Note: Descriptions are shown in the official language in which they were submitted.
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IKB KINASE, SUBUNITS THEREOF, AND METHODS OF USING SAME
This invention was made with government support
under grant number CA50528 awarded by the National
Institutes of Health. The government has certain rights
in the invention.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates generally to
molecular biology and biochemistry and more specifically
to a protein kinase, IKB kinase, which is activated in
response to environmental stresses and proinflammatory
signals to phosphorylate inhibitors of the NF-KB
transcription factors and to methods of using the protein
kinase.
BACKGROUND INFORMATION
The induction of gene expression due to
exposure of a cell to a specific stimulus is a tightly
controlled process. Depending on the inducing stimulus,
it can be critical to survival of the cell that one or
more genes be rapidly induced, such that the expressed
gene product can mediate its effect. For example, an
inflammatory response stimulated due to an injury to or
infection of a tissue results in rapid vasodilation in
the area of the injury and infiltration of effector cells
such as macrophages. Vasodilation occurs within minutes
of the response and is due, in part, to the expression of
cytokines in the injured region.
The rapid induction, for example, of an
inflammatory response or an immune response, requires
that the transcription factors involved in regulating
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such responses be present in the cell in a form that is
amenable to rapid activation. Thus, upon exposure to an
inducing stimulus, the response can occur quickly. If,
on the other hand, such transcription factors were not
already present in a cell in an inactive state, the
factors first would have to be synthesized upon exposure
to an inducing stimulus, greatly reducing the speed with
which a response such as an inflammatory response could
occur.
Regulation of the activity of transcription
factors involved in such rapid induction of gene
expression can occur by various mechanisms. For example,
in some cases, a transcription factor that exists in an
inactive state in a cell can be activated by a post-
translational modification such as phosphorylation on one
or more serine, threonine or tyrosine residues. In
addition, a transcription factor can be inactive due to
an association with a regulatory factor, which, upon
exposure to an inducing stimulus, is released from the
transcription factor, thereby activating the
transcription factor. Alternatively, an inactive
transcription factor may have to associate with a second
protein in order to have transcriptional activity.
Rarely, as in the case of glucocorticoids, the
inducing stimulus interacts directly with the inactive
transcription factor, rendering it active and resulting
in the induction of gene expression. More often,
however, an inducing stimulus initiates the induced
response by interacting with a specific receptor present
on the cell membrane or by entering the cell and
interacting with an intracellular protein. Furthermore,
the signal generally is transmitted along a pathway, for
example, from the cell membrane to the nucleus, due to a
series of interactions of proteins. Such signal
transduction pathways allow for the rapid transmission of
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an extracellular inducing stimulus such that the
appropriate gene expression is rapidly induced.
Although the existence of signal transduction
pathways has long been recognized and many of the
cellular factors involved in such pathways have been
described, the pathways responsible for the expression of
many critical responses, including the inflammatory
response and immune response, have not been completely
defined. For example, it is recognized that various
inducing stimuli such as bacteria or viruses activate
common arms of the immune and inflammatory responses.
However, differences in the gene products expressed also
are observed, indicating that these stimuli share certain
signal transduction pathways but also induce other
pathways unique to the inducing stimulus. Furthermore,
since inducing agents such as bacteria or viruses
initially stimulate different signal transduction
pathways, yet induce the expression of common genes, some
signal transduction pathways must converge at a point
such that the different pathways activate common
transcription factors.
A clearer understanding of the proteins
involved in such pathways can allow a description, for
example, of the mechanism of action of a drug that is
known to interfere with the expression of genes regulated
by a particular pathway, but the target of which is not
known. In addition, the understanding of such pathways
can allow the identification of a defect in the pathway
that is associated with a disease such as cancer. For
example, the altered expression of cell adhesion
molecules is associated with the ability of a cancer cell
to metastasize. However, the critical proteins involved
in the signal transduction pathway leading to expression
of cell adhesion molecules have not been identified.
Thus, a need exists to identify the proteins involved in
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signal transduction pathways, particularly those proteins
present at the convergence point of different initial
pathways that result in the induction, for example, of
gene products involved in the inflammatory and immune
responses. The present invention satisfies this need and
provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic
acid molecules encoding full length human serine protein
kinases, designated IKB kinase (IKK) subunits IKKa and
IKKR. The disclosed IKK subunits share substantial
sequence homology and are activated in response to
proinflarnmatory signals to phosphorylate proteins (IKB's)
that inhibit the activity of the NF-KB transcription
factor.
For example, the invention provides a nucleic
acid molecule having the nucleotide sequence shown as SEQ
ID NO: 1, which encodes a cytokine inducible IKE kinase
subunit designated IKKa, particularly the sequence shown
as nucleotides -35 to 92 in SEQ ID NO: 1, and nucleic
acid molecules encoding the amino acid sequence shown as
SEQ ID NO: 2, as well as nucleotide sequences
complementary thereto. In addition, the invention
provides a nucleic acid molecule having the nucleotide
sequence shown as SEQ ID NO: 14, which encodes a second
cytokine inducible IKB kinase subunit, designated IKK(3,
and nucleic acid molecules encoding the amino acid
sequence shown as SEQ ID NO: 15, as well nucleotide
sequences complementary thereto. The invention also
provides vectors comprising the nucleic acid molecules of
the invention and host cells containing such vectors.
In addition, the invention provides nucleotide
sequences that bind to a nucleic acid molecule of the
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invention, including to nucleotides -35 to 92 as shown in
SEQ ID NO: 1. Such nucleotide sequences of the invention
are useful as probes, which can be used to identify the
presence of a nucleic acid molecule encoding an IKK
5 subunit in a sample, and as antisense molecules, which
can be used to inhibit the expression of a nucleic acid
molecule encoding an IKK subunit.
The present invention also provides isolated
full length human IKK subunits, which can phosphorylate
an IKB protein. For example, the invention provides an
IKKa polypeptide having the amino acid sequence shown as
SEQ ID NO: 2, particularly the amino acid sequence
comprising amino acids 1 to 31 at the N-terminus of the
polypeptide of SEQ ID NO: 2. In addition, the invention
provides an IKK~ polypeptide having the amino acid
sequence shown as SEQ ID NO: 15. The invention also
provides peptide portions of an IKK subunit, including,
for example, peptide portions comprising one or more
contiguous amino acids of the N-terminal amino acids
shown as residues 1 to 31 in SEQ ID NO: 2. A peptide
portion of an IKK subunit can comprise the kinase domain
of the IKK subunit or can comprise a peptide useful for
eliciting production of an antibody that specifically
binds to an IKB kinase or to the IKK subunit.
Accordingly, the invention also provides anti-IKK
antibodies that specifically bind to an IKK complex
comprising an IKK subunit, particularly to the IKK
subunit, for example, to an epitope comprising at least
one of the amino acids shown as residues 1 to 31 of SEQ
ID NO: 2, and also provides IKK subunit-binding fragments
of such antibodies. In addition, the invention provides
cell lines producing anti-IKK antibodies or IKK-binding
fragments thereof.
The invention also provides isolated IKB kinase
complexes. As disclosed herein, an IKK complex can have
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an apparent molecular mass of about 900 kDa or about 300
kDa. An IKK complex is characterized, in part, in that
it comprises an IKKa subunit, an IKKR subunit, or both
and can phosphorylate an IxB protein.
The present invention further provides methods
for isolating an IKK complex or an IKK subunit, as well
as methods of identifying an agent that can alter the
association of an IKK complex or an IKK subunit with a
second protein that associates with the IKK in vitro or
in vivo. Such a second protein can be, for example,
another IKK subunit; an IKB protein, which is a substrate
for IKK activity and is involved in a signal transduction
pathway that results in the regulated expression of a
gene; a protein that is upstream of the IKB kinase in a
signal transduction pathway and regulates IKK activity;
or a protein that acts as a regulatory subunit of the IxB
kinase or of an IKK subunit and is necessary for full
activation of the IKK complex. An agent that alters the
association of an IKK subunit with a second protein can
be, for example, a peptide, a polypeptide, a
peptidomimetic or a small organic molecule. Such agents
can be useful for modulating the level of phosphorylation
of IKB in a cell, thereby modulating the activity of
NF-KB in the cell and the expression of a gene regulated
by NF-KB.
The invention also provides methods of
identifying proteins that can interact with an IKB
kinase, including with an IKK subunit, such proteins
which can be a downstream effector of the IKK such as a
member of the IKB family of proteins or an upstream
activator or a regulatory subunit of an IKK. Such
proteins that interact with an IKK complex or the IKK
subunit can be isolated, for example, by coprecipitation
with the IKK or by using the IKK subunit as a ligand, and
can be involved, for example, in tissue specific
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regulation of NF-KB activation and consequent tissue
specific gene expression.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID
NO: 1; lower case letter) and deduced amino acid sequence
(SEQ ID NO: 2; upper case letters) of full length human
IKKa subunit of an IKK complex. Nucleotide positions are
indicated to the right and left of the sequence; the "A"
of the ATG encoding the initiator methionine is shown as
position 1. Underlined amino acid residues indicate the
peptide portions of the protein ("peptide 1" and
"peptide 2") that were sequenced and used to design
oligonucleotide probes. The asterisk indicates the
sequence encoding the STOP codon.
Figure 2 shows a nucleotide sequence (SEQ ID
NO: 14) encoding a full length IKK(3 polypeptide (see
Figure 3). Numbers to the left and right of the sequence
indicate nucleotide position number. The initiator ATG
codon is present at nucleotides 36-38 and the first stop
codon (TGA) is present at nucleotides 2304-2306.
Figure 3 shows an alignment of the deduced
amino acid sequences of IKKa ("a", SEQ ID NO: 2) and IKK(3
("R", SEQ ID NO: 15). Numbers to the right of the
sequences indicate the respective amino acid positions.
Underlined amino acid residues indicate peptide portions
of the IKKR subunit that were sequenced and used to
search an EST database (see Example III). Vertical bars
between amino acid residues indicate identical amino
acids; two dots between amino acid residues indicates
very similar amino acids (e.g., Glu and Asp; Arg and Lys)
and one dot between amino acid residues indicates a
lesser degree of similarity. A dot within an amino acid
sequence indicates a space introduced to maintain
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sequence homology. The kinase domains in the N-terminal
half of the sequences and helix-loop-helix domains in the
C-terminal half of the sequences are bracketed and the
leucine residues involved in the leucine zippers are
indicated by the filled circles above the IKKa sequence.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides isolated nucleic
acid molecules encoding polypeptide subunits of human
serine protein kinase complex, the IKB kinase (IKK)110 which is activated in
response to proinflammatory signals
and phosphorylates proteins (IKB's) that bind to and
inhibit the activity of NF-KB transcription factors. For
example, the invention provides an isolated nucleic acid
molecule (SEQ ID NO: 1) encoding a full length human IKKa
subunit having the amino acid sequence shown as SEQ ID
NO: 2 (Figure 1). In addition, the invention provides an
isolated nucleic acid molecule (SEQ ID NO: 14; Figure 2)
encoding a full length human IKK(3 subunit having the
amino acid sequence shown as SEQ ID NO: 15 (Figure 3).
As used herein, the term "isolated," when used
in reference to a nucleic acid molecule of the invention,
means that the nucleic acid molecule is relatively free
from contaminating lipids, proteins, nucleic acids or
other cellular material normally associated with a
nucleic acid molecule in a cell. An isolated nucleic
acid molecule of the invention can be obtained, for
example, by chemical synthesis of the nucleotide sequence
shown as SEQ ID NO: 1 or SEQ ID NO: 14 or by cloning the
molecule using methods such as those disclosed in
Examples II and III. In general, an isolated nucleic
acid molecule comprises at least about 30% of a sample
containing the nucleic acid molecule, and generally
comprises about 500 or 70% or 90% of a sample, preferably
95% or 98% of the sample. Such an isolated nucleic acid
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molecule can be identified by comparing, for example, a
sample containing the isolated nucleic acid molecule with
the material from which the sample originally was
obtained. Thus, an isolated nucleic acid molecule can be
identified, for example, by comparing the relative amount
of the nucleic acid molecule in fraction of a cell lysate
obtained following gel electrophoresis with the relative
amount of the nucleic acid molecule in the cell, itself.
IKKa and IKKE3 have been designated IKK subunits
because they are components of an approximately 900 kDa
complex having IKB kinase (IKK) activity and because they
share substantial nucleotide and amino acid sequence
homology. As disclosed herein, IKKa and IKKQ are related
members of a family of IKK catalytic subunits (see
Figure 3). The 900 kDa IKB kinase complex can be
isolated in a single step, for example, by
immunoprecipitation using an antibody specific for an IKK
subunit or by using metal ion chelation chromatography
methods (see Example IV). A 300 kDa IKK complex also can
be isolated as disclosed herein and has kinase activity
for an IKB substrate (see Example III).
Nucleic acid molecules related to SEQ ID NO: 1
previously have been described (Connelly and Marcu, Cell.
Mol. Biol. Res. 41:537-549 (1995)).
herein by reference). For example, Connelly and Marcu
describe a 3466 base pair (bp) nucleic acid molecule
(GenBank Accession #U12473; Locus MMU 12473)),
which encodes a full
length mouse polypeptide having an apparent molecular
mass of 85 kiloDaltons (kDa) and designated CHUK. A 2146
bp nucleic acid molecule (GenBank Accession #U22512;
Locus HSU 22512)),
which encodes a portion of the polypeptide
shown in SEQ ID NO: 2 also was described. However, the
amino acid sequence deduced from #U22512 lacks amino
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acids 1 to 31 as shown in SEQ ID NO: 2 and, therefore, is
not a full length protein. In addition, several
nucleotide differences occur in SEQ ID NO: 1 as compared
to the sequence of #U22512, including nucleotide changes
5 that encode different amino acids at positions 543, 604,
679, 680, 684 and 685 of SEQ ID NO: 2; silent nucleotide
changes also occur at codons 665 and 678. The
polypeptides encoded by the nucleotide sequences of
GenBank Accession #U12473 and #U22512 share about 950
10 identity at the amino acid level and are substantially
similar to that shown in SEQ ID NO: 2. No function has
been demonstrated for the polypeptides described by
Connelly and Marcu, although Regnier et al. (Cell
90:373-383 (1997)) recently have confirmed that human
CHUK corresponds to IKKa, as disclosed herein.
A nucleic acid molecule of the invention is
exemplified by the nucleotide sequences shown as SEQ ID
NO: 1, which encodes a full length human IKKa (SEQ ID
NO: 2; Figure 1), the activity of which is stimulated by
a cytokine or other proinflammatory signal, and as SEQ ID
NO: 14, which encodes a full length IKK(3 (SEQ ID NO: 15).
Due to the degeneracy of the genetic code and in view of
the disclosed amino acid sequence of a full length human
IKKa (SEQ ID NO: 2) and of the IKK(3 (SEQ ID NO: 15),
additional nucleic acid molecules of the invention would
be well known to those skilled in the art. Such nucleic
acid molecules, respectively, have a nucleotide sequence
that is different from SEQ ID NO: 1 but, nevertheless,
encodes the amino acid sequence shown as SEQ ID NO: 2, or
have a nucleotide sequence that is different from SEQ ID
NO: 14 but, nevertheless, encodes the amino acid sequence
shown as SEQ ID NO: 15. Thus, the invention provides a
nucleic acid molecule comprising a nucleotide sequence
encoding the amino acid sequence of a full length human
IKKa as shown in SEQ ID NO: 2 or of IKKQ as shown in SEQ
ID NO: 15.
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As used herein, reference to "a nucleic acid
molecule encoding an IKK subunit" indicates 1) the
polynucleotide sequence of one strand of a double
stranded DNA molecule comprising the nucleotide sequence
that codes for the IKK subunit and can be transcribed
into an RNA that encodes the IKK subunit, or 2) an RNA
molecule, which can be translated into an IKK subunit.
It is recognized that a double stranded DNA molecule also
comprises a second polynucleotide strand that is
complementary to the coding strand and that the
disclosure of a polynucleotide sequence comprising a
coding sequence necessarily discloses the complementary
polynucleotide sequence. Accordingly, the invention
provides polynucleotide sequences, including, for
example, polydeoxyribonucleotide or polyribonucleotide
sequences that are complementary to the nucleotide
sequence shown as SEQ ID NO: 1 or as SEQ ID NO: 14, or to
a nucleic acid molecule encoding an IKK catalytic subunit
having the amino acid sequence shown as SEQ ID NO: 2 or
as SEQ ID NO: 15, respectively.
As used herein, the term "polynucleotide" is
used in its broadest sense to mean two or more
nucleotides or nucleotide analogs linked by a covalent
bond. The term "oligonucleotide" also is used herein to
mean two or more nucleotides or nucleotide analogs linked
by a covalent bond, although those in the art will
recognize that oligonucleotides generally are less than
about fifty nucleotides in length and, therefore, are a
subset within the broader meaning of the term
"polynucleotide."
In general, the nucleotides comprising a
polynucleotide are naturally occurring
deoxyribonucleotides, such as adenine, cytosine, guanine
or thymine linked to 2'-deoxyribose, or ribonucleotides
such as adenine, cytosine, guanine or uracil linked to
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ribose. However, a polynucleotide also can comprise
nucleotide analogs, including non-naturally occurring
synthetic nucleotides or modified naturally occurring
nucleotides. Such nucleotide analogs are well known in
the art and commercially available, as are
polynucleotides containing such nucleotide analogs (Lin
et al., Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et
al., Biochemistry 34:11363-11372 (1995); Pagratis et al.,
Nature Biotechnol. 15:68-73 (1997)). The covalent bond
linking the nucleotides of a polynucleotide generally is
a phosphodiester bond. However, the covalent bond also
can be any of numerous other bonds, including a
thiodiester bond, a phosphorothioate bond, a peptide-like
bond or any other bond known to those in the art as
useful for linking nucleotides to produce synthetic
polynucleotides (see, for example, Tam et al., Nucl.
Acids Res. 22:977-986 (1994); Ecker and Crooke,
BioTechnoloqy 13:351360 (1995)).
Where it is desired to synthesize a
polynucleotide of the invention, the artisan will know
that the selection of particular nucleotides or
nucleotide analogs and the covalent bond used to link the
nucleotides will depend, in part, on the purpose for
which the polynucleotide is prepared. For example, where
a polynucleotide will be exposed to an environment
containing substantial nuclease activity, the artisan
will select nucleotide analogs or covalent bonds that are
relatively resistant to the nucleases. A polynucleotide
comprising naturally occurring nucleotides and
phosphodiester bonds can be chemically synthesized or can
be produced using recombinant DNA methods, using an
appropriate polynucleotide as a template. In comparison,
a polynucleotide comprising nucleotide analogs or
covalent bonds other than phosphodiester bonds generally
will be chemically synthesized, although an enzyme such
as T7 polymerase can incorporate certain types of
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nucleotide analogs and, therefore, can be used to produce
such a polynucleotide recombinantly from an appropriate
template (Jellinek et al., supra, 1995).
The invention also provides nucleotide
sequences that can specifically hybridize to a nucleic
acid molecule of the invention. Such hybridizing
nucleotide sequences are useful, for example, as probes,
which can hybridize to a nucleic acid molecule encoding
an IKK catalytic subunit and allow the identification of
the nucleic acid molecule in a sample. A nucleotide
sequence of the invention is characterized, in part, in
that it is at least nine nucleotides in length, such
sequences being particularly useful as primers for the
polymerase chain reaction (PCR), and can be at least
fourteen nucleotides in length or, if desired, at least
seventeen nucleotides in length, such nucleotide
sequences being particularly useful as hybridization
probes, although such sequences also can be used for PCR.
A nucleotide sequence of the invention can comprise at
least six nucleotides 5' to nucleotide position 92 as
shown in SEQ ID NO: 1 (Figure 1), preferably at least
nine nucleotides 5' to position 92, or more as desired,
where SEQ ID NO: 1 is shown in the conventional manner
from the 5'-terminus (Figure 1; upper left) to the
31-terminus. Such nucleotide sequences of the invention
are particularly useful in methods of diagnosing a
pathology, for example, a human disease, characterized by
aberrant IKK activity. For convenience, such nucleotide
sequences can comprise a kit, which can be made
commercially available and can provide a standardized
diagnostic assay.
A nucleic acid molecule encoding an IKKa such
as the nucleotide sequence shown in SEQ ID NO: 1 diverges
from the sequence encoding the mouse homolog (GenBank
Accession #U12473) in the region encoding amino acid 30.
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Thus, a nucleotide sequence comprising nucleotides 88 to
90 as shown in SEQ ID NO: 1, which encodes amino acid 30
of human IKKa, can be particularly useful, for example,
for identifying the presence of a nucleic acid molecule
encoding a human IKKa in a sample. Furthermore, based on
a comparison of SEQ ID NO: 1 with SEQ ID NO: 14, the
skilled artisan readily can select nucleotide sequences
that can hybridize with a nucleic acid molecule encoding
a human IKKa or a human IKK(3 or both by designing the
sequence to contain conserved or non-conserved nucleotide
sequences, as desired. For example, selection of a
nucleotide sequence that is highly conserved among SEQ ID
NO: 1 and SEQ ID NO: 14 can allow the identification of
related members of the IKK subunit family of proteins.
In comparison, selection of a nucleotide sequence that is
present, for example, in SEQ ID NO: 14, but that is not
present in SEQ ID NO: 1 or that shares only minimal
homology can allow identification of the expression of
SEQ ID NO: 14 in a cell, irrespective of whether SEQ ID
NO: 1 also is expressed in the cell. It should be
recognized, however, that a nucleotide sequence of the
invention readily is identifiable in comparison to
GenBank Accession #U12473 or #U22512 in that a nucleotide
sequence of the invention is not the nucleotide sequence
of GenBank Accession #U12473 or #U22512.
