Note: Descriptions are shown in the official language in which they were submitted.
2148898
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PATENT
A.~O~N~Y DOCKET NO: 00786/217CAl
p54 STRESS-ACTIVATED PROTEIN KINASES
Statement as to Federally Sponsored Research
This invention was funded at least in part by the
following grants from the United States Government:
GM46577 to JMK, DK17776 and DK41513 to JA (DK41513
transferred to JMK as of 1/94), and the U.S. Government
has certain rights in the invention.
Background of the Invention
The field of the invention is second messenger
protein kinases which regulate activation of
transcription factors, which in turn modulate cellular
responses to extracellular stimuli.
The extracellular signal-regulated kinases (ERKs)
are a family of proline-directed kinases that are
activated via concomitant Tyr and Thr phosphorylation and
share sequence homology in the Ser/Thr protein kinase
catalytic domain. They include the mitogen activated
20 protein kinases, or MAPKs. Stimuli for the activation of
these kinases are diverse, and induce discrete second
messenger pathways to effect specific cellular responses.
ERKs participate in the regulation of other protein
kinases and several transcription factors including c-
25 Jun, c-Myc, c-Fos, ATF-2, and p62TCF/Elk-l. These
functions indicate that the ERKs mediate the expression
of genes in response to extracellular agonists.
The first well characterized members of this
kinase family were p42 and p44 MAPKs, which are
30 stimulated by insulin and require both Tyr and Thr
phosphorylation for activity (Sturgill et al, Nature
334:715-718, 1988; Anderson et al., Nature 343:651-653,
1990). They are also stimulated by a variety of
mitogens, phorbol esters, and activated ras.
2148~9~
The ERKs share sequence homology in the Ser/Thr
protein kinase catalytic domain, as mentioned above, and
this functions to phosphorylate c-Jun on serine and
threonine residues that have been localized to c-Jun
5 tryptic peptides termed X and Y. X and Y are located
near the N-terminal trans-activation domain (Pulverer et
al., Nature 353:670-673, 1992). Phosphorylation of these
peptides regulates transactivating binding activity, and
thus their function as promoters of gene expression.
Summary of the Invention
The invention features a molecule consisting of
either DNA or amino acids encoding a p54 stress-activated
protein kinase (SAPK), or a biologically active fragment
thereof, characterized by its ability to modulate
15 transcription pathways in response to extracellular
stress stimuli. By "biologically active fragment" it is
meant that the fragment can exert a physiological effect
(e.g., binding to its biological substrate,
phosphorylation, causing an antigenic response, etc.) in
20 vivo or in vitro.
In preferred embodiments, the molecule of the
invention is a polypeptide at least 95% identical to
p54aI, p54aII, p54~I, p54~II, or p54y, preferably from a
mammalian source (e.g., SEQ ID N0s: 1, 2, 3, 4, or 5).
25 Any polypeptide sequence containing an "X" is intended to
denote a position that could be any amino acid. By
"polypeptide", it is meant any chain of amino acids,
regardless of length or post-translational modification
(e.g., glycosylation or phosphorylation).
Other preferred embodiments include polypeptides
that are substantially identical to SEQ ID NOs 1, 2, 3,
4, 5, 6, 7, and 8. By "substantially identical", it is
meant an amino acid sequence which differs only by
conservative amino acid substitutions, for example,
35 substitution of one amino acid for another of the same
21~8~8
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class (e.g., valine for glycine, arginine for lysine,
etc.) or by one or more non-conservative amino acid
substitutions, deletions, or insertions located at
positions of the amino acid sequence which do not destroy
5 the biological function of the polypeptide.
In one preferred embodiment, the molecule of the
invention is a polypeptide fragment that contains a
biologically active portion of a p54 polypeptide, at
least 10 amino acids in length. Examples include, but
10 are not limited to, the ATP binding site, which includes
Y 55 Gly33, 35, and 38; the site of regulatory
phosphorylation by upstream activators (Thr183-Pro-
Tyrl85); and the SAPK catalytic domain (between amino
acids 22 and 321; see Fig. 1).
In another preferred embodiment the polypeptide or
a fragment thereof is useful for producing antibodies
which specifically bind to a p54 stress-activated protein
kinase (e.g., SEQ ID NOs 6, 7, and 8). In this context,
fragment means at least the smallest antigenic epitope,
20 generally at least 10 contiguous amino acids.
The invention also features a DNA molecule
encoding a p54 stress-activated protein kinase
polypeptide, its degenerate variants, or a fragment
thereof including at least 30 contiguous nucleotides.
25 The DNA sequence may be 90% identical to p54~I (e.g., SEQ
ID N0: 9), p54~II (e.g., SEQ ID N0: 10), p54~I (e.g., SEQ
ID N0: 11), p54~II (e.g., SEQ ID N0: 12), or p54y (e.g.,
SEQ ID N0: 13), or be a fragment of any of the above
nucleotide sequences containing at least 30 contiguous
30 nucleic acids, or a degenerate variant thereof. By
"degenerate variant" it is meant any nucleotide sequence
that encodes the same amino acid sequence as the
polypeptide translated from the DNA, or a substantially
identical polypeptide.
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The molecules of the invention are preferably
derived from a mammal, more preferably from a rat or
human.
The invention also includes DNA molecules which
5 hybridize under stringent conditions to one or more of
the DNAs encoding p54 SAPKs (SEQ ID NOs 9, 10, 11, 12,
and 13). By "stringent conditions" it is meant
conditions under which molecules without significant
homology (e.g., at least 90%) to the DNAs of the
10 invention could not hybridize (protocols to determine
stringency and melting point of DNA sequences are well
known to those skilled in the art and may be found in
Sambrook et al. (eds), Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, 1989; hereby
15 incorporated by reference). Included in the term
"DNA(s)" are the sequences of both strands of double
stranded DNA.
The invention also features methods of screening
potentially therapeutic compounds involving applying test
20 compounds in physiologically relevant concentrations in a
suitable excipient to cultured cells, with or without
previous, prior, or concurrent stress-activated protein
kinase-activating stimuli (e.g., TNF-~, IL-l-~,
sphingomyelinase, heat shock, etc.), preparing cellular
25 extracts, and assaying the isolated recombinant stress-
activated protein kinase for c-Jun kinase activity. By
"physiologically relevant" it is meant a concentration
that is achievable during non-toxic administration to a
human patient. By suitable excipient it is meant a non-
30 toxic or non-injurious solvent or carrier for the
molecules of the invention or test compounds used in the
screening assay.
In another embodiment, the cells treated with the
test compound and with or without previous, prior, or
35 concurrent SAPK activating stimuli are extracted, and
` 2148898
combined with inactive recombinant stress-activated
protein kinase. The recombinant SAPK is then isolated
and assayed for c-Jun kinase activating ability.
The advantages and uses of the invention are in
5 the treatment and prevention of inflammation and the
deleterious effects of hypoxia, heat stress, reperfusion
injury, and other tissue insults which are currently
difficult to manage clinically. The molecules of the
invention may be useful for reducing inflammation in such
10 chronic disorders as autoimmune diseases or allergies,
and in acute conditions such as anaphylactic shock, or
soft tissue injury where swelling may aggravate the
condition (e.g, around broken bones). other uses include
prophylactic treatment of patients about to undergo
15 surgeries where there is a high likelihood of ischemia-
reperfusion injury (e.g., vascular surgery, organ
transplants, etc.), or treatment of sepsis and fever.
Additionally, these molecules could be used to up-
regulate AP-l expression via c-Jun phosphorylation, and
20 thus enhance levels of IL-2 expression as a cancer
therapeutic.
The molecules of the invention may also be useful
for drug design based on an underst~n~;ng of the enzymes'
structure and function (e.g., the polypeptide's binding
25 site on c-Jun), or as indicators in an assay to screen
large numbers of drugs for beneficial effects on the
conditions listed above.
Other features and advantages of the invention
will be apparent from the following description of the
30 preferred embodiments, and from the claims.
Brief Description of the Drawings
Figure 1: Deduced amino acid sequences (SEQ ID
NOs: 1, 2, 3, 4, 5, 6, 7, and 8) alignment of p54
stress-activated protein kinase (SAPK) cDNAs (single
21~8898
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letter code). Sequences determined by protein
microsequencing are underlined, and the black diamonds
flank the catalytic domains of the SAPKs.
Figures 2 a) and b): Specificity of anti p54 SAPK
5 antisera for p54 SAPKs. a) In vitro translation of ERKs.
RNAs (2~g) transcribed from plasmids containing the cDNAs
of rat p54~I (lane 1), p54~ (lane 2), and HA-epitope-
tagged p44 MAPK (lane 3) were translated using rabbit
reticulocyte lysates. Lane 4 shows lysate programmed
10 with water; a 43-kDa background band is indicated by an
open arrowhead. b) Aliquots of the translated proteins
were immunoprecipitated in RIPA buffer with antisera
raised against bacterially expressed p54~. Lanes are the
same as in the left panel (lanes 1, 2, and 3).
Figures 3 a) and b): HepG2 cells were treated
with TNF-~ (lOOng/ml, 15 min). Cell extracts were
prepared and depleted of SAPKs by exhaustive
immunoprecipitation for the times indicated (a) or for 4
hrs (b), at which time extracts were subjected to GST-c-
20 Jun chromatography as a means of determining the c-Jun
kinase activity remaining in the extracts. As a control,
immunodepletions were done with preimmune serum or
without serum (b). Activity in the immunoprecipitates
and on the c-Jun columns was measure (a). The results
25 indicate that around 70% of the c-Jun kinase activity
present in the extracts can be removed with the anti-SAPK
serum.
Figures 4 a) and b): Comparative activation of
p42/44 MAPKs (open bars) and p54 SAPKs (filled bars)
30 showing that NIH3T3 cell (a) and HT-29 cell (b) p54 SAPKs
are poorly activated by mitogens and strongly activated
by heat stress and cycloheximide.
Figure 5 a) and b): Activation of c-Jun
phosphorylation in situ by various stimuli. a) c-Jun
35 phosphorylated in vivo in response to cycloheximide (lane
2148898
1) and heat shock (lane 2); retardation of the Jun
polypeptide upon SDS-PAGE (compare lanes 1 and 2 with
non-phosphorylated c-Jun in lane 3). The c-Jun
polypeptide is indicated with an arrowhead. b) Two
5 dimensional tryptic phosphopeptide mapping of c-Jun
polypeptides immunoprecipitated from control (left), or
heat shock-treated (right) HepG2 cells indicates enhanced
phosphorylation of peptides X and Y in response to heat
shock. The origin is marked with an arrowhead. The
10 dotted circle indicates the position of the xylene cyanol
marker.
Figures 6 a) and b): Detection and isolation of
c-Jun kinases activated by heat shock and other stimuli
on GST-c-Jun immobilized on glutathione agarose (b);
15 comparison with activation of p54 SAPKs (a). The black
and white bars each represent results from one
experiment.
Figures 7 a) and b): Activation of SAPKs and c-
Jun kinases by tunicamycin. p54 SAPK activity is shown
20 in a, closed circles (mean + SD for triplicate
determinations), total c-Jun kinases binding to
immobilized GST-c-Jun, a, open circles; b shows the fold
activation of the p42/p44 MAPKs by tunicamycin.
Figure 8 a) and b): Comparison of activation of
25 p54 SAPKs and p42/44 MAPKs by various stimuli in human
CCD-18Co cells. a) Activation of p54 SAPKs by TNF-~ and
other stimuli. Mean + SD for triplicate determinations
is shown. b) Parallel assays for relative activation of
p42/44 MAPKs by the same stimuli shown in a).
Figure 9 a) and b): Comparison of activation of
p54 SAPKs and p42/44 MAPKs by various stimuli in primary
porcine hepatocytes. a) Activation of p54 SAPKs by TNF-
~ and other stimuli. Mean + SD for triplicate
determinations is shown. b) Comparison of fold
2148898
activation by various stimuli of p54 SAPKs (filled bars)
and p42/44 MAPKs (open bars).
