl8a, and Bluescript M13 (Stratagene, La Jolla, Calif.). Typical
fusion
expression vectors which may be used are discussed above, e.g. pGEX (Amrad
Corp.,
Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5
(Pharmacia, Piscataway, NJ). Examples of inducible non-fusion expression
vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET lld (Studier et al., Gene
Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, California
(1990)
60-89).
Yeast and fungi host cells suitable for carrying out the present invention
include, but are not limited to Saccharomyces cerevisae, the genera Pichia or
Kluyveromyces and various species of the genus Aspergillus. Examples of
vectors for
expression in yeast S. cerivisae include pYepSecl (Baldari. et al., (1987)
Embo J. 6:229-234),
pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,
(1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for
the
transformation of yeast and fungi are well known to those of ordinary skill in
the art (see
Hinnen et al., PNAS USA 75:1929, 1978; Itoh et al., J. Bacteriology 153:163,
1983, and Cullen
et al. (Bio/Technology 5:369, 1987).
Given the teachings provided herein, promoters, terminators, and methods
for introducing expression vectors of an appropriate type into plant, avian,
and insect cells
may also be readily accomplished. For example, within one embodiment, the
proteins of
the invention may be expressed from plant cells (see Sinkar et al., J. Biosci
(Bangalore)
11:47-58, 1987, which reviews the use of Agrobacterium rhizogenes vectors; see
also
Zambryski et al., Genetic Engineering, Principles and Methods, Hollaender and
Setlow
(eds.), Vol. VI, pp. 253-278, Plenum Press, New York, 1984, which describes
the use of
expression vectors for plant cells, including, among others, pAS2022, pAS2023,
and
pAS2034).
Insect cells suitable for carrying out the present invention include cells and
cell lines from Bombyx or Spodotera species. Baculovirus vectors available for
expression of
proteins in cultured insect cells (SF 9, SF 21, and T.ni-High Five cells)
include the pAc series
(Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series
(Lucklow, V.A., and
Summers, M.D., (1989) Virology 170:31-39) and pBAC PAK.
Alternatively, the proteins of the invention may also be expressed in
non-human transgenic animals such as mice, rats, rabbits, sheep and pigs (see
Hammer et al.
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(Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983),
Brinster et al. (Proc
Natl. Acad. Sci USA 82:44384442, 1985), Palmiter and Brinster (Cell. 41:343-
345, 1985) and
U.S. Patent No. 4,736,866).
The invention further provides a recombinant expression vector for the
transcription and translation in invertebrate animals including, but not
limited to,
zebrafish, xenopus and drosophila.
The proteins of the invention may also be prepared by chemical synthesis
using techniques well known in the chemistry of proteins such as solid phase
synthesis
(Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in
homogenous solution
(Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and
II,
Thieme, Stuttgart).
N-terminal or C-terminal fusion proteins comprising the SMAK protein of
the invention conjugated with other molecules, such as proteins may be
prepared by fusing,
through recombinant techniques, the N-terminal or C-terminal of the protein,
and the
sequence of a selected protein or selectable marker protein with a desired
biological
function. The resultant fusion proteins contain the SMAK protein fused to the
selected
protein or marker protein as described herein. Examples of proteins which may
be used to
prepare fusion proteins include green fluorescent protein (GFP), yellow
fluorescent protein,
blue f luorescent p rotein, a lka line p h osphata se, i mmunoglobulins,
glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.
Phosphorylated or activated SMAK proteins of the invention may be
prepared using the method described in Reedijk et al. The EMBO Journal
11(4):1365, 1992.
III. Utility of the Nucleic Acid Molecules and Proteins of the Invention
Diagnostic Uses
The nucleic acid molecules of the invention allow those skilled in the art to
construct nucleotide probes for use in the detection of nucleic acid sequences
in biological
materials. Suitable probes include nucleic acid molecules based on nucleic
acid sequences
encoding at least 6 sequential amino acids from regions of the SMAK protein as
shown in
Figure 2. A nucleotide probe may be labelled with a detectable substance such
as a
radioactive label which provides for an adequate signal and has sufficient
half-life such
as 32P, 3H, 14C or the like. Other detectable substances which may be used
include antigens
that are recognized by a specific labelled antibody, fluorescent compounds,
enzymes,
antibodies specific for a labelled antigen, and luminescent compounds. An
appropriate
label may be selected having regard to the rate of hybridization and binding
of the probe to
the nucleotide to be detected and the amount of nucleotide available for
hybridization.
Labelled probes may be hybridized to nucleic acids on solid supports such as
nitrocellulose
filters or nylon membranes as generally described in Sambrook et al, 1989,
Molecular
Cloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may be used to
detect
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genes, preferably in human cells, that encode the SMAK protein. The nucleotide
probes
may be useful in the diagnosis of disorders of cell transformation such as
muscle atrophy.
The nucleotide probes may also be used as a diagnostic tool on tissue biopsies
to assess the
transformed state of the cell. The probes may also be used in in situ
hybridization of early
embryos to assess both the onset and pattern of expression during development.
SMAK proteins of the invention can be used to prepare antibodies specific for
the SMAK proteins that may be used to detect the SMAK protein in a biological
sample.
Antibodies can be prepared which bind a distinct epitope in an unconserved
region of the
protein. An unconserved region of the protein is one which does not have
substantial
sequence homology to other proteins.
Conventional methods can be used to prepare the antibodies. For example, by
using a peptide of the SMAK protein, polyclonal antisera or monoclonal
antibodies can be
made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can
be
immunized with an immunogenic form of the peptide which elicits an antibody
response in
the mammal. Techniques for conferring immunogenicity on a peptide include
conjugation to
carriers or other techniques well known in the art. For example, the protein
or peptide can
be administered in the presence of adjuvant. The progress of immunization can
be monitored
by detection of antibody titers in plasma or serum. Standard ELISA or other
immunoassay
procedures can be used with the immunogen as antigen to assess the levels of
antibodies.
Following immunization, antisera can be obtained and, if desired, polyclonal
antibodies
isolated from the sera. The preparation of anti-SMAK antibodies is described
in Example
1.
To produce monoclonal antibodies, antibody producing cells (lymphocytes)
can be harvested from an immunized animal and fused with myeloma cells by
standard
somatic cell fusion procedures thus immortalizing these cells and yielding
hybridoma cells.
Such techniques are well known in the art, (e.g., the hybridoma technique
originally
developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other
techniques
such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4,
72 (1983)),
the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et
al.
Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-
96), and
screening of combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)).
Hybridoma cells can be screened immunochemically for production of antibodies
specifically reactive with the peptide and the monoclonal antibodies can be
isolated.
Therefore, the invention also contemplates hybridoma cells secreting
monoclonal antibodies
with specificity for the SMAK protein as described herein.
The term "antibody" as used herein is intended to include fragments thereof
which also specifically react with a SMAK protein, or peptide thereof, having
the
activity of the SMAK protein. Antibodies can be fragmented using conventional
techniques
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and the fragments screened for utility in the same manner as described above.
For example,
F(ab')2 fragments can be generated by treating antibody with pepsin. The
resulting F(ab')2
fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
Chimeric antibody derivatives, i.e., antibody molecules that combine a
non-human animal variable region and a human constant region are also
contemplated
within the scope of the invention. Chimeric antibody molecules can include,
for example,
the antigen binding domain from an antibody of a mouse, rat, or other species,
with human
constant regions. Conventional methods may be used to make chimeric antibodies
containing
the immunoglobulin variable region which recognizes the gene product of SMAK
antigens of
the invention (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A.
81,6851
(1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No.
4,816,567; Boss
et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent
Publication EP171496;
European Patent Publication 0173494, United Kingdom patent GB 2177096B). It is
expected
that chimeric antibodies would be less immunogenic in a human subject than the
corresponding non-chimeric antibody.
Monoclonal or chimeric antibodies specifically reactive with a protein of the
invention as described herein can be further humanized by producing human
constant region
chimeras, in which parts of the variable regions, particularly the conserved
framework
regions of the antigen-binding domain, are of human origin and only the
hypervariable
regions are of non-human origin. Such immunoglobulin molecules may be made by
techniques
known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-
7312 (1983);
Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth.
Enzymol., 92, 3-16
(1982)), and PCT Publication W092/06193 or EP 0239400). Humanized antibodies
can also
be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham,
Middlesex, Great
Britain.)
Specific antibodies, or antibody fragments, reactive against proteins of the
invention may also be generated by screening expression libraries encoding
immunoglobulin
genes, or portions thereof, expressed in bacteria with peptides produced from
the nucleic
acid molecules of the present invention. For example, complete Fab fragments,
VH regions
and FV regions can be expressed in bacteria using phage expression libraries
(See for
example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246,
1275-1281 (1989);
and McCafferty et al. Nature 348, 552-554 (1990)). Alternatively, a SCID-hu
mouse, for
example the model developed by Genpharm, can be used to produce antibodies, or
fragments
thereof.
Antibodies specifically reactive with the SMAK protein, or derivatives
thereof, such as enzyme conjugates or labeled derivatives, may be used to
detect the SMAK
protein in various biological materials, for example they may be used in any
known
immunoassays which rely on the binding interaction between an antigenic
determinant of
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the SMAK protein, and the antibodies. Examples of such assays are
radioimmunoassays,
enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation,
latex
agglutination, hemagglutination, and histochemical tests. Thus, the antibodies
may be
used to detect and quantify the SMAK protein in a sample in order to determine
its role in
particular cellular events or pathological states, and to diagnose and treat
such
pathological states.
In particular, the antibodies of the invention may be used in
immuno-histochemical analyses, for example, at the cellular and sub-
subcellular level, to
detect the SMAK protein, to localise it to particular cells and tissues and to
specific
subcellular locations, and to quantitate the level of expression.
Cytochemical techniques known in the art for localizing antigens using light
and electron microscopy may be used to detect the SMAK protein. Generally, an
antibody of
the invention may be labelled with a detectable substance and the SMAK protein
may be
localised in tissue based upon the presence of the detectable substance.
Examples of
detectable substances include various enzymes, fluorescent materials,
luminescent materials
and radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
biotin, alkaline phosphatase, b-galactosidase, or acetylcholinesterase;
examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; and
examples of
suitable radioactive material include radioactive iodine I125, I131 or
tritium. Antibodies
may also be coupled to electron dense substances, such as ferritin or
colloidal gold, which
are readily visualised by electron microscopy.
Indirect methods may also be employed in which the primary
antigen-antibody reaction is amplified by the introduction of a second
antibody, having
specificity for the antibody reactive against the SMAK protein. By way of
example, if the
antibody having specificity against the SMAK protein is a rabbit IgG antibody,
the second
antibody may be goat anti-rabbit gamma-globulin labelled with a detectable
substance as
described herein.
Where a radioactive label is used as a detectable substance, the SMAK
protein may be localized by autoradiography. The results of autoradiography
may be
quantitated by determining the density of particles in the autoradiographs by
various
optical methods, or by counting the grains.
As discussed herein, the SMAK protein likely plays a role in apoptosis and
actin stress fiber dissolution in cells. Therefore, the above described
methods for detecting
nucleic acid molecules and SMAK proteins of the invention, can be used to
monitor cell
death. It would also be apparent to one skilled in the art that the above
described methods
may be used to study the expression of the SMAK protein and, accordingly, will
provide
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further insight into the role of the SMAK protein in cells.