A nucleotide sequence of the invention can
comprise a portion of a coding sequence of a nucleic acid
molecule encoding an IKK subunit or of a sequence
complementary thereto, depending on the purpose for which
the nucleotide sequence is to be used. In addition, a
mixture of a coding sequence and its complementary
sequence can be prepared and, if desired, can be allowed
to anneal to produce double stranded molecules.
The invention also provides antisense nucleic
acid molecules, which are complementary to a nucleic acid
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molecule encoding an IKK subunit and can bind to and
inhibit the expression of the nucleic acid molecule. As
disclosed herein, expression of an antisense molecule
complementary to the nucleotide sequence shown in SEQ ID
5 NO: 1 inhibited the cytokine inducible expression of an
NF-KB dependent reporter gene in a cell (Example II.B.).
Thus, an antisense molecule of the invention can be
useful for decreasing IKK activity in a cell, thereby
reducing or inhibiting the level of NF-KB mediated gene
10 expression. These experiments were performed twenty-four
hours after the cells were transfected (Example II.B.).
Expression of the antisense molecule in the cell also
resulted in a decreased level of IKKa activity as
compared to vector transfected control cells, indicating
15 that the IKKa has a relatively short half life.
Antisense nucleic acid molecules specific for IKKa or for
IKKQ or for both can be designed based on the criteria
discussed above for the selection of hybridizing
nucleotide sequences.
An antisense nucleic acid molecule of the
invention can comprise a sequence complementary to the
entire coding sequence of an IKK catalytic subunit such
as a sequence complementary to SEQ ID NO: 1 or SEQ ID NO:
14, provided the antisense sequence is not complementary
in its entirety to the sequences of GenBank Accession
#U12473 or #U22512. In addition, a nucleotide sequence
complementary to a portion of a nucleic acid molecule
encoding an IKK subunit can be useful as an antisense
molecule, particularly a nucleotide sequence
complementary to nucleotides -35 to 92 of SEQ ID NO: 1
or, for example, a nucleotide sequence comprising at
least 9 nucleotides on each side of the ATG encoding the
initiator methionine (complementary to positions -9 to 12
of SEQ ID NO: 1) or, if desired, at least 17 nucleotides
on each side of the ATG codon (complementary to positions
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-17 to 20 of SEQ ID NO: 1), or to the corresponding
sequences of SEQ ID NO: 14.
Antisense methods involve introducing the
nucleic acid molecule, which is complementary to and can
hybridize to the target nucleic acid molecule, into a
cell. An antisense nucleic acid molecule can be a
chemically synthesized polynucleotide, which can be
introduced into the target cells by methods of
transfection, or can be expressed from a plasmid or viral
vector, which can be introduced into the cell and stably
or transiently expressed using well known methods (see,
for example, Sambrook et al., Molecular Clonincr: A
laboratory manual (Cold Spring Harbor Laboratory Press
1989); Ausubel et al., Current Protocols in Molecular
Biology (Green Publ., NY 1989)).
One in the art would
know that the ability of an antisense (or other
hybridizing) nucleotide sequence to specifically
hybridize to the target nucleic acid sequence depends,
for example, on the degree of complementarity shared
between the sequences, the GC content of the hybridizing
molecules, and the length of the antisense nucleic acid
sequence, which can be at least ten nucleotides in
length, generally at least thirty nucleotides in length
or at least fifty nucleotides in length, and can be up to
the full length of a nucleotide sequence of SEQ ID NO: 1
or SEQ ID NO: 14 or a nucleotide sequence encoding an IKK
subunit as shown in SEQ ID NO: 2 or in SEQ ID NO: 15 (see
Sambrook et al., supra, 1989).
The invention also provides vectors comprising
a nucleic acid molecule of the invention and host cells,
which are appropriate for maintaining such vectors.
Vectors, which can be cloning vectors or expression
vectors, are well known in the art and commercially
available. An expression vector comprising a nucleic
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acid molecule of the invention, which can encode an IKK-a
or can be an antisense molecule, can be used to express
the nucleic acid molecule in a cell.
In general, an expression vector contains the
expression elements necessary to achieve, for example,
sustained transcription of the nucleic acid molecule,
although such elements also can be inherent to the
nucleic acid molecule cloned into the vector. In
particular, an expression vector contains or encodes a
promoter sequence, which can provide constitutive or, if
desired, inducible expression of a cloned nucleic acid
sequence, a poly-A recognition sequence, and a ribosome
recognition site, and can contain other regulatory
elements such as an enhancer, which can be tissue
specific. The vector also contains elements required for
replication in a procaryotic or eukaryotic host system or
both, as desired. Such vectors, which include plasmid
vectors and viral vectors such as bacteriophage,
baculovirus, retrovirus, lentivirus, adenovirus, vaccinia
virus, semliki forest virus and adeno-associated virus
vectors, are well known and can be purchased from a
commercial source (Promega, Madison WI; Stratagene, La
Jolla CA; GIBCO/BRL, Gaithersburg MD) or can be
constructed by one skilled in the art (see, for example,
Meth. Enzymol., Vol. 185, D.V. Goeddel, ed. (Academic
Press, Inc., 1990); Jolly, Canc Gene Ther. 1:51-64
(1994); Flotte, J. Bioenerg. Biomemb. 25:37-42 (1993);
Kirshenbaum et al., J. Clin. Invest 92:381-387 (1993)).
A nucleic acid molecule, including a vector,
can be introduced into a cell by any of a variety of
methods known in the art (Sambrook et al., supra, 1989,
and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, MD (1994)).
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Such methods include, for example, transfection, lipofection,
microinjection, electroporation and infection with
recombinant vectors or the use of liposomes.
Introduction of a nucleic acid molecule by
infection with a viral vector is particularly
advantageous in that it can efficiently introduce the
nucleic acid-molecule into a cell ex vivo or in vivo.
Moreover, viruses are very specialized and typically
infect and propagate in specific cell types. Thus, their
natural specificity can be used to target the nucleic
acid molecule contained in the vector to specific cell
types. For example, a vector based on HIV-1 can be used
to target an antisense IKK subunit molecule to HIV-1
infected cells, thereby reducing the phosphorylation of
IKB, which can decrease the high level of constitutive
NF-KB activity present in HIV-1 infected cells. Viral or
non-viral vectors also can be modified with specific
receptors or ligands to alter target specificity through
receptor mediated events.
A nucleic acid molecule also can be introduced
into a cell using methods that do not require the initial
introduction of the nucleic acid molecule into a vector.
For example, a nucleic acid molecule encoding an IKK
catalytic subunit can be introduced into a cell using a
cationic liposomes, which also can be modified with
specific receptors or ligands as described above
(Morishita et al., J. Clin. Invest.,
91:2580-2585 (1993); see, also,
Nabel et al., supra, 1993)). In addition, a nucleic acid
molecule can be introduced into a cell using, for
example, adenovirus-polylysine DNA complexes (see, for
example, Michael et al., J. Biol. Chem., 268:6866-6869
(1993)).
Other methods of introducing a nucleic acid molecule into
a cell such that the encoded IKK subunit or antisense
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19
nucleic acid molecule can be expressed are well known
(see, for example, Goeddel, supra, 1990).
Selectable marker genes encoding, for example,
a polypeptide conferring neomycin resistance (NeoR) also
are readily available and, when linked to a nucleic acid
molecule of the invention or incorporated into a vector
containing the nucleic acid molecule, allows for the
selection of cells that have incorporated the nucleic
acid molecule. Other selectable markers such as that
conferring hygromycin, puromycin or ZEOCIN (Invitrogen)
resistance are known to those in the art of gene transfer
can be used to identify cells containing the nucleic acid
molecule, including the selectable marker gene.
A "suicide" gene also can be incorporated into
a vector so as to allow for selective inducible killing
of a cell containing the gene. A gene such as the herpes
simplex virus thymidine kinase gene (TK) can be used as a
suicide gene to provide for inducible destruction of such
cells. For example, where it is desired to terminate the
expression of an introduced nucleic acid molecule
encoding IKK or an antisense IKK subunit molecule in
cells containing the nucleic acid molecule, the cells can
be exposed to a drug such as acyclovir or gancyclovir,
which can be administered to an individual.
Numerous methods are available for transferring
nucleic acid molecules into cultured cells, including the
methods described above. In addition, a useful method
can be similar to that employed in previous human gene
transfer studies, where tumor infiltrating lymphocytes
(TILs) were modified by retroviral gene transduction and
administered to cancer patients (Rosenberg et al., ew
Engl. J. Med. 323:570-578 (1990)). In that Phase I
safety study of retroviral mediated gene transfer, TILs
were genetically modified to express the Neomycin
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resistance (NeoR) gene. Following intravenous infusion,
polymerase chain reaction analyses consistently found
genetically modified cells in the circulation for as long
as two months after administration. No infectious
5 retroviruses were identified in these patients and no
side effects due to gene transfer were noted in any
patients. These retroviral vectors have been altered to
prevent viral replication by the deletion of viral gag,
pol and env genes. Such a method can also be used ex
10 vivo to transduce cells taken from a subject (see
Anderson et al., U.S. Patent No. 5,399,346, issued
March 21, 1995).
When retroviruses are used for gene transfer,
15 replication competent retroviruses theoretically can
develop due to recombination of retroviral vector and
viral gene sequences in the packaging cell line utilized
to produce the retroviral vector. Packaging cell lines
in which the production of replication competent virus by
20 recombination has been reduced or eliminated can be used
to minimize the likelihood that a replication competent
retrovirus will be produced. Hence, all retroviral
vector supernatants used to infect cells will be screened
for replication competent virus by standard assays such
as PCR and reverse transcriptase assays.
To function properly, a cell requires the
precise regulation of expression of nearly all genes.
Such gene regulation is accomplished by activation or
repression of transcription by various transcription
factors, which interact directly with regulatory
sequences on nuclear DNA. The ability of transcription
factors to bind DNA or activate or repress transcription
is regulated in response to external stimuli. In the
case of the transcription factor NF-xB, critical factors
involved in the signaling pathway mediating its
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21
activation have not been identified (Verma, et al., Genes
Devel. 9:2723-2735 (1995); Baeuerle and Baltimore, Cell
87:13-20 (1996)).
NF-KB is a member of the Rel family of
transcription factors, which are present in most if not
all animal cells (Thanos and Maniatis, Cell 80:629-532
(1995)). Rel proteins, which include, for example, RelA
(p65), c-Rel, p50, p52 and the Drosophila dorsal and Dif
gene products, are characterized by region of about 300
amino acids sharing approximately 35o to 61% homology
("Rel homology domain"). The Rel homology domain
includes DNA binding and dimerization domains and a
nuclear localization signal. Rel proteins are grouped
into one of two classes, depending on whether the protein
also contains a transcriptional activation domain
(Siebenlist et al., Ann, Rev. Cell Biol. 10:405-455
(1994)).
Rel proteins can from homodimers or
heterodimers, which can be transcriptionally activating
depending on the presence of a transactivation domain.
The most common Rel/NF-KB dimer, which is designated
"NF-xB," is a p50/p65 heterodimer that can activate
transcription of genes containing the appropriate KB
binding sites. p50/p65 NF-KB is present in most cell
types and is considered the prototype of the Rel/NF-KB
family of transcription factors. Different dimers vary
in their binding to different xB elements, kinetics of
nuclear translocation and levels of expression in a
tissue (Siebenlist et al., supra, 1994). As used herein,
the term "Rel/NF-KB" is used to refer generally to the
Rel family of transcription factors, and the term "NF-xB"
is used to refer specifically to the Rel/NF-KB factor
consisting of a p50/p65 heterodimer.
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22
NF-KB originally was identified by its ability
to bind a specific DNA sequence present in the
immunoglobulin K light chain gene enhancer, the "xB
element" (Sen and Baltimore, Cell 46:705-709 (1986)).
The KB element has been identified in numerous cellular
and viral promotors, including promotors present in human
immunodeficiency virus-i (HIV-1); immunoglobulin
superfamily genes such as the MHC class 1(H-2x) gene;
cytokine genes such as the tumor necrosis factor a
(TNFa), interleukin-1(3 (IL-1(3), IL-2, IL-6 and the
granulocyte-macrophage colony stimulating factor (GM-CSF)
gene; chemokine genes such as RANTES and IL-8; and cell
adhesion protein genes such as E-selectin. The KB
element exhibits dyad symmetry and each half site of the
element likely is bound by one subunit of an NF-KB dimer.
In the absence of an appropriate signaling
stimulus, a Rel/NF-xB is maintained in the cytoplasm in
an inactive form complexed with an IxB protein.
Rel/NF-KB transcriptional activity is induced by numerous
pathogenic events or stresses, including cytokines,
chemokines, viruses and viral products, double stranded
RNA, bacteria and bacterial products such as
lipopolysaccharide (LPS) and toxic shock syndrome
toxin-l, mitogens such as phorbol esters, physical and
oxidative stresses, and chemical agents such as okadaic
acid and cycloheximide (Thanos and Maniatis, supra, 1995;
Siebenlist et al., supra, 1994). Significantly, the
expression of genes encoding agents such as TNFa, IL-1,
IL-6, interferon-(3 and various chemokines, which induce
NF-KB activity, are, themselves, induced by NF-KB,
resulting in amplification of their signal by a positive,
self-regulatory loop (Siebenlist et al., supra, 1994).
Phorbol esters, which activate T cells, also activate
NF-KB and immunosuppressants such as cyclosporin A
inhibit activation of T cells through T cell receptor
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23
mediated signals (Baldwin, Ann. Rev. Immunol. 14:649-681
(1996)).
Regulation of specific genes by NF-KB can
require interaction of NF-KB with one or more other DNA
binding proteins. For example, expression of E-selectin
requires an interaction of NF-KB, the bZIP protein ATF-2
and HMG-I(Y), and expression of the IL-2 receptor a gene
requires an interaction of NF-KB, HMG-I(Y) and the
ets-like protein, ELF-1 (Baldwin, supra, 1996).
The numerous agents that induce activation of
NF-KB likely act through various converging signal
transduction pathways, including pathways involving
activation of protein kinase C, Raf kinase and tyrosine
kinases. The ability of antioxidants to inhibit NF-KB
activation by various inducing agents suggests that
reactive oxygen species are a converging point of such
pathways (Siebenlist et al., supra, 1994).
Upon activation by an appropriate inducing
agent, a Rel/NF-xB dimer is translocated into the
nucleus, where it can activate gene transcription. The
subcellular localization of a Rel/NF-KB is controlled by
specific inhibitory proteins ("inhibitors of Rel/NF-xB"
or "IxB's"), which noncovalently bind the Rel/NF-xB and
mask its nuclear localization signal (NLS), thereby
preventing nuclear uptake. Various IxB's, including, for
example, IKBa, IxBR, Bcl-3 and the Drosophila cactus gene
product, have been identified (Baeuerle and Baltimore,
supra, 1996). In addition, Rel precursor proteins, such
as p105 and p100, which are precursors of p50 and p52,
respectively, function as IKB's (Siebenlist et al.,
supra, 1994). IxBa and IxBR are expressed in most cell
types and generally bind p65- and c-Rel-containing
Rel/NF-KB dimers. Other IxB's appear to be expressed in
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24
a tissue specific manner (Thompson et al., Cell
80:573-582 (1995)).
IxB proteins are characterized by the presence
of 5 to 8 ankyrin repeat domains, each about 30 amino
acids, and a C-terminal PEST domain. For example, IxBa
contains a 70 amino acid N-terminal domain, a 205 amino
acid internal domain containing the ankyrin repeats, and
a 42 amino acid C-terminal domain containing the PEST
domain (Baldwin, supra, 1996). Although IxB proteins
interact through their ankyrin repeats with the Rel
homology domain of Rel/NF-xB dimers, binding of
particular IxB proteins with particular Rel/NF-KB
proteins appears to be relatively specific. For example,
IxBa and IxB(3 associate primarily with RelA- and c-Rel-
containing Rel/NF-KB dimers, thereby blocking their
nuclear localization signal. The binding of an IxB to
NF-xB also interferes with the ability of NF-KB to bind
DNA. However, whereas IxBa is phosphorylated following
exposure of cells to tumor necrosis factor (TNF), IL-1,
bacterial lipopolysaccharide (LPS) or phorbol esters,
IxBR is phosphorylated in certain cell types only in
response to LPS or IL-1 (Baldwin, supra, 1996). However,
in other cell types, IxB(3 is phosphorylated in response
to the same signals that induce IxBa, although with
slower kinetics than IKBa (DiDonato et al., Mol. Cell.
Biol. 16:1295-1304 (1996)).
Formation of a complex between an IKB protein
and a Rel protein is due to an interaction of the ankyrin
domains with a Rel homology domain (Baeuerle and
Baltimore, supra, 1996). Upon exposure to an appropriate
stimulus, the IxB portion of the complex is rapidly
degraded and the Rel/NF-KB portion becomes free to
translocate to the cell nucleus. Thus, activation of a
Rel/NF-xB does not require de novo protein synthesis and,
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therefore, occurs extremely rapidly. Consequently,
activation of gene expression due to a Rel/NF-xB can be
exceptionally rapid and provides an effective means to
respond to an external stimulus. Such a rapid response
5 of Rel/NF-KB transcription factors is particularly
important since these factors are involved in the
regulation of genes involved in the immune, inflammatory
and acute phase responses, including responses to viral
and bacterial infections and to various stresses.
10 Upon exposure of a cell to an appropriate
inducing agent, IxBa, for example, is phosphorylated at
serine residue 32 (Ser-32) and Ser-36 (Haskill et al.,
Cell 65:1281-1289 (1991)). Phosphorylation of IxBa
triggers its rapid ubiquitination, which results in
15 proteasome-mediated degradation of the inhibitor and
translocation of active NF-KB to the nucleus (Brown et
al., Science 267:1485-1488 (1995); Scherer et al., Proc.
Natl. Acad. Sci., USA. 92:11259-11263 (1995); DiDonato et
al., supra, 1996; DiDonato et al., Mol. Cell. Biol.
20 15:1302-1311 (1995); Baldi et al., J. Biol. Chem.
271:376-379 (1996)). The same mechanism also accounts
for IxB(3 degradation (DiDonato et al., supra, 1996).
Rel/NF-KB activation can be transient or
persistent, depending on the inducing agent and the IxB
25 that is phosphorylated. For example, exposure of a cell
to particular cytokines induces IxBa phosphorylation and
degradation, resulting in NF-KB activation, which induces
the expression of various genes, including the gene
encoding IxBa. The newly expressed IxBa then binds to
NF-KB in the nucleus, resulting in its export to the
cytoplasm and inactivation and, therefore, a transient
NF-xB mediated response. In comparison, bacterial LPS
induces IxB(3 phosphorylation, resulting in NF-KB
activation. However, the IxBR gene is not induced by
CA 02281955 1999-08-20
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26
NF-KB and, as a result, activation of NF-xB is more
persistent (Thompson et al., supra, 1995).
A constitutively active multisubunit kinase of
approximately 700 kDa phosphorylates IKBa at Ser-32 and
Ser-36 and, in some cases, requires polyubiquitination
for activity (Chen et al., Cell 84:853-862 (1996); Lee et
al., Cell 88:213-222 (1997)). The mitogen-activated
protein kinase/ERK kinase kinase-1 (MEKK1) phosphorylates
several proteins that copurify with this complex and have
molecular weights of approximately 105 kDa, 64 kDa and
54 kDa; three other copurifying proteins having molecular
weights of about 200 kDa, 180 kDa and 120 kDa are
phosphorylated in the absence of MEKK1 (Lee et al.,
supra, 1997). However, a catalytically inactive MEKK1
mutant, which can block TNFa mediated activation of the
jun kinase, does not block NF-KB activation (Liu et al.,
Cell 87:565-576 (1996)).
Overexpression of MEKK1 also induces the site-
specific phosphorylation of IxBa in vivo and can directly
activate IKBa in vitro by an ubiquitin-independent
mechanism. However, MEKKi did not phosphorylate IKBa at
Ser-32 and Ser-36 in the in vitro experiments, indicating
that it is not an IKBa kinase, but may act upstream of
IKBa kinase in a signal transduction pathway (Lee et al.,
supra, 1997).
In addition to the above described ubiquitin
dependent kinase 700 kDa complex, an ubiquitin
independent 700 kDa complex, as well as an ubiquitin
independent 300 kDa kinase complex phosphorylates IKBa
Ser-32 and Ser-36, but not a mutant containing threonines
substituted for these serines (Baeuerle and Baltimore,
supra, 1996). The specific polypeptides responsible for
the IKB kinase activity of these complexes have not been
described.
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27
A double stranded RNA-dependent protein kinase
(PKR) that phosphorylates IKBa in vitro has been
described (Kumar et a l . , Proc. Natl Acad. i USA
91:6288-6292 (1994)). Moreover, an antisense PKR DNA
molecule prevented NF-KB activation by double stranded
RNA, but did not prevent NF-KB activation by TNFa (Maran
et al., Science 265:789-792 (1995)). Casein kinase II
(CKII) also can interact with and phosphorylate IKBa,
although weakly as compared to CKII phosphorylation of
casein, and the Ser-32 and Ser-36 residues in IKBa
represent CKII phosphorylation sites (Roulston et al.,
supra, 1995). However, all of the inducers of NF-KB
activity do not stimulate these protein kinases to
phosphorylate IKB, indicating that, if they are involved
in NF-KB activation, these kinases, like MEKK1, operate
upstream of the IxB kinase. Thus, a rapidly stimulated
IKB kinase that directly phosphorylates IKBa on Ser-32
and Ser-36 and results in activation of NF-KB has not
been identified.