Figure 10: Activation of HepG2 cell p54 SAPKs by
TNF-~ and S. aureus sphingomyelinase. SMase =
5 sphingomyelinase.
Figures 11 a) and b): Comparison of activation of
p54 SAPKs and p42/44 MAPKs by IL-l-~. EL-4 murine thyoma
cells were treated with 20 ng/ml recombinant human IL-l-
~. Cells were extracted as described in the legend of
10 Table 2. SAPKs (a and filled bars, b) and p42/44 MAPKs
(b) were assayed in standard experimental paradigms. For
a) data are mean + SD for triplicate determinations. For
b) data are presented as percent of control for
comparative purposes.
Figures 12 a) and b): Effect of ischemia/
reperfusion on MAPK and SAPK activation in vivo.
Activation of p54 SAPK (a) and p42/44 MAPKs (b) was
measured at times from 0-90 min after initiating
reperfusion of rat kidneys made ischemic for 45 min. as
20 described in the Methods section. MAPKs experienced
rapid activation and inactivation (b), while SAPKs were
rapidly activated and remained at elevated levels for
periods well in excess of 90 min.
Figure 13: Bacterially expressed rat SAPK-~ was
25 expressed as a GST fusion protein and purified on
glutathione agarose. The SAPK was treated with buffer
alone, or with an extract from HepG2 cells treated with
TNF and prepared as described in the Table 2 legend. The
SAPK was recovered with glutathione beads and assayed for
30 c-Jun kinase activity. As an additional control, blank
beads were exposed to cell extracts (extract alone bar).
214~8
g
Description of the Preferred Embodiments
Methods
Cloning
p54 SAP kinase was purified to homogeneity from
5 livers of cycloheximide injected rats (Kyriakis and
Avruch, J. Biol. Chem. 265:17355-17363) and the sequences
of several tryptic peptides were determined following RP-
HPLC. Two of these peptides (HRDLKPSN and
MLVIDPDKRISVDEAL) were homologous to protein kinase
10 subdomains VIb and XI, respectively (Hanks et al .,
Science, 241:42-52, 1988) and were used to design
degenerate sense and antisense primers, respectively. A
467 bp fragment was amplified by PCR from rat brain cDNA
and used as a probe to screen 250,000 plaques of a rat
15 brain cDNA library in AZAP (Stratagene). Twenty seven
positive plaques were purified and representatives
sequenced on both strands using nested deletion series.
Antisera/Antibodies
To generate antisera, the p54 SAPK-~ isoform was
20 subcloned into pGEX-KG and expressed as a glutathione S-
transferase (GST) fusion protein. Expression p54 SAPK-
~polypeptide was induced with IPTG (50~g/ml) and the
fusion purified to homogeneity by glutathione-agarose
chromatography, followed by thrombin cleavage to release
25 the kinase moiety from the fusion protein. This material
was then used as an immunogen and antisera collected and
tested. The antisera tested have been shown to cross-
react with human, mouse, rat, and pig tissues. The SAPK
sera react with several human cell lines, including human
30 hepatocellular carcinoma cells (HepG2), human colon
fibroblasts (CCD-18Co), human monocytic lymphoma cells
(U937), and human colon carcinoma cells (HT-29).
Monoclonal antibodies may be made by fusing immune
B cells from the spleen with tumor cells to produce
21 18898
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hybridomas specifically secreting each antibody, using
methods well known in the art (see, for instance, Coligan
et al., eds. Current Protocols in Immunology, John Wiley
and Sons, 1992; Kohler et al., Nature 256: 495, 1975;
5 Hammerling et al., in Monoclonal Antibodies and T Cell
Hybridomas, Elsevier, NY, 1981)
Peptide antisera were generated using standard
methods to the p54~, p54~, and p54y classes using the
least conserved region of the molecules (SEQ ID NOs: 6,
10 7, and 8) to enhance the probability of class-
specificity.
Cell Cul ture Treatments and Assay
Confluent cultures of NIH3T3 cells were treated
with H22 (5 mM, 15 min) phorbol-12-myristate-13-acetate
(PMA, 500 nM, 20 min) FGF (10 ng/ml, 20 min), A23187
(100 nM 20 min), heat (42C, 30 min) or cycloheximide.
HT-29 cells were treated with EGF (50 ng/ml, 20 min) or
heat shock (42C, 30 min). Cells were washed three times
in PBS and lysed in ice-cold lysis buffer (20 mM Na-
20 Hepes, pH 7.5, 2 mM EGTA, 1 mM DTT, 1 mM Na3V04. 50 mM ~-
glycerophosphate, 1% (w/v) Triton X-100, 10% (v/v)
glycerol, 2 ~M leupeptin, 10 kallikrein-inhibiting
units/ml aprotinin, 200 ~m PMSF). Extracts were
normalized to identical protein concentration (1 mg/ml).
25 A portion (1 ml) was immunoprecipitated with 5/13-99, and
assayed for GST-Jun kinase activity as follows. To 40 ~l
of a 1:1 suspension of p54 SAPK beads were added 20 ~l
0.2 mg/ml GST-c-Jun-1-135 (Adler et al., (1992), Proc.
Natl. Acad. Sci. U.S.A., 89:5341-5345) or 0.01 mg/ml
30 holo-c-Jun (Pulverer et al., supra; Pulverer et al.,
(1993), Oncogene, 8:407-415). ATP (100 ~M) and MgCl2 (10
mM) were added to start the reaction. The reaction was
allowed to proceed for 20 min at 30C at which time the
reaction was stopped with SDS sample buffer and the
- 21 188~
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mixtures resolved by SDS-PAGE. The 40-kDa GST-Jun band
was excised and counted by liquid scintillation
spectroscopy (mean + SD for triplicate determinations are
shown in the figures). Another portion (1 ml) was
5 assayed for p42/p44 MAPK activity by Mono-Q
chromatography, and by a myelin basic protein (MBP)
kinase activity assay (Ahn et al., (1991), J . Biol .
Chem ., 266:4220-4227). A peak of stimulated MBP kinase
activity was always detected eluting between 200 and 350
10 nM NaCl. Total p42/p44 MAPK activity was taken as that
contained in those fractions and is shown as percent of
control for comparative purposes.
U937 cells were labeled with 32p orthophosphate (1
mCi/ml) and treated with cycloheximide heat stress or
15 vehicle. c-Jun was immunoprecipitated using a polyclonal
antibody specific for the C-terminal 15 amino acids of c-
Jun and subjected to SDS-PAGE (Pulverer et al.). HepG2
cells were labeled with 32p as above and subjected to heat
shock. c-Jun was immunoprecipitated and, after SDS-PAGE,
20 was subjected to two dimensional tryptic phosphopeptide
mapping. In other experiments, U937 cells were treated
with actinomycin-D (10 ~g/ml, 30 min), anisomycin (10
~g/ml, 30 min) emetine (10 ~g/ml, 30 min), puromycin (10
~g/ml, 30 min) or heat stress (42C, 30 min). Lysates
25 were prepared and were passed over columns of immobilized
GST-Jun. After washing away unbound proteins, Mg/y32P-ATP
was added and phosphorylation of the immobilized c-Jun
was assayed as described above. Parallel aliquots were
subjected to immunoprecipitation and assayed for p54
30 SAPK. In both assays, 1 U of kinase activity transferred
1 pmol P04/min from ATP to GST-Jun. HT29 cells were
treated with various concentrations of tunicamycin for 5
h. Lysis, assay of p42/p44 MAPK activity, and p54 SAPK
immune complex kinase assay were done by standard
35 methods. Detection of total tunicamycin-stimulated c-Jun
21~88g8
- 12 -
kinase activity in HT-29 cells isolated on columns of
immobilized c-Jun was performed as described above.
CCD-18Co cells, HepG2 cells, or primary porcine
hepatocytes were cultured to 80% confluence, serum
5 starved (0.5% serum, 16 hours) and treated with the
following agonists as shown in Figures 16 and 17: heat
shock (42C, 30 min), PMA (200 nM, 20 min), EGF (100
ng/ml, 20 min) or TNF-~ (50 ng/ml, 10 or 20 min).
Extracts were prepared and p54 SAPKs were
10 immunoprecipitated and assayed. Parallel assays of
p42/p44 MAPKs were performed. For treatment with
sphingomyelinase, 100 mU/ml S. aureus sphingomyelinase
were added to HepG2 cells as known in the art (Dressler
et al., (1992), Science, 255:1715-1718) for 15 min. lU
15 S. aureus sphingomyelinase hydrolyzed 1 ~mole TNP-
sphingomyelin/min at pH 7.4, 37C.
Ischemia/Reperfusion
Male Sprague-Dawley rats, 120-150 g each, were
anesthetized with sodium pentobarbital (65 mg/kg). In
20 order to induce ischemia, the renal artery of one kidney
was clamped for 45 min. The contralateral kidney served
as a control. Reperfusion was accomplished by releasing
the clamp and allowing blood flow for 0 to 90 min.
Control and ischemia/reperfusion kidneys were harvested
25 and homogenized in lysis buffer (20 mM Hepes, pH 7.4, 2
mM EGTA, 50 mM ~-glycerophosphate, 1 mM DTT, 250 mM
sucrose, 400 ~M PMSF, 2 ~M leupeptin, 2 ~M aprotinin).
After centrifugation for 1 hr at 100,000 x g, the
supernatants were collected and Na3VO4 added to 0.1%
(w/v). Immunoprecipitation and SAPK assay were as
described in the Table 2 legend.
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Isolated clones
p54 protein kinase was isolated from rat liver
that had been stimulated with cycloheximide, using the
above methods (also described in Kyriakis and Avruch,
5 supra; hereby incorporated by reference) and amino acid
sequences were derived from peptides generated by tryptic
digests. These sequences aligned with the consensus
Ser/Thr protein kinase catalytic domain of known MAPKs
(Hanks et al . ) . Two of these peptide sequences were used
10 to design overlapping degenerate oligonucleotide probes
for use in a nested PCR reaction from which a 467 bp cDNA
was generated using rat brain cDNA as the template. This
probe was used to screen a rat brain cDNA library from
which 27 clones were isolated and sequenced. The cDNAs
15 encoded five separate polypeptides (Fig. 1, SEQ ID NOs 1-
5). This was quite unexpected, since it was assumed that
only one peptide had been purified to homogeneity from
the rat liver in this and previous work (Kyriakis and
Avruch). One set of clones (p54~I, SEQ ID NO. 9) encoded
20 a protein that contained all of the tryptic peptides
derived from rat liver p54 kinase. Two additional sets
(p54~I/II, SEQ ID NOs 11 and 12; and p54~, SEQ ID NO. 13)
encoded closely related polypeptides (88-90% identity,
respectively to ~I, Fig. 1). A fourth group of cDNAs
(p54~II, SEQ ID NO. 10) was identical to ~I kinase except
for a region of 71 base pairs which results in the
substitution of 15 amino acids in subdomains IX and X
(see Fig. 1). The ~I and ~II RNAs are most likely
derived from the same gene by alternate splicing. The
30 predicted proteins encoded by the full length clones are:
~I, 48,076 Da; ~II, 47,986 Da; and ~I, 48,095 Da.
Northern blotting analysis of several tissues revealed
ubiquitous, low expression of all three gene classes.
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ProPerties of p54 SAPKs
The distinct characteristics of the isolated p54
clones and expressed and native polypeptides have led to
the nomenclature of Stress-_ctivated Protein _inases
(SAPKs), which will be used throughout to distinguish
them from the MAPKs.
Sequence alignment of the catalytic domains of the
p54 SAPK clones with those of the known mammalian MAPKs,
and with the yeast MAPK homologs RSS1, HOG-l FUS3, SLT-2,
10 spk-1 and erk-l (Courchesne et al., (1989), Cell,
58:1107-1119; Brewster et al., (1993), Science, 259:1760-
1763; Levin et al ., ( 1993), J. NIH Res., 5:49-52) shows
that the p54 polypeptides exhibit nearly equal identity
to the mammalian 44 kDa MAPK (43-44% sequence identity)
15 and the kinases from lower eukaryotes (41-44% identity).