Therapeutic Uses
The SMAK protein of the invention is likely involved in the regulation of
cell signalling pathways that control cell death. Accordingly, the present
invention
provides a method of modulating cell death or apoptosis comprising
administering an
effective amount of a SMAK protein or a nucleic acid encoding a SMAK protein
to a cell or
animal in need thereof. The term "effective amount" as used herein means an
amount
effective, at dosages and for periods of time necessary to achieve the desired
results.
In another aspect the present invention provides a method of modulation of
cell proliferation. In one embodiment, the invention provides a method of
inhibiting or
reducing cell proliferation, such as in neoplasia, by administering to a cell
or animal an
effective amount of an agent that promotes the expression or the biological
activity of the
SMAK protein.
In another embodiment, the present invention provides a method of inducing
cell proliferation by administering to a cell or an animal an effective amount
of an agent
that inhibits the expression or the biological activity of the SMAK protein.
Agents that
inhibit the activity of the SMAK protein include antibodies to SMAK protein.
Agents that
inhibit the expression of the SMAK gene include antisense oligonucleotides to
a SMAK
nucleic acid sequence. Both of these are described above.
In addition to antibodies and antisense oligonucleotides, other substances
that inhibit SMAK protein expression or activity may also be identified. S ub
st a nce s
which affect SMAK protein activity can be identified based on their ability to
bind to the
SMAK protein. Therefore, the invention also provides methods for identifying
substances
which are capable of binding to the SMAK protein. In particular, the methods
may be used
to identify substances which are capable of binding to, and in some cases
activating (i.e.,
phosphorylating) and in other cases deactivating the SMAK protein of the
invention.
Substances which can bind with the SMAK protein of the invention may be
identified by reacting the SMAK protein with a substance which potentially
binds to the
SMAK protein, and assaying for complexes, for free substance, or for non-
complexed SMAK
protein, or for activation of the SMAK protein. In particular, a yeast two
hybrid assay
system may be used to identify proteins which interact with the SMAK protein
(Fields, S.
and Song, O., 1989, Nature, 340:245-247).
Conditions which permit the formation of substance and SMAK protein
complexes may be selected having regard to factors such as the nature and
amounts of the
substance and the protein.
The substance-protein complex, free substance or non-complexed proteins may
be isolated by conventional isolation techniques, for example, salting out,
chromatography,
electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel
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electrophoresis, agglutination, or combinations thereof. To facilitate the
assay of the
components, antibody against the SMAK protein or the substance, or labelled
SMAK
protein, or a labelled substance may be utilized. The antibodies, proteins, or
substances may
be labelled with a detectable substance as described above.
Substances which bind to and activate the SMAK protein of the invention
may be identified by assaying for phosphorylation of the tyrosine residues of
the protein.
Substances which bind to and inactivate the SMAK protein of the invention
may be identified by assaying for reduction in phosphorylation of the fully
activated
protein.
The SMAK protein, or the substance used in the method of the invention may
be insolubilized. For example, the SMAK protein or substance may be bound to a
suitable
carrier. Examples of suitable carriers are agarose, cellulose, dextran,
Sephadex, Sepharose,
carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic
film, plastic
tube, glass beads, polyamine-methyl vinyl-ether-malefic acid copolymer, amino
acid
copolymer, ethylene-malefic acid copolymer, nylon, silk, etc. The carrier may
be in the
shape of, for example, a tube, test plate, beads, disc, sphere etc.
The insolubilized protein or substance may be prepared by reacting the
material with a suitable insoluble carrier using known chemical or physical
methods, for
example, cyanogen bromide coupling.
The proteins or substance may also be expressed on the surface of a cell using
the methods described herein.
The invention also contemplates a method for assaying for an agonist or
antagonist of the binding of the SMAK protein with a substance which is
capable of binding
with the SMAK protein. The agonist or antagonist may be an endogenous
physiological
substance or it may be a natural or synthetic substance. Substances which are
capable of
binding with the SMAK protein may be identified using the methods set forth
herein.
It will be understood that the agonists and antagonists that can be assayed
using the methods of the invention may act on one or more of the binding sites
on the protein
or substance including agonist binding sites, competitive antagonist binding
sites,
non-competitive antagonist binding sites or allosteric sites.
The invention also makes it possible to screen for antagonists that inhibit
the
effects of an agonist of the interaction of the SMAK protein with a substance
which is
capable of binding to the SMAK protein. Thus, the invention may be used to
assay for a
substance that competes for the same binding site of the SMAK protein.
The methods described above may be used to identify a substance which is
capable of binding to an activated SMAK protein, and to assay for an agonist
or antagonist
of the binding of activated SMAK protein, with a substance which is capable of
binding
with activated SMAK protein. An activated (i.e. phosphorylated) the SMAK
protein may
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be prepared using the methods described (for example in Reedijk et al. The
EMBO Journal,
11(4):1365, 1992) for producing a tyrosine phosphorylated protein.
It will also be appreciated that intracellular substances which are capable
of binding to the SMAK protein may be identified using the methods described
herein.
The invention further provides a method for assaying for a substance that
affects an SMAK protein regulatory pathway comprising administering to a non-
human
animal or to a cell, or a tissue of an animal, a substance suspected of
affecting a SMAK
protein regulatory pathway, and quantitating the SMAK protein or nucleic acids
encoding
the SMAK protein, or examining the pattern and/or level of expression of SMAK
protein, in
the non-human animal or tissue, or cell. SMAK protein may be quantitated and
its
expression may be examined using the methods described herein.
The substances identified by the methods described herein, may be used for
modulating SMAK protein regulatory pathways and accordingly may be used in the
treatment of conditions involving perturbation of SMAK protein signalling
pathways. In
particular, the substances may be particularly useful in the treatment of
disorders of cell
death.
As stated previously, SMAK protein may be involved in cell proliferation
and inhibitors of the SMAK protein may be useful in modulating disorders
involving cell
proliferation such as neoplasia. In contrast, stimulators of the SMAK protein
may be useful
in the modulation of disorders requiring reduction of proliferation.
Accordingly, substances
that stimulate the SMAK protein (for example, identified using the methods of
the
invention) may be used to stimulate cell death or apoptosis. Substances which
stimulate
apoptosis may be useful in the treatment of cancer.
Peptide Mimetics
The present invention also include peptide mimetics of the SMAK protein of
the invention. For example, a peptide derived from a binding domain of SMAK
will
interact directly or indirectly with an associated molecule in such a way as
to mimic the
native binding domain. Such peptides may include competitive inhibitors,
enhancers,
peptide mimetics, and the like. All of these peptides as well as molecules
substantially
homologous, complementary or otherwise functionally or structurally equivalent
to these
peptides may be used for purposes of the present invention.
"Peptide mimetics" are structures which serve as substitutes for peptides in
interactions between molecules (See Morgan et al (1989), Ann. Reports Med.
Chem.
24:243-252 for a review). Peptide mimetics include synthetic structures which
may or may
not contain amino acids and/or peptide bonds but retain the structural and
functional
features of a peptide, or enhancer or inhibitor of the invention. Peptide
mimetics also
include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA
89:9367); and
peptide libraries containing peptides of a designed length representing all
possible
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sequences of amino acids corresponding to a peptide of the invention.
Peptide mimetics may be designed based on information obtained by
systematic replacement of L-amino acids by D-amino acids, replacement of side
chains with
groups having different electronic properties, and by systematic replacement
of peptide
bonds with amide bond replacements. Local conformational constraints can also
be
introduced to determine conformational requirements for activity of a
candidate peptide
mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to
stabilize or
promote reverse turn conformations and to help stabilize the molecule. Cyclic
amino acid
analogues may be used to constrain amino acid residues to particular
conformational states.
The mimetics can also include mimics of inhibitor peptide secondary
structures. These
structures can model the 3-dimensional orientation of amino acid residues into
the known
secondary conformations of proteins. Peptoids may also be used which are
oligomers of
N-substituted amino acids and can be used as motifs for the generation of
chemically
diverse libraries of novel molecules.
Peptides of the invention may also be used to identify lead compounds for
drug development. The structure of the peptides described herein can be
readily determined
by a number of methods such as NMR and X-ray crystallography. A comparison of
the
structures of peptides similar in sequence, but differing in the biological
activities they
elicit in target molecules can provide information about the structure-
activity relationship
of the target. Information obtained from the examination of structure-activity
relationships can be used to design either modified peptides, or other small
molecules or
lead compounds which can be tested for predicted properties as related to the
target
molecule. The activity of the lead compounds can be evaluated using assays
similar to those
described herein.
Information about structure-activity relationships may also be obtained from
co-crystallization studies. In these studies, a peptide with a desired
activity is
crystallized in association with a target molecule, and the X-ray structure of
the complex is
determined. The structure can then be compared to the structure of the target
molecule in its
native state, and information from such a comparison may be used to design
compounds
expected to possess desired activities.
Pharmaceutical Compositions
All of the above described substances (such as the SMAK protein, the nucleic
acid encoding the SMAK protein, antibodies to the SMAK protein, antisense
oligonucleotides to the nucleic acid molecules and substances that modulate
the SMAK
protein activity) may be formulated into pharmaceutical compositions for
adminstration to
subjects in a biologically compatible form suitable for administration in
vivo. By
"biologically compatible form suitable for administration in vivo" is meant a
form of the
substance to be administered in which any toxic effects are outweighed by the
therapeutic
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effects. The substances may be administered to living organisms including
humans, and
animals. Administration of a therapeutically active amount of the
pharmaceutical
compositions of the present invention is defined as an amount effective, at
dosages and for
periods of time necessary to achieve the desired result. For example, a
therapeutically
active amount of a substance may vary according to factors such as the disease
state, age,
sex, and weight of the individual, and the ability of antibody to elicit a
desired response in
the individual. Dosage regima may be adjusted to provide the optimum
therapeutic
response. For example, several divided doses may be administered daily or the
dose may be
proportionally reduced as indicated by the exigencies of the therapeutic
situation.
The active substance may be administered in a convenient manner such as by
injection (subcutaneous, intravenous, etc.), oral administration, inhalation,
transdermal
application, or rectal administration. Depending on the route of
administration, the active
substance may be coated in a material to protect the compound from the action
of enzymes,
acids and other natural conditions which may inactivate the compound. If the
active
substance is a nucleic acid encoding a SMAK protein or an antisense
oligonucleotide it may
be delivered using techniques known in the art. Recombinant molecules
comprising an
antisense sequence or oligonucleotide may be directly introduced into cells or
tissues in vivo
using delivery vehicles such as retroviral vectors, adeno viral vectors and
DNA virus
vectors. They may also be introduced using physical techniques such as
microinjection and
electroporation or chemical methods such as co-precipitation and incorporation
of DNA
into liposomes.
The compositions described herein can be prepared by per se known methods
for the preparation of pharmaceutically acceptable compositions which can be
administered to subjects, such that an effective quantity of the active
substance is combined
in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are
described,
for example, in Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the
compositions include, albeit not exclusively, solutions of the substances in
association with
one or more pharmaceutically acceptable vehicles or diluents, and contained in
buffered
solutions with a suitable PH and iso-osmotic with the physiological fluids.
The reagents suitable for applying the methods of the invention to identify
substances that affect the SMAK protein may be packaged into convenient kits
providing
the necessary materials packaged into suitable containers. The kits may also
include
suitable supports useful in performing the methods of the invention.
Experimental Models
The invention also provides methods for studying the function of the SMAK
protein. Cells, tissues and non-human animals that express or over-express
SMAK protein
may be prepared by transfecting cells, tissues or oocytes (to prepare
transgenic animals)
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with a recombinant expression vector of the invention (as described
previously). In
particular, transgenic technology (via nuclear oocyte microinjection of naked
DNA) will
assay the effect of over expression or alterations of the SMAK protein
expression in various
developmental systems, including bone development, neurogenesis, mammary
development,
lung epithelial development.