A putative serine-threonine protein kinase has
been identified in mouse cells by probing for nucleic
acid molecules that encode proteins containing a
consensus helix-loop-helix domain, which is involved in
protein-protein interactions (Connelly and Marcu, supra,
1995). This putative kinase, which is ubiquitously
expressed in various established cell lines, but
differentially expressed in normal mouse tissues, was
named CHUK (conserved helix-loop-helix ~biquitous kinase;
GenBank Accession #U12473). In addition, a nucleic acid
molecule (GenBank Accession #U22512) encoding a portion
of a human CHUK protein that is 931 identical at the
nucleotide level (95% identical at the amino acid level)
with the mouse CHUK also was identified. However,
neither the function of a CHUK protein in a cell nor a
potential substrate for the putative kinase was
described.
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28
The present invention provides an isolated IKB
kinase (IKK), including isolated full length IKK
catalytic subunits. For example, the invention provides
an isolated 300 kDa or 900 kDa complex, which comprises
an IKKa or an IKK(3 subunit and has IKB kinase activity
(see Examples I, III and IV). In addition, the invention
provides is an isolated human IKKa catalytic subunit (SEQ
ID NO: 2; Example II), which contains a previously
undescribed N-terminal amino acid sequence and
essentially the C-terminal region of human CHUK (Connelly
and Marcu, supra, 1995) and phosphorylates IKBa on Ser-32
and Ser-36 and IKB(3 on Ser-19 and Ser-23 (DiDonato et
al., supra, 1996; see, also, Regnier et al., supra,
1997). The invention also provides an isolated IKK(3
catalytic subunit (SEQ ID NO: 15; Example III), which
shares greater than 50% amino acid sequence identity with
IKKa, including conserved homology in the kinase domain,
helix-loop-helix domain and leucine zipper domain.
As used herein, the term "isolated," when used
in reference to an IKB kinase complex or to an IKK
catalytic subunit of the invention, means that the
complex or the subunit is relatively free from
contaminating lipids, proteins, nucleic acids or other
cellular material normally associated with an IKK in a
cell. An isolated 900 kDa IKB kinase complex or 300 kDa
complex can be isolated, for example, by
immunoprecipitation using an antibody that binds to an
IKK catalytic subunit (see Examples III and IV). In
addition, an isolated IKK subunit can be obtained, for
example, by expression of a recombinant nucleic acid
molecule such as SEQ ID NO: 1 or SEQ ID NO: 14, or can be
isolated from a cell by a method comprising affinity
chromatography using ATP or IKB as ligands (Example I) or
using an anti-IKK subunit antibody. An isolated IKK
complex or IKK subunit comprises at least 30% of the
material in a sample, generally about 50% or 70% or 90%
CA 02281955 1999-08-20
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29
of a sample, and preferably about 95% or 980 of a sample,
as described above with respect to nucleic acids.
The amino acid sequences for MEKK1 (GenBank
Accession # U48596; locus RNU48596), PKR (GenBank
Accession # M35663; locus HUMP68A) and CKII (GenBank
Accession # M55268 J02924; locus HUMAlCKII) are different
from the sequences of the IKK subunits disclosed herein
(SEQ ID NO: 2 and SEQ ID NO: 15) and, therefore, are
distinguishable from the present invention. In addition,
a full length human IKKa of the invention is
distinguishable from the partial human CHUK polypeptide
sequence in that the partial human CHUK polypeptide
(Connelly and Marcu, supra, 1995; GenBank Accession
#22512) lacks amino acids 1 to 31 as shown in SEQ ID
NO: 2. As disclosed herein, a polypeptide having the
amino acid sequence of the partial human CHUK polypeptide
does not have IKB kinase activity when expressed in a
cell, indicating that some or all of amino acid residues
1 to 31 are essential for kinase activity.
A full length IKK catalytic subunit of the
invention is exemplified by human IKKa, which has an
apparent molecular mass of about 85 kDa and
phosphorylates IKBa on Ser-32 and Ser-36. An IKK
catalytic subunit of the invention also is exemplified by
IKKQ, which is an 87 kDa polypeptide that shares
substantial amino acid sequence homology with IKKa
(Figure 3). As used herein, the term "full length," when
used in reference to an IKK subunit of the invention,
means a polypeptide having an amino acid sequence of an
IKK subunit expressed normally in a cell. Such a
normally expressed IKK polypeptide begins with a
methionine residue at its N-terminus (Met-1; Figure 3),
the Met-1 being encoded by the initiator ATG (AUG) codon,
and ends as a result of the termination of translation
due to the presence of a STOP codon. A full length human
CA 02281955 1999-08-20
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IKK catalytic subunit can be a native IKK polypeptide,
which is isolated from a cell, or can be produced using
recombinant DNA methods such as by expressing the nucleic
acid molecule shown as SEQ ID NO: 1 or SEQ ID NO: 14.
5 The apparent molecular mass of an isolated IKK
subunit can be measured using routine methods such as
polyacrylamide gel electrophoresis performed in the
presence of sodium dodecyl sulfate (SDS-PAGE) or column
chromatography performed under reducing and denaturing
10 conditions. In addition, the ability of an IKK subunit
to phosphorylate IKBa on Ser-32 and Ser-36 can be
identified using the methods disclosed herein.
With regard to the disclosed 85 kDa and 87 kDa
apparent molecular masses of human IKKa and IKK(3, it is
15 recognized that the apparent molecular mass of a
previously unknown protein as determined, for example, by
SDS-PAGE is an estimate based on the relative migration
of the unknown protein as compared to the migration of
several other proteins having known molecular masses.
20 Thus, one investigator reasonably can estimate, for
example, that an unknown protein has an apparent
molecular mass of 82 kDa, whereas a second investigator,
looking at the same unknown protein under substantially
similar conditions, reasonably can estimate that the
25 protein has an apparent molecular mass of 87 kDa.
Accordingly, reference herein to an IKB kinase having an
apparent molecular mass of "about 85 kDa" indicates that
the kinase migrates by SDS-PAGE in an 8% gel under
reducing conditions in the range of 80 kDa to 90 kDa,
30 preferably in the range of 82 kDa to 87 kDa.
Furthermore, reference herein to an 87 kDa IKK(3 indicates
that IKK(3 has a relatively higher apparent molecular mass
than the 85 kDa apparent molecular mass of IKKa.
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31
An IKK catalytic subunit of the invention is
exemplified by the isolated full length polypeptide
comprising the amino acid sequence shown as SEQ ID NO: 2
or SEQ ID NO: 15. In addition, the invention provides
peptide portions of an IKK subunit polypeptide, wherein
such peptide portions contain at least three contiguous
amino acids as shown in SEQ ID NO: 2 or SEQ ID NO: 15,
and generally contain at least six contiguous amino acids
or, if desired, at least nine contiguous amino acids, as
provided herein. Thus, the invention provides peptide
portions of IKKa, containing, for example, at least three
contiguous amino acids of SEQ ID NO: 2, including amino
acid residue 30, preferably at least four contiguous
amino acids, including amino acid residue 30, and more
preferably at least six contiguous amino acids, including
amino acid residue 30. The invention also provides a
peptide portion of IKK(3 comprising at least three
contiguous amino acids, generally six contiguous amino
acids, and preferably ten contiguous amino acids of SEQ
ID NO: 15. It is recognized, however, that a peptide of
the invention does not consist of a polypeptide disclosed
as GenBank Accession #U12473 or #U22512.
A peptide portion of an IKK subunit generally
is a tripeptide or larger, preferably a hexapeptide or
larger, and more preferably a decapeptide or larger, up
to a contiguous amino acid sequence having a maximum
length that lacks one or more N-terminal or C-terminal
amino acids of the full length polypeptide (SEQ ID NO: 2
or SEQ ID NO: 15). Thus, a peptide portion of IKKa
having the amino acid sequence shown as SEQ ID NO: 2 can
be from three amino acids long to 744 amino acids long,
which is one residue less than the full length
polypeptide, except as provided above.
A peptide portion of an IKK subunit polypeptide
of the invention can be produced by any of several
CA 02281955 2007-02-09
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32
methods well known in the art. For example, a peptide
portion of an IKK subunit can be produced by enzymatic
cleavage of an IKK subunit protein, which has been
isolated from a cell, using a proteolytic enzyme such as
trypsin, chymotrypsin, Lys-C or the like, or combinations
of such enzymes. Such proteolytic cleavage products can
be isolated using methods as disclosed in Example I, to
obtain peptide portions of IKKa and IKK(3, for example. A
peptide portion of an IKK subunit also-can be produced
using methods of solution or solid phase peptide
synthesis or can be expressed from a nucleic acid
molecule such as a portion of the coding region of the
nucleic acid sequence shown as SEQ ID NO: 1 or SEQ ID
NO: 14, or can be purchased from a commercial source.
A peptide portion of an IKK subunit can
comprise the kinase domain of the IKK subunit and,
therefore, can have the ability to phosphorylate an IKB
protein. For example, a peptide portion of SEQ ID NO: 2
comprising amino acids 15 to 301 has the characteristics
of a serine-threonine protein kinase domain (Hanks and
Quinn, Meth. Enzymol. 200:38-62 (1991)).
Such a peptide
portion of an IKK subunit can be examined for kinase
activity by determining that it can phosphorylate IKBa at
Ser-32 and Ser-36 or IKBR at Ser-19 and Ser-23, using
methods as disclosed herein. In addition, a peptide
portion of an IKK subunit can comprise an immunogenic
amino acid sequence of the polypeptide and, therefore,
can be useful for eliciting production of an antibody
that can specifically bind the IKK subunit or to an IKK
complex comprising the subunit, particularly to an
epitope comprising amino acid residue 30 as shown in SEQ
ID NO: 2 or to an epitope of SEQ ID NO: 15, provided said
epitope is not present in a CHUK protein. Accordingly,
the invention also provides anti-IKK antibodies, which
specifically bind to an epitope of an IKK complex,
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33
particularly an IKK catalytic subunit, and to IKK subunit
binding fragments of such antibodies. In addition, the
invention provides cell lines producing anti-IKK
antibodies or IKK-binding fragments of such antibodies.
As used herein, the term "antibody" is used in
its broadest sense to include polyclonal and monoclonal
antibodies, as well as antigen binding fragments of such
antibodies. With regard to an anti-IKK antibody of the
invention, the term "antigen" means an IKK catalytic
subunit protein, polypeptide or peptide portion thereof,
or an IKK complex comprising an IKK catalytic subunit
protein, polypeptide or peptide portion thereof. Thus,
it should be recognized that, while an anti-IKK antibody
can bind to and, for example, immunoprecipitate an IKK
complex, the antibody specifically binds an epitope
comprising at least a portion of an IKK catalytic
subunit. An antibody of the invention also can be used
to immunoprecipitate an IKK subunit, free of the IKK
complex.
An anti-IKK antibody, or antigen binding
fragment of such an antibody, is characterized by having
specific binding activity for an epitope of an IKK
subunit of at least about 1 x 105 M"1, generally, at least
about 1 x 106 M-1. Thus, Fab, F(ab' ) 2, Fd and Fv fragments
of an anti-IKK antibody, which retain specific binding
activity for an IKK subunit, are included within the
definition of an antibody. In particular, an anti-IKK
antibody can react with an epitope comprising the
N-terminus of IKKa or with an epitope of IKK(3, but not to
a polypeptide having an amino acid sequence shown as
residues 32 to 745 of SEQ ID NO: 2.
The term "antibody" as used herein includes
naturally occurring antibodies as well as non-naturally
occurring antibodies, including, for example, single
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34
chain antibodies, chimeric, bifunctional and humanized
antibodies, as well as antigen-binding fragments thereof.
Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be
produced recombinantly or can be obtained, for example,
by screening combinatorial libraries consisting of
variable heavy chains and variable light chains as
described by Huse et al., Science 246:1275-1281 (1989).
These and
other methods of making, for example, chimeric,
humanized, CDR-grafted, single chain, and bifunctional
antibodies are well known to those skilled in the art
(Winter and Harris, Immunol. Today 14:243-246 (1993);
Ward et al., Nature 341:544-546 (1989) ; Harlow and Lane,
Antibodies: A laboratory manual (Cold Spring Harbor
Laboratory Press, 1988); Hilyard et al., Protein
Enaineerina: A practical anproach (IRL Press 1992);
Borrabeck, Antibody Engineering, 2d ed. (Oxford
University Press 1995)).
An anti-IKK antibody of the invention can be
raised using an isolated IKK subunit or a peptide portion
thereof and can bind to a free, uncomplexed form of IKK
subunit or can bind to IKK subunit when it is associated
with a 300 kDa or 900 kDa IKK complex. In addition, an
anti-IKK antibody of the invention can be raised against
an isolated 300 kDa or 900 kDa IKB kinase complex, which
can be obtained as disclosed herein. For convenience, an
antibody of the invention is referred to generally herein
as an "anti-IxB kinase antibody" or an "anti-IKK
antibody." However, the skilled recognize that the
various antibodies of the invention will have unique
antigenic specificities, for example, for a free or
complexed IKK subunit, or both, or for a 300 kDa or
900 kDa IxB kinase complex, or both.
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Anti-IKK antibodies can be raised using as an
immunogen an isolated full length IKK catalytic subunit,
which can be prepared from natural sources or produced
recombinantly, or a peptide portion of an IKK subunit as
5 defined herein, including synthetic peptides as described
above. A non-immunogenic peptide portion of an IKK
catalytic subunit can be made immunogenic by coupling the
hapten to a carrier molecule such bovine serum albumin
(BSA) or keyhole limpet hemocyanin (KLH), or by
10 expressing the peptide portion as a fusion protein.
Various other carrier molecules and methods for coupling
a hapten to a carrier molecule are well known in the art
and described, for example, by Harlow and Lane, supra,
1988). It is recognized that, due to the apparently high
15 amino acid sequence identity of the full length human
IKKa and mouse CHUK, the amino acid sequences of IKKa
polypeptides, as well as IKK(3 polypeptides, likely are
highly conserved among species, particularly among
mammalian species. However, antibodies to highly
20 conserved proteins have been raised successfully, for
example, in chickens. Such a method can be used to
obtain an antibody to an IKK subunit, if desired.
Particularly useful antibodies of the invention
include antibodies that bind with the free, but not the
25 complexed, form of an IKK subunit or, alternatively, with
the complexed, but not free, form of an IKK subunit.
Antibodies of the invention also include antibodies that
bind with the 300 kDa IKB kinase complex or the 900 kDa
IKB kinase complex or both. It should be recognized,
30 however, that an antibody specific for the 300 kDa or
900 kDa IKB kinase complex need not recognize an IKK
subunit epitope in order to be encompassed within the
claimed invention, since, prior to the present
disclosure, the 300 kDa and 900 kDa IKK complexes were
35 not known (see DiDonato et al., Nature 388:548-554
(1997)).
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Antibodies of the invention that bind to an
activated IKK but not to an inactive IKK, and,
conversely, those that bind to an inactive form of the
kinase but not to the activated form also are
particularly useful. For example, an IKK can be
activated by phosphorylation of an IKK subunit and,
therefore, an antibody that recognizes the phosphorylated
form of the IKK, but that does not bind to the
unphosphorylated form can be obtained. In addition, IKK
can be activated by release of a regulatory subunit and,
therefore, an antibody that recognizes a form of the IKK
complex that is not bound to the regulatory subunit can
be obtained. Such antibodies are useful for identifying
the presence of active IKK in a cell.
An anti-IKK antibody is useful, for example,
for determining the presence or level of an IKK or of an
IKK subunit in a tissue sample, which can be a lysate or
a histological section. The identification of the
presence or level of an IKK or an IKK subunit in the
sample can be made using well known immunoassay and
immunohistochemical methods (Harlow and Lane, supra,
1988). An anti-IKK antibody also can be used to
substantially purify an IKB kinase or an IKK subunit from
a sample. In addition, an anti-IKK antibody can be used
in a screening assay to identify agents that alter the
activity of an IKB kinase.
A kit incorporating an anti-IKK antibody, which
can be specific for the active or inactive form of IKB
kinase or can bind to an IKK complex or to an IKK
subunit, regardless of the activity state, can be
particularly useful. Such a kit can contain, in addition
to an anti-IKK antibody, a reaction cocktail that
provides the proper conditions for performing the assay,
control samples that contain known amounts of an IKK or
IKK subunit and, if desired, a second antibody specific
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for the anti-IKK antibody. Such an assay also should
include a simple method for detecting the presence or
amount of an IKK or an IKK subunit in a sample that is
bound to the anti-IKK antibody.
A protein such as anti-IKK antibody, as well as
an IKK subunit or a peptide portion thereof, can be
labeled so as to be detectable using methods well known
in the art (Hermanson, "Bioconjugate Techniques"
(Academic Press 1996);
Harlow and Lane, 1988; chap. 9). For example,
a protein can be labeled with various detectable moieties
including a radiolabel, an enzyme, biotin or a
fluorochrome. Reagents for labeling a protein such as an
anti-IKK antibody can be included in a kit containing the
protein or can be purchased separately from a commercial
source.
Following contact, for example, of a labeled
antibody with a sample such as a tissue homogenate or a
histological section of a tissue, specifically bound
labeled antibody can be identified by detecting the
particular moiety. Alternatively, a labeled second
antibody can be used to identify specific binding of an
unlabeled anti-IKK antibody. A second antibody generally
will be specific for the particular class of the first
antibody. For example, if an anti-IKB kinase antibody is
of the IgG class, a second antibody will be an anti-IgG
antibody. Such second antibodies are readily available
from commercial sources. The second antibody can be
labeled using a detectable moiety as described above.
When a sample is labeled using a second antibody, the
sample is first contacted with a first antibody, which is
an anti-IKK antibody, then the sample is contacted with
the labeled second antibody, which specifically binds to
the anti-IKK antibody and results in a labeled sample.
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Methods for raising polyclonal antibodies, for
example, in a rabbit, goat, mouse or other mammal, are
well known in the art (see Example V). In addition,
monoclonal antibodies can be obtained using methods that
are well known and routine in the art (Harlow and Lane,
supra, 1988). Essentially, spleen cells from a mouse
immunized with an IKK complex or an IKK subunit or
peptide portion thereof can be fused to an appropriate
myeloma cell line such as SP/02 myeloma cells to produce
hybridoma cells. Cloned hybridoma cell lines can be
screened using a labeled IKK subunit to identify clones
that secrete anti-IKK monoclonal antibodies. Hybridomas
expressing anti-IKK monoclonal antibodies having a
desirable specificity and affinity can be isolated and
utilized as a continuous source of the antibodies, which
are useful, for example, for preparing standardized kits
as described above. Similarly, a recombinant phage that
expresses, for example, a single chain anti-IKK also
provides a monoclonal antibody that can used for
preparing standardized kits.
A monoclonal anti-IKK antibody can be used to
prepare anti-idiotypic antibodies, which present an
epitope that mimics the epitope recognized by the
monoclonal antibody used to prepare the anti-idiotypic
antibodies. Where the epitope to which the monoclonal
antibody includes, for example, a portion of the IKK
catalytic subunit kinase domain, the anti-idiotypic
antibody can act as a competitor of IKB and, therefore,
can be useful for reducing the level of phosphorylation
of IKB and, consequently, the activity of NF-KB.
The present invention further provides methods
of identifying an agent that can alter the association of
an IKK catalytic subunit with a second protein, which can
be an upstream activator, a downstream effector such as
IKB, an interacting regulatory protein of the IKK
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39
subunit, or an interacting subunit associated with the
300 kDa or 900 kDa IKB kinase complex. As used herein,
the term "associate" or "association," when used in
reference to an IKK subunit and a second protein means
that the IKK subunit and the second protein have a
binding affinity for each other such that they form a
bound complex in vivo or in vitro, including in a cell in
culture or in a reaction comprising substantially
purified reagents. For convenience, the term "bind" or
"interact" is used interchangeably with the term
"associate."
The affinity of binding of an IKK subunit and a
second protein such as an IKB or another IKK subunit or
other subunit present in an IKK complex is characterized
in that it is sufficiently specific such that a bound
complex can form in vivo in a cell or can form in vitro
under appropriate conditions as disclosed herein. The
formation or dissociation of a bound complex can be
identified, for example, using the two hybrid assay or
demonstrating coimmunoprecipitation of the second protein
with the IKK subunit, as disclosed herein, or using other
well known methods such as equilibrium dialysis. Methods
for distinguishing the specific association of an IKK
subunit and a second protein from nonspecific binding to
the IKK subunit are known in the art and, generally,
include performing the appropriate control experiments to
demonstrate the absence of nonspecific protein binding.
As used herein, the term "second protein"
refers to a protein that specifically associates with an
IKK subunit ("first protein"). Such a second protein is
exemplified herein by IKB proteins, including IKBa and
IKB(3, which are substrates for IKB kinase activity and
are downstream of the IKB kinase in a signal transduction
pathway that results in the regulated expression of a
gene. In addition, such second proteins are exemplified
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by the proteins that, together with the IKK subunits,
form a 300 kDa or 900 kDa IKB kinase complex, which
coimmunoprecipitates using an anti-IKK antibody (see
Example IV). Furthermore, since IKK subunits such as
5 IKKa and IKKR interact with each other to form homodimers
or heterodimers, a second protein also can be a second
IKK subunit, which can be the same as or different from
the "first" protein.
Agents that alter the association of an IKK
10 catalytic subunit and a second protein such as IKB
protein or an IKK regulatory subunit can be extremely
valuable, for example, for limiting excessive cytokine
expression as occurs in an acute phase response by
preventing the activation of NF-KB, thereby preventing
15 NF-KB mediated induction of cytokine gene expression.
Where, in a drug screening assay of the invention, the
second protein is an IKB, the IKK subunit can be any
protein involved in IKB kinase activity, including, for
example, mouse CHUK (Connelly and Marcu, supra, 1995;
20 GenBank Accession #12473), which, prior to the present
disclosure, was not known to have the ability to
associate with IKB or to have IKB kinase activity.