By contrast, p44 MAPK is closer in sequence to the yeast
kin~ces (between 49-52% for the S. cerevisiae enzymes and
56% identity for Spk-l) than it is to the p54 SAPKs.
From these and other comparative data, we conclude that
20 none of the MAP kinase-like genes identified thus far in
lower eukaryotes is likely to be a functional homologue
of the p54 SAPKs. All of the p54 isoforms contain the
sequence Thr183-Pro-Tyrl85 in an analogous position to the
Thr and Tyr residues of p42/p44 MAPKs (Payne et al.,
(1991) EMBO J. 10:885-892) that must be phosphorylated
for activity. These residues in the p42/p44 MAPKs are
phosphorylated by the _APK or ERK _inases (MEKs), a
family of dual specificity protein Tyr/Thr kinases (Ahn
et al.; G6mez et al., (1991) Nature, 253:170-173;
30 Nakielny et al., (1992), EMBO J., 11:2123-2129).
However, in vitro, neither purified dephosphorylated
liver p54 SAPK nor bacterially expressed p54 SAPK
isoforms are phosphorylated or reactivated by p42/p44
MAPK-specific MEKs, suggesting the existence of a
35 specific p54 SAPK kinase.
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- 15 -
In vivo administration of cycloheximide activates
p54 SAPK, and we have also characterized the stimuli and
signal transduction pathways that activate p54 SAPKs in
cultured cells. A polyclonal antiserum (5/13-99), raised
5 against the prokaryotic recombinant p54 SAPK ~-isoform,
immunoblots p54 SAPK and immunoprecipitates in vitro
translated p54~ and p54~ polypeptides but not p44 MAP
kinase (Fig. 2). Purified rat liver p54 SAPK
phosphorylates c-Jun exclusively at Ser73 and Ser63 in the
10 c-Jun trans activation domain, two sites located on c-Jun
tryptic phosphopeptides designated X and Y, respectively
(Pulverer et al.; Smeal et al., (1991), Nature, 354:494-
496). The anti-p54 SAPK antiserum (5/13-99)
immunoprecipitates from HT-29 human colon carcinoma cells
15 a protein kinase activity that phosphorylates recombinant
GST-c-Jun, as well as full-length c-Jun, selectively at
tryptic peptides X and Y. Pretreatment of NIH3T3 cells
or HT-29 cells with cycloheximide substantially augments
this c-Jun (X/Y) kinase activity. Thus, the p54
20 antiserum is reactive with p54 SAPKs but not p42 MAPK or
p44 MAPK. Moreover, p54 SAPK activity, measured as a c-
Jun "X/Y" kinase, is stimulated by cycloheximide in
tissue culture as well as in vivo.
SAPKs are the major c-Jun kinase activated by TNF-
25 ~. SAPK immunodepletion experiments removed 60-70% of
the recombinant c-Jun kinase activity induced in TNF-
~treated cells (Fig. 3). This indicates that the SAPKs
account for around 70% of the c-Jun-associated c-Jun
kinase activity.
30 Requlation of p54 SAPKs
Effects of NAPR Activators
We examined the regulation of the p54 SAPK, in
NIH3T3 and human HT-29 cells, by agents known to regulate
p42/p44 MAPKs. The activity of p54 SAPKs was measured in
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an immune complex kinase assay using GST-c-Jun as a
substrate, whereas the p42/p44 MAPKs in the same extracts
were assayed, after Mono-Q anion exchange chromatography,
using MBP as a substrate. Figure 4 compares the
5 activation of each set of kinases in response to the
various treatments to the baseline values. As expected,
NIH3T3 cell p42/44 MAPKs are strongly activated by
mitogen (10-fold activation by FGF) and phorbol esters
(6-fold), and are activated to a lesser extent by Ca2+
10 influx stimulated by the ionophore A23187 (2-fold). In
addition, the p42/p44 MAPKs are activated by H2O2 and
cycloheximide (4- and 6-fold, respectively). In striking
contrast, the NIH3T3 cell p54 SAPKs are not activated by
FGF, phorbol esters, H22 or by Ca2+ ionophore (top panel,
15 filled bars). A similar result is seen for HT-29 cells
(bottom panel, filled bars). In these cells, EGF
substantially stimulates the p42/p44 MAPKs (6 fold,
bottom panel, open bars) while only slightly stimulating
p54 SAPKs (bottom panel, filled bars).
These results suggest that signals generated by
activation of receptor tyrosine kinases and phospholipase
C are not likely to represent the primary regulatory
input to the p54 SAPKs. Therefore, stimuli other than
mitogens were investigated to see if they could activate
25 p54 SAPKs more vigorously than they activated the p42/p44
MAPKs. Cycloheximide, although capable of strongly
activating the NIH3T3 cell p54 SAPK (10-fold), also gives
substantial (5-fold) activation of endogenous p42/p44
MAPKs. By contrast, heat shock (42C, 30 min) gives a
30 large induction of NIH3T3 cell p54 SAPK activity (7-
fold), while p42/p44 MAPK activity is only slightly (1.5-
fold) stimulated (Fig. 4, top panel). In HT-29 cells as
well, heat shock dramatically stimulates HT-29 cell p54
SAPKs (8.4 fold, Fig. 4, bottom panel, filled bars) while
35 only modestly stimulating p42/p44 MAPKs (2.9 fold, Fig.
21~89~
-
- 17 -
4, bottom, open bars). Activation of p54 SAPK by heat
shock (42C) is evident by 10 min, maximal at 30 min, and
declines slightly after 1 hr.
Effect of p54 SAP~ Activation on c-~un
Since the p54 SAPKs are potent c-Jun kinases in
vitro, we inquired whether stimuli such as heat shock and
cycloheximide, that activate p54 SAPKs preferentially,
also increase the phosphorylation of c-Jun in situ. 32p_
labeled U937 and HepG2 cells were exposed to heat shock
10 or cycloheximide, and the phosphorylation of endogenous
c-Jun was examined. Both treatments induce a robust
phosphorylation of c-Jun, accompanied by a dramatic
retardation of c-Jun mobility on SDS-PAGE (Fig. 5a).
This retardation is characteristic of phosphorylation on
15 tryptic peptides X and Y (corresponding to Ser 73 and 63,
respectively) within the c-Jun N-terminal trans
activation domain (Pulverer et al.); the induction of X/Y
phosphorylation was verified directly by tryptic peptide
maps of 32P-c-Jun isolated from heat-shocked 32P-labeled
20 HepG2 cells (Fig. 5b). Activation of c-Jun N-terminal
phosphorylation by heat shock represents a previously
unrecognized mode of c-Jun regulation. The likelihood
that p54 SAPKs contribute substantially to the c-Jun
phosphorylation elicited by heat shock and cycloheximide
25 in situ is supported by the ability of the
immunoprecipitated p54 SAPK activated by cycloheximide to
phosphorylate recombinant c-Jun in vitro exclusively on
tryptic 32p peptides that co-migrate with tryptic peptides
X and Y (see Fig. 3).
30 Effects of Protein Synthesis Inhibitors on p54 SAPRs
Maneuvers that increase c-Jun X/Y phosphorylation
in situ, such as W light or PMA, have been shown to
activate c-Jun kinases that bind tightly to immobilized
21~8898
- 18 -
GST-Jun (Adler et al .; Hibi et al ., Genes & Dev., 7: 2135-
2148, 1993). Extracts from heat-shocked or
cycloheximide-treated cells also contain activated c-Jun
kinase(s) that bind to immobilized GST-c-Jun (Fig. 6,
5 right panel). Moreover, two other inhibitors of
polypeptide chain elongation, emetine and especially
anisomycin, also activate both p54 SAPK and GST-Jun-bound
c-Jun kinase (Fig. 6), whereas puromycin and the RNA
synthesis inhibitor, actinomycin D, are each unable to
10 activate either c-Jun kinase (Fig. 6, right panel) or p54
SAPK activity (Fig. 6, left panel). A summary of these
results is presented in Table 1, below. Thus, heat
shock, a variety of protein synthesis inhibitors as well
as the glycosylation inhibitor, tunicamycin (see below),
15 alter p54 SAPK, and the GST-Jun-bound c-Jun kinase in
parallel, suggesting that p54 SAPK is likely to be one of
the physiologic Jun kinases activated by this class of
perturbations.
TABLE 1
Activation of p54s and c-Jun-associated k;n~s~c by
protein and RNA synthesis inhibitors.
c-Jun-associated
Treatmentp54 MAPK (mU) kinase (mU)
Control 31.0 14.5
25 Anisomycin 370.0 385.0
Cycloheximide 166.0 243.0
Heat-shock 168.0 180.0
Emetine 75.0 85.0
Puromycin 19.3 21.5
30 Actinomycin-D 22.0 24.0
Activation of p54s and c-Jun-associated kinases by
protein and RNA synthesis inhibitors. U937 cells were
treated with anisomycin (lO~g/ml, 30 min), cycloheximide
21~8~98
,
- 19 -
(200~M, 60 min), heat shock (42C, 30 min), or
actinomycin D (lO~g/ml, 30 min). Extracts were prepared
as described in the methods, and parallel aliquots were
subjected to GST-c-Jun chromatography/assay or
5 immunoprecipitation and assay for p54. In both assays, 1
unit of kinase activity transferred 1 pmol PO4/min from
ATP to GST-c-Jun. Shown are mean results for two
experiments.
Polypeptide Misfolding Stimulates p54 SAPRs
Although heat shock and protein synthesis
inhibitors could activate p54 SAPK through entirely
unrelated mechanisms, a shared property of heat shock and
translational inhibitors (but not the RNA synthesis
inhibitor, actinomycin D), is their ability to promote
15 polypeptide misfolding and denaturation. Consequently,
we investigated whether the accumulation of misfolded
polypeptides might be an initial stimulus common to
protein synthesis inhibitors and heat shock that leads to
the preferential activation of p54 SAPK. Tunicamycin
20 inhibits the synthesis and proper folding of proteins
destined for membrane insertion or secretion through
inhibition of N-linked glycosylation in the Golgi.
Increasing doses of tunicamycin added to HT29 cells
promotes a striking activation (up to 12-fold) of p54
25 SAPK activity (Fig. 7, top, filled circles), whereas the
p42/p44 MAPKs, assayed after separation by Mono-Q
chromatography, are only modestly activated (Fig. 7,
bottom). In addition, tunicamycin stimulates total HT-29
Jun kinases which bind to GST-c-Jun with a similar dose
30 response to p54 SAPK activation (Fig. 7, top, open
circles). The activation of the p54 SAPKs by
tunicamycin, together with the data in Figures 4 and 6
support the idea that cellular stresses which result in
the accumulation of misfolded polypeptides, can, to a
35 degree greater than mitogens, generate a signal to
activate the p54 SAPKs. By contrast, the p42/p44 MAPKs
21~8898
-
- 20 -
are more strongly activated by mitogenic signaling,
relative to stress signaling.
i nf~c l'NF--~ and IL~
In mammals, integration of the multicellular and
5 inter-organ response to a variety of "stressful" noxious
stimuli is mediated by a diverse array of inflammatory
cytokines, such as TNF-~ and IL-1-~. TNF-~ was initially
detected by its ability to induce the hemorrhagic
necrosis of some transplantable tumors in inbred mice
(Buetler et al., Ann. Rev. Biochem., 57:505-517, 1988;
Goeddel et al., Cold Spring Harbor Symp. Quant. Biol.,
51:597-609, 1986). The cellular responses to
inflammatory cytokines are quite diverse and cell-
specific, and are directed at optimizing overall host
15 defense against infection. For example, TNF-a acts on
adipose tissue to inhibit insulin action and energy
storage; on liver to yield the protein secretory pattern
known as the acute phase response; on neutrophils to
enhance superoxide radical production and cytotoxic
20 efficacy; and on T cells to promote the secretion of
additional cytokines, e.g. IL-2 and IL-6 (see Buetler et
al. and Goeddel et al. for review). The multifarious
actions of TNF-~ are due in part to TNF-~-directed
programs of gene expression. Notably, TNF-~ has been
25 shown to be a potent activator of the trans activation
function and autoinduction of c-Jun (Brenner et al.,
Nature, 337:661-663, 1989), as well as an activator of
NF-KB (Osborn et al., Proc. Natl. Acad. Sci. U.S.A.,
86:2336-2340, 1989), both of which are needed by
30 lymphocytes for trans activation of the IL-2 gene.