Cells, tissues, and non-human animals lacking in SMAK protein expression or
partially lacking in SMAK protein expression may be developed using
recombinant
expression vectors of the invention having specific deletion or insertion
mutations in the
SMAK gene. A recombinant expression vector may be used to inactivate or alter
the
endogenous gene by homologous recombination, and thereby create a SMAK protein
deficient
cell, tissue or animal.
Null alleles may be generated in cells, such as embryonic stem cells by
deletion mutation. A recombinant SMAK gene may also be engineered to contain
an insertion
mutation which inactivates the SMAK protein. Such a construct may then be
introduced
into a cell, such as an embryonic stem cell, by a technique such as
transfection,
electroporation, injection etc. Cells lacking an intact SMAK gene may then be
identified,
for example by Southern blotting, Northern Blotting or by assaying for SMAK
protein using
the methods described herein. Such cells may then be fused to embryonic stem
cells to
generate transgenic non-human animals deficient in SMAK protein. Germline
transmission
of the mutation may be achieved, for example, by aggregating the embryonic
stem cells
with early stage embryos, such as 8 cell embryos, in vitro; transferring the
resulting
blastocysts into recipient females and; generating germline transmission of
the resulting
aggregation chimeras. Such a mutant animal may be used to define specific cell
populations, developmental patterns and in vivo processes, normally dependent
on SMAK
protein expression.
The present invention also includes the preparation of tissue specific
knock-outs of the SMAK gene.
The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
EXAMPLE 1
SMAK, an Ste20-related kinase.
In an effort to identify protein kinases that exhibit differential expression
during myoblast differentiation, a 1.6 kb partial cDNA insert encoding a mouse
Ste20-like
kinase was used to survey mRNA samples from differentiating myoblasts.
Cloning and Analysis of SMAK
Full length SMAK (Genbank BankIt # 242010) was obtained from an adult
mouse muscle 1 gtll cDNA library following plaque screening (Sambrook, J. et
al., 1989)
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using a partial SMAK cDNA clone obtained through two hybrid screening. Full
length
cDNA inserts were subcloned into pBluescript and sequenced on an ABI automated
sequencer.
Homology searches were performed using NCBI Blast software and homologies are
presented as percent identities. The serine/threonine kinase subdomains were
identified by
alignment of the consensus amino acid sequence of the catalytic domain (Hanks
and Hunter,
1995) to that of SMAK. Multiple alignment analysis was performed using the
MegAlign
program from the DNAStar software package. Further analyses for consensus
protein motifs
were performed using PSORT, Motif Finder and PRO Site.
Northern blot analysis revealed high level expression in myoblasts that was
strongly downregulated following myotube formation (not shown). Therefore, to
further
characterize the mouse kinase, a muscle lambda gtll cDNA library was screened
to isolate
clones containing full length cDNAs. One clone (3E5) was found to contain a
5253 by cDNA
encoding full length protein (Figure 1). Clone 3E5 encoded a 1202 amino acid
polypeptide
and shared over 90% nucleotide identity with sequences from the guinea pig
(Itoh, et al.,
1997) human and rat Ste20-like kinases (SLK) recently deposited in the
database,
suggesting that 3E5 represents the murine homolog. Inspection of the 3'
untranslated region
(UTR) did not reveal the existence of a poly-A tail or a consensus
polyadenylation signal,
suggesting that 3E5 bears a partial 3'UTR. However, several AU-rich motifs,
previously
implicated in growth factor-dependent mRNA turnover (Ross, 1996), are present
in the
3'UTR.
Searches for homologous proteins revealed that the kinase catalytic domain
was closely related to LOK and MSTl/2, members of the Ste20 family of
serine/threonine
kinases. Interestingly, the central portion of 3E5 was found to be highly
homologous to
microtubule and nuclear associated protein (M-NAP). The remaining carboxy
terminal
region of the protein displayed high homology to AT1-46, a cDNA clone isolated
from
astrocyte mRNA. Because of an increasing number of Ste20-related kinases and
to avoid
potential confusion with other family members, the Ste20-like kinase encoded
by clone 3E5
was renamed. Therefore, due to its relatedness to Ste20, M-NAP and ATl-46,
this novel
protein kinase has been termed SMAK (Figures 1 and 2). Searches for known
protein motifs
using PSORT revealed putative nuclear localization signals at positions 15,
422, and 1149.
However, nuclear localization was never observed following transfection and
immunostaining of Myc epitope-tagged SMAK (see Figure 8). A consensus SH3
binding
(P-X-X-P-X) site was found at position 735 (Pawson and Scott, 1997),
suggesting a potential
interaction with SH3 domain-containing proteins. Other than a C-terminal
region
unusually rich in charged amino acids, database scans using MotifFinder failed
to detect
the presence of other consensus protein motifs.
Further analysis of the coding region revealed that SMAK encoded an
N-terminal serine/threonine kinase catalytic domain, with the signature
sequence
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Gly-X-Gly-X-X-Gly identifying subdomain I at amino acid residue 41 (Figure
3A). The
conserved lysine residue within the ATP binding site of subdomain II was found
at amino
acid residue 63. The catalytic core extended further, up to residue 282 and
presented all the
characteristic subdomains of serine/threonine kinases (Hanks and Hunter, 1995)
(Figure
3A). Alignments and database scans revealed that SMAK displayed 74% identity,
in the
kinase domain, to LOK, a novel kinase preferentially expressed in lymphocytes
(Kuramochi, et al., 1997) (Figure 3A). The SMAK kinase domain was also found
to be
related to MST1 and MST2, both Ste20-like kinases (Schinkmann and Blenis,
1997; Katoh,
et al., 1995; Creasy and Chernoff, 1995; Leung et al., 1995; and Manser et
al., 1995). In the
kinase subdomain VIII, characterizing kinase family members, SMAK presented
the Ste20
signature motif, suggesting that it represents a novel family member (Figure
3A). Further
analysis showed that the central portion of SMAK, from residues 339 to 947
(Figure 2B)
shared 70% identity with microtubule and nuclear associated protein (M-NAP).
The
remainder of SMAK, from residues 788 to 926, overlapping slightly with the M-
NAP
domain, displayed 63% identity to AT1-46 (Schaar, et al., 1996) (Figure 3B).
Interestingly,
M-NAP and AT1-46 share significant homology in the overlapping region. The
function of
both M-NAP and AT1-46 proteins is currently unknown. A second region of
homology (71%)
to AT1-46 was observed from SMAK residues 957 to 1171. Interestingly, these
two AT1-46
domains were found to be 56% identical to the C-terminal region of LOK which
also shares
extensive identity to AT1-46 in its C-terminus (Figure 3B). These observations
suggest that
SMAK and LOK may represent members of a new protein kinase family.
Furthermore, the
ATl-46 homology domain might be a novel protein motif likely to be important
for LOK and
SMAK functions. This new motif has been termed the ATH domain, for AT1-46
homology
domain (see Figure 3C).
Example 2
Expression of SMAK in tissues and cell lines
To gain insights into the role of SMAK, adult human RNA samples were
surveyed for SMAK expression by Northern blotting.
Cell culture and Transfections.
C2C12 cells were maintained in Dulbecco's modified Eagle medium
supplemented with 15% fetal calf serum (FCS) and induced to differentiate in
DMEM
containing 2% horse serum. NIH3T3, P19 and 293 cells were grown in DMEM
containing 10%
FCS. 3T3 cells were strayed in DMEM supplemented with 0.5% FCS and
subsequently
stimulated with growth medium where indicated. The cultures were transfected
by
Lipofectamine (Gibco/BRL) according to the manufacturer's instructions using 2
~g of
plasmid DNA.
SMAK expression analysis.
For expression analysis, total RNA from various adult mouse tissues and cell
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lines was prepared (Sambrook et al., 1989) and poly-A+ RNA was isolated by one
round of
selection through oligo dT cellulose (Sambrook et al., 1989). A total of 4 mg
of poly-A+
RNA for each cell line was subjected to Northern blot analysis using a SMAK-
specific
probe. Analysis of human tissues and cell line RNA was performed by Northern
hybridization of a SMAK probe to Multiple Tissue Northerns (Clontech)
containing 2 ~g of
poly-A+ RNA per lane. Human RNA filters were washed under reduced stringency
(0.2x
SSC/0.1% SDS/55°C). Analysis of SMAK protein expression was performed
by lysing
cultures (150 mM NaCI, 50 mM Tris, pH 7.4, 1 mM EDTA, 1% Triton-X 100 and 1
mg/ml of
each aprotinin, pepstatin and leupeptin) and subjecting 20 ~g of total lysate
to western blot
analysis using anti-SMAK rabbit polyclonal antibody (Sambrook et al., 1989).
Reactive
proteins were detected by ECL (Amersham) using a goat anti-rabbit horse radish
peroxidase (HRP) labeled secondary antibody. SMAK polyclonal antibodies were
generated by immunization of New Zealand rabbits using purified GST-SMAK95-
551,
encompassing part of the kinase and M-NAP domains. Specific immunoreactivity
to murine
SMAK protein was observed in 293 cells transfected with full length and
truncated SMAK
expression vectors.
For expression studies and immunolocalization experiments, HA- or
Myc-tagged pcDNA3 (Invitrogen) expression vectors bearing full length, kinase
dead or
truncated SMAK, were constructed using standard cloning procedures (Sambrook
et al.,
1989). The kinase inactive mutant was generated through site directed PCR-
mediated
mutagenesis. The SMAKD3' truncation (residues 1-950) was obtained by removing
the last
263 amino acids through XhoI restriction enzyme digestion and religation .
Following
transfection into C2C12, the cultures were fixed for 10 minutes in 4%
paraformaldehyde and
SMAK protein was detected using 9E10 monoclonal antibodies in conjunction with
FITC-labeled secondary antibodies.
Analysis of poly-A+ RNA from various tissues with a SMAK-specific probe
revealed the existence of at least three distinct isoforms of about 6, 7 and 8
kb (Figure 4A).
All isoforms were expressed at similar levels with the exception that testes
and colon
tissues were found to express relatively higher levels of the 5 and 6 kb
isoforms. Analysis of
mouse tissues, including heart, skeletal muscle and brain, showed a similar
pattern of
expression (not shown). RNA analysis of mouse cell lines showed that SMAK was
not
expressed in undifferentiated P19 embryocarcinoma cells (Figure 4B),
suggesting a role for
SMAK in more differentiated cell types. In contrast to tissue samples which
predominantly
expressed the 7 kb mRNA (Figure 3C), the other murine cell lines surveyed
displayed
similar levels of all three isoforms (Figure 4B).
Analysis of human tumor cell lines showed that the chronic myelogenous
leukemia cell line K-562 and the colon adenocarcinoma cell line SW480
expressed high
levels of SMAK mRNA, relative to normal blood lymphocytes and colon tissue,
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respectively (Figure 4C). Whether high levels of SMAK correlates with these
specific
tumor phenotypes remains to be clarified. Other human tumor cell lines were
found tc
express SMAK at levels that were comparable to normal human tissues of the
same origin.