In addition, a second protein can be a protein
that is upstream of IKB kinase in a signal transduction
25 pathway and associates with the IKK complex, particularly
with an IKK catalytic subunit of the IKK complex. Such a
second protein, which can be an upstream activator of the
IKB kinase, can be identified using routine methods for
identifying protein-protein interactions as disclosed
30 herein. Such second proteins can be, for example, MEKK1
or PKR or CKII, each of which has been reported to be
involved in a pathway leading to phosphorylation of IKB
and activation of NF-KB, but neither of which has the
characteristics expected of the common IKB kinase present
35 at the point where the various NF-KB activation pathways
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converge (see, for example, Lee et al., supra, 1997), or
can be the NF-KB-inducing kinase (NIK), which reportedly
is upstream from IKK in an NF-KB activation pathway
(Regnier et al., supra, 1997; Malinin et al., Nature
385:540-544 (1997)).
A second protein also can be a regulatory
protein, which associates with an IKK catalytic subunit
in an IKK complex, either constitutively as part of a
300 kDa or 900 kDa complex or in response to activation
of a pathway leading to IKK activation. Such a
regulatory protein can inhibit or activate IKK activity
depending, for example, on whether the regulatory protein
is associated with IKK and whether the regulatory protein
associates with an IKK catalytic subunit in a free form
or as part of an IKK complex. The regulatory protein
also can be important for "docking" a catalytic IKK
subunit to its substrate. The ability of a regulatory
protein to associate with or dissociate from an IKK
subunit or IKK complex can depend, for example, on the
relative phosphorylation state of the regulatory protein.
It is recognized that an upstream activator of IKK also
can interact with such a regulatory protein, thereby
indirectly inhibiting or activating the IKK.
As disclosed herein, two copurifying proteins
were isolated by ATP and IxB affinity chromatography and
identified by SDS-PAGE (Example I). Partial amino acid
sequences were determined and cDNA molecules encoding the
proteins were obtained (see Examples I, II and III). One
of the proteins has an apparent molecular mass of 85 kDa.
Expression in a cell of a cDNA molecule encoding the
85 kDa protein resulted in increased NF-KB activity
following cytokine induction as compared to control
cells, whereas expression of the antisense of this cDNA
decreased the basal NF-KB activity in the cells and
prevented cytokine induction of NF-KB activity.
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Immunoprecipitation of the 85 kDa protein resulted in
isolation of the IKK complex, the kinase activity of
which was stimulated rapidly in response to TNF or to
IL-1. Based on these functional analyses, the 85 kDa
protein was determined to be a component of the 900 kDa
IKB kinase complex and has been designated IKKa (SEQ ID
NO: 2). The second protein, which copurified with the 85
kDa IKB kinase, has an apparent molecular mass of 87 kDa
and shares greater than 50% amino acid sequence identity
with IKKa and has been designated IKK(3 (SEQ ID NO: 15).
The ability of the 85 kDa and 87 kDa IKK
subunits to associate with other proteins such as a
regulatory subunit as well as with IKB is suggested, for
example, by the presence in the IKB kinase of two
different protein binding domains, a helix-loop-helix
domain and a leucine zipper domain (see Connelly and
Marcu, supra, 1995; see, also, Figure 3). While the
leucine zipper motif mediates homotypic and heterotypic
interactions between IKKa and IKKG3, the helix-loop-helix
motif serves as a binding site for regulatory proteins
necessary for IKB kinase activation.
A screening assay of the invention provides a
means to identify an agent that alters the association of
an IKK complex or an IKK catalytic subunit with a second
protein such as the regulatory subunits discussed above.
As used herein, the term "modulate" or "alter" when used
in reference to the association of an IKK and a second
protein, means that the affinity of the association is
increased or decreased with respect to a steady state,
control level of association, i.e., in the absence of an
agent. Agents that can alter the association of an IKK
with a second protein can be useful for modulating the
level of phosphorylation of IKB in a cell, which, in
turn, modulates the activity of NF-KB in the cell and the
expression of a gene regulated by NF-KB. Such an agent
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can be, for example, an anti-idiotypic antibody as
described above, which can inhibit the association of an
IKK and IKB. A peptide portion of IKBa comprising amino
acids 32 to 36, but containing substitutions for Ser-32
and Ser-36, is another example of such an agent, since
the peptide can compete with IKBa binding to IKK, as is
the corresponding peptide of IKBR.
A screening assay of the invention also is
useful for identifying agents that directly alter the
activity of an IKK. While such an agent can act, for
example, by altering the association of an
IKK complex or IKK catalytic subunit with a second
protein, the agent also can act directly as a specific
activator or inhibitor of IKK activity. Specific protein
kinase inhibitors include, for example, staurosporin, the
heat stable inhibitor of cAMP-dependent protein kinase,
and the MLCK inhibitor, which are known in the art and
commercially available. A library of molecules based,
generally, on such inhibitors or on ATP or adenosine can
be screened using an assay of the invention to obtain
agents that desirably modulate the activity of an IKK
complex or an IKK subunit.
As disclosed herein, IKK activity can be
measured by identifying phosphorylation, for example, of
IxBa, either directly or using an antibody specific for
the Ser-32 and Ser-36 phosphorylated form of IKBa. An
antibody that binds to IKBa that is phosphorylated on
Ser-32, for example, can be purchased from a commercial
source (New England Biolabs; Beverly MA). Cultured cells
can be exposed to various agents suspected of having the
ability to directly alter IKK activity, then aliquots of
the cells either are collected or are treated with a
proinflammatory stimulus such as a cytokine, and
collected. The collected cells are lysed and the kinase
is immunoprecipitated using an anti-IKK antibody. A
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44
substrate such as IKBa or IKB(3 is added to the
immunocomplex and the ability of the IKK to phosphorylate
the substrate is determined as described above. If
desired, the anti-IKK antibody first can be coated onto a
plastic surface such as in 96 well plates, then the cell
lysate is added to the wells under conditions that allow
binding of IKK by the antibody. Following washing of the
wells, IKK activity is measured as described above. Such
a method is extremely rapid and provides the additional
advantage that it can be automated for high through-put
assays.
A screening assay of the invention is
particularly useful to identify, from among a diverse
population of molecules, those agents that modulate the
association of an IKK complex or an IKK catalytic subunit
and another protein (referred to herein as a "second
protein") or that directly alter the activity of IKK.
Methods for producing libraries containing diverse
populations of molecules, including chemical or
biological molecules such as simple or complex organic
molecules, peptides, proteins, peptidomimetics,
glycoproteins, lipoproteins, polynucleotides, and the
like, are well known in the art (Huse, U.S. Patent No.
5,264,563, issued November 23, 1993; Blondelle et al.,
Trends Anal. Chem. 14:83-92 (1995); York et al., Science
274:1520-1522 (1996); Gold et al., Proc. Natl. Acad.
Sci., USA 94:59-64 (1997); Gold, U.S. Patent No.
5,270,163, issued December 14, 1993). Such libraries
also can be obtained from commercial sources.
Since libraries of diverse molecules can
contain as many as 1014 to 1015 different molecules, a
screening assay of the invention provides a simple means
for identifying those agents in the library that can
modulate the association of an IKK and a second protein
or can alter the activity of an IKK. In particular, a
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screening assay of the invention can be automated, which
allows for high through-put screening of randomly
designed libraries of agents to identify those particular
agents that can modulate the ability of an IKK and a
5 second protein to associate or that alter the activity of
the IKK.
A drug screening assay of the invention
utilizes an IKK complex, which can be isolated as
disclosed herein; or an IKK subunit, which can be
10 expressed, for example, from a nucleic acid molecule
encoding the amino acid sequence shown in SEQ ID NO: 2 or
in SEQ ID NO: 15; or can be purified as disclosed herein;
or can utilize an IKK subunit fusion protein such as an
IKKa-glutathione-S-transferase (GST) or IKKR-histidine6
15 (HIS6) fusion protein, wherein the GST or HIS6 is linked
to the IKK subunit and comprises a tag (see Example VI).
The IKK or IKK subunit fusion protein is characterized,
in part, by having an affinity for a solid substrate as
well as having the ability to specifically associate with
20 an appropriate second protein such as an IKB protein.
For example, when an IKK catalytic subunit is used in a
screening assay, the solid substrate can contain a
covalently attached anti-IKK antibody, provided that the
antibody binds the IKK subunit without interfering with
25 the ability of the IKK subunit to associate with the
second protein. Where an IKKa-GST fusion protein, for
example, is used in such a screening assay, the solid
substrate can contain covalently attached glutathione,
which is bound by the GST tag component of the fusion
30 protein. If desired, the IKK subunit or IKK subunit
fusion protein can be part of an IKK complex in a drug
screening assay of the invention.
A drug screening assay to identify an agent
that alters the association of an IKK complex or an IKK
35 subunit and a second protein can be performed by
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allowing, for example, the IKK complex or IKK subunit,
which can be a fusion protein, to bind to the solid
support, then adding the second protein, which can be an
IKB such as IKBa, and an agent to be tested, under
conditions suitable for the association of the IKK and
IKBa in the absence of a drug (see Example VI). As
appropriate, the IKK can be activated or inactivated as
disclosed herein and, typically, the IKK or the second
protein is detectably labeled so as to facilitate
identification of the association. Control reactions,
which contain or lack either, the IKK component, or the
IKB protein, or the agent, or which substitute the IKB
protein with a second protein that is known not to
associate specifically with the IKK, also are performed.
Following incubation of the reaction mixture, the amount
of IKBa specifically bound to the IKK in the presence of
an agent can be determined and compared to the amount of
binding in the absence of the agent so that agents that
modulate the association can be identified.
An IKK subunit such as IKKa or IKK~ used in a
screening assay can be detectably labeled with a
radionuclide, a fluorescent label, an enzyme, a peptide
epitope or other such moiety, which facilitates a
determination of the amount of association in a reaction.
By comparing the amount of specific binding of an IKK
subunit or an IKK complex and IKB in the presence of an
agent as compared to the control level of binding, an
agent that increases or decreases the binding of the IKK
and the IKB can be identified. In comparison, where a
drug screening assay is used to identify an agent that
alters the activity of an IKK, the detectable label can
be, for example, Y-32P-ATP, and the amount of 32P-IKB can
be detected as a measure of IKK activity. Thus, the drug
screening assay provides a rapid and simple method for
selecting agents that desirably alter the association of
an IKK and a second protein such as an IKB or for
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altering the activity of an IKK. Such agents can be
useful, for example, for modulating the activity of NF-xB
in a cell and, therefore, can be useful as medicaments
for the treatment of a pathology due, at least in part,
to aberrant NF-KB activity.
The method for performing a drug screening
assay as disclosed herein also provides a research tool
for identifying a target of a drug that is or can be used
therapeutically to ameliorate an undesirable inflammatory
or immune response, but for which the target of the drug
is not known. Cytokine restraining agents, for example,
are a class of agents that can alter the level of
cytokine expression (U.S. Patent No. 5,420,109, issued
May 30, 1995) and can be used to treat various
pathologies, including patho-immunogenic diseases such as
rheumatoid arthritis and those induced by exposure to
bacterial endotoxin such as occur in septic shock (see,
also, W096/27386, published September 12, 1996).
The specific cellular target upon which a
cytokine restraining agent acts has not been reported.
However, the myriad of pathologic effects ameliorated by
such agents are similar to various pathologies associated
with aberrant NF-KB activity, suggesting that cytokine
restraining agents may target an effector molecule in a
NF-KB signal transduction pathway. Thus, one potential
target of a cytokine restraining agent can be an IKB
kinase, particularly an IKK catalytic subunit of the
kinase. Accordingly, a screening assay of the invention
can be used to determine whether a cytokine restraining
agent alters the activity of IKB kinase or alters the
association of an IKK and a second protein such as IxB.
If it is determined that a cytokine restraining agent has
such an effect, the screening assay then can be used to
screen a library of cytokine regulatory agents to
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identify those having desirable characteristics, such as
those having the highest affinity for the IKK.
The invention also provides a method of
obtaining an isolated IKK complex or an IKK catalytic
subunit. For example, a 300 kDa or a 900 kDa IKK
complex, comprising an IKKa subunit can be isolated from
a sample by immunoprecipitation using an anti-IKKa
antibody or by tagging the IKKa and using an antibody
specific for the tag (see Examples III and IV). In
addition, an IKK catalytic subunit can be isolated from a
sample by 1) incubating the sample containing the IKK
subunit with ATP, which is immobilized on a matrix, under
conditions suitable for binding of the IKK subunit to the
ATP; 2) obtaining from the immobilized ATP a fraction of
the sample containing the IKK subunit; 3) incubating the
fraction containing the IKK subunit with an IKB, which is
immobilized on a matrix, under conditions suitable for
binding of the IKK subunit to the IKB; and 4) obtaining
from the immobilized IxB an isolated IKK catalytic
subunit. Such a method of isolating an IKK subunit is
exemplified herein by the use of ATP affinity
chromatography and IxBa affinity chromatography to
isolate IKKa or IKK(3 from a sample of HeLa cells (see
Example I).
The skilled artisan will recognize that a
ligand such as ATP or an IKB or an anti-IKK antibody also
can be immobilized on various other matrices, including,
for example, on magnetic beads, which provide a rapid and
simple method of obtaining a fraction containing an ATP-
or an IKB-bound IKK complex or IKK subunit or an anti-IKB
kinase-bound IKK from the remainder of the sample.
Methods for immobilizing a ligand such as ATP or an IKB
or an antibody are well known in the art (Haystead et
al., Eur. J. Biochem. 214:459-467 (1993);
see, also, Hermanson,
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supra, 1996). Similarly, the artisan will recognize that
a sample containing an IKK complex or an IKK subunit can
be a cell, tissue or organ sample, which is obtained from
an animal, including a mammal such as a human, and
prepared as a lysate; or can be a bacterial, insect,
yeast or mammalian cell lysate, in which an IKK catalytic
subunit is expressed from a recombinant nucleic_acid
molecule. As disclosed herein, a recombinantly expressed
IKKa or IKK(3 such as a tagged IKKa or IKK(3 associates
into an active 300 kDa and 900 kDa IKK complex (see
Examples III and IV).
The invention also provides a method of
identifying a second protein that associates with an IKK
complex, particularly with an IKK subunit. A
transcription activation assay such as the yeast two
hybrid system is particularly useful for the
identification of protein-protein interactions (Fields
and Song, Nature 340:245-246 (1989))-
In addition, the two
hybrid assay is useful for the manipulation of protein-
protein interaction and, therefore, also is useful in a
screening assay to identify agents that modulate the
specific interaction.
A transcription activation assay such as the
two hybrid assay also can be performed in mammalian cells
(Fearon et al., Proc. Natl. Acad. Sci.. USA 89:7958-7962
(1992)).
However, the yeast two hybrid system provides a
particularly useful assay due to the ease of working with
yeast and the speed with which the assay can be
performed. Thus, the invention also provides methods of
identifying proteins that can interact with an IKK
subunit, including proteins that can act as upstream
activators or downstream effectors of IKK activity in a
signal transduction pathway mediated by the IKK or
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proteins that bind to and regulate the activity of the
IKK. Such proteins that interact with an IKK catalytic
subunit can be involved, for example, in tissue specific
regulation of NF-KB activation or constitutive NF-KB
5 activation and consequent gene expression.
The conceptual basis for a transcription
activation assay is predicated on the modular nature of
transcription factors, which consist of functionally
separable DNA-binding and trans-activation domains. When
10 expressed as separate proteins, these two domains fail to
mediate gene transcription. However, the ability to
activate transcription can be restored if the DNA-binding
domain and the trans-activation domain are bridged
together through a protein-protein interaction. These
15 domains can be bridged, for example, by expressing the
DNA-binding domain and trans-activation domain as fusion
proteins (hybrids), where the proteins that are appended
to these domains can interact with each other. The
protein-protein interaction of the hybrids can bring the
20 DNA-binding and trans-activation domains together to
create a transcriptionally competent complex.
One adaptation of the transcription activation
assay, the yeast two hybrid system, uses S. cerevisiae as
a host cell for vectors that express the hybrid proteins.
25 For example, a yeast host cell containing a reporter lacZ
gene linked to a LexA operator sequence can be used to
identify specific interactions between an IKK subunit and
a second protein, where the DNA-binding domain is the
LexA binding domain, which binds the LexA promoter, and
30 the trans-activation domain is the B42 acidic region.
When the LexA domain is bridged to the B42
transactivation domain through the interaction of the IKK
subunit with a second protein, which can be expressed,
for example, from a cDNA library, transcription of the
35 reporter lacZ gene is activated. In this way, proteins
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that interact with the IKK subunit can be identified and
their role in a signal transduction pathway mediated by
the IKK can be elucidated. Such second proteins can
include additional subunits comprising the 300 kDa or
900 kDa IKK complex.
In addition to identifying proteins that were
not previously known to interact with an IKK,
particularly with an IKKa or IKK(3 subunit, a
transcription activation assay such as the yeast two
hybrid system also is useful as a screening assay to
identify agents that alter association of an IKK subunit
and a second protein known to bind the IKK. Thus, as
described above for in vitro screening assays, a
transcription activation assay can be used to screen a
panel of agents to identify those agents particularly
useful for altering the association of an IKK subunit and
a second protein in a cell. Such agents can be
identified by detecting an altered level of transcription
of a reporter gene, as described above, as compared to
the level of transcription in the absence of the agent.
For example, an agent that increases the interaction
between an IKK subunit and IKB can be identified by an
increased level of transcription of the reporter gene as
compared to the control level of transcription in the
absence of the agent. Such a method is particularly
useful because it identifies an agent that alters the
association of an IKK subunit and a second protein in a
living cell.
In some cases, an agent may not be able to
cross the yeast cell wall and, therefore, cannot enter
the yeast cell to alter a protein-protein interaction.
The use of yeast spheroplasts, which are yeast cells that
lack a cell wall, can circumvent this problem (Smith and
Corcoran, In Current Protocols in Molecular Biology (ed.
Ausubel et al.; Green Publ.; NY 1989)).
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In addition, an
agent, upon entering a cell, may require "activation" by
a cellular mechanism that may not be present in yeast.
Activation of an agent can include, for example,
metabolic processing of the agent or a modification such
as phosphorylation of the agent, which can be necessary
to confer activity upon the agent. In this case, a
mammalian cell line can be used to screen a panel of
agents (Fearon et al., supra, 1992).
An agent that alters the catalytic activity of
an IKK or that alters the association of an IKK subunit
or IKK complex and a second protein such as an IxB or an
IKK regulatory subunit or an upstream activator of an IKK
can be useful as a drug to reduce the severity of a
pathology characterized by aberrant NF-xB activity. For
example, a drug that increases the activity of an IKK or
that increases the affinity of_an IKK catalytic subunit
and IKBa can increase the amount of IxBa phosphorylated
on Ser-32 or Ser-36 and, therefore, increase the amount
of active NF-xB and the expression of a gene regulated by
NF-xB, since the drug will increase the level of
phosphorylated IxBa in the cell, thereby allowing NF-KB
translocation to the nucleus. In contrast, a drug that
decreases or inhibits the catalytic activity of an IKK or
the association of an IKK catalytic subunit and IxBa can
be useful where it is desirable to decrease the level of
active NF-KB in a cell and the expression of a gene
induced by activated NF-xB. It should be recognized that
an antisense IKK subunit molecule of the invention also
can be used to decrease IKK activity in a cell by
reducing or inhibiting expression of the IKK subunit or
by reducing or inhibiting its responsiveness to an
inducing agent such as TNFa, 11-1 or phorbol ester (see
Example II). Accordingly, the invention also provides
methods of treating an individual suffering from a
pathology characterized by aberrant NF-xB activity by
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administering to the individual an agent that modulates
the catalytic activity of an IKK or that alters the
association of an IKK subunit and a second protein such
as IKB or a subunit of a 300 kDa or 900 kDa IKK complex
that interacts with the IKK subunit.
An agent that decreases the activity of an IKK
or otherwise decreases the amount of IKB phosphorylation
in a cell can reduce or inhibit NF-KB mediated gene
expression, including, for example, the expression of
proinflammatory molecules such as cytokines and other
biological effectors involved in an inflammatory, immune
or acute phase response. The ability to reduce or
inhibit such gene expression can be particularly valuable
for treating various pathological conditions such as
rheumatoid arthritis, asthma and septic shock, which are
characterized or exacerbated by the expression of such
proinflammatory molecules.
Glucocorticoids are potent anti-inflammatory
and immunosuppressive agents that are used clinically to
treat various pathologic conditions, including autoimmune
diseases such as rheumatoid arthritis, systemic lupus
erythematosis and asthma. Glucocorticoids suppress the
immune and inflammatory responses, at least in part, by
increasing the rate of IicBa synthesis, resulting in
increased cellular levels of IKBa, which bind to and
inactivate NF-KB (Scheinman et al., Science 270:283-286
(1995); Auphan et al., Science 270:286-290 (1995)).
Thus, glucocorticoids suppress NF-KB mediated expression
of genes encoding, for example, cytokines, thereby
suppressing the immune, inflammatory and acute phase
responses. However, glucocorticoids and glucocorticoid-
like steroids also are produced physiologically and are
required for normal growth and development.
Unfortunately, prolonged treatment of an individual with
higher than physiological amounts of glucocorticoids
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produces clinically undesirable side effects. Thus, the
use of an agent that alters the activity of an IKK or
that alters the association of an IKK complex or IKK
subunit and a second protein, as identified using a
method of the invention, can provide a means for
selectively altering NF-KB activity without producing
some of the undesirable side effects associated with
glucocorticoid treatment.