The profile of physiologic and cellular responses
to TNF-~ and IL-l-~ led us to inquire whether these
agents could activate p54 SAPK. Human CCD-18Co colon
fibroblasts are acutely responsive to TNF-~ (Goeddel et
21~8~98
-
- 21 -
al . ); in these cells, TNF-~ elicited a striking
activation of p54 SAPKs, whereas EGF and PMA were without
noteworthy effect (Fig. 8a). The 10-fold stimulation of
p54 SAPK by TNF-~ was slightly greater than that provoked
5 by heat stress. Comparing the responses of p42/p44 MAPKs
in CCD-18Co cells, it is clear that heat stress and TNF-a
are far more potent activators of p54 SAPK than are EGF
and PMA, while the converse is true for the p42/44 MAPKs
(Fig. 8b).
Liver is also a target tissue for TNF-~ (Buetler
et al ., and Goeddel et al . ); addition of TNF-~ to primary
cultures of freshly isolated porcine hepatocytes
stimulates p54 SAPK activity by ~ 6-fold (Fig. 9a),
whereas EGF increases p54 SAPK activity 3-fold. In these
15 cells, EGF and PMA activate the p42/p44 MAPKs and the p54
SAPKs to a comparable degree. However, as is seen in
CCD-18Co cells, heat shock and TNF-a are much more potent
activators of p54 SAPK than they are p42/p44 MAPK
activators (Fig. 9b). The present results are thus
20 consistent with recent reports showing that TNF-
~activates p42/p44 MAPKs (Van Lint et al., J. Biol. Chem.,
267:25916-25921, 1992); however, it is clear that the
p42/p44 MAP kinases are much more potently activated by
ligands like EGF and FGF, that operate through receptor
25 tyrosine kinase, whereas the ability of TNF-~ to activate
the p54 SAPKs greatly exceeds the ability of FGF and EGF
to activate the p54 SAPK, at least in most cell types.
Little is known of the molecular mechanisms of
TNF-~ signaling. TNF-~ binds to one of two receptors,
30 55-kDa and 70-kDa, whose intracellular extensions show no
homology with receptors whose signal transduction
mechanisms are better understood (Loetscher et al., Cell,
61:351-359, 1990; Heller et al ., Proc. Natl . Acad . sci .
U.S .A., 87:6151-6155, 1990). TNF-~ has been shown to
35 stimulate rapid sphingomyelin hydrolysis and the
2148898
- 22 -
accumulation of ceramide, through the activation of a
neutral sphingomyelinase (Dressler et al.; Yang et al.,
J. Biol . Chem., 268:20520-20523, 1993; Dobrowsky et al.,
J. Biol. Chem., 267:5048-5051, 1992; Dbaibo et al., J.
5 Biol . Chem., 268:17762-17766, 1993; Schutze et al., Cell,
71:42-52, 1992). Ceramide has been proposed to serve as
a second messenger for TNF-~, analogous to the role
envisioned for diacylglycerol in the action of hormones
that activate phosphatidylinositol-specific phospholipase
10 C enzymes (Dressler, et al.; Yang, Z., et al.;
Dobrowsky, et al.; Dbaibo, et al.; Schutze et al.; and
Sch~tze, et al . ) . Many of the responses to TNF-~,
including growth inhibition, apoptosis, activation of
heterotrimeric forms of protein phosphatase-2A,
15 activation of a membrane bound Ser/Thr kinase and,
possibly, activation of NF-~B can be elicited by the
addition of various ceramide derivatives or bacterial
sphingomyelinase to intact or permeabilized cells
(Dressler et al.; Yang, et al.; Dobrowsky, et al.;
20 Dbaibo, et al .; and Schutze et al . ) . Based on these
considerations, we compared the ability of TNF-~, IL-1-
~and S. aureus sphingomyelinase treatment of HepG2 or EL-4
cells to alter p54 SAPK activity. TNF-~ stimulates a
robust activation of the p54 SAPKs (15-fold) in these
25 cells, and a substantial activation of p54 SAPKs (5.3-
fold) is also evident in response to sphingomyelinase
(Fig. 10). These results are consistent with the
possibility that sphingomyelin hydrolysis, as is known to
occur in response to inflammatory cytokines, may be an
30 early step in the p54 SAPK signal transduction pathway.
When EL-4 murine thyoma cells were treated with 20
ng/ml recombinant human IL-1-~, p54 SAPK activity
increased over 4-fold, while p42/44 MAPK activity
remained unchanged by the treatment (Fig. 11 a and b).
35 This strong induction of SAPK activity without
- 2148898
- 23 -
concomitant activation of MAPKs mirrors the results with
cytokine TNF-~, and indicates a similar inflammation-
mediated pathway.
Ischemia/Reperfusion Induces p54 SAP~s
An additional distinction for the p54 SAPKs from
the p42/44 MAPKs is the time course of activation in
response to reperfusion of ischemic tissue. Rat kidney
was made ischemic in vivo by the methods described above.
The results of this experiment (Fig. 12) showed that the
10 p54 SAPKs and the p42/44 MAPKs both were activated
immediately upon reperfusion of the kidney, and that the
activation of p54 SAPKs peaked after about 20 min (Fig.
12a), while the p42/44 MAPKs peaked at about 5 min after
initiation of reperfusion (Fig. 12b). The duration of
15 p54 SAPK activation was also considerably longer, lasting
considerably longer than 90 min before returning to
control levels, whereas p42/44 MAPK activation returned
to control levels within 20 min.
These findings have important implications in the
20 pathogenesis of chronic problems such as acute renal
failure, infarction arising from ischemic heart disease,
and surgical induction of ischemia/reperfusion injury.
The p54 SAPKs clearly play a more significant role in
tissue stresses such as ischemia than do the p42/44
25 MAPKs, and their effects are more sustained. These
results are the first to delineate a signal transduction
cascade specifically activated during reperfusion of
ischemic tissue. It is known that c-fos and some heat
shock protein genes are transcribed during reperfusion,
30 and that ischemia results in the generation of oxygen
radicals and can lead to tissue damage. The data
presented here indicate that SAPKs are activated during
reperfusion, and SAPKs target c-Jun. Tissue repair
genes, such as the collagenase gene, are regulated by AP-
2148898
~.
- 24 -
1, and thus, SAPK activation may represent the initiation
of repair processes at the molecular level.
Therapeutic Drug Screening with pS4 SAPKs
The experimental approaches described above can be
S used to screen compounds to identify those with
therapeutic potential. Two examples of possible
screening protocols follow. These examples are not
intended to be limiting.
Exampl e
Human (or other mammalian) cell lines such as
HepG2, CCD-18Co, U937, and HT-29 can be treated with any
test compound, and treated with SAPK activators (e.g.,
TNF-~, IL~ , ATP-depletion/refeeding analogous to
ischemia/reperfusion, etc.). The order of treatment can
15 be test compound, then SAPK-activator; SAPK activator
then test compound, or both treatments simultaneously.
Treatment times can vary from 1 min to several hours,
depending on the time necessary to induce changes in the
SAPK pathway.
The cells are then extracted, and the endogenous
SAPKs are immunoprecipitated as described above. The
SAPKs are then tested in a st~n~rd c-Jun kinase assay
for activity. This method of assaying for SAPK
activation can also be employed with tissue samples
25 obtained from patient biopsy, and has been used
successfully to detect the activity of SAPKs from samples
of rat kidney. In this way, therapeutics can be
evaluated for their ability to activate or inhibit the
SAPK pathway.
30 Example 2
Any of the human or rat SAPK clones may be used in
an expression vector for recombinant bacterial (or viral,
etc.) expression of inactive forms of the SAPK. Because
of the high homology between rat and human SAPKs, either
21~8898
-
- 25 -
may be used in the assay for therapeutic compounds.
Human cell lines such as HepG2, CCD-18Co, U937, and HT-29
can be treated with any compound to evaluate its toxicity
and efficacy as a therapeutic in modulating the SAPK/c-
5 Jun kinase pathway.
Cells are treated with the test compound either
alone or with a SAPK activating compound (such as TNF-~,
etc.; the activator may be applied before or concurrently
with the application of the test compound), and then
10 extracted to produce a cytoplasmic lysate. This extract
is then combined with the recombinantly expressed,
inactive SAPK. The SAPK can be expressed as a GST fusion
protein so that it is easily purified on glutathione
agarose. After a suitable length of time (minutes to
15 hours) for interaction between the cell lysate and the
recombinant inactive SAPK, the recombinant SAPK is
isolated by binding to glutathione beads (leaving behind
endogenous SAPKs) or by immunoprecipitation with an anti-
glutathione antibody (commercially available; any other
20 isolation technique may be used also), and assayed for c-
Jun kinase activity. If there is no c-Jun kinase
activity, and the test compound was applied to the cells
alone, then it may be concluded that this compound does
not activate SAPKs. If the test compound was applied
25 with a SAPK activator, then it may be concluded that the
test compound inhibits the activation of the SAPK
pathway. If c-Jun kinase activity is detected, and the
test compound was applied to the cells alone, then the
compound activates the SAPK pathway. If the test
30 compound was applied with a SAPK activator, and c-Jun
kinase activity is detected, then it may be concluded
that a) the compound does not inhibit the SAPK pathway,
b) the compound may enhance SAPK activity (which can be
quantitatively evaluated), or c) the drug has no effect.
214~898
- 26 -
Both of these screening systems allow rapid and
extensive testing of many compounds, regardless of their
preconceived potential therapeutic use, and may
facilitate identification of therapeutics which
5 accelerate tissue repair (increased SAPK pathway
activation), or prevent or alleviate allergy, excess
swelling, anaphylaxis, etc. (decreased SAPK pathway
activation). An example of this assay is shown in Figure
13, using TNF-~ as the test compound, and showing
10 controls with the expressed SAPK alone, and the HepG2
cell extract alone to demonstrate that the recombinant
form is inactive as expressed, and that the cell extract
has no intrinsic activity in the assay.
Summary of Results
This invention identifies the molecular structures
of a new subfamily of proline-directed protein kinases
whose upstream activators include heat stress, protein
synthesis inhibitors, ischemia/reperfusion injury, and
the inflammatory cytokines, TNF-~ and IL-1-~. This array
20 of regulatory inputs suggests that this kinase subfamily
may serve to monitor and form part of the cellular
response to a variety of intracellular and extracellular
stress signals. The p42/p44 MAPKs, although capable of
being activated by these stress treatments, are to a much
25 greater degree activated by mitogens (see Table 2 for
summary of results). The mitogenic agents such as EGF,
FGF and PMA, which have been shown to activate the
p42/p44 MAPKs through a Ras and c-Raf-l-dependent
pathway, activate the SAPKs weakly if at all. This
30 suggests that the SAPKs, although reguiring both Ser/Thr
and Tyr phosphorylation for activity (Kyriakis et al.,
(1991), J. Biol . Chem., 266:10043-10046) as do the
p42/p44 MAPKs (Anderson et al . ), lie on an entirely
distinct signal transduction pathway. Consistent with
21~8898
-
- 27 -
this conclusion is our inability, using highly purified
MEK, to reactivate phosphatase-2A inactivated rat liver
SAPK, or to activate prokaryotic recombinant SAPK-~ under
conditions that provide near complete MEK activation of
5 comparable preparations of purified or recombinant
p42/p44 MAPK. The SAPKs are thus the proline-directed
kinase elements in what is likely to be a new protein
kinase cascade, the second such pathway uncovered in
mammalian cells. Three independently regulated protein
10 kinase cascades upstream of distinct proline-directed
kinases (FUS/RSS; HOG-1; MPR-l) have been uncovered thus
far in S. cerevisiae (Levin et al.). We propose that the
SAPK cascade is an important, perhaps central, component
of the cellular response system to TNF-~, analogous to
15 the role of the MAPK cascade in the response to
activation of receptor tyrosine kinases. Lipid
regulators derived from sphingomyelin hydrolysis may
participate at one of the earlier intracellular steps
upstream of the SAPKs; a similar role for phospholipid-
20 derived mediators upstream of the MAPKs has recently beenproposed (Cai et al., (1993), Mol. Cell. Biol., 13:7645-
7651).