To facilitate the biochemical characterization and to gain insights into
SMAK functions, anti-SMAK antibodies were developed. Rabbit polyclonal
antibodies
were generated against a GST-SMAK fusion protein encompassing residues 95 to
551 and
used in western blot analysis of C2C12 cultures. Specific reactivity to murine
SMAK protein
was observed at approximately 220 and 170 kDa in control 293 cells expressing
full length
SMAK or a C-terminal truncation (SMAKD3'), respectively (Figure 5A). No signal
was
detected in control vector transfected 293 cells. The p220 SMAK is relatively
larger than
the predicted molecular weight of 148 kDa, suggesting that SMAK is subject to
post-translational modifications (Figure 5A). In contrast to Northern
blotting, Western
analysis of C2C12 myoblast cultures revealed 6 SMAK immunoreactive
polypeptides
(Figure 5A). Some of these may represent breakdown products or, alternatively,
they might
reflect post-translational modifications. Interestingly, SMAK protein
expression decreased
as myogenic differentiation progressed. A marked reduction was observed 3 days
following
the onset of differentiation, well after the majority of the myoblasts have
fused into
multinucleated myotubes (not shown). Alternatively, the observed decrease in
SMAK
expression could be due to growth factor depletion which was used to initiate
differentiation. Furthermore, the 3' UTR of cDNA clone 3E5 displays several AU-
rich
motifs potentially mediating growth factor-dependent stability of SMAK mRNA.
To test
whether the downregulation of SMAK during C2C12 differentiation could be
attributed to
growth factor depletion, NIH 3T3 cells were serum-starved for 24 hours,
stimulated with
10% serum and analyzed for SMAK expression by western blotting. As shown in
Figure 4B,
serum starvation of 3T3 cells resulted in a rapid decrease in SMAK expression.
Following
the stimulation of 3T3 starved cultures for 24 hours, a SMAK reactive species
of
approximately 140 kDa was induced, likely representing one of several isoforms
(Figure
5B). In contrast to C2C12, 3T3 cells were found to express only 3 SMAK protein
isoforms.
With the exception of p220 SMAK, these isoforms did not correspond to any of
the ones
detected in C2C12 cells, suggesting differential splicing or processing among
cell types.
Example 3
Cellular Distribution of SMAK
Because our anti-SMAK antibodies failed to detect antigen in fixed cells, a
Myc epitope tag expression vector carrying full length SMAK was constructed
and
transfected into C2C12 myoblasts. To evaluate the cellular distribution of
SMAK,
ianmunostaining was performed using 9E10 monoclonal antibodies on fixed
cultures following
transient transfection. Interestingly, 9E10-positive cells displayed high
concentration of
Myc-SMAK protein in distinct cytosolic domains, predominantly at the periphery
of the
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cells (Figure 8A).The distribution of these domains appeared suggestive of
scaffold
structures such as growth factor receptors or focal adhesion complex from
which SMAK
signaling could occur. Transfection of the kinase dead mutant SMAKK63R did not
alter the
cellular distribution (Figure 8B).
Example 4
Modulation of SMAK activity by extracellular stimuli
As shown in Figure 6A, bacterially expressed GST-SMAK and
immunoprecipitated Myc-SMAK autophosphorylated and efficiently phosphorylated
MBP and histone H1 in vitro. However, SMAK did not phosphorylate the JNK and
p38
substrates GST-Jun and GST-ATF2, respectively. Therefore, all subsequent
assays were
performed using the substrate histone H1. Interestingly, when compared to full
length
GST-SMAK, immunoprecipitated Myc-SMAK from transfected 293 cells displayed
markedly higher autophosphorylation activity (Figure 6A). Treatment of active
GST-SMAK (Figure 6B) or immunoprecipitated Myc-SMAK (not shown) with calf
intestinal phosphatase (CIAP) caused a marked decrease in GST-SMAK activity,
suggesting that its activity is regulated by phosphorylation. Furthermore
these results
indicate that SMAK is active in a hyperphosphorylated state.
Various growth factors, stress-inducing agents and apoptotic triggers have
been demonstrated to affect the activity of several MAP kinases such as ERK1/2
as well as
stress-activated kinases (Fanger et al., 1997; Davis, R.J., 1993; Xia, Z. et
al., 1995; Hughes,
D.A., 1995). To test whether SMAK activity could be modulated by such factors,
a Myc
epitope-tagged SMAK was transfected into 293 cells followed by stimulation
with various
factors and stress inducing agents in the absence of serum (see Figure 7A).
The activity of
SMAK following stimulation was evaluated by in vitro kinase assays.
Interestingly, none of
the factors tested were found to significantly modulate SMAK kinase activity
in
overexpressing 293 cells (Figure 7A), suggesting that SMAK is a component of a
novel
signaling pathway.
To gain insights into potential downstream effectors of SMAK, the relative
kinase activity of JNK1 isoforms or the mitogen-activated protein kinases
ERK1/2 was
determined by immunoprecipitations and in vitro kinase assays following
transient
transfections of Myc-SMAK. As shown in figure 6C, JNK1 activity was found to
be
upregulated 3- to 5-fold relative to vector-transfected cells, suggesting that
c-jun
amino-terminal kinasel (JNKl) is activated by SMAK overexpression. However,
JNK
activation by SMAK overexpression was about 3-fold less than that observed
following UV
irradiation (Figure 7C, lane 2). In contrast to JNKl, immunoprecipitated
ERKl/2 were found
to be inactive following SMAK transfection, suggesting that components of the
mitogen
response pathways are not markedly affected by SMAK overexpression (Figure
7C).
Materials and Methods For Immunoprecipitations and in vitro kinase assays of
Examples 3
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and 4
For growth factor and stress agent stimulation, transfected 293 cells were
serum starved in DMEM for 1 hour prior to exposure to the stimuli. The cells
were then
exposed to the agonists as indicated in the figure legends and lysed as
described above. For
SMAK expression analysis, 20 mg of total cell lysate was subjected to western
blotting with
anti-SMAK antibodies as described above. For in vitro kinase assays, 100 mg of
total cell
lysate was immunoprecipitated with 1mg of 9E10 monoclonal antibodies and 20m1
of
G-protein sepharose (Pharmacia) for 2 hours at 4°C. Immunoprecipitates
were washed
three times with NETN (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1 mM EDTA, 0.1%
Nonidet P-40) and once with kinase assay buffer (20 mM Tris-HCl, pH 7.5, 15 mM
MgCl2, 10
mM NaF, 10 mM b-glycerophosphate, 1 mM sodium orthovanadate). Reactions (20
ml) were
initiated by the addition of 5 mCi of [g32P] ATP and 3 mg of histone Hl. After
a 30 minute
incubation at 30°C, reactions were terminated by the addition of 4X SDS
sample buffer and
ml aliquots were fractionated by 12% SDS-PAGE. Gels were stained, dried and
exposed
15 to X-ray films.
Recombinant GST-SMAK fusion protein was purified using glutathione
sepharose as described (Pharmacia) from 10 ml cultures. GST-SMAK immobilized
on beads
was assayed directly for kinase activity on various substrates or treated with
CIAP prior to
kinase assays.
20 DISCUSSION OF EXAMPLES 1-4
The present inventors have cloned and characterized a murine Ste20-related
protein kinase designated SMAK. Database searches revealed that the cloned
cDNA
encodes a protein kinase highly related to LOK, a Ste20-related protein kinase
preferentially expressed in lymphocytes (Kuramochi, S. et al., 1997) . Because
it was found
to be related to Ste20 and shared extensive homology to microtubule and
nuclear associated
protein (M-NAP) and to AT1-46, we have termed this kinase SMAK. Analysis of
the kinase
domain shows that SMAK is also related to MST1/2 (Schinkmann, K and Blenis,
J., 1997;
Katoh, M. et al., 1995; Creasy, C.L. and Chernoff, J., 1995) and more
distantly related to
Ste20, a yeast kinase involved in the pheromone response pathway (Zhao, Z.S.
et al.,
1995). Furthermore, SMAK showed 70% identity to M-NAP and 63% and 71% identity
to
ATl-46 in two distinct carboxy terminal domains. Interestingly, LOK also
display extensive
homology to AT1-46 in its C-terminal domain, suggesting that AT1-46, SMAK and
LOK
represent members of a new protein family. Furthermore, the ATl-46 homology
(ATH)
domain may represent a novel protein motif required for SMAK and LOK function.
Further
analysis of SMAK protein showed the presence of an SH3-binding motif and a
coiled-coil
structure in the C-terminal region. Northern blot analysis demonstrated that
SMAK is
ubiquitously expressed in adult tissues and its expression appears to be
restricted to more
differentiated cell types.
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Purified GST-SMAK fusion protein showed that it autophosphorylates
efficiently and that it can phosphorylate exogenous substrates. However, the
JNK and p38
substrates c-Jun and ATF2, respectively, were not phosphorylated by SMAK.
Interestingly,
alkaline phosphatase treatment of recombinant SMAK lead to a substantial
decrease in
kinase activity in vitro, suggesting that its activity is regulated by
phosphorylation.
Immunoprecipitations and in vitro kinase assays following overexpression of
SMAK in 293
cells showed that it activated isoforms of JNK1. As for SMAK, the Ste20-
related kinase
p65PAK also activates the stress response pathway (Fanger, G.R. et al., 1997)
. Similarly,
activation of the related kinases MST1/2 activates the p38 MAPK pathway
(Greasy, C.L.
and Chernoff J., 1995; Graves, J.D. et al., 1998). However, treatment of
transfected 293 cells
with various growth factors and stress-inducing agents did not result in any
significant
changes in SMAK activity, suggesting that it is part of a novel pathway. One
possibility is
that SMAK possesses low kinase activity and is activated by
autophosphorylation in
vitro, making it difficult to observe any significant effects. Alternatively,
SMAK is
constitutively active and is downregulated through a novel signaling pathway.
Immunolocalization of Myc epitope-tagged SMAK protein revealed that it is
localized predominantly to the periphery of C2C12 myoblasts. Interestingly,
SMAK was
found to be localized to distinct cytosolic domains. Whether these represent
larger
signaling complexes involving SMAK remains to be elucidated. One possibility
is that the
putative SH3-binding domain of SMAK mediates interactions with other proteins
of such
complexes.
Example 5
SMAK Overexpression Results in Rapid Induction of Apoptosis
Inspection of SMAK transfected cultures after 24 h revealed the presence of
numerous cells that exhibited extensive cellular shrinkage, membrane blebbing,
and loss of
substrate adhesion. (Figure 9A). Moreover, several 9E10-positive cellular
fragments,
reminiscent of apoptotic bodies ~n~ere evident following SMAK transfection
into C2C12 cells
(Figure 9A). Similar results were observed following transfection of SMAK into
COS-1,
HeLa, NIH3T3, and 293 cells (not shown). Taken together, these data suggested
that forced
expression of SMAK induced an apoptotic response.
To determine whether SMAK-transfected cells were exhibiting a bona fide
apoptotic response, double staining was performed using 9E10 and FITC-labeled
annexin V
or terminal transferase dUTP nick end labeling (TUNEL) for the detection of
early and late
apoptotic stages, respectively. Double staining experiments performed 24 hours
following
Myc-SMAK transfection revealed the colocalization of FITC-annexin V, marking
early
apoptotic cells, and Myc-tagged SMAK protein (Figures 9C and 9D). Furthermore,
over 75%
of TUNEL-positive cells also expressed the transfected Myc-SMAK protein
(Figures 9G and
9H). However, 48 hours after transfection, virtually no SMAK-expressing cells
were
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detectable. Taken together, these results demonstrate that forced expression
of SMAK in
C2C12 myoblasts triggered an apoptotic response. In addition, overexpression
of the
catalytically inactive mutant SMAKK63R also induced an apoptotic response
(Figures 9E
and 9F) raising the possibility that regions outside the kinase domain were of
functional
significance.