Inappropriate regulation of Rel/NF-KB
transcription factors is associated with various human
diseases. For example, many viruses, including human
immunodeficiency virus-i (HIV-1), herpes simplex virus-1
(HSV-1) and cytomegalovirus (CMV) contain genes regulated
by a KB regulatory element and these viruses, upon
infecting a cell, utilize cellular Rel/NF-KB
transcription factors to mediate viral gene expression
(Siebenlist et al., supra, 1994). Tat-mediated
transcription from the HIV-1 enhancer, for example, is
decreased if the NF-KB and SP1 binding sites are deleted
from the enhancer/promotor region, indicating that Tat
interacts with NF-KB, SP1 or other transcription factors
bound at this site to stimulate transcription (Roulston
et al., Microbiol. Rev. 59:481-505 (1995)). In addition,
chronic HIV-1 infection, and progression to AIDS, is
associated with the development of constitutive NF-KB DNA
binding activity in myeloid cells (Roulston et al.,
supra, 1995). Thus, a positive autoregulatory loop is
formed, whereby HIV-1 infection results in constitutively
active NF-KB, which induces expression of HIV-1 genes
(Baeuerle and Baltimore, Cell 87:13-20 (1996).
Constitutive NF-KB activation also may protect cells
against apoptosis, preventing clearance of virus-infected
cells by the immune system (Liu et al., supra, 1996).
An agent that decreases the activity of an IKK
or that alters the association of an IKK and a second
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protein such that IKB phosphorylation is decreased can be
useful for reducing the severity of a viral infection
such as HIV-1 infection in an individual by providing
increased levels of unphosphorylated IxB in virus-
5 infected cells. The unphosphorylated IxB then can bind
to NF-xB in the cell, thereby preventing nuclear
translocation of the NF-KB and viral gene expression. In
this way, the rate of expansion of the virus population
can be limited, thereby providing a therapeutic advantage
10 to the individual.
In addition, the decreased level of NF-xB
activity may allow the virus-infected cell to undergo
apoptosis, resulting in a decrease in the viral load in
the individual. As such, it can be particularly useful
15 to treat virus-infected cells ex vivo with an agent
identified using a method of the invention. For example,
peripheral blood mononuclear cells (PBMCs) can be
collected from an HIV-1 infected individual and treated
in culture with an agent that decreases the activity of
20 an IKK or alters the association of an IKK complex or an
IKK catalytic subunit with an IKB. Such a treatment can
be useful to purge the PBMCs of the virus-infected cells
by allowing apoptosis to proceed. The purged population
of PBMCs then can be expanded, if desired, and
25 readministered to the individual.
Rel/NF-xB proteins also are involved in a
number of different types of cancer. For example, the
adhesion of cancer cells to endothelial cells is
increased due to treatment of the cancer cells with IL-1,
30 suggesting that NF-KB induced the expression of cell
adhesion molecules, which mediated adherence of the tumor
cells to the endothelial cells; agents such as aspirin-,
which decrease NF-xB activity, blocked the adhesion by
inhibiting expression of the cell adhesion molecules
35 (Tozawa et al., Cancer Res. 55:4162-4167 (1995)). These
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results indicate that an agent that decreases the
activity of an IKK or that decrease the association of an
IKK and IKB or of an IKK subunit and a second protein,
for example, a second protein present in an IKK complex,
can be useful for reducing the likelihood of metastasis
of a tumor in an individual.
As discussed above for virus-infected cells,
constitutive NF-KB activation also may protect tumor
cells against programmed cell death as well as apoptosis
induced by chemotherapeutic agents (Liu et al., supra,
1996; Baeuerle and Baltimore, Cell 87:13-20 (1996)).
Thus, an agent that decreases IKK activity or that
decreases the association of IKK and IKB also can be
useful for allowing programmed cell death to occur in a
tumor cell by increasing the level of unphosphorylated
IKB, which can bind NF-KB and decrease the level of
active NF-KB in the tumor cell.
The following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
IDENTIFICATION AND CHARACTERIZATION OF
A HUMAN IKB KINASE COMPLEX AND IKK SUBUNITS
This example provides a method for identifying
and isolating a cytokine responsive protein kinase
complex that phosphorylates IxB, which regulates NF-KB
activity, and catalytic subunits of the protein kinase
complex.
A. Kinase assays:
Kinase assays were performed using GST fusion
proteins containing amino acid residues 1 to 54 of IKB.
The fusion proteins were linked to glutathione SEPHAROSE
and the beads were used directly in the assays. At
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earlier stages in the purification of the IKK activity,
the beads were washed prior to loading onto the gel to
minimize contributions from other proteins. In some of
the later characterization of highly purified material,
soluble fusion protein was used.
Three distinct substrates for the IKK activity
were used: 1) substrate "WT" contained amino acid
residues 1 to 54 of IKBa; 2) substrate "AA" contained
amino acid residues 1 to 54 of IKBa, except that Ser-32
(S32) and S36 were replaced with Ala-32 (A32) and A36,
respectively; and 3) substrate "TT" contained amino acid
residues 1 to 54 of IKBa, except that S32 and S36 were
replaced with Thr-32 (T32) and T36, respectively
(DiDonato et al., MQl. Cell. Biol. 16:1295-1304 (1996)).
Each substrate was expressed as a GST fusion protein.
The physiologic, inducible IKB kinase is specific for S32
and S36 (WT) in IKBa, but does not recognize the TT or AA
mutants (DiDonato et al., Mol. Cell. Biol. 16:1295-1304
(1996)).
Kinase assays were carried out in 20 mM HEPES
(pH 7.5-7.6), 20 mM ~-glycerophosphate (Q-GP), 10 mM
MgCl2, 10 mM PNPP, 100 M Na3VO4, 2 mM dithiothreitol
(DTT), 20 M ATP, 10 g/ml aprotinin. NaCl concentration
was 150-200 mM and the assays were carried out at 30 C
for 30 min. Fractionation was performed by SDS-PAGE,
followed by quantitation by phosphoimager analysis.
B. Purification of IKK complex and IKK subunits:
The protein purification buffer (Buffer A)
consisted of 20 mM Tris (pH 7.6, measured at RT), 20 mM
NaF, 20 mM R-GP, 1 mM PNPP, 500 M Na3VOq, 2 mM DTT,
2.5 mM metabisulfite, 5 mM benzamidine, 1 mM EDTA, 0.5 mM
EGTA, 1 mM PMSF, and 10o glycerol. Brij-35 was added as
indicated. Cell lysis buffer was Buffer A containing an
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additional 19 mM PNPP, 20 mM R-GP and 500 M Na3VO41 and
20 g/ml aprotinin, 2.5 g/ml leupeptin, 8.3 g/ml
bestatin, 1.7 g/ml pepstatin.
Purification was performed using 5 to 130
liters of HeLa S3 cells. For illustration, the procedure
for a 15 liter preparation is presented. All
purification steps were performed in a cold room at 4 C.
In order to activate the IKK, cells were
stimulated with TNFa prior to purification. TNFa was
either recombinant TNFa, which was purchased from R&D
Systems and used at 20 ng/ml, or HIS6-tagged TNFa, which
was expressed and partially purified from E. coli and
used at 5 g/ml. TNFa-induced HeLa S3 cell killing
activity assays were performed in the presence of
cycloheximide and indicated that the partially purified
HIS6-tagged TNFa had approximately one-tenth the activity
of the commercial TNFa.
Fifteen liters of HeLa S3 cells were grown in
suspension in high glucose Dulbecco's modified Eagle's
medium supplemented with 10% calf serum, 2 mg/ml
L-glutamine, 100 U/ml penicillin/streptomycin, 0.11 mg/ml
sodium pyruvate, and 1X nonessential amino acids (Irvine
Scientific; Irvine CA). Cell density was approximately
5 x 105 cells/ml at the time of collection. Cells were
concentrated 10-fold by centrifugation. stimulated for
5 min with TNFa at 37 C, then diluted with 2.5 volumes of
ice cold phosphate buffered saline (PBS) containing 50 mM
NaF and pelletted at 2000 x g. The cell pellet was
washed once with ice cold PBS/50 mM NaF, then suspended
in lysis buffer, quick frozen in liquid nitrogen and
stored at -80 C.
For purification of IKB kinase, cells were
thawed and cytoplasmic extract prepared. Lysis was
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achieved by 40 strokes in an all glass Dounce homogenizer
(pestle A) in lysis buffer containing 0.05% NP-40 on ice.
The homogenate was centrifuged at 12,000 rpm for 19 min
in a Beckmana' SS34 rotor at 4 C.
Supernatant was collected and centrifuged at
38,000 rpm for 80 min in a Beckman 50.1 Ti rotor at 4 C.
The supernatant (S100 fraction) was quick frozen in
liquid nitrogen and stored at -80 C. Small aliquots of
S100 material, prepared from either unstimulated HeLa
cells or from TNFa stimulated cells, were purified in a
single passage over a SUPEROSE 6 gel filtration column
(1.0 x 30 cm; Pharmacia; Uppsalla Sweden) equilibrated in
Buffer A containing 0.1% Brij-35 and 300 mM NaCl and
eluted at a flow rate of 0.3 ml/min. 0.6 ml fractions
were collected and kinase assays were performed on an
aliquot of each fraction. The high molecular weight
material (fractions 16-20) contained TNFa-inducible IKK
activity, which is specific for the WT substrate.
110 ml of S100 material (900 mg of protein;
Bio-Rad Protein Assay) was pumped onto a Q-SEPHAROSE FAST
FLOW column (56 ml bed volume, 2.6 cm ID) equilibrated at
2 ml/min with Buffer A containing 0.1% Brij-35. After
the sample was loaded, the column was washed with 100 ml
of Buffer A containing 0.1% Brij-35 and 100 mM NaCl, then
a linear NaCl gradient was run from 100-300 mM. The
gradient volume was 500 ml and the flow rate was
2 ml/mi.n. Ten ml fractions were collected and the kinase
assay was performed on those fractions that eluted during
the gradient. Fractions corresponding to the
TNFa-inducible IKK activity (fractions 30-42; i.e., 20-32
of the gradient portion) were pooled. The pooled
material contained 40 mg of protein.
The pooled material was diluted to 390 ml by
addition of Buffer A containing 0.1 % Brij-35 and loaded
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onto a pre-equilibrated 5 ml HITRAP Q column (Pharmacia)
at a flow rate of 4 ml/min. Following sample loading,
the column was washed with 20 ml of Buffer A containing
0.1 % Brij-35. The protein was eluted at 1 ml/min
5 isocratically in Buffer A containing 0.1 o Brij-35 and
300 mM NaCl and 1 ml fractions were collected. Protein-
containing fractions were identified using the BioRad
assay and were collected and pooled to yield 4 ml of
solution. Previously performed control experiments
10 demonstrated that the IKK activity directly correlated
with protein concentration.
The pooled material was diluted 1:1 with ATP
column buffer (20 mM HEPES (pH 7.3), 50 mM R-GP, 60 mM
MgClz, 1 mM Na2VO4, 1.5 mM EGTA, 1 mM DTT, 10 g/ml
15 aprotinin), then passed 4 times over a y-ATP affinity
column having 4 ml bed volume (Haystead et al., supra,
1993); the column had been prewashed with 2 M NaCl, 0.25%
Brij-35 and equilibrated with 10 bed volumes of ATP
column buffer containing 0.05% Brij-35 at a flow rate of
20 0.5 ml/min. Following loading of the sample, the column
was washed with 10 ml of ATP column buffer containing
0.05% Brij-35, then with 10 ml ATP column buffer
containing 0.05% Brij-35 and 250 mM NaCl.
Bound material was eluted in 10 ml of ATP
25 column buffer containing 0.05 % Brij-35, 250 mM NaCl and
10 mM ATP (elution buffer). Elution was performed by
passing 5 ml of elution buffer through the column,
allowing the column to incubate, capped, for 20 min, then
passing an additional 5 ml of elution buffer through the
30 column. The samples were pooled to yield 10 ml.
The 10 ml pooled sample from the ATP column was
diluted with 30 ml Buffer A containing 0.1 % Brij-35 and
loaded onto a 1 ml HITRAP Q column (Pharmacia) at
1 ml/min. The column was eluted at 0.4 ml/min with
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Buffer A containing 0.1 % Brij-35 and 300 mM NaCl.
0.2 ml fractions were collected and the four protein-
containing fractions were pooled (0.5 mg). The pooled
material was concentrated to 200 l on a 10K NANOSEP
concentrator (Pall/Filtron) and loaded onto a SUPEROSE 6
gel filtration column (1.0 x 30 cm). The SUPEROSE 6
column was equilibrated in Buffer A containing 0.1 a
Brij-35 and 300 mM NaCl and run at a flow rate of 0.3
ml/min; 0.6 ml fractions were collected. Fractions 17,
18 and 19 contained kinase activity.
Based on silver stained SDS-PAGE gels, the
final purified material consisted of approximately 20 g
to 40 g of total protein, of which approximately 2 g
corresponded to the 85 kDa band, later designated IKKa
(see Example II). A second band migrating at 87 kDa was
later designated IKK(3 (see Example III). The total time
from the thawing of the S100 material until the
collection of fractions from the gel filtration column
was 24 hours.
C. Confirmation of IKK purification:
Since the 85 kDa IKKa band identified by the
kinase assay following the above procedure contained only
about 100 of the total purified protein, three additional
criteria were used to confirm that the identified band
was an intrinsic component of the IKK complex.
In one procedure, the elution profile of the
SUPEROSE 6 column was analyzed by silver stained
8% SDS-PAGE gels, then compared to the kinase activity
profile. For this analysis, 0.3 ml fractions were
collected from the SUPEROSE 6 column, then separated by
8% SDS-PAGE and silver stained. This comparison
confirmed that a single band of 85 kDa correlated
precisely with the elution of IKK activity.
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In a second procedure, the IKK activity was
further purified on a substrate affinity column at 4 C.
A GST fusion protein was prepared containing the A32/A36
1 to 54 amino acid sequence of IKBa repeated 8 times
(GST-(8X-AA)). The GST-(8X-AA) then was covalently
linked to a CNBr activated SEPHAROSE 4B resin to produce
the substrate affinity resin.
IKK-containing material was diluted into
Buffer A to yield a final concentration of 70 mM NaCl,
0.025% Brij-35, then added to the substrate affinity
resin at a ratio of 4:1 (solution:swollen beads). The
resin was suspended and the mixture rotated gently
overnight in a small column at 4 C. The resin was
allowed to settle for 30 min, then the column was eluted
by gravity. The column was washed with 4 bed volumes
Buffer A containing 0.02a Brij-35, then the resin was
suspended with 1.1 bed volumes of Buffer A containing
600 mM NaCl and 0.1 % Brij-35. The resin was allowed to
settle for 40 min, then gravity elution was performed.
The column was washed with an additional 1.1 bed volumes
of Buffer A containing 600 mM NaCl and 0.1 o Brij-35 and
the two fractions were pooled.
The IKBa substrate affinity column was used for
two separate experiments. In one experiment, the
material that eluted from the final SUPEROSE 6 column was
further purified on the IKBa substrate affinity column.
In the second experiment, material obtained after the
initial Q-SEPHAROSE column was purified on the IKBa
substrate affinity column. The Q-SEPHAROSE bound
fraction then was further purified on the ATP column and
the SUPEROSE 6 column (see above)-
Analysis of the purified material from these
two experiments by silver stained SDS-PAGE gels revealed
different protein profiles. However, comparison of these
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profiles revealed only two bands common to both
preparations, one of which was confirmed to be the same
85 kDa IKKa band that was identified by the SUPEROSE 6
profile analysis and cofractionated with IKB kinase
activity. The other band, which was 87 kDa in size,
later was identified as IKKR. In several different
experiments, the 85 kDa protein and 87 kDa protein were
specifically purified by the substrate affinity column in
what appeared to be an equimolar ratio.
In a third procedure, purified IKK was treated
with excess phosphatase, which inactivates the IKK, then
reactivated by addition of a semi-purified HeLa extract.
Phosphatase inactivation was performed by adding excess
protein phosphatase 2A catalytic domain (PP2A) to
purified IKB kinase in 50 mM Tris (pH 7.6), 50 mM NaCl,
1 mM MgC121 then equilibrating the reaction for 60 min at
30 C. 1.25 M okadaic acid was added to completely
inactivate the phosphatase and the phosphatase
inactivated material was used in standard kinase assays
and to perform the reactivation and phosphorylation
procedure.
Cytoplasmic extract was prepared using HeLa S3
cells. The cells were stimulated with TNFa for 5 min,
then harvested in lysis buffer containing 0.1 % NP-40 and
0.15 M NaCl. Reactivation was performed at 30 C in
kinase buffer for 60 min in the absence of (y-32P)ATP.
Samples containing only cold ATP were used for kinase
activity assays. Reactivation by the HeLa cell extract
was performed in the presence of (Y-3zP)ATP, then the
sample was separated by 8% SDS-PAGE and examined by
autoradiography. A band of approximately 86 kDa was
phosphorylated in the reactivated material and,
associated with the reactivation procedure, was
restoration of the IKK activity.
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D Partial amino acid sequences of IKKa and IKKl3
Following SDS-PAGE as described above, the
85 kDa IKKa and 87 kDa IKKQ bands were excised from the
gel and submitted for internal peptide sequencing
analysis. From the IKKa polypeptide, the sequences of
two proteolytic fragments were identified, as follows:
KIIDLLPK (SEQ ID NO: 3) and KHR(D/A)LKPENIVLQDVG(P/G)K
(SEQ ID NO: 4). Where a residue could not be
unambiguously determined, an "X" was used to indicate no
amino acid could be determined and parentheses were used
to delimit amino acids that could not be distinguished.
Since Lys-C protease was used to digest the protein, the
presence of lysine residues at the N-termini of the
peptides was inferred. From the 87 kDa IKK(3 band, the
sequences of five proteolytic fragments were determined
(see Figure 3, underlined; see, also, Example III).
EXAMPLE II
IDENTIFICATION AND CHARACTERIZATION
OF A FULL LENGTH HUMAN IKKa SUBUNIT
This example provides methods for isolating a
nucleic acid molecule encoding the IKKa subunit and for
characterizing the functional activity of the subunit.
A. Cloning of cDNA encoding human IKKa:
Degenerate oligonucleotide (length) sequences
of the amino acid sequences of two peptide fragments (SEQ
ID NOS: 3 and 4) of the IKKa (see Figure 1) were searched
in the GenBank DNA sequence database. This search
revealed that nucleotide sequences encoding both peptide
fragments were present in a partial cDNA encoding a
portion of a protein designated human CHUK (GenBank
Accession #U22512; Connelly and Marcu, supra, 1995).
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Based on the human CHUK cDNA sequence, PCR
primers were prepared corresponding to the 51-terminus
(51-CCCC AT TACCAGCATCGGGAA-3'; SEQ ID NO: 5) and
3'-terminus (3'-CCCCTCGAGTTCTGTTAACCAACT-5'; SEQ ID
5 NO: 6). SEQ ID NO: 5 also contains a Nde I restriction
endonuclease site (underlined) and an ATG (AUG)
methionine codon (bold) and SEQ ID NO: 6 also contains an
Xho I site. RNA was isolated from HeLa cells and first
strand cDNA was prepared and used for a template by PCR
10 using SEQ ID NOS: 5 and 6 as primers. The resulting
2.1 kilobase (kb) fragment was gel purified, 32P-labeled
using oligo-dT and random primers, and used to screen a
human fetal brain library (Clontech; Palo Alto CA) under
high stringency conditions (50o formamide, 42 C; Sambrook
15 et al., supra, 1989).
In order to obtain the 5'-end of the cDNA
encoding IKKa, positive plaques from above were screened
by PCR using two internal primers,
(5'-CATGGCACCATCGTTCTCTG-3'; SEQ ID NO: 7), which is
20 complementary to the sequence including the Ban I site
around position 136 of SEQ ID NO: 1, and
(5'-CTCAAAGAGCTCTGGGGCCAGATAC-3'; SEQ ID NO: 8), which is
complementary to the sequence including the Sac I site
around position 475, and a vector specific primer
25 (TCCGAGATCTGGACGAGC-3'; SEQ ID NO: 9), which is
complementary to vector sequences at the 5'-end of the
cDNA insert. The longest PCR product was selected and
sequenced by the dideoxy method.
DNA sequencing revealed that the cloned IKKa
30 cDNA contained an additional 31 amino acids at the
N-terminus as compared to human CHUK. The human IKKa
shares a high amount of sequence identity with a protein
designated mouse CHUK (GenBank Accession #U12473;
Connelly and Marcu, supra, 1995). Although the mouse
35 CHUK contains a domain having characteristics of a
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serine-threonine protein kinase, no functional activity
of the protein was reported and no potential substrates
were identified. The putative serine-threonine protein
kinase domain of human CHUK was truncated at the
N-terminus.
B. Expression of human IKKa or of an antisense IKKa
nucleic acid in a cell:
The full length IKKa cDNA and a cDNA encoding
the L31 human CHUK protein (Connelly and Marcu, supra,
1995) were subcloned into the Nde I and Xho I sites of a
bacterial expression vector encoding a carboxy terminal
FLAG epitope and HIS6 tag. Mammalian cell expression
vectors were constructed by cleaving the bacterial
expression vector with Nde I and Hind III, to release the
cDNA inserts, converting the ends of the inserts to blunt
ends using Klenow polymerase, and ligating the cDNA
inserts encoding the full length IKKa or the 031 human
CHUK into pCDNA3 (Invitrogen).