2148~98
- 28 -
TABLE 2
Activation of p54 and MAPKs P42/44 In various cell
lines by various treatments. ND = not done.
p54 Activity p42/44
5 Cells Treatment (mU) MAPK
Activity
NIH3T3 Control 5.8 + 0.6 71.4
PMA 5.8 + 0.9 409.7
FGF 8.5 + 0.6 795.5
A23187 8.9 + 0.7 172.0
H2O2 5.9 + 0.1 272.2
Heat shock38.8 + 1.7 169.7
Cycloheximide57.1 + 4.1 419.9
HT-29 Control 20.7 + 1.7 95.0
EGF 35.7 + 1.4 576.0
Heat shock172.0 + 28.5 281.0
HT-29 Control 69.4 + 7.5 195.5
Tunicamycin803.9 + 77.1 480.9
CCD-18Co Control 7.0 + 1.0 12.2
PMA 8.8 + 0.7 601.0
EGF 13.0 + 2.1 394.0
Heat shock48.0 + 1.3 83.7
TNF-a 60.0 + 5.0 117.6
25 Primary Control 179 + 13 1294
Porcine PMA 479 + 58 3529
Hepatocytes EGF 665 + 58 4882
Heat shock697 + 49 2427
TNF-~, 10 min 1008 + 31 3064
TNF-~, 20 min 924 + 38 ND
HepG2 Control 29.8 + 4.3 ND
TNF-~ 434.8 + 36.4 ND
Sphingomyelinase 159.0 + 17.3 ND
Confluent cultures of NIH3T3 cells were
35 treated with H22 (5 mM, 15 min) phorbol-12-myristate-13-
acetate (PMA, 500 nM, 20 min) FGF (10 ng ml~1, 20 min),
A23187 (100 nM 20 min), heat (42C, 30 min) or
cycloheximide (200 ~M, 60 min). HT-29 cells were treated
with EGF (50 ng ml~ , 20 min), heat shock (42C, 30 min)
40 or tunicamycin (50 ~ ml~l). CCD-18Co Cells (100%
confluent) were treated with PMA 200 nM, 15 min), EGF (50
ng ml~1, 15 min), heat shock (42C, 40 min) or TNF-~ (50
ng ml~l, 20 min). Primary porcine hepatocytes were
treated the same as the CCD-18Co cells. HepG2 cells (80%
45 confluent) were treated with TNF-~ (50 ng ml~1, 15 min) or
2148898
- 29 -
S. aureus sphingomyelinase (100 U ml~l). lU 5. aureus
sphingomyelinase will hydrolyze 1 ~mole TNP-sphingomyelin
min~1 at pH 7.4, 37C. Cell lysis and immunoprecipitation
were performed as described in Kyriakis et al, Nature
5 358:417-421, 1992 (hereby incorporated by reference).
Precipitates were assayed for GST-Jun kinase activity as
follows. To 40 ~l of a 1:1 suspension of protein G
Sepharose beads containing immunocomplexed p54 were added
20 ~1 0.2 mg ml~l GST-C-Jun-1-135 (26,37) or 0.01 mg ml~
10 holo-c-Jun. [y-32P]ATP (100 ~M) and MgC12 (10 mM) were
added to start the reaction. The reaction was allowed to
proceed for 20 min at 30C at which time the reaction was
stopped with SDS sample buffer and the mixtures resolved
by SDS-PAGE. The 40-kDa GST-Jun band was excised and
15 counted by liquid scintillation spectrometry. p54 kinase
assays were performed in triplicate. Mean + SD are
shown. Another portion of extracts (1 ml) was subjected
to Mono-Q chromatography; fractions were assayed for MAPK
p42/44 activity as MBP kinase activity. A peak of
20 stimulated MBP kinase activity was always detected
eluting between 200 and 350 mM NaCl; total MAPK p42/44
activity was taken as MBP kinase activity in those
fractions combined. 1 U p54 activity will transfer 1
pmol min~1 PO4 from ATP to c-Jun. 1 U MAPK p42/44 will
25 transfer 1 pmol min~1 P04 from ATP to MBP.
A central conundrum in the field of signal
transduction is how agonists such as EGF and TNF-~ can
exert such different effects while apparently only
activating one MAPK (the p42/p44 MAPK) pathway. The data
30 in Figures 16 and 17, taken in combination with the known
differences in SAPK and p42/p44 MAPK substrate
specificity, suggest that signaling specificity may arise
in part from the differential recruitment of signaling
pathways such as the SAPK and p42/p44 MAPK pathways.
Whatever the identity of the elements that couple
heat shock, protein synthesis inhibition, the cytokine
receptor(s), and other cellular stresses to the SAPKs,
the results presented here support the contention that
the c-Jun polypeptide is a downstream target of the
40 SAPKs. The SAPKs are a group of c-Jun N-terminal
kinases. These kinases and members of the related
superfamily focus a broad variety of regulatory signals,
including phorbol esters, Ha-Ras, ultraviolet radiation
21~8898
-
- 30 -
(Pulverer et al.; Smeal et al.; Adler et al.; Hibi et
al .; Binétruy et al .; Devary et al ., ( 1992 ), Cel l,
71:1081-1091), heat shock, protein synthesis inhibitors
and cytokines into the phosphorylation of the c-Jun trans
5 activation domain. The multiplicity of these c-Jun
kinases and their activating inputs attest to the
diversity of c-Jun function, and the complexity of its
regulation. The role of c-Jun phosphorylation in the
heat shock response and in the actions of TNF-~ remain to
10 be clarified. The consequences of c-Jun mediated
transcriptional trans activation exhibit a great deal of
cell specificity, and whether the outcome of MAPK- or
SAPK-mediated c-Jun transactivation is mitogenesis,
growth inhibition or a stable new phenotype resulting
lS from altered gene expression is likely to depend on the
array of signaling components acting in concert with AP-
1.
Uses of the Invention
The extracellular signal regulated family of
20 kinases are activated in response to different
extracellular stimuli, and this specificity allows cells
to diverge in their function in order to target a
response to a given stimulus. The molecules of the
invention can intervene in or stimulate a basic pathway
25 modulating transcription of proteins that mediate an
inflammatory or cell-stress response. Their use in the
treatment and prevention of inflammation and the
deleterious effects of hypoxia, heat stress, reperfusion
injury, and other tissue insults may resolve many
30 difficulties that arise with current therapies that rely
on amelioration of the aftermath of damage instead of
being able to redirect the course of the syndrome.
An additional use for these molecules may be in
the upregulation of IL-2, which has proven useful in the
21~88~8
- 31 -
treatment of cancer. AP-1 activation is known to induce
IL-2, and since c-Jun is a component of the AP-1 dimer,
p54 phosphorylation of c-Jun would be expected to
modulate AP-l levels. p54 immunodepletion experiments
5 have been shown to have a major effect on the
phosphorylation of c-Jun.
A use for the p54 kinases is as a template for
drug design, or a functional component for therapeutic
drug assays. Many functional motifs of the molecules are
10 already known (e.g., the ATP-binding site, sites of
regulatory phosphorylation, c-Jun binding site, etc.)
which, if an effective antagonist or agonist could be
derived, could play an important role in therapies for
conditions such as are listed above. Assays to screen
15 for such drugs exist, and are described above.
Monitoring the enzymatic activity following incubation
with potentially therapeutic compounds such as was done
with the drugs described herein would allow a simple,
fast method to determine efficacy and dose-response for
20 up or down regulation of the SAPKs. Large numbers of
candidate compounds can be screened easily and evaluated
for specificity, efficacy, and toxicity. Such
information would allow rational evaluation of many drugs
for their ability to up- or down-regulate cellular
25 responses to physiological stresses, and be useful in
clinical management of inflammation, ischemia, and many
other stimuli that activate the SAPKs.
The molecules of the invention may be useful for
reducing inflammation in such chronic disorders as
30 autoimmune diseases or allergies, and in acute conditions
such as anaphylactic shock, or soft tissue injury where
swelling may aggravate the condition (e.g, around broken
bones). Other uses include prophylactic treatment of
patients about to undergo surgeries where there is a high
35 likelihood of ischemia-reperfusion injury (e.g., vascular
2148898
-
- 32 -
surgery, organ transplants, etc.), or treatment of sepsis
and fever.
For such conditions as mentioned above, a
systemic application of the molecules of the invention,
5 or antibodies derived from them, is generally desirable,
although local application may be more appropriate in
certain cases. Transport of the molecules to their site
of action may be effected by, for example, liposome
delivery systems, antisense technology, plasmid or
10 retroviral vectors, or any of a number of other methods
known in the art. The vehicle for application may be any
excipient compatible with the molecules and the health of
the patient.
214889~
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: KYRIAKIS, JOHN M.
AVRUCH, JOSEPH
BANERJEE, PAPIA
WOODGETT, JAMES R.
(ii) TITLE OF INVENTION: p54 STRESS-ACTIVATED PROTEIN
KINASES
(iii) NUMBER OF SEQUENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FISH & RICHARDSON
(B) STREET: 225 FRANKLIN STREET
(C) CITY: BOSTON
(D) STATE: MA
(E) COUNTRY: US
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COM~ K: 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: US UNASSIGNED
(B) FILING DATE: 04-MAY-1994
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CLARK, PAUL C.