To gain insights into potential downstream effectors of SMAK, plasmids
expressing c-Jun, Elk1 and CREB Gal4 fusions (Pathdetect plasmid system;
Stratagene) were
co-transfected with SMAK expression vectors together with a Gal4-Luciferase
reporter
gene. Transfection of SMAK with Gal4-Jun resulted in a 5 to 7-fold increase in
luciferase
activity relative to Gal4-Jun alone (Figure 10). This observation suggested
that SMAK
overexpression activated the c-jun amino-terminal kinase (JNK) pathway.
However, JNK
activation by SMAK overexpression was about 3-fold less than that observed
with MEK
kinase (MEKK), an upstream regulator of JNK . Relative to either effector
transfected
alone, a modest increase (2 to 3-fold) in reporter gene activity was observed
when SMAK
was co-transfected with Gal4-Elk or Gal4-CREB (Figure 4). Therefore, SMAK
overexpression appeared to predominantly activate the JNK pathway. Supporting
this, in
vitro kinase assays revealed that active JNK1 was readily immunoprecipitated
from
SMAK-transfected 293 cells whereas ERKl/2 remained inactive (not shown).
Example 6
SMAK Overexpression Induces Actin Stress Fiber Dissolution
Focal adhesions are large protein complexes particularly prominent in
cultured cells involved in cellular adherence to the substratum (Burridge and
Chrzanowska-Wodnicka, 1996; Craig and Johnson, 1996; Jockusch et al., 1995;
Schaller and
Parsons, 1994). Focal adhesion complexes are enriched for proteins such as
paxillin, vinculin
and focal adhesion kinase (FAK) which modulate focal contact dynamics. In
addition, the
assembly of focal adhesions is regulated by the Rho family of small GTPases,
which recruit
and modulate the activity of several protein kinases through direct
interaction (Burridge
and Chrzanowska-Wodnicka, 1996; Nobes and Hall, 1995). The relatedness to
Ste20
kinases such as p65PAK and the cellular distribution of SMAK to distinct
cytosolic foci
predominantly along the periphery of the cells raised the possibility that
SMAK may
modulate focal adhesion dynamics. To investigate this possibility, actin
stress fibers in
SMAK-transfected C2C12 cells were detected by phalloidin staining. For
comparison, cells
were transfected with activated RhoA, which promotes stress fiber and focal
complex
assembly (Nobes and Hall, 1995), as well as activated Rac1 and p65PAK, both
previously
shown to induce actin stress fiber disassembly (Nobes and Hall, 1995; Van
Aelst and
D'Souza-Schorey, 1997).
Transfected myoblasts overexpressing SMAK showed almost a complete
absence of stress fibers together with redistribution of actin to the cell
periphery (Figures
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11A and 11B). This observation suggested that SMAK induced stress fiber
dissolution and
actin reorganization. The catalytically inactive SMAK mutant SMAKK63R also
promoted
stress fiber dissolution, suggesting that SMAK-mediated actin reorganization
was
independent of kinase activity (not shown). The disassembly of stress fibers
induced by
SMAK was comparable to that observed for the activated form of Racl, Rac1G12V
(Figures
11C and 11D). Similarly PAKDE, an activated form of p65PAK (Manser et al.,
1997), also
mediated the dissolution of stress fibers in C2C12 myoblasts (Figures 10E and
10F).
Overexpression of the stress fiber inducer RhoAGI4V in C2C12 cells similarly
induced
apoptosis (see Figures 12E and 12F). Taken together, these data raise the
possibility that
Rho GTPases and p65PAK represent potential regulators or effectors of SMAK.
The observation that Rho GTPases, p65PAK, and SMAK promoted stress
fiber reorganization in C2C12 myoblasts prompted us to examine whether they
localized to
similar sites within the cell. Myoblasts were transfected with Myc epitope-
tagged versions
of both SMAK and p65PAK and immunolocalized using 9E10 antibodies. The small
GTPases
RhoA and Racl were similarly transfected. Interestingly, PAK and SMAK
displayed a
very similar pattern of staining with reactivity at the periphery of
transfected myoblasts
(Figures 12A and 12B). Similarly, transfection of the dominant negative
RhoATI9N also
resulted in distinct domains of reactivity predominantly at the periphery of
the cells
(Figure 12C). In contrast, Racl displayed a uniform cytosolic distribution
(Figure 12D). In
contrast to HeLa cells, overexpression of the activated RhoAGI4V resulted in
morphological changes characteristic of apoptosis in myoblasts (Figures 12E
and 12F).
Interestingly, overexpression of p65PAK in C2C12 myoblasts also resulted in
characteristic
apoptotic morphology (see arrow Figure 12B). Whether this is due to cellular
retraction
following stress fiber dissolution or activation of PAK through caspase
proteolytic cleavage
is unclear.
Recently, aPAK (hPAK1) was shown to colocalize with Rac1 and Cdc42 to
peripheral focal complexes in transfected HeLa cells. In addition,
overexpression of aPAK
in HeLa cells induces the loss of actin stress fibers (Manser et al., 1997).
In C2C12 myoblasts,
the cellular distribution of Myc epitope-tagged versions of p65PAK, RhoATI9N
and SMAK
are strikingly similar (Figures 12A-F). Therefore, these observations raise
the possibility
that PAK, RhoA and SMAK are part of the same complex. To test this, we
constructed a
HA-tagged SMAK expression vector and performed cotransfections experiments
with
Myc-tagged PAK or RhoATI9N plasmid vectors. Transient transfections and double
labeling experiments clearly indicate that PAK and RhoA colocalized with SMAK
at
peripheral as well as internal sites (Figures 12G-L). Therefore, our data are
consistent with
the hypothesis that PAK, RhoA, and SMAK are components of a peripheral focal
adhesion
complex.
Example 7
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Induction of Apoptosis and Actin Disassembly are Separable Activities
The observation that the kinase inactive SMAKK63R promoted actin
reorganization suggested that stress fiber dissolution by SMAK was independent
of kinase
activity. To investigate this hypothesis, we constructed vectors expressing
Myc-tagged
wildtype and mutant SMAK truncations and performed phalloidin staining of
transfected
C2C12 cells. Two of the vectors, SMAKOC and SMAKK63Rt1C contained the wildtype
and
mutant SMAK kinase domain from residues 1-372, extending slightly into the M-
NAP
region, 66 residues short of the caspase cleavage site (see Figure 13 for
plasmid vectors).
Strikingly, this truncation displayed about a 10-increase in activity compared
to full
length SMAK in an in vitro kinase assay using both immunoprecipitated Myc-SMAK
kinase
(see Figure 14J) and recombinant GST-SMAK fusion proteins purified from
bacteria (not
shown). Therefore, these data strongly suggest that the carboxy-terminal
region contains a
negative autoregulatory domain that normally inhibits kinase activity.
Relative to wildtype SMAK, overexpression of SMAKOC in C2C12 cells
resulted in a markedly increased rate of apoptosis as evidenced by a large
increase in
numbers of cells exhibiting cellular shrinkage and membrane blebbing 16 hours
following
transfection (Figures 14A and 14B). By contrast forced expression of the
kinase-inactive
SMAKK63ROC did not induce cell death, suggesting that the enhanced apoptotic
response
was due to activation of the kinase domain (Figure 14C). However,
overexpression of
SMAKK63ROC did not result in any apparent loss of stress fibers following
transfection
(Figure 14D). Consistent with the observation that SMAK-mediated actin
reorganization
was independent of kinase activity, a mutant lacking the kinase domain up to
residue 372
(termed SMAIC~N, see Figure 13), strongly promoted stress fiber disassembly
(Figures 14E
and 14F). In addition, SMAKON-transfected cultures contained a high proportion
of
retracting cells, suggesting that the SMAK C-terminal domain relative to full
length
SMAK is a potent effector of stress fiber dissolution.
To evaluate the rate of apoptosis induced by the various Myc-tagged SMAK
vectors, the proportion of annexin V and 9E10 double positive cells was
measured relative to
the total number of 9E10-positive cells. As shown in Figure 14I, cultures
transfected with
the active SMAK~C displayed about a 6-fold increase in numbers of double-
positive
apoptotic cells 16 hours post-transfection compared to cultures transfected
with
SMAICOCK63R or control vector. Transfection with SMAKON resulted in a
frequency of
double positive apoptotic cells that was about 3-fold less than that observed
in cultures
transfected with SMAKOC and similar to that observed in cultures transfected
with full
length SMAK (not shown). Transfection with SMAK4C resulted in a 3-fold higher
rate of
induction of apoptosis relative to cells transfected with SMAK4N (Figure 14G).
In
summary, these results suggest that both N- and C-terminal domains of SMAK
were
capable of inducing apoptosis. Forced expression of the N-terminal kinase
domain resulted
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in a rapid and efficient induction of apoptosis that was dependent on kinase
activity. By
contrast, forced expression of the C-terminal actin disassembling region
resulted in
stress-fiber dissolution followed by a delayed induction of apoptosis
presumably due to
cellular retraction and loss of adhesion.
Example 8
The ATH Domain Mediates Actin Disassembly
To further delineate the domains that mediate stress fiber disassembly, a
series of Myc-SMAK deletions and truncations were generated and evaluated for
their
ability to reorganize actin stress fibers (Figure 13). SMAK~3' bears a carboxy-
terminal
deletion of 263 amino acids, removing part of the ATH domain. Construct pXXl.2
encompasses SMAK amino acids 551-950 spanning part of the kinase domain
extending into
the M-NAP region. Construct pBg2631 contains amino acids 856-1202, extending
to the end
of the ATH domain. Finally, pXh2973 encodes the last C-terminal 263 amino
acids of the
ATH region that were deleted in SMAK~3'. Expression plasmids were transiently
transfected into C2C12 cells and the cultures fixed and processed for 9E10 and
phalloidin
staining.
Consistent with the notion that the carboxyl 263 amino acids negatively
regulates actin polymerization, overexpression of SMAK~3' led to a markedly
increased
density of actin stress fibers (Table 1). Transfection of SMAIC03'
nevertheless resulted in an
apoptotic response marked by shrinkage and membrane blebbing (not shown).
Transfection
of pXXl.2 resulted in a nuclear associated staining pattern together with an
increased
density of strongly staining stress fibers, but not in an induction of
apoptosis (Table 1, data
not shown). Therefore, we conclude that the ATH region of SMAK is required to
effect actin
reorganization and the subsequent induction of apoptosis. Transfection of
pBg2631, bearing a
partial deletion of the M-NAP region, but encompassing the ATH domain,
efficiently
effected the disassembly of actin stress fibers and thereafter induced
apoptosis (Table 1).
Taken together, transfection of deletion constructs mapped the stress fiber
disassembling domain of SMAK to the last carboxy-terminal 263 amino acids. To
confirm
this, pXh2973 was constructed and transfected into C2C12 cells. Phalloidin
staining of
pXh2973-expressing cells revealed a dramatic loss of actin fibers (Figures 13G
and 13H and
Table 1). In addition, most cells expressing Xh2973 displayed a morphology
reminiscent of
cellular retraction, indicating that SMAK-mediated disassembly of actin fibers
lead to a
loss of substrate adherence. Furthermore, the presence of 9E10-positive
apoptotic cells
observed in Xh2973-transfected cultures supports the contention that the loss
of actin stress
fibers irrevocably leads to cell death. Therefore, we conclude that the ATH
domain is
necessary and sufficient to induce stress fiber dissolution.
Example 9
Caspase-3 Cleavage Stimulates Kinase Activation
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Inspection of the SMAK protein sequence revealed the presence of a caspase-3
consensus cleavage site D-T-Q-D436 at amino acid position 436. This
observation together
with the finding that SMAK mediated apoptosis and stress fiber disassembly
through
distinct domains, raised the possibility that SMAK represents a novel caspase-
3 substrate.