Alternatively, the IKKa cDNA and L31 cDNA were
subcloned into the Bst XI site of the pRcpactin vector
(DiDonato et al., supra, 1996). Orientation of the
inserts (sense or antisense) was determined by
restriction endonuclease mapping and partial sequence
using vector-specific primers. Vector containing the
cDNA's inserted in the sense orientation were examined
for expression of the encoded product by immunoblot
analysis using an antibody specific for the FLAG epitope.
Transfection experiments were performed to
determine the effect of expressing the cloned IKKa in
HeLa cells or of expressing the cloned IKKa cDNA in the
antisense orientation. One day prior to performing the
transfections, HeLa cells were split into 35 mm dishes to
approximately 50o confluency. Cells were transfected
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with 0.25 g of a lugiferase reporter gene containing an
IL-8 promotor (Eckman et al., Amer. Soc. Clin. Invest.
96:1269-1279 (1995))
along with eithar 1 g pCDNA3 (Invitrogen, La
Jolla CA; vector control), 1 g pRcRactin-IKKa-AA (sense
orientation), 1 g pRcQactin-IKKa-K (antisense), or
0.1 Mg pCDNA-IKKa-K using the LIPOFECTAMINE method as
recommended by the manufacturer (GIBCO/BRL, Gaithersburg
MD). Total DNA concentrations were kept constant by
addition of empty pRc(3actin DNA.
Transfected cells were incubated in DMEM
containing 10% FBS for 24 hr. The cells then were washed
and the growth medium was replaced with DMEM containing
0.10i FBS. Cells either were left untreated, or were
treated with 20 ng/ml TNFa, 20 ng/ml IL-la, or 100 ng/ml
TPA (phorbol ester) for 3.5 hr. Cells were harvested by
scraping and washed once with PBS, then lysed in 100 l
PBS containing 1% TRITON-X100. Luciferase assays were
performed using 20 g1 of lysate (DiDonato et al., supra,
1995). The protein concentration of each extract was
determined using the BIORAD protein assay kit and
luciferase activity was normalized according to the
protein concentrations.
NF-xB is known to induce expression for the
IL-8 promotor. Thus, as expected, treatment of the
vector transfected control cells with TNFa, IL-la or TPA
resulted in a 3- to 5-fold increase in normalized
luciferase activity. In comparison, in cells transfected
with the cDNA encoding IKKa, treatment with TNFa, IL-la
or TPA potentiated induction of luciferase activity 5- to
6-fold above the level of induction observed in the
vector transfected cells. These results indicate that
expression of IKKa in cells increased the amount of NF-KB
activated in response to the inducing agents.
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In cells transfected with the vector expressing
the antisense IKKa n`ucleic acid molecule, transcription
of the luciferase reporter gene induced by IL-1 or TNFa
was at the limit of detection, indicating transcription
was almost completely inhibited due to expression of the
r=-
anti!3ense IKKa. This result indicates that the native
IKKa is turned over relatively rapidly in the cells.
Furthermore, treatment of the cells with the various
inducing agents had no effect on the level of luciferase
expression of control reporter genes, which are not
responsive to NF-KB, as compared to the untreated cells.
Other appropriate control experiments were performed in
parallel. These results demonstrate the an expression of
an antisense IKKa nucleic acid molecule in a cell can
specifically inhibit NF-KB mediated gene expression.
EXAMPLE III
IDENTIFICATION AND CHARACTERIZATION
OF A FULL LENGTH HUMAN IKK(3 SUBUNIT
This example provides methods for isolating a
nucleic acid molecule encoding an IKK(3 catalytic subunit
of IKK and characterizing the activity of the IKK(3
subunit.
A. Cloning of IKK(3 cDNA:
IKK(3 was purified following SDS-PAGE and
subjected to internal peptide sequencing (Example I).
Five peptide sequences were obtained as follows:
KIIDLGYAK (SEQ ID NO: 10);
KXVHILN(M/Y)(V/G)(T/N/R/E)(G/N)TI(H/I/S) (SEQ ID NO: 11);
KXXIQQD(T/A)GIP (SEQ ID NO: 12); KXRVIYTQL (SEQ ID
NO: 13); and KXEEVVSLMNEDEK (SEQ ID NO: 20), where amino
acid residues that could not be unambiguously determined
are indicated by an "X" and where amino acids that could
not be distinguished are shown in parentheses. These
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peptide sequences were used to screen the NCBI EST
database and a 336 base pair EST (EST29518; Accession No.
AA326115) encoding SEQ ID NOS: 12 and 13 was identified.
This EST was determined to correspond to amino acid
residues 551 to 661 of SEQ ID NO: 15.
cDNA corresponding to the EST was obtained by
PCR using first strand HeLa cDNA as a template and used
to probe a human fetal brain library (Clontech). A 1 kb
fragment was identified and used as a probe to screen a
plasmid based B cell library (Invitrogen). A 3 kb cDNA
insert was isolated and sequenced (Figure 2; SEQ ID
NO: 14) and encoded the full length IKK(3 (SEQ ID NO: 15),
including all five proteolytic fragments (see Figure 3).
Comparison of the amino acid sequences of IKKa
and IKKP revealed greater than 50o amino acid identity
(Figure 3). In addition, SEQ ID NO: 15 contains a kinase
domain, which shares 65% amino acid identity with IKKa, a
leucine zipper and a helix-loop-helix domain. Based on
the sequence homology and domain structure, the
polypeptide (SEQ ID NO: 15) was determined to be a member
of the IKK catalytic subunit family of proteins with IKKa
and, therefore, was designated IKK(3.
B. Characterization of IKKQ:
This section describes the results of various
assays characterizing IKKR activity, particularly with
regard to its association with IKKa. In addition,
northern blot analysis revealed that IKKR and IKKa are
coexpressed in most tissues examined, including pancreas,
kidney, skeletal muscle, lung, placenta, brain, heart,
peripheral blood lymphocytes, colon, small intestine,
prostate, thymus and spleen.
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1. IKKQ kinase activity
The kinase activity associated with IKKP was
characterized using HeLa or 293 cells transiently
transfected with an HA-tagged IKK(3 expression vector.
5 Transfected cells were stimulated with 20 ng/ml TNF for
10 min and HA-IKK(3 was isolated by immunoprecipitation
using anti-HA antibody (Kolodziej and Young, Meth.
Enzymol. 194:508-519 (1991)). The immune complexes were
tested for the ability to phosphorylate wild type (wt)
10 and mutant forms of IxBa and IKBR (see Example I).
Similarly to the purified IKK complex and the
complex associated with IKKa, the IKK(3 immune complex
phosphorylated wt IKBa and IKB(3, but not mutants in which
the inducible phosphorylation sites (Ser-32 and Ser-36
15 for IKBa and Ser-19 and Ser-23 for IKBa) were replaced
with either alanines or threonines. However, a low level
of residual phosphorylation of full length IKBa(A32/A36)
was observed due to phosphorylation of sites in the
C-terminal portion of the protein (DiDonato et al.,
20 supra, 1997). Single substitution mutants, IKBa(A32) and
IKB(A36), were phosphorylated almost as efficiently as
wt IKBa, indicating that IKK(3-associated IKK activity can
phosphorylate IKBa at both Ser-32 and Ser-36.
The response of IKK(3-associated kinase activity
25 to various stimuli also was examined in HeLa cells
transiently transfected with the HA-IKK(3 expression
vector. After 24 hr, the cells were stimulated with
either 10 ng/ml IL-1, 20 ng/ml TNF or 100 ng/ml TPA, then
HA-IKK(3 immune complexes were isolated by
30 immunoprecipitation and IKK activity was measured. TNF
and IL-1 potently stimulated IKKP-associated kinase
activity, whereas the response to TPA was weaker. The
kinetics of IKK(3 activation by either TNF or IL-1
essentially were identical to the kinetics of activation
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of the IKKa-associated IxB kinase measured by a similar
protocol.
2. Functional interactions between IKKa and IKKR
As shown in Example I, IKKa and IKK(3 copurified
in about a 1:1 ratio through several chromatographic
steps, suggesting that the two proteins interact with
each other. The ability of the IKK subunits to interact
in a functional complex and the effect of each subunit on
the activity of the other subunit was examined using
293 cells transfected with expression vectors encoding
Flag(M2)-IKKa or M2-IKKa and HA-IKKO, either alone or in
combination (see Hopp et al., BioTec noloav 6:1204-1210
(1988)). After 24 hr, samples of the cells were
stimulated with TNF, lysates were prepared from
stimulated and unstimulated cells, and one portion of the
lysates was precipitated with anti-Flag antibodies
(Eastman KodakTM Co.; New Haven CT) and another portion was
precipitated with anti-HA antibodies. The IKK activity
associated with the different immune complexes and their
content of IKKca and IKKR were measured.
Considerably more basal IKK activity was
precipitated with HA-IKKR than with Flag-IKKa. However,
the activity associated with HA-IKKR was further elevated
upon coexpression of M2-IKKa and the low basal activity
associated with Flag-IKKa was strongly augmented by
coexpression of IKK(3. Immunoblot analysis revealed that
the potentiating effect of such coexpression was not due
to changes in the level of expression of IKKa or IKK(i.
The levels of IKK activities associated with
IKKa and IKKR were compared more precisely by
transfecting 293 cells with increasing amounts of HA-IKKa
or HA-IKK(3 expression vectors (0.1 to 0.5 g/106 cells,)
and determining the kinase activities associated with the
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two proteins in cell lysates prepared before or after TNF
stimulation (20 ng/ml, 5 min); GST-IKBa(1-54) was used as
substrate. The level of expression of each protein was
determined by immunoblot analysis and used to calculate
the relative levels of specific IKK activity.
The HA-IKKa-associated IKK had a low level of
basal specific activity, whereas expression of HA-IKKP
resulted in high basal specific activity that was
increased when higher amounts of HA-IKKR were expressed.
However, the specific IKK activity associated with either
IKKa or IKKQ isolated from TNF-stimulated cells was very
similar and was not considerably affected by their
expression level. These results indicate that titration
of a negative regulator or formation of a constitutively
active IKK complex can occur due to overexpression of
IKK(3 .
The ability of IKKa and IKKR to physically
interact was examined. Immunoblot analysis demonstrated
that precipitation of HA-IKKR using an anti-HA antibody
coprecipitated both endogenous IKKa and coexpressed
Flag-IKKa, as indicated by the higher amount of
coprecipitating IKKa detected after cotransfection with
Flag-IKKa. Similarly, immunoprecipitation of Flag-IKKa
with anti-Flag(M2) antibody resulted in coprecipitation
of cotransfected HA-IKKQ. Exposure of the cells to TNF
had no significant effect on the association of IKKa and
IKK~.
The interaction between IKKa and IM was
further examined by transfecting HeLa cells with various
amounts (0.1 to 1.0 g/106 cells) of the HA-IKK(3 vector.
After 24 hr, the cells were incubated for 5 min in the
absence or presence of 20 ng/ml TNF, then lysed. The
lysates were examined for IKK activity and for the amount
of HA-IKK(3 and endogenous IKKa. Expression of increasing
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amounts of HA-IKK(3 resulted in higher basal levels of IKK
activity and increasing amounts of coprecipitated IKKa.
The level of TNF stimulated IKK activity increased only
marginally in response to IKKR overexpression and TNF had
no effect on the association of IKK(3 and IKKa.
Since the results described above revealed that
HA-IKK(3 associates with endogenous IKKa to generate a
functional cytokine-regulated IKK complex, this
association was examined further by transfecting HeLa
cells with either empty expression vector or small
amounts (1 g/60 mm plate) of either HA-IKKa or HA-IKKP
vectors. After 24 hr, samples of the transfected cell
populations were stimulated with 20 ng/ml TNF for 5 min,
then cell lysates were prepared and separated by gel
filtration on a SUPEROSE 6 column. One portion of each
column fraction was immunoprecipitated with a polyclonal
antibody specific for IKKa and assayed for
IKKa-associated IKK activity, while a second portion was
precipitated with anti-HA antibody and examined for
HA-IKK(3- or HA-IKKa-associated IKK activity. Relative
specific activity was determined by immunoprecipitating
the complexes, separating the proteins by SDS-PAGE,
blotting the proteins onto IMOBILON membranes (Millipore;
Bedford MA), immunoblotting with anti-HA antibody and
quantitating the levels of IKB phosphorylation and HA-
tagged proteins by phosphoimaging. The results
demonstrated that endogenous IKKa-associated IKK activity
exists as two complexes, a larger complex of
approximately 900 kDa and a smaller one of approximately
300 kDa. Stimulation with TNF increased the IKK activity
of both complexes, although the extent of increase was
considerably greater for the 900 kDa complex.
HA-IKK(3-associated IKK activity had exactly the
same distribution as the IKKa-associated activity,
eluting at 900 kDa and 300 kDa and, again, the extent of
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TNF responsiveness was considerably greater for the
900 kDa complex. Comparison to the IKKa-associated
activity in cells transfected with the empty vector
indicated that HA-IKK(3 expression produced a modest,
approximately 2-fold increase in the relative amount of
IKK activity associated with the smaller 300 kDa complex.
These results indicate that the 300 kDa IKK complex, like
the 900 kDa complex, contains both IKKa and IKKR.
However, the 300 kDa lacks other subunits present in the
900 kDa complex. When IKKR was overexpressed, the
relative amount of the smaller complex increased,
indicating that some of the subunits that are unique to
the larger complex are present in a limited amount.
3. Both IKKa and IKKQ contribute to IKK activity
The relative contribution of IKKa and IKK(3 to
IKK activity was examined by constructing mutant subunits
in which the lysine (K) codon present at position 44 of
each subunit was substituted with a codon for either
methionine (M) or alanine (A) codon, respectively.
Similar mutations in other protein kinases render the
enzymes defective in binding ATP and, therefore,
catalytically inactive (Taylor et al., Ann. Rev. Cell
Biol. 8:429-462 (1992)). The activity of the IKK mutants
was compared to the activity of their wild type (wt)
counterparts by cell-free translation in reticulocyte
lysates using GST-IKBa(1-54) as a substrate. Translation
of IKKa(KM) resulted in formation of IKB kinase having
only slightly less activity than the IKK formed by
translation of wt IKKa. In comparison, translation of
IKKR(KA) did not generate IKK activity. Translation of
wt IKKP generated IKB kinase activity as expected.
The activities of the different proteins also
was examined by transient transfection in mammalian
cells. Expression and immunoprecipitation of HA-IKKa(KM)
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resulted in isolation of cytokine stimulated IKK activity
that, after TNF stimulation, was 2-to 3-fold lower than
the activity of IKK formed by wt HA-IKKa isolated from
TNF-stimulated cells. Similarly, expression and
5 immunoprecipitation of HA-IKKR resulted in formation of a
cytokine responsive IKK activity that, after TNF
stimulation, was 3- to 5-fold lower than the activity of
IKK generated by wt HA-IKK~ isolated from TNF stimulated
cells. In contrast to results obtained by overexpression
10 of wt HA-IKKR, however, overexpression of HA-IKKR(KA) did
not result in the generation of basal IKK activity.
Immunoprecipitation experiments revealed that IKKa(KM)
associates IKK(3 and that IKK(3(KA) associates with IKKa
and that both IKKa and IKK5 undergo homotypic
15 interactions as efficiently as they undergo heterotypic
interactions.
Autophosphorylation of wt and kinase-defective
HA-IKKa and HA-IKK(3 was examined in transiently
transfected HeLa cells. HeLa cells expressing these
20 proteins were treated with TNF for 10 min, then cell
lysates of TNF treated or untreated cells were
immunoprecipitated with HA antibodies and the immune
complexes were subjected to a phosphorylation reaction
(DiDonato et al., supra, 1997). Both wt HA-IKKa and
25 wt HA-IKKR were phosphorylated and their
autophosphorylation was enhanced in TNF-stimulated
extracts. In contrast, the kinase-defective IKKa or IKKE3
mutants did not exhibit significant autophosphorylation.
4. The role of the LZ and HLH motifs in IKKa and IKKQ
30 IKKa and IKKR both contain leucine zipper (LZ)
and helix-loop-helix (HLH) motifs, which are known to
mediate protein-protein interactions through their
hydrophobic surfaces. The role of the LZ motif in the
IKK subunit interaction was examined using an IKKa
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mutant in which the L462 and L469 residues within the LZ
region were substituted with serine residues. The role
of the HLH motif was examined using an HLH mutant of IKKa
containing a substitution of L605 with arginine (R) and
of F606 with proline (P). The activity of the IKKa LZ-
and HLH- mutants was examined by transient transfection in
293 cells, either alone or in the presence of
cotransfected Flag-IKKa.
Expression of wt HA-IKKa generated substantial
IKK activity that was isolated by immunoprecipitation
with anti-HA, whereas very little IKK activity was
generated in cells transfected with either the
HA-IKKa(LZ)- or HA-IKKa(HLH)- mutant. Coexpression of the
mutant IKK subunits with Flag-IKKR resulted in a
substantial increase in the IKK activity isolated by
immunoprecipitation of HA-IKKa, but had no effect on the
very low activity that coprecipitated with HA-IKKa(LZ)-.
However, coexpression of Flag-IKK(3 did stimulate the low
level of IKK activity associated with HA-IKKa(HLH)-.
Probing of the HA immune complexes with anti-Flag(M2)
antibodies indicated that both wt HA-IKKa and
HA-IKKa(HLH)- associated with similar amounts of
Flag-IKK(3, but that the HA-IKKa(LZ)- mutant did not
associate with Flag-IKK(3. These results indicate that
the lower IKB kinase activity associated with the
IKKa(LZ)" mutant is due to a defect in its ability to
interact with IKKQ. The lower IKB kinase activity of the
IKKa(HLH)- mutant, on the other hand, likely is due to a
defect in the ability to interact with a second,
undefined protein, since the HLH mutant can interact with
IKK(3.
5. Both IKKa and IKKR are necessary for NF-KB activation
The contribution of IKKa and IKKR to NF-KB
activation was examined using HeLa cells transfected with
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expression vectors encoding HA-tagged wt IKKa, IKKa(KM),
wt IKK(3 and IKK(3(KA); an HA-JNK1 vector was used as a
control. NF-KB activation was assessed by examining the
subcellular distribution of RelA(p65) by indirect
immunofluorescence.
HeLa cells were grown on glass cover slips in
growth medium, then transfected with 1 g plasmid DNA by
the lipofectamine method. After 24 hr, samples of cells
were stimulated with 20 ng/ml TNF for 30 min, then
stimulated or unstimulated cells were washed with PBS and
fixed with 3.5o formaldehyde in PBS for 15 min at room
temperature (RT). The fixed cells were permeablized with
0.02% NP-40 in PBS for 1 min, then incubated with 100%
goat serum at 4 C for 12 hr. The cells then were washed
3 times with PBS and incubated with a mixture of a rabbit
anti-NF-xB p65 (Re1A) antibody (1:100 dilution; Santa
Cruz Biotech) and a mouse monoclonal anti-HA antibody in
PBS containing 1% BSA and 0.2% TRITON X-100 at 37 C for
2 hr. Cells then were washed 3 times with PBS containing
0.2% TRITON X-100 and incubated for 2 hr at RT with
secondary antibodies, fluorescein-conjugated goat
affinity purified anti-mouse IgG-IgM and rhodamine-
conjugated IgG fraction goat anti-rabbit IgG (1:200
dilution; Cappel). Cells were washed 4 times with PBS
containing 0.2% TRITON X-100, then covered with a drop of
gelvatol mounting solution and viewed and photographed
using a Zeiss Axioplan microscope equipped for
epifluorescence with the aid of fluoroscein and rhodamine
specific filters.
Double staining with both anti-RelA and anti-HA
revealed that expression of moderate amounts of either
wt IKKa or wt IKKR did not produce considerable
stimulation of RelA nuclear translocation. In addition,
the wt IKK proteins did not interfere with the nuclear
translocation of RelA induced by TNF treatment. However,
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expression of similar levels of either IKKa(KM) or
IKKR(KA), as determined by the intensity of the
fluorescent signal, inhibited the nuclear translocation
of RelA in TNF-treated cells. Expression of HA-JNKi had
no effect on the subcellular distribution of RelA. Since
the subcellular distribution of RelA is dependent on the
state and abundance of IKB, these results indicate that
expression of either IKKa(KM) or IKK(3(KA) inhibits the
induction of IKB phosphorylation and degradation by TNF.
EXAMPLE IV
ISOLATION OF IKB KINASE COMPLEX
This example demonstrates a method for
isolating the 900 kDa IKB kinase complex comprising an
IKKa polypeptide.
Proteins that associate with IKKa in vivo were
isolated by immunoprecipitation using HIS6 and FLAG
epitope tags. The HIS6-FLAG-IKKa (HF-IKKa) encoding
construct was prepared using a double stranded
oligonucleotide, 51-AGCTTGCGCGTATGGCTTCGGGTCATCACCATCACCA
TCACGGTGACTACAAGGACGACGATGACAAAGGTGACATCGAAGGTAGAGGTCA-3'
(SEQ ID NO: 16), which encodes six histidine residues
(HIS6), the FLAG epitope and the factor Xa site in
tandem. The oligonucleotide was inserted using HindIII-
NdeI site in frame with the N-terminus of the IKKa coding
sequence in the BLUESCRIPT KS plasmid (Stratagene;
La Jolla CA). The HindIII-Notl fragment of this plasmid,
which contains the HF-IKKa cDNA sequence, was subcloned
into the pRc~actin mammalian expression vector, which
contains a nucleic acid sequence conferring neomycin
resistance, to produce plasmid pRC-HF-IKKa. Expression
of the HF-IKKa polypeptide was confirmed by western blot
analysis using anti-FLAG antibodies.