(B) REGISTRATION NUMBER: 30,162
(C) REFERENCE/DOCKET NUMBER: 00786/217001
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617/542-5070
(B) TELEFAX: 617/542-8906
(C) TELEX: 200154
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
2148898
-
- 34 -
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Ser Asp Ser Lys Ser Asp Gly Gln Phe Tyr Ser Val Gln Val Ala
1 S 10 15
Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Gln Leu Lys Pro Ile
Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Phe Asp Thr Val Leu
Gly Ile Asn Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln
Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Leu Lys Cys Val
Asn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys
Thr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp
100 105 110
Ala Asn Leu Cys Gln Val Ile His Met Glu Leu Asp His Glu Arg Met
115 120 125
Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser
130 135 140
Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys
145 150 155 160
Ser Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala
165 170 175
Cys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile
195 200 205
Trp Ser Val Gly Cys Ile Met Ala Glu Met Val Leu His Lys Ser Cys
210 215 220
Ser Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln
225 230 235 240
Leu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro Thr Val
245 250 255
Arg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys Phe Glu
260 265 270
Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu Arg Asp Lys
275 280 285
Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile
290 295 300
2148898
Asp Pro Asp Lys Arg Ile Ser Val ABP Glu Ala Leu Arg His Pro Tyr
305 310 315 320
le Thr Val Trp Tyr Asp Pro Ala Glu Ala Glu Ala Pro Pro Pro Gln
325 330 335
le Tyr Asp Ala Gln Leu Glu Glu Arg Glu His Ala Ile Glu Glu Trp
340 345 350
Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Trp Glu Glu Arg Ser Lys
355 360 365
Asn Gly Val Lys Asp Gln Pro Ser Asp Ala Ala Val Ser Ser Lys Ala
370 375 380
Thr Pro Ser Gln Ser Ser Ser Ile Asn Asp Ile Ser Ser Met Ser Thr
385 390 395 400
Glu His Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Asp Ala Ser Thr
405 410 415
Gly Pro Leu Glu Gly Cys Arg
420
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 423 amino acids
~B TYPE: amino acid
C STRANDEDNESS: single
D, TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Asp Ser Lys Ser Asp Gly Gln Phe Tyr Ser Val Gln Val Ala
1 5 10 15
Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Gln Leu Lys Pro Ile
Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Phe Asp Thr Val Leu
Gly Ile Asn Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln
Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Leu Lys Cys Val
sn Hi~ Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys
hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp
100 105 110
Ala Asn Leu Cys Gln Val Ile His Met Glu Leu Asp His Glu Arg Met
115 120 125
Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser
130 135 140
Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys
145 150 155 160
21~8898
- 36 -
er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala
165 170 175
ys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile
195 200 205
Trp Ser Val Gly Cys Ile Met Gly Glu Leu Val Lys Gly Cys Val Ile
210 215 220
Phe Gln Gly Thr Asp His Ile Asp Gln Trp Asn Lys Val Ile Glu Gln
225 230 235 240
eu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro Thr Val
245 250 255
rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys Phe Glu
260 265 270
Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu Arg Asp Lys
275 280 285
Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile
290 295 300
Asp Pro Asp Lys Arg Ile Ser Val Asp Glu Ala Leu Arg His Pro Tyr
305 310 315 320
le Thr Val Trp Tyr Asp Pro Ala Glu Ala Glu Ala Pro Pro Pro Gln
325 330 335
le Tyr Asp Ala Gln Leu Glu Glu Arg Glu His Ala Ile Glu Glu Trp
340 345 350
Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Trp Glu Glu Arg Ser Lys
355 360 365
Asn Gly Val Lys Asp Gln Pro Ser Asp Ala Ala Val Ser Ser Lys Ala
370 375 380
Thr Pro Ser Gln Ser Ser Ser Ile Asn Asp Ile Ser Ser Met Ser Thr
385 390 395 400
Glu His Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Asp Ala Ser Thr
405 410 415
Gly Pro Leu Glu Gly Cys Arg
420
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 426 amino acids
B TYPE: amino acid
,C STRANDEDNESS: single
l,DI TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ser Lys Ser Lys Val Asp Asn Gln Phe Tyr Ser Val Glu Val Gly
1 5 10 15
2148898
- 37 -
sp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile
Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Val Leu
Asp Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln
Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val
sn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys
hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp
100 105 110
Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met
115 120 125
Ser Tyr Leu Leu Tyr Gln Met Leu Ser Ala Ile Lys His Leu His Ser
130 135 140
Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys
145 150 155 160
er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala
165 170 175
ly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile
195 200 205
Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Arg His Lys Ile Leu
210 215 220
Phe Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln
225 230 235 240
eu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val
245 250 255
rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Leu Thr Phe Pro
260 265 270
Lys Leu Phe Pro Asp Ser Leu Phe Pro Ala Asp Ser Glu His Asn Lys
275 280 285
Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile
290 295 300
Asp Pro Ala Lys Arg Ile Ser Val Asp Asp Ala Leu Gln His Pro Tyr
305 310 315 320
le Asn Val Trp Tyr Asp Pro Ala Glu Val Glu Ala Pro Pro Pro Gln
325 330 335
le Tyr Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp
340 345 350
ys Glu Leu Ile Tyr Lys Glu Val Met Asn Ser Glu Glu Lys Thr Lys
355 360 365
21~8898
- 38 -
Asn Gly Val Val Lys Gly Gln Pro Ser Pro Ser Gly Ala Ala Val Asn
370 375 380
Ser Ser Glu Ser Leu Pro Pro Ser Ser Ser Val Asn Asp Ile Ser Ser
385 390 395 400
Met Ser Thr Asp Gln Thr Leu Ala Ser Asp Thr Asp Ser Ser Leu Glu
405 410 415
la Ser Ala Gly Pro Leu Gly Cys Cys Arg
420 425
2) INFORMATION FOR SEQ ID NO:4:
(i) ~U~N~: CHARACTERISTICS:
'A' LENGTH: 385 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
D, TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Ser Lys Ser Lys Val Asp Asn Gln Phe Tyr Ser Val Glu Val Gly
1 5 10 15
sp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile
Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Val Leu
Asp Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln
Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val
sn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys
hr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp
100 105 110
Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met
115 120 125
Ser Tyr Leu Leu Tyr Gln Met Leu Ser Ala Ile Lys His Leu His Ser
130 135 140
Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys
145 150 155 160
er Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala
165 170 175
ly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile
195 200 205
Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Arg His Lys Ile Leu
210 215 220
2148Q38
- 39 -
Phe Pro Gly Arg Asp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln
225 230 235 240
eu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val
245 250 255
rg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Leu Thr Phe Pro
260 265 270
Lys Leu Phe Pro Asp Ser Leu Phe Pro Ala Asp Ser Glu His Asn Lys
275 280 285
Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile
290 295 300
Asp Pro Ala Lys Arg Ile Ser Val Asp Asp Ala Leu Gln His Pro Tyr
305 310 315 320
le Asn Val Trp Tyr Asp Pro Ala Glu Val Glu Ala Pro Pro Pro Gln
325 330 335
le Tyr Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp
340 345 350
ys Glu Leu Ile Tyr Lys Glu Val Met Asn Ser Glu Glu Lys Thr Lys
355 360 365
sn Gly Val Val Lys Gly Gln Pro Ser Pro Ser Xaa Xaa Gly Ala Ala
370 375 380
Val
385
(2) INFORM~TION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
A' LENGTH: 411 amino acids
B TYPE: amino acid
,C STRANDEDNESS: single
,DI TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Ser Arg Ser Ly~ Arg Asp Asn Asn Phe Tyr Ser Val Glu Ile Ala