To investigate this possibility, full length wildtype SMAK and the
caspase-3 cleavage site mutant SMAKD436N were translated in vitro in the
presence of
35S-methionine. The translation products were incubated with crude lysates
from bacteria
expressing recombinant caspase-3, or alternatively, with lysates from Rat1-
Myc/ER cells
triggered to undergo apoptosis , then analyzed by SDS-PAGE. As shown in Figure
15A, in
vitro translated SMAK displayed a 'complex banding pattern due to the presence
of
incomplete translation products. Nevertheless, caspase-3 cleavage products of
approximately 133 and 60 kDa were observed following incubation of SMAK with
recombinant caspase-3 (Figure 15A; lanes 3 and 7). Mutation of the cleavage
site at residue
436 resulted in an inability of caspase-3 digestion to liberate the 133 and 60
kDa fragments
(Figure 15A; lanes 4 and 8). Addition of the caspase-3 inhibitor Z-DEVD-fmk,
completely
inhibited the release of both fragments from wildtype SMAK indicating that
cleavage is
mediated by a caspase-3-like activity (Figure 15A; lanes 5). Lastly,
incubation of wildtype
SMAK or SMAKD436N with an apoptotic cell lysate resulted in a similar banding
pattern,
which was abrogated by the addition of Z-DEVD-fmk (Figure 15A; lanes 6-9).
Therefore,
we conclude that caspase-3 specifically cleaves SMAK in vitro at residue 436.
The cleavage of SMAK by a caspase-3-like activity following incubation
with an apoptotic cell lysate raised the possibility that SMAK is an in vivo
substrate for
caspase-3 during an apoptotic response. To address this, Rat1-Myc/ER cells
expressing a
Myc-estrogen receptor fusion were induced to undergo apoptosis by the addition
of
-estradiol and extracts subjected to Western blot analysis using different
anti-SMAK
polyclonal antibodies. Western analysis with anti-SMAKl antibody, directed
against the
kinase domain and the M-NAP region, revealed that the 133 and 60 kDa SMAK
cleavage
products increased over time following the onset of Myc-induced apoptosis
(Figure 15B).
Analysis of the same extracts with anti-SMAK2, a kinase domain-specific
antibody,
resulted in the detection of the 60 kDa cleavage fragment 12 hours following
the addition of
-estradiol (Figure 15C; lanes 4 and 5). Anti-SMAK2 antibodies did not detect
the 133 kDa
fragment, suggesting that it represents the C-terminal domains of SMAK (not
shown).
Western blot analysis of caspase-3-treated N-terminal Myc-tagged SMAK with
antibody
9E10 also detected a 60 kDa product (data not shown). Similarly, release of
the 60 kDa
product from the endogenous SMAK protein was observed when NIH3T3 cells were
exposed
to apoptotic triggers such as TNF- and UV irradiation (Figure 15C; lanes 6-9).
Therefore, we
conclude that caspase-3 cleavage of SMAK in vivo releases the kinase domain as
an
N-terminal 60 kDa product and this cleavage represent a common step in
response to various
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apoptotic stimuli. As shown previously, SMAKOC was found to display higher
activity
than wild type SMAK in an in vitro kinase assay. Surprisingly, the cleavage
site mutant
SMAKD436N showed no kinase activity in vitro, suggesting that the caspase-3
cleavage
site is required for SMAK activity. One possible explanation is that
overexpression of
SMAK in cells induces actin fiber disassembly which triggers an apoptotic
response
resulting in caspase activation and subsequent SMAK cleavage in a feedback
loop.
Therefore, the D436N mutation results in a non-cleavable inactive kinase.
To investigate the effect of caspase-3 cleavage on SMAK kinase activity,
various SMAK mutants were transfected into 293 cells, immunoprecipitated and
assayed for
kinase activity following treatment with recombinant caspase-3 in vitro. As
shown in
Figure 15D, exposure of wildtype or truncated SMAK proteins to recombinant
caspase-3
resulted in a marked increase in kinase activity, suggesting that caspase-
mediated
cleavage of SMAK activated protein kinase activity. By contrast, only a slight
increase in
kinase activity was observed following incubation of SMAKD436N with caspase-3.
This
small increase is likely due to limited non-caspase proteolytic degradation,
as breakdown
products were observed following incubation of in vitro translated SMAKD436N
with the
caspase-3 inhibitor Z-DEVD-fmk (see Figure 15A). Shown in Figure 15E is a
Western blot
demonstrating expression of the different SMAK mutants used in the kinase
assays (Figure
15D) following transfection into 293 cells.
Materials and Methods For Examples 5-9
Cell Culture, Transfection, and Luciferase Assays
C2C12 cells were maintained in Dulbecco's modified Eagle medium
supplemented with 15% fetal calf serum (FCS). Rat1-Myc/ER, NIH3T3, and 293
cells were
grown in DMEM containing 10% FCS. For luciferase assays and stimulation of
293, the cells
were plated at 1 x 105 / 35 mm dish 24 hours prior to transfection. The
cultures were
transfected by Lipofectamine (Gibco/BRL) according to the manufacturer's
instructions using
1 ~g of Gal4-Luciferase reporter, 1 ~g of SMAK expression vector and 100 ng of
effector
plasmid. For luciferase assays, 293 cells were harvested 18-20 hours following
transfection
and the cells were lysed using reporter lysis buffer (Gibco/BRL). Equivalent
portions of
extracts were assayed on a Lumat-100 luminometer using 100 ~1 of Luciferase
assay reagents
(Promega). The average of five independent experiments, performed in duplicate
and
normalized to protein concentration, is shown.
Northern Analysis, Immunofluorescence, and Apoptosis Assays
Analysis of SMAK protein expression was performed by lysing the cultures
(150 mM NaCI, 50 mM Tris, pH 7.4, 1 mM EDTA, 1% Triton-X 100 and 1 ~g/ml of
each
aprotinin, pepstatin and leupeptin) and subjecting 20 ~,g of total lysate to
western blot
analysis using anti-SMAK rabbit polyclonal antibodies . Reactive proteins were
detected
by ECL (Amersham) using a goat anti-rabbit horse radish peroxidase (HRP)
labeled
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secondary antibody. SMAK polyclonal antibodies were generated by immunization
of New
Zealand rabbits using purified GST-SMAK95-551 (anti-SMAKl) encompassing part
of the
kinase and M-NAP domains, or GST-SMAK1-93 (anti-SMAK2; kinase domain).
Specific
immunoreactivity to murine SMAK protein was observed in 293 cells transfected
with full
length and truncated SMAK expression vectors.
For expression studies and immunolocalization experiments, HA- or
Myc-tagged pcDNA3 (Invitrogen) expression vectors bearing full length or
truncated SMAK
were constructed using standard cloning procedures . Briefly, SMAK~C was
constructed by
deleting amino acids 373-1202, leaving the kinase domain. The SMAKON deletion
was
generated by deletion of the first 372 amino acids of SMAK. The kinase dead
version,
SMAKK63R, and the cleavage mutant SMAKD436N, were obtained through PCR-based
mutagenesis of the ATP binding site at residue 63 and the aspartic acide at
position 436,
respectively. Plasmid SMAK03' was created by deleting the last c-terminal 263
amino
acids of Myc-SMAK. Plasmid pXXl.2 encompasses amino acids 551-950. The
expression
vector pBg2631 contains amino acids 856-1202. Finally pXh2973 was generated by
inserting
a fragment encompassing the last 263 amino acids of SMAK into Myc-tagged
pcDNA3.
Following transfection into C2C12, the cultures were fixed for 10 minutes in
4% paraformaldehyde and SMAK protein was detected using 9E10 or rabbit anti-HA
(Santa Cruz) antibodies in conjunction with FITC- or TRITC-labeled secondary
antibodies.
Actin stress fibers were detected using 220 nM TRITC-phalloidin (Sigma) on
fixed cultures
for 15 minutes. Cells undergoing apoptosis were detected using annexin V-FITC
(Oncor) or by
TL1NEL staining (Oncor) according to the manufacturer's specifications.
Caspase-3-expressing bacterial lysates or Ratl-Myc/ER lysates were
prepared according to Song et al. (1997). Cleavage assays on
immunoprecipitated or in vitro
labeled (Promega TNT System) SMAK proteins were performed as described (Song
et al.,
1997).
Immunoprecipitations and In Vitro Kinase Assays
Bacterially expressed GST-SMAK and immunoprecipitated Myc-SMAK
autophosphorylated and efficiently phosphorylated MBP and histone Hl in vitro
(not
shown). However, SMAK did not phosphorylate the JNK and p38 substrates GST-Jun
and
GST-ATF2, respectively. Therefore, all assays were performed using histone Hl
or MBP.
For in vitro kinase assays, of total cell lysate were immunoprecipitated with
1 ~g of 9E10
monoclonal antibodies and 20 ~1 of G-protein sepharose (Pharmacia) for 2 h at
4oC.
Immunoprecipitates were washed three times with NETN (50 mM Tris-HCl, pH 7.5,
150
mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40) and once with kinase assay buffer (20
mM
Tris-HCl, pH 7.5, 15 mM MgCl2, 10 mM NaF, 10 mM b-glycerophosphate, 1 mM
sodium
orthovanadate). Reactions (20 ~l) were initiated by the addition of 5 ~Ci of
[g32P] ATP and
3 ug of histone Hl. After a 30 min incubation at 30°C, reactions were
terminated by the
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addition of 4X SDS sample buffer and 20 ~tl aliquots were fractionated by 12%
SDS-PAGE.
The gels were stained, dried and exposed to X-ray films. For coupled caspase-3
cleavage/kinase assays, immunoprecipitated SMAK proteins were incubated at
37°C in the
presence or absence of caspase-3 lysates and then washed extensively with NETN
and
subjected to kinase assays using histone H1 as above.
Discussion of Examples 5-9
SMAK Induces Apoptosis and Stress Fiber Disassembly
Immunolocalization of Myc epitope-tagged SMAK protein revealed that it
was localized predominantly to the periphery of C2C12 myoblasts. These regions
are
highly enriched in focal adhesion proteins such as FAK and vinculin (Craig and
Johnson,
1996; Jockusch et al., 1995). Transfected cells exhibited cellular shrinkage
and membrane
blebbing suggestive of programmed cell death. Staining of the transfected
cultures for
specific apoptotic markers such as membrane inversion and DNA fragmentation
indicated
that SMAK rapidly induced an apoptotic response. Similarly, the recently
identified but
unrelated ZIP, RIP, and ASKl kinases also induce cell death (Ichijo et al.,
1997; Kawai et
al., 1998; Stanger et al., 1995). However, an inactive form of SMAK was still
able to elicit
an apopotic response, suggesting that SMAK induced programmed cell death in a
kinase-independent manner. This notion was confirmed by the observation that
the
C-terminal ATH region (lacking the kinase domain), was sufficient to induce an
apoptotic
response, but with delayed kinetics.
Interestingly, apoptotic membrane blebbing is regulated in part by myosin
light chain (MLC) phosphorylation and inhibitors of MLC kinase decrease
membrane
blebbing (Mills et al., 1998). In addition, MLC kinase activity is regulated
by the
Rho-binding kinase ROK suggesting a role for p21-activated kinases in the
process of
cytoskeletal reorganization during apoptosis. Supporting this is the
observation that
overexpression of p65PAK mutants in Fas-triggered Jurkat cells induces cell
death but
without the formation of apoptotic bodies (Rudel and Bokoch, 1997). Extensive
membrane
blebbing was induced following transfection of the SMAK kW ase domain and this
effect was
kinase-dependent. Therefore, an interesting possibility is that SMAK has a
unique role in
the regulation of cellular remodeling during programmed cell death.