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pRC-HF-IKKa was transfected into human
embryonic kidney 293 cells and transfected cells were
selected for growth in the presence of G418. A low basal
level of IKK activity was detected in cells expressing
HF-IKKa and IKK activity increased several fold when the
cells were treated with TNFa. This result indicates that
the HF-IKKa expression in 293 cells is associated with
IKK activity in the cells and that such IKK activity is
inducible in response to TNFa.
A 293 cell line that expresses HF-IKKa was
selected and expanded to approximately 4 x 108 cells. The
cells were treated with 10 ng/ml TNFa for 5 min, then
harvested in ice cold PBS by centrifugation at 2500 x g.
The cell pellet was washed with ice cold PBS, resuspended
in lysis buffer (20 mM Tris, pH 7.6), 150 mM NaCl,
1% TRITON X-100, 20 mM R-glycerophosphate, 2 mM PNPP,
1 mM Na3VO4, 5 mM ~-mercaptoethanol, 1 mM EDTA, 0.5 mM
EGTA, 1 mM PMSF, 3 g/ml pepstatin, 3 g/ml leupeptin,
10 g/ml bestatin and 25 g/ml aprotinin), and lysed by
20 strokes in a glass Dounce homogenizer (pestle A).
The homogenate was centrifuged at 15,000 rpm in
a Beckman SS34 rotor for 30 min at 4 C. The supernatant
was collected, supplemented with 20 mM imidazole and
300 mM NaCl, then mixed with 0.5 ml of a 50o slurry of
Ni-NTA (nickel nitrilotriacetic acid; Qiagen, Inc.;
Chatsworth CA) and stirred for 4 hr at 4 C. Following
incubation, the resin was pelleted at 200 x g and the
supernatant was removed. The resin was washed 3 times
with 50 ml binding buffer containing 25 mM imidazole.
Proteins bound to the resin were eluted in 2 ml
binding buffer containing 150 mM imidazole and 20 mM DTT.
The eluate was mixed with 100 l of a 50o slurry of
anti-FLAG antibody coupled to SEPHAROSE resin using the
AMINOLINK PLUS immobilization kit (Pierce Chem. Co.;
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Rockford IL) and stirred for 4 hr at 4 C. The resin was
pelleted at 1000 x g, the supernatant was removed, and
the resin was washed with 10 ml binding buffer (without
imidazole). Proteins bound to the resin then were eluted
5 with 1o SDS or with FLAG peptide and examined by 100i SDS-
PAGE.
Silver staining revealed the presence of seven
proteins, including the HF-IKKa, which was confirmed by
western blot analysis using anti-FLAG antibody. The
10 copurified proteins had apparent molecular masses of
about 100 kDa, 63 kDa, 60 kDa, 55 kDa, 46 kDa and 29 kDa;
the endogenous 87 kDa IKKR comigrates with the HA-IKKa
protein. These results indicate that IKKa, along with
some or all of the copurifying proteins, comprise the
15 900 kDa IKB kinase complex.
EXAMPLE V
ANTI-IKK ANTISERA
This example provides a method of producing
anti-IKK antisera.
20 Anti-IKKa antibodies were raised in rabbits
using either His-tagged IKKa expressed in E. col.i or the
IKKa peptide ERPPGLRPGAGGPWE (SEQ ID NO: 17) or
TIIHEAWEEQGNS (SEQ ID NO: 18) as an immunogen. Anti-IKK(3
antibodies were raised using the peptide SKVRGPVSGSPDS
25 (SEQ ID NO: 19). The peptides were conjugated to keyhole
limpet hemocyanin (Sigma Chemical Co.; St. Louis MO).
Rabbits were immunized with 250 to 500 g conjugated
peptide in complete Freund's adjuvant. Three weeks after
the primary immunization, booster immunizations were
30 performed using 50 to 100 g immunogen and were repeated
three times, at 3 to 4 week intervals. Rabbits were bled
one week after the final booster and antisera were
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collected. Anti-IKKa antiserum was specific for IKKa and
did not cross react with IKKR.
EXAMPLE VI
USE OF AN IKK SUBUNIT IN A DRUG SCREENING ASSAY
This example describes an assay for screening
for agents such as drugs that alter the association of an
IKK subunit and a second protein that specifically
associates with the IKK subunit.
A GST-IKK subunit fusion protein or HIS6-IKK
subunit fusion protein can be prepared using methods as
described above and purified using glutathione- or metal-
chelation chromatography, respectively (Smith and
Johnson, Gene 67:31-40 (1988);
see, also, Example IV). The fusion
protein is immobilized to a solid support taking
advantage of the ability of the GST protein to
specifically bind glutathione or of the HIS6 peptide
region to chelate a metal ion such as nickel (Ni) ion or
cobalt (Co) ion (Clontech) by immobilized metal affinity
chromatography. Alternatively, an anti-IKK antibody can
be immobilized on a matrix and the IKK-a can be allowed
to bind to the antibody.
The second protein, which can be IxB or a
protein that copurifies with IKK subunit as part of the
900 kDa IxB kinase, for example, can be detectably
labeled with a moiety such as a fluorescent molecule or a
radiolabel (Hermanson, supra, 1996), then contacted in
solution with the immobilized IKK subunit under
conditions as described in Example I, which allow IKB to
specifically associate with the IKK subunit. Preferably,
the reactions are performed in 96 well plates, which
allow automated reading of the reactions. Various agents
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such as drugs then are screened for the ability to alter
the association of the IKK subunit and IKB.
The agent and labeled IKB, for example, can be
added together to the immobilized IKK subunit, incubated
to allow binding, then washed to remove unbound labeled
IKB. The relative amount of binding of labeled IKB in
the absence as compared to the presence of the agent
being screened is determined by detecting the amount of
label remaining in the plate. Appropriate controls are
performed to account, for example, for nonspecific
binding of the labeled IKB to the matrix. Such a method
allows the identification of an agent that alter the
association of an IKK subunit and a second protein such
as IKB.
Alternatively, the labeled IKB or other
appropriate second protein can be added to the
immobilized IKK subunit and allowed to associate, then
the agent can be added. Such a method allows the
identification of agents that can induce the dissociation
of a bound complex comprising the IKK subunit and IKB.
Similarly, a screening assay of the invention can be
performed using the 900 kDa IKK complex, comprising an
IKK subunit.
Although the invention has been described with
reference to the examples provided above, it should be
understood that various modifications can be made without
departing from the spirit of the invention. Accordingly,
the invention is limited only by the claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Regents of the University of California
(ii) TITLE OF INVENTION: I-kappa-B Kinase, Subunits Thereof, and
Methods of Using Same
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Gowling, Strathy & Henderson
(B) STREET: 160 Elgin Street, Suite 2600
(C) CITY: Ottawa
(D). STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP: K1P 1C3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,281,955
(B) FILING DATE: 23-FEB-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/810,131
(B) FILING DATE: 25-FEB-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/061,470
(B) FILING DATE: 09-OCT-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gowling, Strathy & Henderson
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 08-884357CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 233-1781
(B) TELEFAX: (613) 563-9869
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2273 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
= CA 02281955 2000-02-21
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(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 36..2271
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TCGACGGAAC CTGAGGCCGC TTGCCCTCCC GCCCC ATG GAG CGG CCC CCG GGG 53
Met Glu Arg Pro Pro Gly
1 5
CTG CGG CCG GGC GCG GGC GGG CCC TGG GAG ATG CGG GAG CGG CTG GGC 101
Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu Met Arg Glu Arg Leu Gly
15 20
ACC GGC GGC TTC GGG AAC GTC TGT CTG TAC CAG CAT CGG GAA CTT GAT 149
Thr Gly Gly Phe Gly Asn Val Cys Leu Tyr Gln His Arg Glu Leu Asp
25 30 35
CTC AAA ATA GCA ATT AAG TCT TGT CGC CTA GAG CTA AGT ACC AAA AAC 197
Leu Lys Ile Ala Ile Lys Ser Cys Arg Leu Glu Leu Ser Thr Lys Asn
40 45 50
AGA GAA CGA TGG TGC CAT GAA ATC CAG ATT ATG AAG AAG TTG AAC CAT 245
Arg Glu Arg Trp Cys His Glu Ile Gln Ile Met Lys Lys Leu Asn His
55 60 65 70
GCC AAT GTT GTA AAG GCC TGT GAT GTT CCT GAA GAA TTG AAT ATT TTG 293
Ala Asn Val Val Lys Ala Cys Asp Val Pro Glu Glu Leu Asn Ile Leu
75 80 85
ATT CAT GAT GTG CCT CTT CTA GCA ATG GAA TAC TGT TCT GGA GGA GAT 341
Ile His Asp Val Pro Leu Leu Ala Met Glu Tyr Cys Ser Gly Gly Asp
90 95 100
CTC CGA AAG CTG CTC AAC AAA CCA GAA AAT TGT TGT GGA CTT AAA GAA 389
Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn Cys Cys Gly Leu Lys Glu
105 110 115
AGC CAG ATA CTT TCT TTA CTA AGT GAT ATA GGG TCT GGG ATT CGA TAT 437
Ser Gln Ile Leu Ser Leu Leu Ser Asp Ile Gly Ser Gly Ile Arg Tyr
120 125 130
TTG CAT GAA AAC AAA ATT ATA CAT CGA GAT CTA AAA CCT GAA AAC ATA 485
Leu His Glu Asn Lys Ile Ile His Arg Asp Leu Lys Pro Glu Asn Ile
135 140 145 150
GTT CTT CAG GAT GTT GGT GGA AAG ATA ATA CAT AAA ATA ATT GAT CTG 533
Val Leu Gln Asp Val Gly Gly Lys Ile Ile His Lys Ile Ile Asp Leu
155 160 165
GGA TAT GCC AAA GAT GTT GAT CAA GGA AGT CTG TGT ACA TCT TTT GTG 581
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Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser Leu Cys Thr Ser Phe Val
170 175 180
GGA ACA CTG CAG TAT CTG GCC CCA GAG CTC TTT GAG AAT AAG CCT TAC 629
Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu Phe Glu Asn Lys Pro Tyr
185 190 195
ACA GCC ACT GTT GAT TAT TGG AGC TTT GGG ACC ATG GTA TTT GAA TGT 677
Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly Thr Met Val Phe Glu Cys
200 205 210
ATT GCT GGA TAT AGG CCT TTT TTG CAT CAT CTG=CAG CCA TTT ACC TGG 725
Ile Ala Gly Tyr Arg Pro Phe Leu His His Leu Gln Pro Phe Thr Trp
215 220 225 230
CAT GAG AAG ATT AAG AAG AAG GAT CCA AAG TGT ATA TTT GCA TGT GAA 773
His Glu Lys Ile Lys Lys Lys Asp Pro Lys Cys Ile Phe Ala Cys Glu
235 240 245
GAG ATG TCA GGA GAA GTT CGG TTT AGT AGC CAT TTA CCT CAA CCA AAT 821
Glu Met Ser Gly Glu Val Arg Phe Ser Ser His Leu Pro Gln Pro Asn
250 255 260
AGC CTT TGT AGT TTA ATA GTA GAA CCC ATG GAA AAC TGG CTA CAG TTG 869
Ser Leu Cys Ser Leu Ile Val Glu Pro Met Glu Asn Trp Leu Gln Leu
265 270 275
ATG TTG AAT TGG GAC CCT CAG CAG AGA GGA GGA CCT GTT GAC CTT ACT 917
Met Leu Asn Trp Asp Pro Gln Gln Arg Gly Gly Pro Val Asp Leu Thr
280 285 290
TTG AAG CAG CCA AGA TGT TTT GTA TTA ATG GAT CAC ATT TTG AAT TTG 965
Leu Lys Gln Pro Arg Cys Phe Val Leu Met Asp His Ile Leu Asn Leu
295 300 305 310
AAG ATA GTA CAC ATC CTA AAT ATG ACT TCT GCA AAG ATA ATT TCT TTT 1013
Lys Ile Val His Ile Leu Asn Met Thr Ser Ala Lys Ile Ile Ser Phe
315 320 325
CTG TTA CCA CCT GAT GAA AGT CTT CAT TCA CTA CAG TCT CGT ATT GAG 1061
Leu Leu Pro Pro Asp Glu Ser Leu His Ser Leu Gln Ser Arg Ile Glu
330 335 340
CGT GAA ACT GGA ATA AAT ACT GGT TCT CAA GAA CTT CTT TCA GAG ACA 1109
Arg Glu Thr Gly Ile Asn Thr Gly Ser Gln Glu Leu Leu Ser Glu Thr
345 350 355
GGA ATT TCT CTG GAT CCT CGG AAA CCA GCC TCT CAA TGT GTT CTA GAT 1157
Gly Ile Ser Leu Asp Pro Arg Lys Pro Ala Ser Gln Cys Val Leu Asp
360 365 370
GGA GTT AGA GGC TGT GAT AGC TAT ATG GTT TAT TTG TTT GAT AAA AGT 1205
Gly Val Arg Gly Cys Asp Ser Tyr Met Val Tyr Leu Phe Asp Lys Ser
375 380 385 390
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AAA ACT GTA TAT GAA GGG CCA TTT GCT TCC AGA AGT TTA TCT GAT TGT 1253
Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser Arg Ser Leu Ser Asp Cys
395 400 405
GTA AAT TAT ATT GTA CAG GAC AGC AAA ATA CAG CTT CCA ATT ATA CAG 1301
Val Asn Tyr Ile Val Gln Asp Ser Lys Ile Gln Leu Pro Ile Ile Gln
410 415 420
CTG CGT AAA GTG TGG GCT GAA GCA GTG CAC TAT GTG TCT GGA CTA AAA 1349
Leu Arg Lys Val Trp Ala Glu Ala Val His Tyr Val Ser Gly Leu Lys
425 430 435
GAA GAC TAT AGC AGG CTC TTT CAG GGA CAA AGG GCA GCA ATG TTA AGT 1397
Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln Arg Ala Ala Met Leu Ser
440 445 450
CTT CTT AGA TAT AAT GCT AAC TTA ACA AAA ATG AAG AAC ACT TTG ATC 1445
Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys Met Lys Asn Thr Leu Ile
455 460 465 470
TCA GCA TCA CAA CAA CTG AAA GCT AAA TTG GAG TTT TTT CAC AAA AGC 1493
Ser Ala Ser Gln Gln Leu Lys Ala Lys Leu Glu Phe Phe His Lys Ser
475 480 485
ATT CAG CTT GAC TTG GAG AGA TAC AGC GAG CAG ATG ACG TAT GGG ATA 1541
Ile Gln Leu Asp Leu Glu Arg Tyr Ser Glu Gln Met Thr Tyr Gly Ile
490 495 500
TCT TCA GAA AAA ATG CTA AAA GCA TGG AAA GAA ATG GAA GAA AAG GCC 1589
Ser Ser Glu Lys Met Leu Lys Ala Trp Lys Glu Met Glu Glu Lys Ala
505 510 515
ATC CAC TAT GCT GAG GTT GGT GTC ATT GGA TAC CTG GAG GAT CAG ATT 1637
Ile His Tyr Ala Glu Val Gly Val Ile Gly Tyr Leu Glu Asp Gln Ile
520 525 530
ATG TCT TTG CAT GCT GAA ATC ATG GAG CTA CAG AAG AGC CCC TAT GGA 1685
Met Ser Leu His Ala Glu Ile Met Glu Leu Gln Lys Ser Pro Tyr Gly
535 540 545 550
AGA CGT CAG GGA GAC TTG ATG GAA TCT CTG GAA CAG CGT GCC ATT GAT 1733
Arg Arg Gln Gly Asp Leu Met Glu Ser Leu Glu Gln Arg Ala Ile Asp
555 560 565
CTA TAT AAG CAG TTA AAA CAC AGA CCT TCA GAT CAC TCC TAC AGT GAC 1781
Leu Tyr Lys Gln Leu Lys His Arg Pro Ser Asp His Ser Tyr Ser Asp
570 575 580
AGC ACA GAG ATG GTG AAA ATC ATT GTG CAC ACT GTG CAG AGT CAG GAC 1829
Ser Thr Glu Met Val Lys Ile Ile Val His Thr Val Gln Ser Gln Asp
585 590 595
CGT GTG CTC AAG GAG CGT TTT GGT CAT TTG AGC AAG TTG TTG GGC TGT 1877
Arg Val Leu Lys Glu Arg Phe Gly His Leu Ser Lys Leu Leu Gly Cys
600 605 610
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AAG CAG AAG ATT ATT GAT CTA CTC CCT AAG GTG GAA GTG GCC CTC AGT 1925
Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys Val Glu Val Ala Leu Ser
615 620 625 630
AAT ATC AAA GAA GCT GAC AAT ACT GTC ATG TTC ATG CAG GGA AAA AGG 1973
Asn Ile Lys Glu Ala Asp Asn Thr Val Met Phe Met Gln Gly Lys Arg
635 640 645
CAG AAA GAA ATA TGG CAT CTC CTT AAA ATT GCC TGT ACA CAG AGT TCT 2021
Gln Lys Glu Ile Trp His Leu Leu Lys Ile Ala Cys Thr Gln Ser Ser
650 655 660
GCC CGC TCT CTT GTA GGA TCC AGT CTA GAA GGT GCA GTA ACC CCT CAA 2069
Ala Arg Ser Leu Val Gly Ser Ser Leu Glu Gly Ala Val Thr Pro Gln
665 670 675
GCA TAC GCA TGG CTG GCC CCC GAC TTA GCA GAA CAT GAT CAT TCT CTG 2117
Ala Tyr Ala Trp Leu Ala Pro Asp Leu Ala Glu His Asp His Ser Leu
680 685 690
TCA TGT GTG GTA ACT CCT CAA GAT GGG GAG ACT TCA GCA CAA ATG ATA 2165
Ser Cys Val Val Thr Pro Gln Asp Gly Glu Thr Ser Ala Gln Met Ile
695 700 705 710
GAA GAA AAT TTG AAC TGC CTT GGC CAT TTA AGC ACT ATT ATT CAT GAG 2213
Glu Glu Asn Leu Asn Cys Leu Gly His Leu Ser Thr Ile Ile His Glu
715 720 725
GCA AAT GAG GAA CAG GGC AAT AGT ATG ATG AAT CTT GAT TGG AGT TGG 2261
Ala Asn Glu Glu Gln Gly Asn Ser Met Met Asn Leu Asp Trp Ser Trp
730 735 740
TTA ACA GAA T GA 2273
Leu Thr Glu
745
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Glu Arg Pro Pro Gly Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu
1 5 10 15
Met Arg Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Cys Leu Tyr
20 25 30
Gln His Arg'Glu Leu Asp Leu Lys Ile Ala Ile Lys Ser Cys Arg Leu
CA 02281955 2000-02-21
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35 40 45
Glu Leu Ser Thr Lys Asn Arg Glu Arg Trp Cys His Glu Ile Gln Ile
50 55 60
Met Lys Lys Leu Asn His Ala Asn Val Val Lys Ala Cys Asp Val Pro
65 70 75 80
Glu Glu Leu Asn Ile Leu Ile His Asp Val Pro Leu Leu Ala Met Glu
85 90 95
Tyr Cys Ser Gly Gly Asp Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn
100 105 110
Cys Cys Gly Leu Lys Glu Ser Gln Ile Leu Ser Leu Leu Ser Asp Ile
115 120 125
Gly Ser Gly Ile Arg Tyr Leu His Glu Asn Lys Ile Ile His Arg Asp
130 135 140
Leu Lys Pro Glu Asn Ile Val Leu Gln Asp Val Gly Gly Lys Ile Ile
145 150 155 160
His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser
165 170 175
Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu
180 185 190
Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly
195 200 205
Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Arg Pro Phe Leu His His
210 215 220
Leu Gln Pro Phe Thr Trp His Glu Lys Ile Lys Lys Lys Asp Pro Lys
225 230 235 240
Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Glu Val Arg Phe Ser Ser
245 250 255
His Leu Pro Gln Pro Asn Ser Leu Cys Ser Leu Ile Val Glu Pro Met
260 265 270
Glu Asn Trp Leu Gln Leu Met Leu Asn Trp Asp Pro Gln Gln Arg Gly
275 280 285
Gly Pro Val Asp Leu Thr Leu Lys Gln Pro Arg Cys Phe Val Leu Met
290 295 300
Asp His Ile Leu Asn Leu Lys Ile Val His Ile Leu Asn Met Thr Ser
305 310 315 320
Ala Lys Ile Ile Ser Phe Leu Leu Pro Pro Asp Glu Ser Leu His Ser
325 330 335
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Leu Gln Ser Arg Ile Glu Arg Glu Thr Gly Ile Asn Thr Gly Ser Gln
340 345 350
Glu Leu Leu Ser Glu Thr Gly Ile Ser Leu Asp Pro Arg Lys Pro Ala
355 360 365
Ser Gln Cys Val Leu Asp Gly Val Arg Gly Cys Asp Ser Tyr Met Val
370 375 380
Tyr Leu Phe Asp Lys Ser Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser
385 390 395 400
Arg Ser Leu Ser Asp Cys Val Asn Tyr Ile Val Gln Asp Ser Lys Ile
405 410 415
Gln Leu Pro Ile Ile Gln Leu Arg Lys Val Trp Ala Glu Ala Val His
420 425 430
Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln
435 440 445
Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys
450 455 460
Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gln Leu Lys Ala Lys Leu
465 470 475 480
Glu Phe Phe His Lys Ser Ile Gln Leu Asp Leu Glu Arg Tyr Ser Glu
485 490 495
Gln Met Thr Tyr Gly Ile Ser Ser Glu Lys Met Leu Lys Ala Trp Lys
500 505 510
Glu Met Glu Glu Lys Ala Ile His Tyr Ala Glu Val Gly Val Ile Gly
515 520 525
Tyr Leu Glu Asp Gln Ile Met Ser Leu His Ala Glu Ile Met Glu Leu
530 535 540
Gin Lys Ser Pro Tyr Gly Arg Arg Gln Gly Asp Leu Met Glu Ser Leu
545 550 555 560
Glu Gln Arg Ala Ile Asp Leu Tyr Lys Gln Leu Lys His Arg Pro Ser
565 570 575
Asp His Ser Tyr Ser Asp Ser Thr Glu Met Val Lys Ile Ile Val His
580 585 590
Thr Val Gln Ser Gln Asp Arg Val Leu Lys Glu Arg Phe Gly His Leu
595 600 605
Ser Lys Leu Leu Gly Cys Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys
610 615 620
Val Glu Val Ala Leu Ser Asn Ile Lys Glu Ala Asp Asn Thr Val Met
CA 02281955 2000-02-21
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625 630 635 640
Phe Met Gln Gly Lys Arg Gln Lys Glu Ile Trp His Leu Leu Lys Ile
645 650 655
Ala Cys Thr Gln Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu
660 665 670
Gly Ala Val Thr Pro Gln Ala Tyr Ala Trp Leu Ala Pro Asp Leu Ala
675 680 685
Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gln Asp Gly Glu
690 695 700
Thr Ser Ala Gln Met Ile Glu Glu Asn Leu Asn Cys Leu Gly His Leu
705 710 715 720
Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gln Gly Asn Ser Met Met
725 730 735
Asn Leu Asp Trp Ser Trp Leu Thr Glu
740 745
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Lys Ile Ile Asp Leu Leu Pro Lys
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "Xaa is aspartic acid or
alanine (D/A)."