1 5 10 15
Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu Lys Pro Ile
Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp Ala Ile Leu
Glu Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Asn Gln
Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met Lys Cys Val
Asn His Lys Asn Ile Ile Gly Leu Leu Asn Val Phe Thr Pro Gln Lys
Ser Leu Glu Glu Phe Gln Asp Val Tyr Ile Val Met Glu Leu Met Asp
100 105 110
2148898
- 40 -
Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu Asp His Glu Arg Met
115 120 125
Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser
130 135 140
Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys
145 150 155 160
Ser Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala
165 170 175
Gly Thr Ser Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Leu
195 200 205
Trp Ser Val Gly Cys Ile Met Gly Glu Met Val Cys Leu Lys Ile Leu
210 215 220
Phe Pro Gly Arg Asp Tyr Ile ABP Gln Trp Asn Lys Val Ile Glu Gln
225 230 235 240
Leu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val
245 250 255
Arg Thr Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Tyr Ser Phe Glu
260 265 270
Lys Leu Phe Pro Asp Val Leu Phe Pro Ala Asp Ser Glu His A~n Lys
275 280 285
Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile
290 295 300
Asp Ala Ser Lys Arg Ile Ser Val Asp Glu Ala Leu Gln His Pro Tyr
305 310 315 320
Ile Asn Val Trp Tyr Asp Pro Ser Glu Ala Glu Ala Pro Pro Pro Lys
325 330 335
Ile Pro Asp Lys Gln Leu Asp Glu Arg Glu His Thr Ile Glu Glu Trp
340 345 350
Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Leu Glu Glu Arg Thr Lys
355 360 365
Asn Gly Val Ile Arg Gly Gln Pro Ser Pro Leu Gly Ala Ala Val Ile
370 375 380
Asn Gly Ser Gln His Pro Val Ser Ser Pro Ser Val Asn Asp Met Ser
385 390 395 400
Ser Met Ser Thr Asp Pro Thr Leu Ala Ser Asp
405 410
(2) lNrOR~ATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 17 amino acids
B TYPE: amino acid
C, STRANDEDNESS: single
,D TOPOLOGY: linear
2148898
- 41 -
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Asp Ala Ala Val Ser Ser Lys Ala Thr Pro Ser Gln Ser Ser Ser
1 5 10 15
Ile
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 21 amino acids
B TYPE: amino acid
,C STRANDEDNESS: single
l,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Gly Ala Ala Val Asn Ser Ser Glu Ser Leu Pro Pro Ser Ser Ser Val
1 5 10 15
Gln Pro Ser Pro Ser
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 20 amino acids
B TYPE: amino acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ser Pro Leu Gly Ala Ala Val Ile Asn Gln Ser Gln His Pro Val Ser
1 5 10 15
Ser Pro Ser Val
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 2629 base pairs
B TYPE: nucleic acid
C, STRANDEDNESS: single
~D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GGAl~CC~ ATGACACTAC ATCATGAGTG ACAGTA~AAG CGATGGCCAG TTTTACAGTG 60
TGCAAGTGGC AGACTCAACT TTCACTGTTC TAAAACGTTA CCAGCAGTTG AAACCAATTG 120
21~88~8
- 42 -
GCTCTGGAGC CCAAGGAATT G~..~lGCTG CTTTTGATAC A~.~-.GGA ATAAATGTTG 180
CTGTCAAGAA GTTAAGTCGT C~....CAGA ACrAAACGCA TGrAAAr-Ar-A GCCTACCGTG 240
AA~ C~. CCTAAAGTGT GTCAATCATA AAAATATAAT TAG~..~..A AA~ ~A 300
rACrArAAAA AACGCTAGAA GAATTCCAAG ATGTGTACTT GGTTATGGAG TTAATGGACG 360
CTAACTTATG TCAGGTTATT CATATGGAGC TGGACCATGA AAGAATGTCA TAC~.C~ ~. 420
AcrAr,ATGCT TTGTGGCATT AAGCACCTGC ATTCAGCTGG CATAATTCAT AGGGATTTGA 480
AGCCTAGCAA CATTGTAGTA AAATCAGACT GTACTCTCAA GATCCTTGAC TTTGGCCTGG 540
CACGGACAGC CTGTACCAAC TTTATGATGA ~.CC~.ATGT GGTAACTCGC TACTATCGGG 600
CTCr-Ar-AAr,T CATCCTGGGC ATGGGCTACA AGGAGAATGT GGACATCTGG ~ ,.CGGC,~ 660
GCATCATGGC AGAAATGGTC CTCrATAAAT C~.~..CCCC AGr-AAr-Ar-AC TATATTGATC 720
AATGGAATAA AGTTATTGAA CAGCTAGGAA CACCATCCGC AGAGTTCATG AAGAAACTTC 780
AGCCAACTGT AAGGAATTAT GTGGAAAAÇA GACrAAAr,TA CCCTGGAATC AAATTTGAAG 840
AG~.~...CC AGATTGGATA TTTCCGTCAG AATCCGAACG Ar7ArAAAATA AAAArAAr,Tc goo
AAGCrAGAr,A .~.~..ATCG AAAATGTTAG TGATTGATCC GGACAAGCGG A.~.~.~,.GG 960
ACGAAGCCTT GCGCCACCCG TATATTACTG TTTGGTATGA CCCCGCTGAA GCAGAAGCGC 1020
rACrACCTCA AATTTATGAT GCCCAGTTGG AArAAAr,AÇA GCATGCGATT GAAGAGTGGA 1080
AAr-AAcTAAT TTAr~AAAr7AA GTGATGGACT GGr7AAr-AAAr7 AAGçAAGAAT GGGGTGAAAG 1140
ACCAGCCTTC AGATGCAGCA GTAAGCAGCA AGGCTACTCC TTCTCAGTCG TCATCCATCA 1200
ATGACATCTC ATCCATGTCC ACTGAGCACA CCCTGGCCTC AGAC'-ACAGAC AGCAGTCTCG 1260
ATGCCTCAAC CGGACCCCTG GAAGGCTGCC GATGAAACCT CGCAGATGGC GCA~..~.~. 1320
GTGAAGGACT CTGGCTTCCA TGGCCCTGAG CACATGGGAG CTGGTGGAAC AAATCAAGAA 1380
GCTCCATGTT CTGCATGTAA rAAArACrAr GCCTTGCCCC CACTCAGTTC CAGTAGGATT 1440
GCCTGCGTAG ACTGTAACAT GAGGCAGACG A.~.~.GGAG AAAAAGTACA AACrArACTG 1500
TTAr,AAATTT TGTTCAAGAT CATTCAGGTG AGCAATTAGA ATAGCCGAGT .~.l..CAAG 1560
.C~.~.GGTG TCCTTGGTGA CAGATCATGT GTAACTGTGG GGACTCGTAT GCATGTGACC 1620
ACAAATGCTT GCTTGAACTT GCCCATGTAG CACTTTGGGA ATCAGTATTT AAATGCCAAA 1680
TAA.~..C~A GGTAGTTCTG CTTCTAGAAT AA.~.~ .AA .C~.C...AG TAATTTGGTG 1740
.~.~.C~ACA AAAAAATArA TTA.~.~.~,. ATGAATTGGC CACTATCATA TTATCATATT 1800
TTACCCACTT TTATGGTATG ATTTATTCTG ~.-..~-AT TTCAGAAGGA ~TATAATTAA 1860
ATTTATTTAA TAAATAAAAC TACAGCTTTT CTTAAATTTG TGA.~-....A GGCTGAGAAT 1920
TACCACTGCT TTATATCGAC A~.~l~.~,.C CTTTAAACTG CCCACTATGG GAAACTTTAC 1980
GTACAGCTTT CTGCATGACA AAGTTCCAAG TTGTATTTCA CTCTGCTTAA CGACTTATGT 2040
CACCTTGAAT CCTr-ACr-ArA CA...C~... TTCTTGGTCC TCTGAACTTG GATCTAGAAT 2100
CCCTCACAGA ACTTCACCTT CTTTATCACA AAGCACCCCA TCTCAGTAGA ATGAATCGGC 2160
21~8898
- 43 -
AGATTCCTGA GCCCCGCTGC CTAATGTAGA GCTGACAGGG TGG~,,CCCC AGAACGGTGG 2220
GTGGGTGCAT C~1~CC~GA GCCCACCCAT CCTTTGCTCC C~ A TTTAAGGTGA 2280
AAGGTGATTG GGTCTCATAG C~C~1~ TGTAGCATTG CCTAACTTGT ~ ACT 2340
r~rArAAGCC ACCACGTCCA GCCAGAGCAC ATG~ ,, AGGAGACCGG GCTTACTTAC 2400
CATGCATGTT TGCTGCTGTC ~ C~ATT TTGTGGAGGC A~1~C~ TCTAAGGGAA 2460
~ C~CAGAT GTTCTAGAAA CATTCAGAAG AACGCAGAAG AAATATTCTA GAGAATTGGG 2520
GGTTCATTCT TGAATATTTT CTGATTTAAA ACTGCTCACC TGAAATTGAT ACTTTCAGAT 2580
CCTGATCTTG TAAATTACTC GAGATTTGGT AAGATGCTGA ~ C~V~ 2629
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
~A LENGTH: 2629 ba~e pair~
~B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
~D TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGA, C~,~, ATGACACTAC ATCATGAGTG ACAGTAAAAG CGATGGCCAG TTTTACAGTG 60
TGCAAGTGGC AGACTCAACT TTCACTGTTC TAAAACGTTA CCAGCAGTTG AAAcrAATTG 120
GCTCTGGAGC CCAAGGAATT ~,,,~,GCTG CTTTTGATAC AG1~ GGA ATAAATGTTG 180
CTGTCAAGAA GTTAAGTCGT C~,,,.~AGA ACrAAACGCA TGrAAA~Ar,A GCCTACCGTG 240
AA~,,~,C~, CCTAAAGTGT GTCAATCATA AAAATATAAT TAG~,,~A AA~ A 300
rArrAr~AAA AACGCTAGAA GAATTCCAAG ATGTGTACTT GGTTATGGAG TTAATGGACG 360
CTAACTTATG TCAGGTTATT CATATGGAGC TGGACCATGA AAGAATGTCA TAC~,C~,~-, 420
ACCAGATGCT TTGTGGCATT AAGCACCTGC ATTCAGCTGG CATAATTCAT AGGGATTTGA 480
AGCCTAGCAA CATTGTAGTA AAATCAGACT GTACTCTCAA GATCCTTGAC TTTGGCCTGG 540
CACGGACAGC CTGTACCAAC TTTATGATGA CTCCCTATGT GGTAACTCGC TACTATCGGG 600
CTCrAr~A~T CATCCTGGGC ATGGGCTACA AGGAGAATGT TGATATCTGG TCAGTGGGTT 660
GCATCATGGG AGAGCTGGTG AAAGGTTGTG TGATATTCCA AGGTACTGAC CATATTGATC 720
AATGGAATAA AGTTATTGAA CAGCTAGGAA CACCATCCGC AGAGTTCATG AAr-AAACTTC 780
AGCCAACTGT AAGGAATTAT GTGGAAAACA GACCAAAGTA CCCTGGAATC AAATTTGAAG 840
AG~ CC AGATTGGATA ,,-CC~,CAG AATCCGAACG A~rAAAATA AAAArAA~TC 900
AAGCCAGAGA 1~ ATCG AAAATGTTAG TGATTGATCC GGACAAGCGG A.~.~.~.GG 960
ACGAAGCCTT GCGCr~CCCG TATATTACTG TTTGGTATGA CCCCGCTGAA GCAGAAGCGC 1020
r~rrACCTCA AATTTATGAT GCCCAGTTGG AA~AAA~A~A GCATGCGATT GAAGAGTGGA 1080
AAGAACTAAT TTArAAA~AA GTGATGGACT GG~AA~AAA~ AAGrAA~AT GGGGTGAAAG 1140
21488~8
- 44 -
ACCAGCCTTC AGATGCAGCA GTAAGCAGCA AGGCTACTCC TTCTCAGTCG TCATCCATCA 1200
ATGACATCTC ATCCATGTCC ACTGAGCACA CCCTGGCCTC Ar,ACAÇAr-AC AGCAGTCTCG 1260
ATGCCTCAAC CGGACCCCTG GAAGGCTGCC GATGAAACCT CGCAGATGGC GCA~ll~l`l 1320
GTGAAGGACT CTGGCTTCCA TGGCCCTGAG CACATGGGAG CTGGTGGAAC AAATCAAGAA 1380
GCTCCATGTT CTGCATGTAA r-AAA~ACGAC GCCTTGCCCC CACTCAGTTC CAGTAGGATT 1440
GCCTGCGTAG ACTGTAACAT GAGGCAGACG ATGTCTGGAG AAAAAGTACA AACCAÇACTG 1500
TTAr-AAATTT TGTTCAAGAT CATTCAGGTG AGCAATTAGA ATAGCCGAGT l~ AAG 1560
l~l~lGGTG TCCTTGGTGA CAGATCATGT GTAACTGTGG GGACTCGTAT GCATGTGACC 1620
ACAAATGCTT GCTTGAACTT GCCCATGTAG CACTTTGGGA ATCAGTATTT AAATGCCAAA 1680
TAA.`llC~A GGTAGTTCTG CTTCTAGAAT AA.~l~llAA ~C~l~lllAG TAATTTGGTG 1740
.~l~lC~ACA AAAAAATpr.A TTA~l~l~l ATGAATTGGC CACTATCATA TTATCATATT 1800
TTACCr-Ar,TT TTATGGTATG ATTTATTCTG l~ ~lAT TTCAGAAGGA ATATAATTAA 1860
ATTTATTTAA TAAATAAAAc TACAGCTTTT CTTAAATTTG TGAl~llllA GGCTGAGAAT 1920
TACCACTGCT TTATATCGAC A~l~l~l~lC CTTTA~ACTG CCCACTATGG GAAACTTTAC 1980