Focal adhesions are a dynamic protein complex involved in cellular
adherence to the substratum (Craig and Johnson, 1996). The turnover of focal
complexes and
actin stress fibers is coupled to the activity of the Rho family of small
GTPases (Burridge
and Chrzanowska-Wodnicka, 1996; Ilic et al., 1997); (Narumiya et al., 1997).
Several
GTP-Rho binding proteins have been identified including p140mDia and the
protein kinases
p160ROCK, ROK a, MRCK a, PKN, and PRK2. These GTP-Rho binding proteins appear
to
regulate cytoskeletal reorganization by promoting stress fiber formation. By
contrast,
overexpression of a-PAK induces loss of both focal adhesions and actin stress
fibers (Manser
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et al., 1997). Overexpression of the tumor suppressor and protein phosphatase
PTEN also
downregulates the formation of focal adhesions (Tamura et al., 1998). Although
SMAK
does not contain any homology to known GTPase binding domains, overexpression
of both
SMAK and an kinase-inactive mutant resulted in actin stress fiber dissolution,
suggesting
that this is a kinase-independent process (Kawai et al., 1998). Moreover, an N-
terminal
deletion of SMAK lacking the kinase domain efficiently promoted stress fiber
disassembly
and induced characteristic apoptotic morphology in transfected C2C12 cells.
Similarly,
exposure of C2C12 myoblasts to 2-chloro adenosine induces disruption of actin
microfilaments and triggers apoptosis (Rufini et al., 1997). One possibility
is that SMAK is
titrating important factors required for the maintenance of focal adhesions or
actin stress
fibers. Functional deletion analysis of SMAK revealed that overexpression of
the
C-terminal ATH domain of SMAK led to stress fiber dissolution and cellular
retraction,
suggesting that this domain negatively regulates stress fiber formation. The
ATH region
may be interfering with regulatory components of focal complexes, supporting a
role for
SMAK in the regulation of stress fiber dynamics.
The Ste20-related kinase, PAK, has been shown to be recruited to focal
adhesion sites by activated Cdc42 and Rac1 (Manser et al., 1997).
Interestingly, SMAK
colocalizes with PAK and RhoA in transfected C2C12 myoblasts, suggesting that
they are
part of the same protein complex. The presence of a putative SH3-binding
domain in the
M-NAP region of SMAK represents an attractive target for docking onto such a
protein
scaffold. The Ste20-related protein kinase HPK1 has been demonstrated to bind
directly to
the SH2/SH3 containing adapter protein Grb2 and to be recruited to the
autophosphorylated EGF receptor (Anafi et al, 1997), providing a mechanism for
cross-talk
between distinct biochemical pathways. Whether activated p2ls impinge on SMAK
activity is currently being investigated. However, preliminary results suggest
that SMAK
and Rac/PAK are components of independent pathways (L. A. Sabourin and M.A.
Rudnicki,
unpublished observations).
The Rho-associated kW ase ROK, has been shown to phosphorylate proteins
of the ezrin/radixin/moesin family (ERM), localized within focal complexes and
to
regulate their association (Matsui et al., 1998). In addition ROK is involved
in the control
of MLC kinase activity and membrane blebbing through direct regulation of
myosin
phosphatase (Kimura et al., 1996). Recently, LIM-kinase has been implicated in
the
control of cytoskeleton reorganization through phosphorylation of cofilin, a
ubiquitous
actin binding protein required for actin depolimerization (Arber et al., 1998;
Yang et al.,
1998). Therefore, to understand its role during actin reorganization, it will
be of interest to
identify SMAK substrates. In addition, the identification of targets for the
ATH domains
will provide valuable clues as to the mechanisms by which SMAK regulates
stress fiber
dynamics.
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Caspase Cleavage of SMAK Releases Distinct Functional Domains
Although there are several kinases that mediate cell growth, only a few
have been identified that trigger apoptosis. Recently, JNKs have been shown to
be
activated by apoptotic triggers such as Fas ligand and TNF-a. ASKl, a MAP
kinase kinase
kinase, has been shown to induce cell death and to activate JNK and p38 MAP
kinase (Ichijo
et al., 1997). Recently, the SMAK related kinases MST1 and PAK2 have been
shown to be
substrates for caspase-3. Caspase-mediated cleavage was demonstrated to
activate their
kinase activity. In addition, MST1 has been shown to activate MKK6, MKK7, p38
and stress
activated protein kinases (Graves et al., 1998). However the mechanisms by
which MST1
activates these kinases are unknown.
Similar to p65PAK and MSTl (Graves et al., 1998; Lee et al., 1998; Lee et al.,
1997; Rudel and Bokoch, 1997), SMAK is a substrate for caspase-3 and is
rapidly cleaved
following the induction of apoptosis. Furthermore, cleavage releases an
activated SMAK
kinase domain and an actin fiber disassembling region that appear to function
independently.
Several studies have demonstrated that actin fiber disassembly and
cytoskeletal rearrangements represent significant steps in the process of
apoptosis
(Brancolini et al., 1997; DeMeester et al., 1998; Ghosh et al., 1997; Kletsas
et al., 1998; Mills
et al., 1998; Palladini et al., 1996; Rufini et al., 1997). Furthermore, actin
has been
demonstrated to be resistant to caspase cleavage during apoptosis, suggesting
that it is
required for cellular remodeling during the apoptotic process and that
reorganization
mechanisms need to be activated (Rice et al., 1998; Song et al., 1997; Villa
et al., 1998).
Therefore, SMAK may represent a novel pro-apoptotic effector for which caspase
cleavage
releases a cytoskeletal disassembling function concomitant with kinase
activation.
Whether the active kinase domain is involved in cytoskeletal remodeling,
apoptotic
signaling or both, remains to be determined. Interestingly, disruption of the
actin filament
network by cytochalasin D activates p53-dependent transcription and apoptosis
Conversely, activation of p53 through SV40 large T antigen inactivation also
leads to
F-actin disassembly (Guenal et al., 1997).
Actin stress fibers are ultimately anchored at focal adhesion sites through
interactions with proteins such as a-actinin, vinculin and talin (Craig and
Johnson, 1996).
Interestingly, overexpression of gelsolin, an actin-regulatory protein found
at focal sites has
been demonstrated to protect Jurkat cells from Fas-induced apoptosis by
preventing changes
in the F-actin morphology and inhibition of caspase-3 (Ohtsu et al., 1997).
However,
endogenous gelsolin protein was found to be a substrate for caspase-3. Caspase-
3-cleaved
gelsolin was demonstrated to destabilize the actin network, causing cellular
retraction,
detachment and apoptosis (Kothakota et al., 1997). Similarly, the product of
the growth
arrest-specific 2 (Gas2) gene is also cleaved by an ICE-like protease activity
during
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apoptosis (Brancolini et al., 1995). Gas2 is known to be associated with the
actin
microfilament network and caspase cleavage induces its actin reorganization
activity
(Brancolini et al., 1995). One attractive possibility is that the caspase-3-
mediated release
of the ATH domain interferes directly with the function of actin-regulatory
proteins such as
gelsolin or Gas2 or alternatively, may promote their proteolytic processing.
The present inventors have cloned and characterized a Ste20-related kinase,
SMAK, which can mediate apoptosis and promote stress fiber dissolution.
Although the
full length protein can mediate both effects, the individual N- and C-
terminal domains
were more efficient at inducing apoptosis and actin reorganization
respectively. We have
shown that SMAK is a substrate for a caspase-3-like activity in vivo during
the process of
apoptosis. Furthermore, caspase-3-mediated cleavage of SMAK increased its
intrinsic
kinase activity. These results raise the interesting possibility that kinases
such as SMAK
and PAK may represent a new class of dual function proteins playing important
roles in the
regulation of the apoptotic response as well as cytoskeleton reorganization.
The
identification of SMAK substrates, regulatory molecules and interacting
partners will
provide further insights into mechanisms underlying its regulation during the
process of
actin reorganization and programmed cell death.
Having illustrated and described the principles of the invention in a
preferred embodiment, it should be appreciated to those skilled in the art
that the
invention can be modified in arrangement and detail without departure from
such
principles. We claim all modifications coming within the scope of the
following claims.
All publications, patents and patent applications referred to herein are
incorporated by reference in their entirety to the same extent as if each
individual
publication, patent or patent application was specifically and individually
indicated to be
incorporated by reference in its entirety.
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Table 1
Expression Actin Kinase Apoptosis Cellular
Vector Disassembly Activity Localization
SMAK + + + CP
SMAK4C - +++ +++ DC
SMAKON + - + DC
SMAK43' - * + + DC
XX1.2 - * - - PN
Bg2631 + - + DC
Xh2974 ++ - + DC
SMAKK63R .~ _
+ CP
Table 1. Actin dissembling and apoptosis-inducing activities of SMAK mutants
and N- and
C-terminal truncations. Following transfection into C2C12 cells, the cultures
were fixed and
stained using anti-Myc and TRITC-phalloidin to evaluate their actin
disassembling
activity. The relative kinase activities were determined by
Immunoprecipitations and in
vitro kinase assays following transfections in 293 cells.
Abbreviations: CP, cell periphery; DC, diffuse cytoplasmic; PN, perinuclear.
* An increased density of stress fibers was observed following transfection of
SMAK43' and
XX1.2.
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DETAILED FIGURE LEGENDS
Figure 1. Nucleotide and amino acid sequence of murine SMAK. The N-terminal
kinase
domain is highlighted in black. The M-NAP and AT1-46 regions are shaded and
boxed,
respectively. The putative SH3-binding motif has been highlighted in white
within the
M-NAP domain.
Figure 2. Deduced amino acid sequence of murine SMAK. The N-terminal kinase
domain is
highlighted in black. The M-NAP and AT1-46 regions are shaded and boxed,
respectively.
The putative SH3-binding motif has been highlighted in white within the M-NAP
domain.
Figure 3. Sequence alignment of SMAK with related proteins. (A) Alignment of
SMAK kinase
domain with that of the Ste20-related kinases human SLK (hSLK), LOK and MST.
The
kinase subdomains are numbered I-XI and conserved residues within the kinase
domain are
marked by an asterisk. The characteristic Ste20 motif in subdomain VIII is
underlined. (B)
Alignment of SMAK with Rat SLK, LOK, M-NAP and ATl-46. In both alignments, the
shaded area represent identical amino acid residues. Numbers on the left
indicate the amino
acid residues. (C) Schematic representation of SMAK and similarity indices (%)
to various
other polypeptides. The catalytic domain (black) is most similar to LOK and
MST1/2, two
Ste20-like kinases. SMAK is highly homologous to microtubule and nuclear
associated
protein (M-NAP; grey) and to AT1-46 (white) in the carboxy terminal region.
Numbers in
brackets represent SMAK1 amino acid residues.
Figure 4. Northern blot analysis of tissues and cell lines. 4 ~g of poly-A+
RNA from various
tissues was analyzed for SMAK expression and normalized for b-actin mRNA
levels.
Northern analysis shows the presence of at least 3 SMAK mRNA species of
approximately
6,7 and 8 kb in length. The 7kb isoform being predominantly expressed in all
the human
tissues surveyed. (B) Expression analysis of murine tissue RNA. 4 ~,g of poly-
A+ RNA from
various human tissues (Clonetech MTN) was analyzed for SMAK mRNA levels and
normalized to b-actin expression. Northern analysis shows the presence of at
least 3 SMAK
mRNA species of approximately 6, 7 and 8 kb in length. The largest isoform
being
predominately expressed in all the murine tissues surveyed. (C) SMAK mRNA
expression in
murine cell lines. 4 ~g of poly-A+ RNA from four murine cell lines were
surveyed for SMAK
mRNA levels. In contrast to adult tissues, cultured cells expressed similar
amounts of all
three SMAK mRNA isoforms. Interestingly, the embryocarcinoma cell line P19
showed no
detectable levels of SMAkl mRNA. (D) Analysis of SMAK expression in various
human cell
lines. 2 ~g of poly-A+ RNA from different human tumor cell lines (Clonetech
MTN) were
analyzed for SMAK expression. Relative to normal blood lymphocytes and colon
tissue,
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respectively, high levels of SMAkl mRNA were observed in K562 cells, a chronic
myelogenous leukemia cell line and SW480, a colon adenocarcinoma.
Figure 5. Expression of SMAK during C2C12 differentiation and serum
stimulation of NIH3T3
fibroblasts. (A) C2C12 cells were induced to differentiate in DMEM containing
2% horse
serum (DM) and harvested at 0, 1, 3 and 5 days following the induction of
differentiation. 20
~g of total cell lysate was analyzed for SMAK expression by immunoblotting
using an
anti-SMAK rabbit polyclonal antibody. A significant decrease in SMAK protein
levels is
observed following the transfer of the cells to differentiation medium.
293/SMAK and
293/SMAK43' represent antibody control lysates from 293 cells transfected with
a full
length and a carboxy terminal truncated version of SMAK. (B) NIH 3T3 cells
were serum
starved for 24 hours and then stimulated with 10% fetal calf serum for 1 day
and total cell
lysates were analyzed for SMAK protein levels. A rapid decrease in SMAK levels
was
observed in serum starved cells. However, following serum stimulation, SMAK
levels were
up-regulated.
Figure 6. In Vitro kinase assay of purified or immunoprecipitated SMAK. (A)
Bacterially
expressed full length SMAK (lane A) or immunoprecipitated Myc-tagged SMAK
(lane B)
were assayed for kinase activity on various substrates. Both showed activity
on MBP and
histone H1. No detectable activity was observed on GST-Jun or GST-ATF2. SMAK
protein
purified from bacteria was approximately 10 times more active on these
substrates relative
to the kinase immunoprecipitated from transfected 293 cells. (B) Inactivation
of SMAK by
calf intestinal alkaline phosphatase (CIAP) treatment. Purified recombinant
GST-SMAK
protein was subjected to 2 and 20 units of CIAP for 30 minutes at 37°C,
washed and assayed for
activity. Decreased kinase activity was observed following CIAP treatment.
Figure 7. Effect of extracellular stimuli on SMAK activity. (A)
Immunoprecipitation and in
vitro kinase assay of transfected Myc-SMAK. Following transfection of 293
cells, the cultures
were starved for 1 hour in serum free medium and then stimulated with various
agents for 15
minutes. The cells were then lysed and assayed for SMAK activity on histone Hl
in vitro.
Lane 1: untransfected 293, lane 2: 10% FCS, lane 3: 300 nM TPA, lane 4: 100
ng/ml EGF, lane 5:
non-starved, lane 6: 10 M A23187, lane 7: UV (200J/m2), lane 8: 100 ng/ml TNF-
a, lane 9: 20
ng/ml ILl-a, lane 10: unstimulated. No change in SMAK activity was detected
after exposure
to the various stimuli. (B) Immunoblot analysis shows that the cultures
expressed equivalent
amounts of transfected SMAK protein. (C) Activation of JNKl by SMAK. 293 cells
were
transiently transfected with Myc-SMAK and JNKl or ERK1/2 was
immunoprecipitated 18
hours later using an anti-human JNK1 (Pharmingen) or ERKl/2 (Santa Cruz)
antibody.
Kinase assay was performed on GST-Jun (JNK) or MBP (ERKl/2).
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Figure 8. Immunolocalization of SMAK in C2C12 myoblasts. C2C12 cells were
transiently
transfected with a Myc-epitope tagged SMAK vector and stained at various times
after
transfection for expression using 9E10 supernatant. Distinct clusters of
staining can be observed
at the periphery of the cells 16 hours after transfection for both the wild
type (A) and
SMAKK63R, a mutant form of SMAK (B). Photomicrographs are shown at 400X.
Figure 9 shows induction of Apoptosis in SMAK Transfected C2C12 Myoblasts
(A) Detection of Myc-SMAK 24 h following transfection revealed that almost all
9E10
labeled cells exhibited membrane blebbing and cell shrinkage suggesting that
SMAK induced
cell death.
(B) Phase contrast photomicrograph of C.
(C and D) Double immunofluorescent staining of Myc-SMAK and annexin V-FITC in
transfected C2C12 cells indicating that SMAK transfection had induced
apoptosis. Arrows
denote identical cells in E and F.
(E and F) Immunodetection of the Myc-SMAKK63R lacking functional kinase
activity in
transfected C2C12 cells and co-localization of annexin V-FITC indicated that
kinase activity
was not required for full length SMAK to induce apoptosis. Arrows denote
identical cells in G
and H.
(G and H) Immunodetection of Myc-SMAK expression in cells undergoing DNA
fragmentation
as detected by TUNEL labeling supports the contention that forced expression
of SMAK
induces apoptosis. Arrows denote identical cells in G and H.
Figure 10 shows SMAK Induces the Stress Activated Signaling Pathway
Gal4-Luciferase reporter and Gal4-Elkl, Gal4-Jun and Gal4-CREB fusions with or
without
SMAK were transiently transfected into 293 cells. Cotransfection of SMAK and
Gal4-Jun
resulted in a 5 to 7-fold increase in luciferase activity. By contrast,
cotransfection of SMAK
and Gal4-Elk and Gal4-CREB resulted in a 2-fold increase in luciferase
activity. The data
shown represent the average +/- SEM from 5 independent experiments using
duplicate
samples, normalized to protein concentrations.
Figure 11. Overexpression of SMAK Induces stress fiber dissolution
(A and B) Expression of Myc-SMAK in transiently transfected C2C12 cells
induced loss of
actin stress fibers as detected by phalloidin staining. Arrow denotes the
identical cell in
panels A and B.
(C and D) Expression of Myc-RacVl2 in C2C12 cells similarly induced loss of
stress fibers as
evidenced by phalloidin staining. Arrow denotes the identical cell in panels C
and D.
(E and F) Expression of transfected Myc-p65PAK also induced loss of actin
stress fibers. Arrow
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denotes the identical cell in panels E and F.
The cells were fixed and stained 16 hours post transfection with anti-Myc
antibody 9E10 and
TRITC-Phalloidin, and photographed at 400X.
Figure 12. Colocalization of SMAK, PAK, and RhoA in transfected C2C12
myoblasts
(A-D) Immunodetection of transfected Myc-tagged SMAK, PAK, RhoANl9 and
Rac1V12,
respectively with antibody 9E10. Note the similar pattern of punctate staining
at the cell
periphery.
(E) Induction of cell death by activated RhoA in C2C12 cells. RhoAVl4
expression was
detected using 9E10 16 hours following transfection.
(F) Phase contrast photomicrograph of E.
(G and H) Double immunodetection of transfected HA-tagged SMAK with a rabbit
anti-HA
antibody and of transfected Myc-RhoANl9 expressing cells using 9E10.
(I) Superposition of G and H showing colocalization of SMAK and RhoA.
(J-L) Double immunodetection of transfected HA-tagged SMAK with a rabbit anti-
HA
antibody and of transfected Myc-PAK expressing cells using 9E10.
(L) Superposition of J and L showing colocalization of SMAK and PAK.
Staining was performed 16 hours following transfection. Photomicrographs are
shown at
400X.
Figure 13. Schematic representation of the plasmid expression vectors used in
this study. The
Ste20 kinase domain (black), M-NAP (grey) and ATH (white) domains are
indicated. SMAK
amino acid residues are indicated at the N- and C-terminal.
Figure 14. Separable SMAK Domains Induce Apoptosis and Stress Fiber
Disassembly
(A and B) Immunodetection and phalloidin staining of Myc-SMAKOC-expressing
C2C12 cells
16 hours post-transfection. Extensive membrane blebbing was observed in
expressing cells.
(C and D) Overexpression of the Myc-SMAKDCK63R mutant did not result in any
morphological changes suggesting that it is kinase-dependent.
(E and F) Forced expression of Myc-SMAK~N induced stress fiber dissolution in
overexpressing cells as shown by phalloidin staining demonstrating that SMAK
may play
important roles in the regulation of actin reorganization and cell death.
(G and H) Transfection of the AT1-46 domain (pXh2973; H and I) resulted in a
loss of strongly
staining actin stress fibers indicating that the actin reorganizing domain of
SMAK is
contained within the ATH region. Photomicrographs are shown at 400X.
(I) Enhanced apoptotic response by SMAKOC overexpression. The percentage of
double
positive annexin V and 9E10 cells relative to the total number of 9E10
positive cells
([(#annexinV and 9E10)/total 9E10] x 100} was evaluated at 16 and 20 hours
following
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transfection. The results revealed an increased rate of cell death by SMAKOC.
(J) The indicated expression vectors were transfected into 293T cells,
immunoprecipitated
using 9E10 antibodies and assayed for kinase activity on myelin basic protein
MBP arrow).
Equivalent aliquots of protein were also subjected to Western blot analysis
for normalization
(upper panel). About 5- to 10-fold higher kinase activity was consistently
observed with
immunoprecipitated SMAKOC.
Figure 15. Activation of SMAK through Caspase-3-Mediated Cleavage (A) Caspase-
3
cleavage of in vitro translated wildtype (WT) and caspase-3 cleavage site
mutant (D436N)
SMAK proteins. A similar pattern of cleavage was observed in the presence of
an apoptotic
cell lysate from induced (2 mM b-estradiol) Ratl-Myc/ER cells. Introduction of
the D436N
mutation in SMAK prevented cleavage and release of the 133 and 60 kDa
fragments.
Addition of the caspase-3 inhibitor Z-DEVD-fmk (50 mM) to the reaction
abolished
cleavage.
(B) Apoptosis-induced cleavage of endogenous SMAK protein in stimulated Ratl-
Myc/ER
cells. The levels of SMAK fragments of 133 and 60 kDa progressively increased
during the
apoptotic response while the levels of the full length p220 were reduced.
(C) N-terminal-specific antibodies identified the 60 kDa fragment as the
kinase domain in
induced Rat1-Myc/ER cells and NIH3T3 cells exposed to apoptotic triggers.
NIH3T3
fibroblasts were exposed to TNF-a (20 ng/ml) plus 10 mM
pyrrolidinedithiocarbamate for 16
hours or UV-irradiated (180 J/m2) and allowed to recover in growth medium for
4 and 15
hours. Release of SMAK kinase domain was clearly evident. No reactivity to the
133 kDa
fragment was observed, suggesting that it bears a C-terminal portion of SMAK.
(D) In vitro caspase-3/kinase assay on immunoprecipitated Myc-SMAK proteins.
Wild type
or mutant Myc-SMAK proteins were immunopurified and subjected to caspase-3-
expressing
lysates, or a control lysate, and assayed
for kinase activity in vitro.
(E) Anti-Myc tag western blot analysis showing expression of all Myc-SMAK
proteins in
transfected 293 cells.
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