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(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 17
(D) OTHER INFORMATION: /note= "Xaa is proline or glycine
(P/G)."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Lys His Arg Xaa Leu Lys Pro Glu Asn Ile Val Leu Gln Asp Val Gly
1 5 10 15
Xaa Lys
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCCCATATGT ACCAGCATCG GGAA 24
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TCAACCAATT GTCTTGAGCT CCCC 24
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
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CATGGCACCA TCGTTCTCTG 20
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CTCAAAGAGC TCTGGGGCCA GATAC 25
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TCCGAGATCT GGACGAGC 18
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Lys Ile Ile Asp Leu Gly Tyr Ala Lys
1 5
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "Xaa is Methionine or
Tyrosine (M/Y). "
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "Xaa is Valine or Glycine
(V/G)
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 10
(D) OTHER INFORMATION: /note= "Xaa is Threonine,
Asparagine, Arginine or Glutamic acid (T/N/R/E)."
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 11
(D) OTHER INFORMATION: /note= "Xaa is Glycine or
Asparagine (G/N)."
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 14
(D) OTHER INFORMATION: /note= "Xaa is Histidine,
Isoleucine or Serine (H/I/S)."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Lys Xaa Val His Ile Leu Asn Xaa Xaa Xaa Xaa Thr Ile Xaa
1 5 10
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "Xaa is Threonine or Alanine
(T/A) . "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
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Lys Xaa Xaa Ile Gln Gln Asp Xaa Gly Ile Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Lys Xaa Arg Val Ile Tyr Thr Gin Leu
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2931 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 36..2304
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CGCGTCCCTG CCGACAGAGT TAGCACGACA TCAGT ATG AGC TGG TCA CCT TCC 53
Met Ser Trp Ser Pro Ser
1 5
CTG ACA ACG CAG ACA TGC GGG GCC TGG GAA ATG AAA GAG CGC CTT GGG 101
Leu Thr Thr Gln Thr Cys Gly Ala Trp Glu Met Lys Glu Arg Leu Gly
15 20
ACA GGG GGA TTT GGA AAT GTC ATC CGA TGG CAC AAT CAG GAA ACA GGT 149
Thr Gly Gly Phe Gly Asn Val Ile Arg Trp His Asn Gln Glu Thr Gly
25 30 35
GAG CAG ATT GCC ATC AAG CAG TGC CGG CAG GAG CTC AGC CCC CGG AAC 197
Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln Glu Leu Ser Pro Arg Asn
40 45 50
CGA GAG CGG TGG TGC CTG GAG ATC CAG ATC ATG AGA AGG CTG ACC CAC 245
Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile Met Arg Arg Leu Thr His
55 60 65 70
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CCC AAT GTG GTG GCT GCC CGA GAT GTC CCT GAG GGG ATG CAG AAC TTG 293
Pro Asn Val Val Ala Ala Arg Asp Val Pro Glu Gly Met Gln Asn Leu
75 80 85
GCG CCC AAT GAC CTG CCC CTG CTG GCC ATG GAG TAC TGC CAA GGA GGA 341
Ala Pro Asn Asp Leu Pro Leu Leu Ala Met Glu Tyr Cys Gln Gly Gly
90 95 100
GAT CTC CGG AAG TAC CTG AAC CAG TTT GAG AAC TGC TGT GGT CTG CGG 389
Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu Asn Cys Cys Gly Leu Arg
105 110 115
GAA GGT GCC ATC CTC ACC TTG CTG AGT GAC ATT GCC TCT GCG CTT AGA 437
Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp Ile Ala Ser Ala Leu Arg
120 125 130
TAC CTT CAT GAA AAC AGA ATC ATC CAT CGG GAT CTA AAG CCA GAA AAC 485
Tyr Leu His Glu Asn Arg Ile Ile His Arg Asp Leu Lys Pro Glu Asn
135 140 145 150
ATC GTC CTG CAG CAA GGA GAA CAG AGG TTA ATA CAC AAA ATT ATT GAC 533
Ile Val Leu Gln Gln Gly Glu Gln Arg Leu Ile His Lys Ile Ile Asp
155 160 165
CTA GGA TAT GCC AAG GAG CTG GAT CAG GGC AGT CTT TGC ACA TCA TTC 581
Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly Ser Leu Cys Thr Ser Phe
170 175 180
GTG GGG ACC CTG CAG TAC CTG GCC CCA GAG CTA CTG GAG CAG CAG AAG 629
Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu Leu Glu Gln Gln Lys
185 190 195
TAC ACA GTG ACC GTC GAC TAC TGG AGC TTC GGC ACC CTG GCC TTT GAG 677
Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe Gly Thr Leu Ala Phe Glu
200 205 210
TGC ATC ACG GGC TTC CGG CCC TTC CTC CCC AAC TGG CAG CCC GTG CAG 725
Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro Asn Trp Gln Pro Val Gln
215 220 225 230
TGG CAT TCA AAA GTG CGG CAG AAG AGT GAG GTG GAC ATT GTT GTT AGC 773
Trp His Ser Lys Val Arg Gln Lys Ser Glu Val Asp Ile Val Val Ser
235 240 245
GAA GAC TTG AAT GGA ACG GTG AAG TTT TCA AGC TCT TTA CCC TAC CCC 821
Glu Asp Leu Asn Gly Thr Val Lys Phe Ser Ser Ser Leu Pro Tyr Pro
250 255 260
AAT AAT CTT AAC AGT GTC CTG GCT GAG CGA CTG GAG AAG TGG CTG CAA 869
Asn Asn Leu Asn Ser Val Leu Ala Glu Arg Leu Glu Lys Trp Leu Gln
265 270 275
CTG ATG CTG ATG TGG CAC CCC CGA CAG AGG GGC ACG GAT CCC ACG TAT 917
Leu Met Leu Met Trp His Pro Arg Gln Arg Gly Thr Asp Pro Thr Tyr
280 285 290
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GGG CCC AAT GGC TGC TTC AAG GCC CTG GAT GAC ATC TTA AAC TTA AAG 965
Gly Pro Asn Gly Cys Phe Lys Ala Leu Asp Asp Ile Leu Asn Leu Lys
295 300 305 310
CTG GTT CAT ATC TTG AAC ATG GTC ACG GGC ACC ATC CAC ACC TAC CCT 1013
Leu Val His Ile Leu Asn Met Val Thr Gly Thr Ile His Thr Tyr Pro
315 320 325
GTG ACA GAG GAT GAG AGT CTG CAG AGC TTG AAG GCC AGA ATC CAA CAG 1061
Val Thr Glu Asp Glu Ser Leu Gln Ser Leu Lys Ala Arg Ile Gln Gln
330 335 340
GAC ACG GGC ATC CCA GAG GAG GAC CAG GAG CTG CTG CAG GAA GCG GGC 1109
Asp Thr Gly Ile Pro Glu Glu Asp Gln Glu Leu Leu Gln Glu Ala Gly
345 350 355
CTG GCG TTG ATC CCC GAT AAG CCT GCC ACT CAG TGT ATT TCA GAC GGC 1157
Leu Ala Leu Ile Pro Asp Lys Pro Ala Thr Gln Cys Ile Ser Asp Gly
360 365 370
AAG TTA AAT GAG GGC CAC ACA TTG GAC ATG GAT CTT GTT TTT CTC TTT 1205
Lys Leu Asn Glu Gly His Thr Leu Asp Met Asp Leu Val Phe Leu Phe
375 380 385 390
GAC AAC AGT AAA ATC ACC TAT GAG ACT CAG ATC TCC CCA CGG CCC CAA 1253
Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln Ile Ser Pro Arg Pro Gln
395 400 405
CCT GAA AGT GTC AGC TGT ATC CTT CAA GAG CCC AAG AGG AAT CTC GCC 1301
Pro Glu Ser Val Ser Cys Ile Leu Gln Glu Pro Lys Arg Asn Leu Ala
410 415 420
TTC TTC CAG CTG AGG AAG GTG TGG GGC CAG GTC TGG CAC AGC ATC CAG 1349
Phe Phe Gln Leu Arg Lys Val Trp Gly Gln Val Trp His Ser Ile Gln
425 430 435
ACC CTG AAG GAA GAT TGC AAC CGG CTG CAG CAG GGA CAG CGA GCC GCC 1397
Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln Gln Gly Gln Arg Ala Ala
440 445 450
ATG ATG AAT CTC CTC CGA AAC AAC AGC TGC CTC TCC AAA ATG AAG AAT 1445
Met Met Asn Leu Leu Arg Asn Asn Ser Cys Leu Ser Lys Met Lys Asn
455 460 465 470
TCC ATG GCT TCC ATG TCT CAG CAG CTC AAG GCC AAG TTG GAT TTC TTC 1493
Ser Met Ala Ser Met Ser Gln Gln Leu Lys Ala Lys Leu Asp Phe Phe
475 480 485
AAA ACC AGC ATC CAG ATT GAC CTG GAG AAG TAC AGC GAG CAA ACC GAG 1541
Lys Thr Ser Ile Gln Ile Asp Leu Glu Lys Tyr Ser Glu Gln Thr Glu
490 495 500
TTT GGG ATC ACA TCA GAT AAA CTG CTG CTG GCC TGG AGG GAA ATG GAG 1589
Phe Gly Ile Thr Ser Asp Lys Leu Leu Leu Ala Trp Arg Glu Met Glu
505 510 515
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CAG GCT GTG GAG CTC TGT GGG CGG GAG AAC GAA GTG AAA CTC CTG GTA 1637
Gln Ala Val Glu Leu Cys Gly Arg Glu Asn Glu Val Lys Leu Leu Val
520 525 530
GAA CGG ATG ATG GCT CTG CAG ACC GAC ATT GTG GAC TTA CAG AGG AGC 1685
Glu Arg Met Met Ala Leu Gin Thr Asp Ile Val Asp Leu Gln Arg Ser
535 540 545 550
CCC ATG GGC CGG AAG CAG GGG GGA ACG CTG GAC GAC CTA GAG GAG CAA 1733
Pro Met Gly Arg Lys Gln Gly Gly Thr Leu Asp Asp Leu Glu Glu Gln
555 560 565
GCA AGG GAG CTG TAC AGG AGA CTA AGG GAA AAA CCT CGA GAC CAG CGA 1781
Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu Lys Pro Arg Asp Gln Arg
570 575 580
ACT GAG GGT GAC AGT CAG GAA ATG GTA CGG CTG CTG CTT CAG GCA ATT 1829
Thr Glu Gly Asp Ser Gln Glu Met Val Arg Leu Leu Leu Gln Ala Ile
585 590 595
CAG AGC TTC GAG AAG AAA GTG CGA GTG ATC TAT ACG CAG CTC AGT AAA 1877
Gln Ser Phe Glu Lys Lys Val Arg Val Ile Tyr Thr Gln Leu Ser Lys
600 605 610
ACT GTG GTT TGC AAG CAG AAG GCG CTG GAA CTG TTG CCC AAG GTG GAA 1925
Thr Val Val Cys Lys Gln Lys Ala Leu Glu Leu Leu Pro Lys Val Glu
615 620 625 630
GAG GTG GTG AGC TTA ATG AAT GAG GAT GAG AAG ACT GTT GTC CGG CTG 1973
Glu Val Val Ser Leu Met Asn Glu Asp Glu Lys Thr Val Val Arg Leu
635 640 645
CAG GAG AAG CGG CAG AAG GAG CTC TGG AAT CTC CTG AAG ATT GCT TGT 2021
Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn Leu Leu Lys Ile Ala Cys
650 655 660
AGC AAG GTC CGT GGT CCT GTC AGT GGA AGC CCG GAT AGC ATG AAT GCC 2069
Ser Lys Val Arg Gly Pro Val Ser Gly Ser Pro Asp Ser Met Asn Ala
665 670 675
TCT CGA CTT AGC CAG CCT GGG CAG CTG ATG TCT CAG CCC TCC ACG GCC 2117
Ser Arg Leu Ser Gln Pro Gly Gln Leu Met Ser Gln Pro Ser Thr Ala
680 685 690
TCC AAC AGC TTA CCT GAG CCA GCC AAG AAG AGT GAA GAA CTG GTG GCT 2165
Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys Ser Glu Glu Leu Val Ala
695 700 705 710
GAA GCA CAT AAC CTC TGC ACC CTG CTA GAA AAT GCC ATA CAG GAC ACT 2213
Glu Ala His Asn Leu Cys Thr Leu Leu Glu Asn Ala Ile Gln Asp Thr
715 720 725
GTG AGG GAA CAA GAC CAG AGT TTC ACG GCC CTA GAC TGG AGC TGG TTA 2261
Val Arg Glu Gln Asp Gln Ser Phe Thr Ala Leu Asp Trp Ser Trp Leu
730 735 740
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CAG ACG GAA GAA GAA GAG CAC AGC TGC CTG GAG CAG GCC TCA T 2304
Gln Thr Glu Glu Glu Glu His Ser Cys Leu Glu Gln Ala Ser
745 750 755
GATGTGGGGG GACTCGACCC CCTGACATGG GGCAGCCCAT AGCAGGCCTT GTGCAGTGGG 2364
GGGACTCGAC CCCCTGACAT GGGGCTGCCT GGAGCAGGCC GCGTGACGTG GGGCTGCCTG 2424
GCCGTGGCTC TCACATGGTG GTTCCTGCTG CACTGATGGC CCAGGGGTCT CTGGTATCCA 2484
GATGGAGCTC TCGCTTCCTC AGCAGCTGTG ACTTTCACCC AGGACCCAGG ACGCAGCCCT 2544
CCGTGGGCAC TGCCGGCGCC TTGTCTGCAC ACTGGAGGTC CTCCATTACA GAGGCCCAGC 2604
GCACATCGCT GGCCCCACAA ACGTTCAGGG GTACAGCCAT GGCAGCTCCT TCCTCTGCCG 2664
TGAGAAAAGT GCTTGGAGTA CGGTTTGCCA CACACGTGAC TGGACAGTGT CCAATTCAAA 2724
TCTTTCAGGG CAGAGTCCGA GCAGCGCTTG GTGACAGCCT GTCCTCTCCT GCTCTCCAAA 2784
GGCCCTGCTC CCTGTCCTCT CTCACTTTAC AGCTTGTGTT TCTTCTGGAT TCAGCTTCTC 2844
CTAAACAGAC AGTTTAATTA TAGTTGCGGC CTGGCCCCAT CCTCACTTCC TCTTTTTATT 2904
TCACTGCTGC TAAAATTGTG TTTTTAC 2931
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 756 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Ser Trp Ser Pro Ser Leu Thr Thr Gln Thr Cys Gly Ala Trp Glu
1 5 10 15
Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Ile Arg Trp
20 25 30
His Asn Gln Glu Thr Gly Glu Gln Ile Ala Ile Lys Gln Cys Arg Gln
35 40 45
Glu Leu Ser Pro Arg Asn Arg Glu Arg Trp Cys Leu Glu Ile Gln Ile
50 55 60
Met Arg Arg Leu Thr His Pro Asn Val Val Ala Ala Arg Asp Val Pro
65 70 75 80
Glu Gly Met Gln Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met
85 90 95
CA 02281955 2000-02-21
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Glu Tyr Cys Gln Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gln Phe Glu
100 105 110
Asn Cys Cys Gly Leu Arg Glu Gly Ala Ile Leu Thr Leu Leu Ser Asp
115 120 125
Ile Ala Ser Ala Leu Arg Tyr Leu His Glu Asn Arg Ile Ile His Arg
130 135 140
Asp Leu Lys Pro Glu Asn Ile Val Leu Gln Gln Gly Glu Gln Arg Leu
145 150 155 160
Ile His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gln Gly
165 170 175
Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu
180 185 190
Leu Leu Glu Gln Gln Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe
195 200 205
Gly Thr Leu Ala Phe Glu Cys Ile Thr Gly Phe Arg Pro Phe Leu Pro
210 215 220
Asn Trp Gln Pro Val Gln Trp His Ser Lys Val Arg Gln Lys Ser Glu
225 230 235 240
Val Asp Ile Val Vai Ser Glu Asp Leu Asn Gly Thr Val Lys Phe Ser
245 250 255
Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Ser Val Leu Ala Glu Arg
260 265 270
Leu Glu Lys Trp Leu Gln Leu Met Leu Met Trp His Pro Arg Gln Arg
275 280 285
Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly Cys Phe Lys Ala Leu Asp
290 295 300
Asp Ile Leu Asn Leu Lys Leu Val His Ile Leu Asn Met Val Thr Gly
305 310 315 320
Thr Ile His Thr Tyr Pro Val Thr Glu Asp Glu Ser Leu Gln Ser Leu
325 330 335
Lys Ala Arg Ile Gln Gln Asp Thr Gly Ile Pro Glu Glu Asp Gln Glu
340 345 350
Leu Leu Gln Glu Ala Gly Leu Ala Leu Ile Pro Asp Lys Pro Ala Thr
355 360 365
Gln Cys Ile Ser Asp Gly Lys Leu Asn Glu Gly His Thr Leu Asp Met
370 375 380
Asp Leu Val Phe Leu Phe Asp Asn Ser Lys Ile Thr Tyr Glu Thr Gln
CA 02281955 2000-02-21
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385 390 395 400
Ile Ser Pro Arg Pro Gln Pro Glu Ser Val Ser Cys Ile Leu Gln Glu
405 410 415
Pro Lys Arg Asn Leu Ala Phe Phe Gln Leu Arg Lys Val Trp Gly Gln.
420 425 430
Val Trp His Ser Ile Gln Thr Leu Lys Glu Asp Cys Asn Arg Leu Gln
435 440 445
Gln Gly Gln Arg Ala Ala Met Met Asn Leu Leu Arg Asn Asn Ser Cys
450 455 460
Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met Ser Gln Gln Leu Lys
465 470 475 480
Ala Lys Leu Asp Phe Phe Lys Thr Ser Ile Gln Ile Asp Leu Glu Lys
485 490 495
Tyr Ser Glu Gln Thr Glu Phe Gly Ile Thr Ser Asp Lys Leu Leu Leu
500 505 510
Ala Trp Arg Glu Met Glu Gln Ala Val Glu Leu Cys Gly Arg Glu Asn
515 520 525
Glu Val Lys Leu Leu Val Glu Arg Met Met Ala Leu Gln Thr Asp Ile
530 535 540
Val Asp Leu Gln Arg Ser Pro Met Gly Arg Lys Gln Gly Gly Thr Leu
545 550 555 560
Asp Asp Leu Glu Glu Gln Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu
565 570 575
Lys Pro Arg Asp Gln Arg Thr Glu Gly Asp Ser Gln Glu Met Val Arg
580 585 590
Leu Leu Leu Gln Ala Ile Gln Ser Phe Glu Lys Lys Val Arg Val Ile
595 600 605
Tyr Thr Gln Leu Ser Lys Thr Val Val Cys Lys Gln Lys Ala Leu Glu
610 615 620
Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu
625 630 635 640
Lys Thr Val Val Arg Leu Gln Glu Lys Arg Gln Lys Glu Leu Trp Asn
645 650 655
Leu Leu Lys Ile Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser
660 665 670
Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gln Pro Gly Gln Leu Met
675 680 685
CA 02281955 2000-02-21
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Ser Gln Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys
690 695 700
Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu Cys Thr Leu Leu Glu
705 710 715 720
Asn Ala Ile Gln Asp Thr Val Arg Glu Gln Asp Gln Ser Phe Thr Ala
725 730 735
Leu Asp Trp Ser Trp Leu Gln Thr Glu Glu Glu Glu His Ser Cys Leu
740 745 750
Glu Gln Ala Ser
755
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 91 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
AGCTTGCGCG TATGGCTTCG GGTCATCACC ATCACCATCA CGGTGACTAC AAGGACGACG 60
ATGACAAAGG TGACATCGAA GGTAGAGGTC A 91
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Arg Pro Pro Gly Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02281955 2000-02-21
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Thr Ile Ile His Glu Ala Trp Glu Glu Gln Gly Asn Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ser Lys Val Arg Gly Pro Val Ser Gly Ser Pro Asp Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Lys Xaa Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu Lys
1 5 10