GTACAGCTTT CTGCATGACA AAGTTCCAAG TTGTATTTCA CTCTGCTTAA CGACTTATGT 2040
CACCTTGAAT CCTr-ACr-AÇA CA.~lC~lll TTCTTGGTCC TCTGAACTTG GATCTAGAAT 2100
CCCTCACAGA ACTTCACCTT CTTTATCACA AAGr-AcccrA TCTCAGTAGA ATGAATCGGC 2160
AGATTCCTGA GCCCCGCTGC CTAATGTAGA GCTGACAGGG TGG~lCCCC AGAACGGTGG 2220
GTGGGTGCAT C~llCC~lGA GCCCACCCAT CCTTTGCTCC C~l`l`ll lA TTTAAGGTGA 2280
AAGGTGATTG GGTCTCATAG C~lllC~.l' TGTAGCATTG CCTAACTTGT ~ lCACT 2340
~-A~AGAAGCC ACCACGTCCA GCCAGAGCAC ATG~l~l~ll AGr-Ar-ACCGG GCTTACTTAC 2400
CATGCATGTT TGCTGCTGTC ~l lllC~ATT TTGTGGAGGC AlllC~llll TCTAAGGGAA 2460
llC~l~AGAT GTTCTAGAAA CATTCAGAAG AACGCAGAAG AAATATTCTA GAGAATTGGG 2520
GGTTCATTCT TGAATATTTT CTGATTTAAA ACTGCTCACC TGAAATTGAT ACTTTCAGAT 2580
CCTGATCTTG TAAATTACTC GAGATTTGGT AAGATGCTGA Gll`l~l~ 2629
~2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
'A LENGTH: 1975 base pairq
B TYPE: nucleic acid
C STRANDEDNESS: ~ingle
D, TOPOLOGY: 1inear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCClC~llAT TCCGGTTTGG AATGTGGCTA ATGAAAGCCC AGTAGGAGGA TTTCTGGGGC 60
AAACAGGTGG ACCAGGATCC TG~l~l~AG GCACGGAATG GCTATTGTGA GAGCGCCACC 120
21~8898
- 45 -
AGCAGGACCA TCGCAGATCT TGGTTATGGC TGCTCACGCA AGAGGCTGTT GATGTAGACC 180
CC~...CCCG TAGATGAGAA ATÇAÇAC~AG CAGTGGTATT TATGAGCCTC CA... AT 240
ACTACTGCAG TGAAC~AACC TTGGATGTGA AAATTGCCTT TTGTCAGGTG .~7.~..C~.. 300
ACAGGTAAAA CAAAGGGATT CGAÇAAAÇA~ GTGGATGTGT ~..~ ~7..~. ÇAAAÇATTAC 360
AACATGAGCA AAAGCAAGGT Ar-ATAACCAG TTCTACAGTG TGGAAGTGGG AGACTCAACC 420
TTCACAGTTC TAAAGCGCTA C~AGAACCTG AAGCCGATCG GCTCTGGGGC TCAGGGAATA 480
~...~.GCTG CGTATGACGC l~.C~lCGAC AGAAATGTGG CCATTAAGAA GCTCAGCAGA 540
CC~..CCAGA ACCAAACTCA TGC~AA~-AGG GCTTACCGGG AGCTGGTCCT CATGAAGTGT 600
GT~-~ACcATA AAAA~-ATTAT TAGCTTATTA AA.~ A ~-Accc~A~-~A AACACTGGAG 660
GAGTTCCAAG ATGTTTACTT AGTGATGGAA CTGATGGACG CCAACTTGTG TCAGGTGATT 720
CAGATGGAGC TG~-AC~AC~-A GCGGATGTCG TACTTGCTGT ACCAGATGCT GTCGGCGATC 780
AAACACCTCC ACTCCGCTGG GATCATCCAC AGGGACTTAA AACCCAGTAA CATCGTAGTC 840
AAGTCTGATT GCACACTGAA AATCCTGGAC TTTGGACTGG CCAGGACAGC GGGCACAAGC 900
TTCATGATGA ~lCCG.ATGT GGTG~C~-A~-~ TATTAcA~-AG CCCCCGAGGT CATCCTGGGC 960
ATGGGCTACA AG~-A~-AACGT G~Ac~TATGG TCTGTGGGCT GCATCATGGG AGAAATGGTT 1020
CGTCAC~AAA .C~l..CC CGGAAGGGAC TATATTGACC AGTGGAAÇAA AGT~ATAGAG 1080
CAGCTAGGAA ~lCC~l~.CC AGAATTCATG AA~AAATTGC AGCCCACCGT ~A~-AAACTAC 1140
GTG~-A~-AACC GGCCCAAGTA TGCAGGCCTC ACCTTCCCCA AG~.~l.lCC AGAl.CC~.C 1200
TTCCCAGCGG ATTCCGAGCA ~AATAAACTT AAAGCCAGCC AAGCCAGGGA ~ 7.CA 1260
AAGATGTTAG TGATTGACCC AGCGAAr-AGG ATATCGGTGG ATGACGCATT GCAGCATCCG 1320
TACATCAACG TTTGGTACGA CCCTGCTGAA GTGGAGGCGC CTCCGCCTCA ~-ATATATGAC 1380
AAGCAATTGG ATGAAAGGGA GcAÇAC~ATC ~AA~AATGGA AAGAACTCAT CTACAAGGAA 1440
GTAATGAACT ~7AGAAGAGAA GACTAA~-AAC GGCGTAGTCA AAGGCCAGCC CTCACCTTCA 1500
GGTGCAGCAG TGAACAGCAG TGAGAGTCTC CCTCCATCCT CAl~-~l~AA CGACATCTCC 1560
TCCATGTCCA CC~-AC~A~-AC CCTCGCATCC GACACTGACA GCAGCCTGGA AGCCTCGGCG 1620
GGACCGCTGG Gll~7llGCAG GTGACTAGCC GCCTGCCTGC ~AAAcc~A~c ~ll~l ~AGG 1680
AGATGACGCC AT~-ATAGAAC ACAGCGCACA TGcAcAçAr-~ CAGAGCTTGT AcAcAcAcAC 1740
A~ArA~ACAC A~AC~CGCAC GCACGCACGC ACGCAAGCAC GCACGCACGC ACAAATGCAC 1800
TCACGCAATG T~-AA~-AA~AA A~AAAGTAGC GA~-A~-AG~GC GAGA~-AGCCA ACGTAAAACT 1860
AAGTTAAATC TTTCTGCGTG ~ll lC~AGA Gll~l~lATC GCAGCTGAGC TGAAATGTAT 1920
ACTTAACTTC TAGTCGCGCT CGCTCGACTT TGGl~lCC~l CCGGCAGTGC TTACT 1975
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1986 base pairs
`- 21~8898
-
- 46 -
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCCTATCCCT CCTTATTCCG GTTTGGAATG TGGCTAATGA AAGCCCAGTA GGAGGATTTC 60
TGGGGCAAAC AGGTGGACCA GGA.C~.G~ TCTCAGGCAC GGAATGGCTA TTGTGAGAGC 120
GC~A~-AGCA G~'AC~-ATCGC AGATCTTGGT TATGGCTGCT CACGCAAGAG GCTGTTGATG 180
TA~ACCCCCT ,CCC~7.AGA T~-A~-AAATCA CACGAGCAGT GGTATTTATG AGCCTCCATT 240
TCTTATACTA CTGCAGTGAA CCAACCTTGG ATGTGAAAAT TGC~.~ 7~ CAGGl~7 ~7~G 300
l~C~.ACAG GTAAAA~AAA GGGATTCGAC AAP~PCGTGG A~ 7~ ~C ~71~71 AAA 360
CATTACAA~A TGAGCAAAAG CAAGGTAGAT AACCAGTTCT ACAGTGTGGA AGTGGGAGAC 420
TCAACCTTCA CAGTTCTAAA GCGCTACCAG AACCTGAAGC CGATCGGCTC TGGGGCTCAG 480
GGAATAGTTT GTGCTGCGTA TGACGCTGTC CTC~-ACA~-AA ATGTGGCCAT TAA~-AAGCTC 540
AG~A~ACCCT TCCA~-AAC~A AACTCATGCC AAGAGGGCTT ACCGGGAGCT GGlCC-~ATG 600
AA~7~ 7LGA AC~ATPAAAA CATTATTAGC TTATTAAATG TCTTTACACC C~A~-AAAA~A 660
CTG~-A~,AGT TC~-AA~-ATGT TTACTTAGTG ATGGAACTGA TGGACGCCAA ~7~7L AG 720
GTGATTCAGA TGGAGCTGGA CCACGAGCGG Ai~lC~7lACT TGCTGTACCA GATGCTGTCG 780
GCGATCAAAC ACCTCCACTC CGCTGGGATC ATCCACAGGG ACTTAAAACC CAGTAACATC 840
GTAGTCAAGT CTGATTGCAC ACTGAAAATC CTGGACTTTG GACTGGCCAG GACAGCGGGC 900
ACAAGCTTCA TGATGACTCC GTATGTGGTG ACGAGATATT ACAGAGCCCC CGAGGTCATC 960
CTGGGCATGG GCTA~-AAr~r-A GAACGTGGAC ATATGGTCTG TGGGCTGCAT CATGGGAGAA 1020
ATG~7..C~7~C A~AAAATCCT ~.~CCCGGA AGGGACTATA TTGACCAGTG ~AA~AAAGTC 1080
ATAGAGCAGC TAGGAACTCC GTGTCCAGAA TTCATGAAGA AATTGCAGCC CACCGTCAGA 1140
AACTACGTGG A~AACCGGCC CAAGTATGCA GGCCTCACCT TCCC~AAGCT ~l..C~AGAT 1200
.CC~.~..CC CAGCGGATTC CGAGCACAAT AAACTTAAAG CCAGCCAAGC CAGGGACTTG 1260
TTGTCAAAGA TGTTAGTGAT TGACCCAGCG AAGAGGATAT CGGTGGATGA CGCATTGCAG 1320
CATCCGTACA TCAACGTTTG GTAC~-ACCCT GCTGAAGTGG AGGCGCCTCC GCCTCAGATA 1380
TATGACAAGC AATTGGATGA AAGGGAGCAC ACCATCGAAG AATGGAAAGA ACTCATCTAC 1440
AAGGAAGTAA TGAACTCAGA AGAGPAGACT AAGAACGGCG TAGTCAAAGG CCAGCCCTCA 1500
CCTTCAGCAC AGGTGCAGCA GTGAACAGCA GTGAGAGTCT CCCTCCATCC TCA.~.~7.~A 1560
AC~A~ATCTC CTCCATGTCC ACCGACCAGA CCCTCGCATC CGACACTGAC AGCAGCCTGG 1620
AAGCCTCGGC GGGACCGCTG G~7..~7..GCA GGTGACTAGC CGCCTGCCTG C~-AAACCCAG 1680
CG.. ~ AG GAGATGACGC CAT~-ATAGAA CACAGCGCAC ATGCACACAC ACAGAGCTTG 1740
21488~8
- 47 -
TArArAçAcA cAçArlAr-AçA r-~rArAcGcA CGCACGCACG CACGCAAGCA CGCACGCACG 1800
rlArAAATGCA CTCACGCAAT GTrAAGAAA~A- AAAAAAGTAG cr-Ar-ArArAr, Cr-AGAGAGCC 1860
AACGTAAAAC TAAGTTAAAT ~,, GCGT G~ C~AG AG~ ~ AT CGCAGCTGAG 1920
CTGAAATGTA TACTTAACTT CTAGTCGCGC TCGCTCGACT TTGG~CCC TCCGGCAGTG 1980
CTTACT 1986
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 1408 base pairs
B) TYPE: nucleic acid
C) STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GCGGCCGAGC GCGGGACGTT GCGGCCGAAA CGCGGAGCCG CGAGCAGGAT TAAGTAGCGG 60
CCCGGCCACC GGCACGGCGC CGCTCTCCGC TACTGGCTTC CAGGTCTCCG TTGGCTGCAC 120
TGCCGGCCGG TTGTTGAATA TTTGGATGAA GCCATTAGAC TAATTGCTTG CCATCATGAG 180
CAGAAGTAAA cGTr~Ar-AAr-~ ATTTTTATAG TGTArAr-ATC GCAGATTCTA CATTCACAGT 240
CcTAAAAcr-A TACCAGAACT TAAAGCCTAT AGGCTCAGGA GCTCAAGGAA TAG.~GC 300
AGCTTATGAT GCTATTCTTG AAAr-AAATGT TGCAATCAAG AAGCTCAGCC GGCCATTTCA 360
GAATCAGACC CATGCTAAGC GAGCCTACCG AGAACTAGTT CTTATGAAGT GTGTTAATCA 420
rAAAAATAT~ ATTGGCCTTT TGAATGTTTT CACACCACAG AAATCCCTAG AAGAATTTCA 480
AGATGTTTAC ATAGTCATGG AGCTCATGGA TGCAAATCTT TGCCAAGTGA TTCAGATGGA 540
GTTAGATCAT rAAAr-AATGT CCTACCTTCT CTATCAAATG ~ GGAA TCAAGCACCT 600
TCACTCTGCT GGAATTATTC ATCGGGACTT AAAGCCTAGT AATATAr,TAG TCAAATCAGA 660
CTGCACTTTG AAGATTCTTG ATTTTGGACT GGCAAGGACT GCAGGAACGA GTTTTATGAT 720
GACGCCTTAC GTGGTAACTC GTTACTACAG AGrAcr-AGAG GTCATTCTCG GCATGGGCTA 780
CAAGGAGAAC GTGGATTTAT GG~ GGG GTGCATTATG Gr-Ar-AAATGG TTTGCCTCAA 840
AA.C~ CCAGGAAGGG ACTATATTGA TCAGTGGAAT AAAGTTATTG AACAGCTCGG 900
AACACCTTGT CCTGAATTCA Tr-AAGAAACT ArAACÇAAr-~ GTAAGGACTT ACGTTGAAAA 960
CAGACCTAAG TACGCTGGCT ATAGCTTTGA GAAACTGTTT CCTGATGTGC ~CC~AGC 1020
TGACTCAGAA çATAAçAAAC TTAAAGCCAG TCAGGCGAGA GA~ AT CTAAAATGCT 1080
GGTr-ATAr-AT GCGTCCAAAA GGAl~CCG~ Ar-ACGAAGCT CTCCAGCACC CGTACATCAA 1140
C~ AT GATCCTTCAG AAGCAGAGGC CCÇArrArrA AAGATCCCTG ACAAGCAGTT 1200
AGATGAAAGG GAGCACACAA TAGAGGAGTG GAAAGAACTG ATATACAAGG AGGTCATGGA 1260
TTTGGAGGAG CGAACTAAGA ATGGCGTCAT AAGAGGGCAG CC~,~,C~,, TAGGTGCAGC 1320
21~88~8
- - 48 -
AGTGATCAAT GGCTCTCAGC ATCCGGTCTC TTCGCCGTCT GTCAATGACA ~.~.-~AAT 1380
GTC~-ACAr-AT CCGACTCTGG CCTCGGAT 1408
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
'A' LENGTH: 51 base pairs
B TYPE: nucleic acid
,C, STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TCAGATGCAG CAGTAAGCAG CAAGGCTACT C~--~.~AGT CGTCATCCAT C 51
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
~'A'~ LENGTH: 63 base pairs
B, TYPE: nucleic acid
C STRANDEDNESS: single
,D,I TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CAGCCCTCAC CTTCAGGTGC AGCAGTGAAC AGCAGTGAGA ~.CC~.CC ATCCTCATCT 60
GTC 63
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: 60 base pairs
~B, TYPE: nucleic acid
C STRANDEDNESS: single
,D, TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
.C~.l.AG GTGCAGCAGT GATCAATGGC TCTCAGCATC CGG.~ C GCCG.`:.~.C 60
What is claimed is:
