U.S. patent application number 10/739413 was filed with the patent office on 2005-03-03 for proteins having serine/threonine kinase domains, corresponding nucleic acid molecules, and their use.
Invention is credited to Dijke, Peter ten, Imamura, Takeshe, Miyazono, Kohei.
Application Number | 20050048607 10/739413 |
Document ID | / |
Family ID | 31192585 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048607 |
Kind Code |
A1 |
Miyazono, Kohei ; et
al. |
March 3, 2005 |
Proteins having serine/threonine kinase domains, corresponding
nucleic acid molecules, and their use
Abstract
The invention relates to the molecule referred to as ALK-1, and
its role as a type I receptor for members of the TGF-.beta. family.
The molecule has a role in the phosphorylation of Smad-5 and Smad1,
which also act as activators of certain genes. Aspects of the
invention relate to this interaction.
Inventors: |
Miyazono, Kohei; (Shiki,
JP) ; Imamura, Takeshe; (Tokyo, JP) ; Dijke,
Peter ten; (Em Hoofddorp, NL) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
Mary Anne Schofield
801 Pennsylvania Avenue, N.W.
Washington
DC
20004-2623
US
|
Family ID: |
31192585 |
Appl. No.: |
10/739413 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10739413 |
Dec 19, 2003 |
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09267963 |
Mar 12, 1999 |
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6692925 |
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09267963 |
Mar 12, 1999 |
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09039177 |
Mar 13, 1998 |
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09039177 |
Mar 13, 1998 |
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08436265 |
Oct 30, 1995 |
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6316217 |
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08436265 |
Oct 30, 1995 |
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PCT/GB93/02367 |
Nov 17, 1993 |
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Current U.S.
Class: |
435/69.1 ;
435/194; 435/196; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
G01N 33/566 20130101;
A61K 38/00 20130101; C07K 14/71 20130101; G01N 2333/495 20130101;
C07K 16/2863 20130101 |
Class at
Publication: |
435/069.1 ;
435/196; 435/320.1; 435/325; 435/194; 536/023.2 |
International
Class: |
C07H 021/04; C12N
009/12; C12N 009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 1992 |
GB |
9224057.1 |
Mar 8, 1993 |
GB |
9304677.9 |
Mar 8, 1993 |
GB |
9304680.3 |
May 28, 1993 |
GB |
9311047.6 |
Jul 2, 1993 |
GB |
9313763.6 |
Aug 3, 1993 |
GB |
9136099.2 |
Oct 15, 1993 |
GB |
9321344.5 |
Claims
1-28. (cancelled)
29 A method for determining if a substance inhibits binding of
TGF-.beta. to Alk-1 comprising contacting a cell that expresses an
Alk-1 with said substance in the presence of TGF-.beta. and
determining if said substance inhibits binding of TGF-.beta. to
Alk-1.
30 The method of claim 29, wherein said substance is an antibody
that binds to TGF-.beta..
31 The method of claim 29, wherein said substance is an antibody
that binds to the extracellular domain of Alk-1.
32 The method of claim 29 wherein said cells that express Alk-1 are
transfected with a nucleic acid molecule which encodes Alk-1.
33 The method of claim 29 wherein said Alk-1 is a constitutively
active Alk-1.
34 The method of claim 29, wherein said Alk-1 is a kinase inactive
Alk-1.
35 The method of claim 32, wherein said Alk-1 is an Alk-1 fusion
polypeptide.
36 The method of claim 35, wherein said Alk-1 is fused to
hemagglutinin.
37 The method of claim 29, wherein said cells that express Alk-1
are transfected with a nucleic acid molecule which encodes a Smad1
or a nucleic acid molecule which encodes a Smad5.
38 The method of claim 37, wherein said Smad1 is a Smad1 fusion
polypeptide.
39 The method of claim 38, wherein said Smad1 is fused to Flag.
40 The method of claim 37, wherein said Smad5 is a Smad5 fusion
polypeptide.
41 The method of claim 40, wherein said Smad5 is fused to Flag.
42 The method of claim 29, wherein inhibition of Smad1 or Smad5
phosphorylation in said cells that express an Alk-1 indicates
inhibition of binding of TGF-.beta. to Alk-1.
Description
FIELD OF THE INVENTION
[0001] This invention relates to proteins having serine/threonine
kinase domains, corresponding nucleic acid molecules, and their
use.
BACKGROUND OF THE INVENTION
[0002] The transforming growth factor-.beta. (TGF-.beta.)
superfamily consists of a family of structurally-related proteins,
including three different mammalian isoforms of TGF-.beta.
(TGF-.beta.1, .beta.2 and .beta.3), activins, inhibins,
mullerian-inhibiting substance and bone morphogenic proteins (BMPs)
(for reviews see Roberts and Sporn, (1990) Peptide Growth Factors
and Their Receptors, Pt.1, Sporn and Roberts, eds. (Berlin:
Springer-Verlag) pp 419-472; Moses et al (1990) Cell 63, 245-247).
The proteins of the TGF-.beta. superfamily have a wide variety of
biological activities. TGF-.beta. acts as a growth inhibitor for
many cell types and appears to play a central role in the
regulation of embryonic development, tissue regeneration,
immuno-regulation, as well as in fibrosis and carcinogenesis
(Roberts and Sporn (199) see above).
[0003] Activins and inhibins were originally identified as factors
which regulate secretion of follicle-stimulating hormone secretion
(Vale et al (1990) Peptide Growth Factors and Their Receptors,
Pt.2, Sporn and Roberts, eds. (Berlin: Springer-Verlag)
pp.211-248). Activins were also shown to induce the differentiation
of haematopoietic progenitor cells (Murata et al (1988) Proc. Natl.
Acad. Sci. USA 85, 2434-2438; Eto et al (1987) Biochem. Biophys.
Res. Commun. 142, 1095-1103) and induce mesoderm formation in
Xenopus embryos (Smith et al (1990) Nature 345, 729-731; van den
Eijnden-Van Raaij et al (1990) Nature 345, 732-734).
[0004] BMPs or osteogenic proteins which induce the formation of
bone and cartilage when implanted subcutaneously (Wozney et al
(1988) Science 242, 1528-1534), facilitate neuronal differentiation
(Paralkar et al (1992) J. Cell Biol. 119, 1721-1728) and induce
monocyte chemotaxis (Cunningham et al (1992) Proc. Natl. Acad. Sci.
USA 89, 11740-11744). Mullerian-inhibiting substance induces
regression of the Mullerian duct in the male reproductive system
(Cate et al (1986) Cell 45, 685-698), and a glial cell line-derived
neurotrophic factor enhances survival of midbrain dopaminergic
neurons (Lin et al (1993) Science 260, 1130-1132). The action of
these growth factors is mediated through binding to specific cell
surface receptors.
[0005] Within this family, TGF-.beta. receptors have been most
thoroughly characterized. By covalently cross-linking
radio-labelled TGF-.beta. to cell surface molecules followed by
polyacrylamide gel electrophoresis of the affinity-labelled
complexes, three distinct size classes of cell surface proteins (in
most cases) have been identified, denoted receptor type I (53 kd),
type II (75 kd), type III or betaglycan (a 300 kd proteoglycan with
a 120 kd core protein) (for a review see Massague (1992) Cell 69
1067-1070) and more recently endoglin (a homodimer of two 95 kd
subunits) (Cheifetz et al (1992) J. Biol. Chem. 267 19027-19030).
Current evidence suggests that type I and type II receptors are
directly involved in receptor signal transduction (Segarini et al
(1989) Mol. Endo., 3, 261-272; Laiho et al (1991) J. Biol. Chem.
266, 9100-9112) and may form a heteromeric complex; the type II
receptor is needed for the binding of TGF-.beta. to the type I
receptor and the type I receptor is needed for the signal
transduction induced by the type II receptor (Wrana et al (1992)
Cell, 71, 1003-1004). The type III receptor and endoglin may have
more indirect roles, possibly by facilitating the binding of ligand
to type II receptors (Wang et al (1991) Cell, 67 797-805;
Lpez-Casillas et al (1993) Cell, 73 1435-1444).
[0006] Binding analyses with activin A and BMP4 have led to the
identification of two co-existing cross-linked affinity complexes
of 50-60 kDa and 70-80 kDa on responsive cells (Hino et al (1989)
J. Biol. Chem. 264, 10309-10314; Mathews and Vale (1991), Cell 68,
775-785; Paralker et al (1991) Proc. Natl. Acad. Sci. USA 87,
8913-8917). By analogy with TGF-.beta. receptors they are thought
to be signalling receptors and have been named type I and type II
receptors.
[0007] Among the type II receptors for the TGF-.beta. superfamily
of proteins, the cDNA for the activin type II receptor (Act RII)
was the first to be cloned (Mathews and Vale (1991) Cell 65,
973-982). The predicted structure of the receptor was shown to be a
transmembrane protein with an intracellular serine/threonine kinase
domain. The activin receptor is related to the C. elegans daf-1
gene product, but the ligand is currently unknown (Georgi et al
(1990) Cell 61, 635-645). Thereafter, another form of the activin
type II receptor (activin type IIB receptor), of which there are
different splicing variants (Mathews et al (1992), Science 225,
1702-1705; Attisano et al (1992) Cell 68, 97-108), and the
TGF-.beta. type II receptor (T.beta.RII) (Lin et al (1992) Cell 68,
775-785) were cloned, both of which have putative serine/threonine
kinase domains.
SUMMARY OF THE INVENTION
[0008] The present invention involves the discovery of related
novel peptides, including peptides having the activity of those
defined herein as SEQ ID Nos. 2, 4, 8, 10, 12, 14, 16 and 18. Their
discovery is based on the realisation that receptor
serine/threonine kinases form a new receptor family, which may
include the type II receptors for other proteins in the TGF-.beta.
superfamily. To ascertain whether there were other members of this
family of receptors, a protocol was designed to clone ActRII/daf I
related cDNAs. This approach made use of the polymerase chain
reaction (PCR), using degenerate primers based upon the amino-acid
sequence similarity between kinase domains of the mouse activin
type II receptor and daf-I gene products.
[0009] This strategy resulted in the isolation of a new family of
receptor kinases called Activin receptor like kinases (ALK's) 1-6.
These cDNAs showed an overall 33-39% sequence similarity with
ActRII and TGF-.beta. type II receptor and 40-92% sequence
similarity towards each other in the kinase domains.
[0010] Soluble receptors according to the invention comprise at
least predominantly the extracellular domain. These can be selected
from the information provided herein, prepared in conventional
manner, and used in any manner associated with the invention.
[0011] Antibodies to the peptides described herein may be raised in
conventional manner. By selecting unique sequences of the peptides,
antibodies having desired specificity can be obtained.
[0012] The antibodies may be monoclonal, prepared in known manner.
In particular, monoclonal antibodies to the extracellular domain
are of potential value in therapy.
[0013] Products of the invention are useful in diagnostic methods,
e.g. to determine the presence in a sample for an analyte binding
therewith, such as in an antagonist assay. Conventional techniques,
e.g. an enzyme-linked immunosorbent assay, may be used.
[0014] Products of the invention having a specific receptor
activity can be used in therapy, e.g. to modulate conditions
associated with activin or TGF-.beta. activity. Such conditions
include fibrosis, e.g. liver cirrhosis and pulmonary fibrosis,
cancer, rheumatoid arthritis and glomeronephritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the alignment of the serine/threonine (S/T)
kinase domains (I-VIII) of related receptors from transmembrane
proteins, including embodiments of the present invention. The
nomenclature of the subdomains is accordingly to Hanks et al
(1988).
[0016] FIGS. 2A to 2D shows the sequences and characteristics of
the respective primers used in the initial PCR reactions. The
nucleic acid sequences are also given as SEQ ID Nos. 19 to 22.
[0017] FIG. 3 is a comparison of the amino-acid sequences of human
activin type II receptor (Act R-II), mouse activin type IIB
receptor (Act R-IIB), human TGF-.beta. type II receptor
(T.beta.R-II), human TGF-.beta. type I receptor (ALK-5), human
activin receptor type IA (ALK-2), and type IB (ALK-4), ALKs 1 &
3 and mouse ALK-6.
[0018] FIG. 4 shows, schematically, the structures for Daf-1, Act
R-II, Act R-IIB, T.beta.R-II, T.beta.R-I/ALK-5, ALK's -1, -2 (Act
RIA), -3, -4 (Act RIB) & -6.
[0019] FIG. 5 shows the sequence alignment of the cysteine-rich
domains of the ALKs, T.beta.R-II, Act R-II, Act R-IIB and daf-1
receptors.
[0020] FIG. 6 is a comparison of kinase domains of serine/threonine
kinases, showing the percentage amino-acid identity of the kinase
domains.
[0021] FIG. 7 shows the pairwise alignment relationship between the
kinase domains of the receptor serine/threonine kinases. The
dendrogram was generated using the Jotun-Hein alignment program
(Hein (1990) Meth. Enzymol. 183, 626-645).
[0022] FIG. 8 depicts the phosphorylation of Smad-5 following
interaction with ALK-1 but not following interaction with
ALK-5.
BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS
[0023] Sequences 1 and 2 are the nucleotide and deduced amino-acid
sequences of cDNA for hALK-1 (clone HP57).
[0024] Sequences 3 and 4 are the nucleotide and deduced amino-acid
sequences of cDNA for hALK-2 (clone HP53).
[0025] Sequences 5 and 6 are the nucleotide and deduced amino-acid
sequences of cDNA for hALK-3 (clone ONF5).
[0026] Sequences 7 and 8 the nucleotide and deduced amino-acid
sequences of cDNA for hALK-4 (clone 11H8), complemented with PCR
product encoding extracellular domain.
[0027] Sequences 9 and 10 are the nucleotide and deduced amino-acid
sequences of cDNA for hALK-5 (clone EMBLA).
[0028] Sequences 11 and 12 are the nucleotide and deduced
amino-acid sequences of cDNA for mALK-1 (clone AM6).
[0029] Sequences 13 and 14 are the nucleotide and deduced
amino-acid sequences of cDNA for mALK-3 (clones ME-7 and ME-D).
[0030] Sequences 15 and 16 are the nucleotide and deduced
amino-acid sequences of cDNA for mALK-4 (clone 8al).
[0031] Sequences 17 and 18 are the nucleotide and deduced
amino-acid sequences of cDNA for mALK-6 (clone ME-6).
[0032] Sequence 19 (B1-S) is a sense primer, extracellular domain,
cysteine-rich region, BamHI site at 5' end, 28-mer, 64-fold
degeneracy.
[0033] Sequence 20 (B3-S) is a sense primer, kinase domain II,
BamHI site at 5' end, 25-mer, 162-fold degeneracy.
[0034] Sequence 21 (B7-S) is a sense primer, kinase domain VIB, S/T
kinase specific residues, BamHI site at 5' end, 24-mer, 288-fold
degeneracy.
[0035] Sequence 22 (E8-AS) is an anti-sense primer, kinase domain,
S/T kinase-specific residues EcoRI site at 5' end, 20-mer, 18-fold
degeneracy.
[0036] Sequence 23 is an oligonucleotide probe.
[0037] Sequence 24 is a 5' primer.
[0038] Sequence 25 is a 3' primer.
[0039] Sequence 26 is a consensus sequence in Subdomain I.
[0040] Sequences 27 and 28 are novel sequence motifs in Subdomain
VIB.
[0041] Sequence 29 is a novel sequence motif in Subdomain VIII.
DESCRIPTION OF THE INVENTION
[0042] As described in more detail below, nucleic acid sequences
have been isolated, coding for a new sub-family of serine/threonine
receptor kinases. The term nucleic acid molecules as used herein
refers to any sequence which codes for the murine, human or
mammalian form, amino-acid sequences of which are presented herein.
It is understood that the well known phenomenon of codon degeneracy
provides for a great deal of sequence variation and all such
varieties are included within the scope of this invention.
[0043] The nucleic acid sequences described herein may be used to
clone the respective genomic DNA sequences in order to study the
genes' structure and regulation. The murine and human cDNA or
genomic sequences can also be used to isolate the homologous genes
from other mammalian species. The mammalian DNA sequences can be
used to study the receptors' functions in various in vitro and in
vivo model systems.
[0044] As exemplified below for ALK-5 cDNA, it is also recognised
that, given the sequence information provided herein, the artisan
could easily combine the molecules with a pertinent promoter in a
vector, so as to produce a cloning vehicle for expression of the
molecule. The promoter and coding molecule must be operably linked
via any of the well-recognized and easily-practised methodologies
for so doing. The resulting vectors, as well as the isolated
nucleic acid molecules themselves, may be used to transform
prokaryotic cells (e.g. E. coli), or transfect eukaryotes such as
yeast (S. cerevisiae), PAE, COS or CHO cell lines. Other
appropriate expression systems will also be apparent to the skilled
artisan.
[0045] Several methods may be used to isolate the ligands for the
ALKs. As shown for ALK-5 cDNA, cDNA clones encoding the active open
reading frames can be subcloned into expression vectors and
transfected into eukaryotic cells, for example COS cells. The
transfected cells which can express the receptor can be subjected
to binding assays for radioactively-labelled members of the
TGF-.beta. superfamily (TGF-.beta., activins, inhibins, bone
morphogenic proteins and mullerian-inhibiting substances), as it
may be expected that the receptors will bind members of the
TGF-.beta. superfamily. Various biochemical or cell-based assays
can be designed to identify the ligands, in tissue extracts or
conditioned media, for receptors in which a ligand is not known.
Antibodies raised to the receptors may also be used to identify the
ligands, using the immunoprecipitation of the cross-linked
complexes. Alternatively, purified receptor could be used to
isolate the ligands using an affinity-based approach. The
determination of the expression patterns of the receptors may also
aid in the isolation of the ligand. These studies may be carried
out using ALK DNA or RNA sequences as probes to perform in situ
hybridisation studies.
[0046] The use of various model systems or structural studies
should enable the rational development of specific agonists and
antagonists useful in regulating receptor function. It may be
envisaged that these can be peptides, mutated ligands, antibodies
or other molecules able to interact with the receptors.
[0047] The foregoing provides examples of the invention Applicants
intend to claim which includes, inter alia, isolated nucleic acid
molecules coding for activin receptor-like kinases (ALKs), as
defined herein. These include such sequences isolated from
mammalian species such as mouse, human, rat, rabbit and monkey.
[0048] The following description relates to specific embodiments.
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
[0049] Preparation of mRNA and Construction of a cDNA Library
[0050] For construction of a cDNA library, poly (A).sup.- RNA was
isolated from a human erythroleukemia cell line (HEL 92.1.7)
obtained from the American Type Culture Collection (ATCC TIB 180).
These cells were chosen as they have been shown to respond to both
activin and TGF-S. Moreover leukaemic cells have proved to be rich
sources for the cloning of novel receptor tyrosine kinases
(Partanen et al (1990) Proc. Natl. Acad. Sci. USA 87, 8913-8917 and
(1992) Mol. Cell. Biol. 12, 1698-1707). (Total) RNA was prepared by
the guanidinium isothiocyanate method (Chirgwin et al (1979)
Biochemistry 18, 5294-5299). mRNA was selected using the poly-A or
poly AT tract mRNA isolation kit (Promega, Madison, Wis., U.S.A.)
as described by the manufacturers, or purified through an oligo
(dT)-cellulose column as described by Aviv and Leder (1972) Proc.
Natl. Acad. Sci. USA 69, 1408-1412. The isolated mRNA was used for
the synthesis of random primed (Amersham) cDNA, that was used to
make a .lambda.gt10 library with 1.times.10.sup.5 independent cDNA
clones using the Riboclone cDNA synthesis system (Promega) and
.lambda.gt10 in vitro packaging kit (Amersham) according to the
manufacturers' procedures. An amplified oligo (dT) primed human
placenta .lambda.ZAPII cDNA library of 5.times.10.sup.5 independent
clones was used. Poly (A).sup.+ RNA isolated from AG1518 human
foreskin fibroblasts was used to prepare a primary random primed
.lambda.ZAPII cDNA library of 1.5.times.10.sup.6 independent clones
using the RiboClone cDNA synthesis system and Gigapack Gold II
packaging extract (Stratagene). In addition, a primary oligo (dT)
primed human foreskin fibroblast .lambda.gt10 cDNA library
(Claesson-Welsh et al (1989) Proc. Natl. Acad. Sci. USA. 86
4917-4912) was prepared. An amplified oligo (dT) primed HEL cell
.lambda.gt11 cDNA library of 1.5.times.10.sup.6 independent clones
(Poncz et al (1987) Blood 69 219-223) was used. A twelve-day mouse
embryo .lambda.EXIox cDNA library was obtained from Novagen
(Madison, Wis., U.S.A.); a mouse placenta .lambda.ZAPII cDNA
library was also used.
[0051] Generation of cDNA Probes by PCR
[0052] For the generation of cDNA probes by PCR (Lee et al (1988)
Science 239, 1288-1291) degenerate PCR primers were constructed
based upon the amino-acid sequence similarity between the mouse
activin type II receptor (Mathews and Vale (1991) Cell 65, 973-982)
and daf-1 (George et al (1990) Cell 61, 635-645) in the kinase
domains II and VIII. FIG. 1 shows the aligned serine/threonine
kinase domains (I-VIII), of four related receptors of the
TGF-.beta. superfamily, i.e. hT.beta.R-II, mActR-IIB, mActR-II and
the daf-1 gene product, using the nomenclature of the subdomains
according to Hanks et al (1988) Science 241, 45-52.
[0053] Several considerations were applied in the design of the PCR
primers. The sequences were taken from regions of homology between
the activin type II receptor and the daf-1 gene product, with
particular emphasis on residues that confer serine/threonine
specificity (see Table 2) and on residues that are shared by
transmembrane kinase proteins and not by cytoplasmic kinases. The
primers were designed so that each primer of a PCR set had an
approximately similar GC composition, and so that self
complementarity and complementarity between the 3' ends of the
primer sets were avoided. Degeneracy of the primers was kept as low
as possible, in particular avoiding serine, leucine and arginine
residues (6 possible codons), and human codon preference was
applied. Degeneracy was particularly avoided at the 3' end as,
unlike the 5' end, where mismatches are tolerated, mismatches at
the 3' end dramatically reduce the efficiency of PCR.
[0054] In order to facilitate directional subcloning, restriction
enzyme sites were included at the 5' end of the primers, with a GC
clamp, which permits efficient restriction enzyme digestion. The
primers utilised are shown in FIG. 2. Oligonucleotides were
synthesized using Gene assembler plus (Pharmacia-LKB) according to
the manufacturers instructions.
[0055] The mRNA prepared from HEL cells as described above was
reverse-transcribed into cDNA in the presence of 50 mM Tris-HCl, pH
8.3, 8 mM MgCl.sub.2, 30 mM KCl, 10 mM dithiothreitol, 2 mM
nucleotide triphosphates, excess oligo (dT) primers and 34 units of
AMV reverse transcriptase at 42.degree. C. for 2 hours in 40 .mu.l
of reaction volume. Amplification by PCR was carried out with a
7.5% aliquot (3 .mu.l) of the reverse-transcribed mRNA, in the
presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 M MgCl.sub.2,
0.01% gelatin, 0.2 mM nucleotide triphosphates, 1 .mu.M of both
sense and antisense primers and 2.5 units of Taq polymerase (Perkin
Elmer Cetus) in 100 .mu.l reaction volume. Amplifications were
performed on a thermal cycler (Perkin Elmer Cetus) using the
following program: first 5 thermal cycles with denaturation for 1
minute at 94.degree. C., annealing for 1 minute at 50.degree. C., a
2 minute ramp to 55.degree. C. and elongation for 1 minute at
72.degree. C., followed by 20 cycles of 1 minute at 94.degree. C.,
30 seconds at 55.degree. C. and 1 minute at 72.degree. C. A second
round of PCR was performed with 3 .mu.l of the first reaction as a
template. This involved 25 thermal cycles, each composed of
94.degree. C. (1 min), 55.degree. C. (0.5 min), 72.degree. C. (1
min)
[0056] General procedures such as purification of nucleic acids,
restriction enzyme digestion, gel electrophoresis, transfer of
nucleic acid to solid supports and subcloning were performed
essentially according to established procedures as described by
Sambrook et al, (1989), Molecular cloning: A Laboratory Manual,
2.sup.nd Ed. Cold Spring Harbor Laboratory (Cold Spring Harbor,
N.Y., USA).
[0057] Samples of the PCR products were digested with BamHI and
EcoRI and subsequently fractionated by low melting point agarose
gel electrophoresis. Bands corresponding to the approximate
expected sizes, (see Table 1: .apprxeq.460 bp for primer pair B3-S
and E8-AS and .apprxeq.140 bp for primer pair B7-S and E8-AS) were
excised from the gel and the DNA was purified. Subsequently, these
fragments were ligated into pUC19 (Yanisch-Perron et al (1985) Gene
33, 103-119), which had been previously linearised with BamHI and
EcoR1 and transformed into E. coli strain DH5.alpha. using standard
protocols (Sambrook et al, supra). Individual clones were sequenced
using standard double-stranded sequencing techniques and the
dideoxynucleotide chain termination method as described by Sanger
et al (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467, and T7 DNA
polymerase.
[0058] Employing Reverse Transcriptase PCR on HEL mRNA with the
primer pair B3-S and E8-AS, three PCR products were obtained,
termed 11.1, 11.2 and 11.3, that corresponded to novel genes. Using
the primer pair B7-S and E8-AS, an additional novel PCR product was
obtained termed 5.2.
1TABLE 1 SEQUENCE SIZE OF DNA IDENTITY FRAGMENT IN SEQUENCE BETWEEN
mActRII/ IDENTITY WITH mActRII NAME INSERT hT.beta.RII SEQUENCE and
OF PCR SIZE CLONES mActRII/hT.beta.RII T.beta.R-II PRODUCT PRIMERS
(bp) (bp) (%) (%) 11.1 B3-S/E8-AS 460 460 46/40 42 11.2 B3-S/E8-AS
460 460 49/44 47 11.3 B3-S/E8-AS 460 460 44/36 48 11.29 B3-S/E8-AS
460 460 ND/100 ND 9.2 B1-S/E8-AS 800 795 100/ND ND 5.2 B7-S/E8-AS
140 143 40/38 60
[0059] Isolation of cDNA Clones
[0060] The PCR products obtained were used to screen various cDNA
libraries described supra. Labelling of the inserts of PCR products
was performed using random priming method (Feinberg and Vogelstein
(1983) Anal. Biochem, 132 6-13) using the Megaprime DNA labelling
system (Amersham). The oligonucleotide derived from the sequence of
the PCR product 5.2 was labelled by phosphorylation with T4
polynucleotide kinase following standard protocols (Sambrook et al,
supra). Hybridization and purification of positive bacteriophages
were performed using standard molecular biological techniques.
[0061] The double-stranded DNA clones were all sequenced using the
dideoxynucleotide chain-termination method as described by Sanger
et al, supra, using T7 DNA polymerase (Pharmacia-LKB) or Sequenase
(U.S. Biochemical Corporation, Cleveland, Ohio, U.S.A.).
Compressions of nucleotides were resolved using 7-deaza-GTP (U.S.
Biochemical Corp.) DNA sequences were analyzed using the DNA STAR
computer program (DNA STAR Ltd. U.K.). Analyses of the sequences
obtained revealed the existence of six distinct putative receptor
serine/threonine kinases which have been named ALK 1-6.
[0062] To clone cDNA for ALK-1 the oligo (dT) primed human placenta
cDNA library was screened with a radiolabelled insert derived from
the PCR product 11.3; based upon their restriction enzyme digestion
patternS, three different types of clones with approximate insert
sizes. of 1.7 kb, 2 kb & 3.5 kb were identified. The 2 kb
clone, named HP57, was chosen as representative of this class and
subjected to complete sequencing. Sequence analysis of ALK-1
revealed a sequence of 1984 nucleotides including a poly-A tail
(SEQ ID No. 1). The longest open reading frame encodes a protein of
503 amino-acids, with high sequence similarity to receptor
serine/threonine kinases (see below). The first methionine codon,
the putative translation start site, is at nucleotide 283-285 and
is preceded by an in-frame stop codon. This first ATG is in a more
favourable context for translation initiation (Kozak (1987) Nucl.
Acids Res., 15, 8125-8148) than the second and third in-frame ATG
at nucleotides 316-318 and 325-327. The putative initiation codon
is preceded by a 5' untranslated sequence of 282 nucleotides that
is GC-rich (80% GC), which is not uncommon for growth factor
receptors (Kozak (1991) J. Cell Biol., 115, 887-903). The 3'
untranslated sequence comprises 193 nucleotides and ends with a
poly-A tail. No bona fide poly-A addition signal is found, but
there is a sequence (AATACA), 17-22 nucleotides upstream of the
poly-A tail, which may serve as a poly-A addition signal.
[0063] ALK-2 cDNA was cloned by screening an amplified oligo (dT)
primed human placenta cDNA library with a radiolabelled insert
derived from the PCR product 11.2. Two clones, termed HP53 and
HP64, with insert sizes of 2.7 kb and 2.4 kb respectively, were
identified and their sequences were determined. No sequence
difference in the overlapping clones was found, suggesting they are
both derived from transcripts of the same gene.
[0064] Sequence analysis of cDNA clone HP53 (SEQ ID No. 3) revealed
a sequence of 2719 nucleotides with a poly-A tail. The longest open
reading frame encodes a protein of 509 amino-acids. The first ATG
at nucleotides 104-106 agrees favourably with Kozak's consensus
sequence with an A at position 3. This ATG is preceded in-frame by
a stop codon. There are four ATG codons in close proximity further
downstream, which agree with the Kozak's consensus sequence (Kozak,
supra), but according to Kozak's scanning model the first ATG is
predicted to be the translation start site. The 5' untranslated
sequence is 103 nucleotides. The 3' untranslated sequence of 1089
nucleotides contains a polyadenylation signal located 9-14
nucleotides upstream from the poly-A tail. The cDNA clone HP64
lacks 498 nucleotides from the 5' end compared to HP53, but the
sequence extended at the 3' end with 190 nucleotides and poly-A
tail is absent. This suggests that different polyadenylation sites
occur for ALK-2. In Northern blots, however, only one transcript
was detected (see below).
[0065] The cDNA for human ALK-3 was cloned by initially screening
an oligo (dT) primed human foreskin fibroblast cDNA library with an
oligonucleotide (SEQ ID No. 23) derived from the PCR product 5.2.
One positive cDNA clone with an insert size of 3 kb, termed ON11,
was identified. However, upon partial sequencing, it appeared that
this clone was incomplete; it encodes only part of the kinase
domain and lacks the extracelluar domain. The most 5' sequence of
ON11, a 540 nucleotide XbaI restriction fragment encoding a
truncated kinase domain, was subsequently used to probe a random
primed fibroblast cDNA library from which one cDNA clone with an
insert size of 3 kb, termed ONF5, was isolated (SEQ ID No. 5).
Sequence analysis of ONF5 revealed a sequence of 2932 nucleotides
without a poly-A tail, suggesting that this clone was derived by
internal priming. The longest open reading frame codes for a
protein of 532 amino-acids. The first ATG codon which is compatible
with Kozak's consensus sequence (Kozak, supra), is at 310-312
nucleotides and is preceded by an in-frame stop codon. The 5' and
3' untranslated sequences are 309 and 1027 nucleotides long,
respectively.
[0066] ALK-4 cDNA was identified by screening a human oligo (dT)
primed human erythroleukemia cDNA library with the radiolabelled
insert of the PCR product 11.1 as a probe. One cDNA clone, termed
11H8, was identified with an insert size of 2 kb (SEQ ID No. 7). An
open reading frame was found encoding a protein sequence of 383
amino-acids encoding a truncated extracellular domain with high
similarity to receptor serine/threonine kinases. The 3'
untranslated sequence is 818 nucleotides and does not contain a
poly-A tail, suggesting that the cDNA was internally primed. cDNA
encoding the complete extracellular domain (nucleotides 1-366) was
obtained from HEL cells by RT-PCR with 5' primer (SEQ ID No. 24)
derived in part from sequence at translation start site of SKR-2 (a
cDNA sequence deposited in GenBank data base, accesion number
L10125, that is identical in part to ALK-4) and 3' primer (SEQ ID
No. 25) derived from 11H8 cDNA clone.
[0067] ALK-5 was identified by screening the random primed HEL cell
.lambda.gt 10 cDNA library with the PCR product 11.1 as a probe.
This yielded one positive clone termed EMBLA (insert size of 5.3 kb
with 2 internal EcoRI sites). Nucleotide sequencing revealed an
open reading frame of 1509 bp, coding for 503 amino-acids. The open
reading frame was flanked by a 5' untranslated sequence of 76 bp,
and a 3' untranslated sequence of 3.7 kb which was not completely
sequenced. The nucleotide and deduced amino-acid sequences of ALK-5
are shown in SEQ ID Nos. 9 and 10. In the 5' part of the open
reading frame, only one ATG codon was found; this codon fulfils the
rules of translation initiation (Kozak, supra). An in-frame stop
codon was found at nucleotides (-54)-(-52) in the 5' untranslated
region. The predicted ATG start codon is followed by a stretch of
hydrophobic amino-acid residues which has characteristics of a
cleavable signal sequence. Therefore, the first ATG codon is likely
to be used as a translation initiation site. A preferred cleavage
site for the signal peptidase, according to von Heijne (1986) Nucl.
Acid. Res. 14, 4683-4690, is located between amino-acid residues 24
and 25. The calculated molecular mass of the primary translated
product of the ALK-5 without signal sequence is 53,646 Da.
[0068] Screening of the mouse embryo .lambda.EX Iox cDNA library
using PCR, product 11.1 as a probe yielded 20 positive clones. DNAs
from the positive clones obtained from this library were digested
with EcoRI and HindIII, electrophoretically separated on a 1.3%
agarose gel and transferred to nitrocellulose filters according to
established procedures as described by Sambrook et al, supra. The
filters were then hybridized with specific probes for human ALK-1
(nucleotide 288-670), ALK-2 (nucleotide 1-581), ALK-3 (nucleotide
79-824) or ALK-4 nucleotide 1178-1967). Such analyses revealed that
a clone termed ME-7 hybridised with the human ALK-3 probe. However,
nucleotide sequencing revealed that this clone was incomplete, and
lacked the 5' part of the translated region. Screening the same
cDNA library with a probe corresponding to the extracelluar domain
of human ALK-3 (nucleotides 79-824) revealed the clone ME-D. This
clone was isolated and the sequence was analyzed. Although this
clone was incomplete in the 3' end of the translated region, ME-7
and ME-D overlapped and together covered the complete sequence of
mouse ALK-3. The predicted amino-acid sequence of mouse ALK-3 is
very similar to the human sequence; only 8 amino-acid residues
differ (98% identity; see SEQ ID No. 14) and the calculated
molecular mass of the primary translated product without the
putative signal sequence is 57,447 Da.
[0069] Of the clones obtained from the initial library screening
with PCR product 11.1, four clones hybridized to the probe
corresponding to the conserved kinase domain of ALK-4 but not to
probes from more divergent parts of ALK-1 to -4. Analysis of these
clones revealed that they have an identical sequence which differs
from those of ALK-1 to -5 and was termed ALK-6. The longest clone
ME6 with a 2.0 kb insert was completely sequenced yielding a 1952
bp fragment consisting of an open reading frame of 1506 bp (502
amino-acids), flanked by a 5' untranslated sequence of 186 bp, and
a 3' untranslated sequence of 160 bp. The nucleotide and predicted
amino-acid sequences of mouse ALK-6 are shown in SEQ ID Nos. 17 and
18. No polyadenylation signal was found in the 3' untranslated
region of ME6, indicating that the cDNA was internally primed in
the 3' end. Only one ATG codon was found in the 5' part of the open
reading frame, which fulfils the rules for translation initiation
(Kozak, supra), and was preceded by an in-frame stop codon at
nucleotides 163-165. However, a typical hydrophobic leader sequence
was not observed at the N terminus of the translated region. Since
there is no ATG codon and putative hydrophobic leader sequence,
this ATG codon is likely to be used as a translation initiation
site. The calculated molecular mass of the primary translated
product with the putative signal sequence is 55,576 Da.
[0070] Mouse ALK-1 (clone AM6 with 1.9 kb insert) was obtained from
the mouse placenta .lambda.ZAPII cDNA library using human ALK-1
cDNA as a probe (see SEQ ID No. 11). Mouse ALK-4 (clone 8al with
2.3 kb insert) was also obtained from this library using human
ALK-4 cDNA library as a probe (SEQ ID No. 15).
[0071] To summarise, clones HP22, HP57, ONF1, ONF3, ONF4 and HP29
encode the same gene, ALK-1. Clone AM6 encodes mouse ALK-1. HP53,
HP64 and HP84 encode the same gene, ALK-2. ONF5, ONF2 and ON11
encode the same gene ALK-3. ME-7 and ME-D encode the mouse
counterpart of human ALK-3. 11H8 encodes a different gene ALK-4,
whilst 8al encodes the mouse equivalent. EMBLA encodes ALK-5, and
ME-6 encodes ALK-6.
[0072] The sequence alignment between the 6 ALK genes and
T.beta.R-II, mActR-II and ActR-IIB is shown in FIG. 3. These
molecules have a similar domain structure; an N-terminal predicted
hydrophobic signal sequence (von Heijne (1986) Nucl. Acids Res. 14:
4683-4690) is followed by a relatively small extracellular
cysteine-rich ligand binding domain, a single hydrophobic
transmembrane region (Kyte & Doolittle (1982) J. Mol. Biol.
157, 105-132) and a C-terminal intracellular portion, which
consists almost entirely of a kinase domain (FIGS. 3 and 4).
[0073] The extracelluar domains of these receptors have
cysteine-rich regions, but they show little sequence similarity;
for example, less than 20% sequence identity is found between
Daf-1, ActR-II, T.beta.R-II and ALK-5. The ALKs appear to form a
subfamily as they show higher sequence similarities (15-47%
identity) in their extracellular domains. The extracellular domains
of ALK-5 and ALK-4 have about 29% sequence identity. In addition,
ALK-3 and ALK-6 share a high degree of sequence similarity in their
extracellular domains (46% identity).
[0074] The positions of many of the cysteine residues in all
receptors can be aligned, suggesting that the extracellular domains
may adopt a similar structural configuration. See FIG. 5 for
ALKs-1,-2,-3 & -5. Each of the ALKs (except ALK-6) has a
potential N-linked glycosylation site, the position of which is
conserved between ALK-1 and ALK-2, and between ALK-3, ALK-4 and
ALK-5 (see FIG. 4).
[0075] The sequence similarities in the kinase domains between
daf-1, ActR-II, T.beta.R-II and ALK-5 are approximately 40%,
whereas the sequence similarity between the ALKs 1 to 6 is higher
(between 59% and 90%; see FIG. 6). Pairwise comparison using the
Jutun-Hein sequence alignment program (Hein (1990) Meth, Enzymol.,
183, 626-645), between all family members, identifies the ALKs as a
separate subclass among serine/threonine kinases (FIG. 7).
[0076] The catalytic domains of kinases can be divided into 12
subdomains with stretches of conserved amino-acid residues. The key
motifs are found in serine/threonine kinase receptors suggesting
that they are functional kinases. The consensus sequence for the
binding of ATP (Gly-X-Gly-X-X-Gly in subdomain I followed by a Lys
residue further downstream in subdomain II) is found in all the
ALKs.
[0077] The kinase domains of daf-1, ActR-II, and ALKs show
approximately equal sequence similarity with tyrosine and
serine/threonine protein kinases. However analysis of the
amino-acid sequences in subdomains VI and VIII, which are the most
useful to distinguish a specificity for phosphorylation of tyrosine
residues versus serine/threonine residues (Hanks et al (1988)
Science 241 42-52) indicates that these kinases are
serine/threonine kinases; refer to Table 2.
2 TABLE 2 SUBDOMAINS KINASE VIB VIII Serine/threonine kinase
consensus DLKPEN G (T/S) XX (Y/F) X Tyrosine kinase consensus
DLAARN XP (I/V) (K/R) W (T/M) Act R-II DIKSKN GTRRYM Act R-IIB
DFKSKN GTRRYM T.beta.R-II DLKSSN GTARYM ALK-I DFKSRN GTKRYM ALK -2,
-3, -4, -5, & -6 DLKSKN GTKRYM
[0078] The sequence motifs DLKSKN (Subdomain VIB) and GTKRYM
(Subdomain VIII), that are found in most of the serine/threonine
kinase receptors, agree well with the consensus sequences for all
protein serine/threonine kinase receptors in these regions. In
addition, these receptors, except for ALK-1, do not have a tyrosine
residue surrounded by acidic residues between subdomains VII and
VIII, which is common for tyrosine kinases. A unique characteristic
of the members of the ALK serine/threonine kinase receptor family
is the presence of two short inserts in the kinase domain between
subdomains VIA and VIB and between subdomains X and XI. In the
intracellular domain, these regions, together with the
juxtamembrane part and C-terminal tail, are the most divergent
between family members (see FIGS. 3 and 4). Based on the sequence
similarity with the type II receptors for TGF-.beta. and activin,
the C termini of the kinase domains of ALKs -1 to -6 are set at
Ser-495, Ser-501, Ser-527, Gln-500, Gln-498 and Ser-497,
respectively.
[0079] mRNA Expression
[0080] The distribution of ALK-1, -2, -3, -4 was determined by
Northern blot analysis. A Northern blot filter with mRNAs from
different human tissues was obtained from Clontech (Palo Alto,
Calif.). The filters were hybridized with .sup.32P-labelled probes
at 42.degree. C. overnight in 50% formaldehyde, 5.times. standard
saline citrate (SSC; 1.times.SSC is 50 mM sodium citrate, pH 7.0,
150 mM NaCl), 0.1% SDS, 50 mM sodium phosphate, 5.times. Denhardt's
solution and 0.1 mg/ml salmon sperm DNA. In order to minimize
cross-hybridization, probes were used that did not encode part of
the kinase domains, but corresponded to the highly diverged
sequences of either 5' untranslated and ligand-binding regions
(probes for ALK-1, -2 and -3) or 3' untranslated sequences (probe
for ALK-4). The probes were labelled by random priming using the
Multiprime (or Mega-prime) DNA labelling system and
[.alpha.-.sup.32P] dCTP (Feinberg & Vogelstein (1983) Anal.
Biochem. 132: 6-13). Unincorporated label was removed by Sephadex
G-25 chromatography. Filters were washed at 65.degree. C., twice
for 30 minutes in 2.5.times.SSC, 0.1% SDS and twice for 30 minutes
in 0.3.times.SSC, 0.1% SDS before being exposed to X-ray film.
Stripping of blots was performed by incubation at 90-100.degree. C.
in water for 20 minutes.
[0081] Our further analysis suggest ALK-1 is endothelial cell
specific.
[0082] The ALK-5 mRNA size and distribution were determined by
Northern blot analysis as above. An EcoR1 fragment of 980 bp of the
full length ALK-5 cDNA clone, corresponding to the C-terminal part
of the kinase domain and 3' untranslated region (nucleotides
1259-2232 in SEQ ID No. 9) was used as a probe. The filter was
washed twice in 0.5.times.SSC, 0.1% SDS at 55.degree. C. for 15
minutes.
[0083] Using the probe for ALK-1, two transcripts of 2.2 and 4.9 kb
were detected. The ALK-1 expression level varied strongly between
different tissues, high in placenta and lung, moderate in heart,
muscle and kidney, and low (to not detectable) in brain, liver and
pancreas. The relative ratios between the two transcripts were
similar in most tissues; in kidney, however, there was relatively
more of the 4.9 kb transcript. By reprobing the blot with a probe
for ALK-2, one transcript of 4.0 kb was detected with a ubiquitous
expression pattern. Expression was detected in every tissue
investigated and was highest in placenta and skeletal muscle.
Subsequently the blot was reprobed for ALK-3. One major transcript
of 4.4 kb and a minor transcript of 7.9 kb were detected.
Expression was high in skeletal muscle, in which also an additional
minor transcript of 10 kb was observed. Moderate levels of ALK-3
mRNA were detected in heart, placenta, kidney and pancreas, and low
(to not detectable) expression was found in brain, lung and liver.
The relative ratios between the different transcripts were similar
in the tested tissues, the 4.4 kb transcript being the predominant
one, with the exception for brain where both transcripts were
expressed at a similar level. Probing the blot with ALK-4 indicated
the presence of a transcript with the estimated size of 5.2 kb and
revealed an ubiquitous expression pattern. The results of Northern
blot analysis using the probe for ALK-5 showed that a 5.5 kb
transcript is expressed in all human tissues tested, being most
abundant in placenta and least abundant in brain and heart.
[0084] The distribution of mRNA for mouse ALK-3 and -6 in various
mouse tissues was also determined by Northern blot analysis. A
multiple mouse tissue blot was obtained from Clontech, Palo Alto,
Calif., U.S.A. The filter was hybridized as described above with
probes for mouse ALK-3 and ALK-6. The EcoRI-PstI restriction
fragment, corresponding to nucleotides 79-1100 of ALK-3, and the
SacI-HpaI fragment, corresponding to nucleotides 57-720 of ALK-6,
were used as probes. The filter was washed at 65.degree. C. twice
for 30 minutes in 2.5.times.SSC, 0.1% SDS and twice for 30 minutes
with 0.3.times.SSC, 0.1% SDS and then subjected to
autoradiography.
[0085] Using the probe for mouse ALK-3, a 1.1 kb transcript was
found only in spleen. By reprobing the blot with the ALK-6 specific
probe, a transcript of 7.2 kb was found in brain and a weak signal
was also seen in lung. No other signal was seen in the other
tissues tested, i.e. heart, liver, skeletal muscle, kidney and
testis.
[0086] All detected transcript sizes were different, and thus no
cross-reaction between mRNAs for the different ALKs was observed
when the specific probes were used. This suggests that the multiple
transcripts of ALK-1 and ALK-3 are coded from the same gene. The
mechanism for generation of the different transcripts is unknown at
present; they may be formed by alternative mRNA splicing,
differential polyadenylation, use of different promotors, or by a
combination of these events. Differences in mRNA splicing in the
regions coding for the extracellular domains may lead to the
synthesis of receptors with different affinities for ligands, as
was shown for mActR-IIB (Attisano et al (1992) Cell 68, 97-108) or
to the production of soluble binding protein.
[0087] The above experiments describe the isolation of nucleic acid
sequences coding for new family of human receptor kinases. The cDNA
for ALK-5 was then used to determine the encoded protein size and
binding properties.
[0088] Properties of the ALKs cDNA Encoded Proteins
[0089] To study the properties of the proteins encoded by the
different ALK cDNAs, the cDNA for each ALK was subcloned into a
eukaryotic expression vector and transfected into various cell
types and then subjected to immunoprecipitation using a rabbit
antiserum raised against a synthetic peptide corresponding to part
of the intracellular juxtamembrane region. This region is divergent
in sequence between the various serine/threonine kinase receptors.
The following amino-acid residues were used:
3 ALK-1 145-166 ALK-2 151-172 ALK-3 181-202 ALK-4 153-171 ALK-5
158-179 ALK-6 151-168
[0090] The rabbit antiserum against ALK-5 was designated VPN.
[0091] The peptides were synthesized with an Applied Biosystems
430A Peptide Synthesizer using t-butoxycarbonyl chemistry and
purified by reversed-phase high performance liquid chromatography.
The peptides were coupled to keyhole limpet haemocyanin
(Calbiochem-Behring) using glutaraldehyde, as described by Guillick
et al (1985) EMBO J. 4, 2869-2877. The coupled peptides were mixed
with Freunds adjuvant and used to immunize rabbits.
[0092] Transient Transfection of the ALK-5 cDNA
[0093] COS-1 cells (American Type Culture Collection) and the R
mutant of Mv1Lu cells (for references, see below) were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum (FBS) and 100 units/ml penicillin and 50 .mu.g 1 ml
streptomycin in 5% CO.sub.2 atmosphere at 37.degree. C. The ALK-5
cDNA (nucleotides (-76)-2232), which includes the complete coding
region, was cloned in the pSV7d vector (Truett et al, (1985) DNA 4,
333-349), and used for transfection. Transfection into COS-1 cells
was performed by the calcium phosphate precipitation method (Wigler
et al (1979) Cell 16, 777-785). Briefly, cells were seeded into
6-well cell culture plates at a density of 5.times.10.sup.5
cells/well, and transfected the following day with 10 .mu.g of
recombinant plasmid. After overnight incubation, cells were washed
three times with a buffer containing 25 mM Tris-HCl, pH 7.4, 138 mM
NaCl, 5 mM KCl, 0.7 mM CaCl.sub.2, 0.5 mM MgCl.sub.2 and 0.6 mM
Na.sub.2HPO.sub.4, and then incubated with Dulbecco's modified
Eagle's medium containing FBS and antibiotics. Two days after
transfection, the cells were metabolically labelled by incubating
the cells for 6 hours in methionine and cysteine-free MCDB 104
medium with 150 .mu.Ci/ml of [.sup.35S]-methionine and
[.sup.35S]-cysteine (in vivo labelling mix; Amersham). After
labelling, the cells were washed with 150 mM NaCI, 25 mM Tris-HCl,
pH 7.4, and then solubilized with a buffer containing 20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1%
deoxycholate, 1.5% Trasylol (Bayer) and 1 mM
phenylmethylsulfonylfluoride (PMSF; Sigma). After 15 minutes on
ice, the cell lysates were pelleted by centrifugation, and the
supernatants were then incubated with 7 .mu.l of preimmune serum
for 1.5 hours at 4.degree. C. Samples were then given 50 .mu.l of
protein A-Sepharose (Pharmacia-LKB) slurry (50% packed beads in 150
mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.2% Triton X100) and incubated
for 45 minutes at 4.degree. C. The beads were spun down by
centrifugation, and the supernatants (1 ml) were then incubated
with either 7 .mu.l of preimmune serum or the VPN antiserum for 1.5
hours at 4.degree. C. For blocking, 10 .mu.g of peptide was added
together with the antiserum. Immune complexes were then given 50
.mu.l of protein A-Sepharose (Pharmacia-LKB) slurry (50% packed
beads in 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.2% Triton X-100)
and incubated for 45 minutes at 4.degree. C. The beads were spun
down and washed four times with a washing buffer (20 mM Tris-HCl,
pH 7.4, 500 mM NaCI, 1% Triton X-100, 1% deoxycholate and 0.2%
SDS), followed by one wash in distilled water. The immune complexes
were eluted by boiling for 5 minutes in the SDS-sample buffer (100
mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS)
in the presence of 10 mM DTT, and analyzed by SDS-gel
electrophoresis using 7-15% polyacrylamide gels (Blobel and
Dobberstein, (1975) J. Cell Biol. 67, 835-851). Gels were fixed,
incubated with Amplify (Amersham) for 20 minutes, and subjected to
fluorography. A component of 53Da was seen. This component was not
seen when preimmune serum was used, or when 10 .mu.g blocking
peptide was added together with the antiserum. Moreover, it was not
detectable in samples derived from untransfected COS-1 cells using
either preimmune serum or the antiserum.
[0094] Digestion with Endoglycosidase F
[0095] Samples immunoprecipitated with the VPN antisera obtained as
described above were incubated with 0.5 U of endoglycosidase F
(Boehringer Mannheim Biochemica) in a buffer containing 100 mM
sodium phosphate, pH 6.1, 50 mM EDTA, 1% Triton X-100, 0.1% SDS and
1% .beta.-mercaptoethanol at 37.degree. C. for 24 hours. Samples
were eluted by boiling for 5 minutes in the SDS-sample buffer, and
analyzed by SDS-polyacrylamide gel electrophoresis as described
above. Hydrolysis of N-linked carbohydrates by endoglycosidase F
shifted the 53 kDa band to 51 kDa. The extracelluar domain of ALK-5
contains one potential acceptor site for N-glycosylation and the
size of the deglycosylated protein is close to the predicted size
of the core protein.
[0096] Establishment of PAE Cell Lines Expressing ALK-5
[0097] In order to investigate whether the ALK-5 cDNA encodes a
receptor for TGF-.beta., porcine aortic endothelial (PAE) cells
were transfected with an expression vector containing the ALK-5
cDNA, and analyzed for the binding of .sup.125I-TGF-.beta.1.
[0098] PAE cells were cultured in Ham's F-12 medium supplemented
with 10% FBS and antibiotics (Miyazono et al., (1988) J. Biol.
Chem. 263, 6407-6415). The ALK-5 cDNA was cloned into the
cytomegalovirus (CMV)-based expression vector pcDNA I/NEO
(Invitrogen), and transfected into PAE cells by electroporation.
After 48 hours, selection was initiated by adding Geneticin (G418
sulphate; Gibco-BRL) to the culture medium at a final concentration
of 0.5 mg/ml (Westermark et al., (1990) Proc. Natl. Acad. Sci. USA
87, 128-132). Several clones were obtained, and after analysis by
immunoprecipitation using the VPN antiserum, one clone denoted
PAE/T.beta.R-1 was chosen and further analyzed.
[0099] Iodination of TGF-.beta.1, Binding and Affinity
Crosslinking
[0100] Recombinant human TGF-.beta.1 was iodinated using the
chloramine T method according to Frolik et al., (1984) J. Biol.
Chem. 259, 10995-11000. Cross-linking experiments were performed as
previously described (Ichijo et al., (1990) Exp. Cell Res. 187,
263-269). Briefly, cells in 6-well plates were washed with binding
buffer (phosphate-buffered saline containing 0.9 mM CaCl.sub.2,
0.49 mM MgCl.sub.2 and 1 mg/ml bovine serum albumin (BSA)), and
incubated on ice in the same buffer with .sup.125I-TGF-.beta.1 in
the presence or absence of excess unlabelled TGF-.beta.1 for 3
hours. Cells were washed and cross-linking was done in the binding
buffer without BSA together with 0.28 mM disuccinimidyl suberate
(DSS; Pierce Chemical Co.) for 15 minutes on ice. The cells were
harvested by the addition of 1 ml of detachment buffer (10 mM
Tris-HC1, pH 7.4, 1 mM EDTA, 10% glycerol, 0.3 mM PMSF). The cells
were pelleted by centrifugation, then resuspended in 50 .mu.l of
solubilization buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 1% Triton X-100, 0.3 mM PMSF, 1% Trasylol) and incubated for
40 minutes on ice. Cells were centrifuged again and supernatants
were subjected to analysis by SDS-gel electrophoresis using 4-15%
polyacrylamide gels, followed by autoradiography.
.sup.125I-TGF-.beta.1 formed a 70 kDa cross-linked complex in the
transfected PAE cells (PAE/T.beta.R-I cells). The size of this
complex was very similar to that of the TGF-.beta. type I receptor
complex observed at lower amounts in the untransfected cells. A
concomitant increase of 94 kDa TGF-.beta. type II receptor complex
could also be observed in the PAE/T.beta.R-I cells. Components of
150-190 kDa, which may represent crosslinked complexes between the
type I and type II receptors, were also observed in the
PAE/T.beta.R-I cells.
[0101] In order to determine whether the cross-linked 70 kDa
complex contained the protein encoded by the ALK-5 cDNA, the
affinity cross-linking was followed by immunoprecipitation using
the VPN antiserum. For this, cells in 25 cm.sup.2 flasks were used.
The supernatants obtained after cross-linking were incubated with 7
.mu.l of preimmune serum or VPN antiserum in the presence or
absence of 10 .mu.g of peptide for 1.5 h at 4.degree. C. Immune
complexes were then added to 50 .mu.l of protein A-Sepharose slurry
and incubated for 45 minutes at 4.degree. C. The protein
A-Sepharose beads were washed four times with the washing buffer,
once with distilled water, and the samples were analyzed by SDS-gel
electrophoresis using 4-15% polyacrylamide gradient gels and
autoradiography. A 70 kDa cross-linked complex was precipitated by
the VPN antiserum in PAE/T.beta.R-1 cells, and a weaker band of the
same size was also seen in the untransfected cells, indicating that
the untransfected PAE cells contained a low amount of endogenous
ALK-5. The 70 kDa complex was not observed when preimmune serum was
used, or when immune serum was blocked by 10 .mu.g of peptide.
Moreover, a coprecipitated 94 kDa component could also be observed
in the PAE/T.beta.R-I cells. The latter component is likely to
represent a TGF-.beta. type II receptor complex, since an
antiserum, termed DRL, which was raised against a synthetic peptide
from the C-terminal part of the TGF-.beta. type II receptor,
precipitated a 94 kDa TGF-.beta. type II receptor complex, as well
as a 70 kDa type I receptor complex from PAE/T.beta.R-I cells.
[0102] The carbohydrate contents of ALK-5 and the TGF-.beta. type
II receptor were characterized by deglycosylation using
endoglycosidase F as described above and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. The
ALK-5 cross-linked complex shifted from 70 kDa to 66 kDa, whereas
that of the type II receptor shifted from 94 kDa to 82 kDa. The
observed larger shift of the type II receptor band compared with
that of the ALK-5 band is consistent with the deglycosylation data
of the type I and type II receptors on rat liver cells reported
previously (Cheifetz et al (1988) J. Biol. Chem. 263, 16984-16991),
and fits well with the fact that the porcine TGF-.beta. type II
receptor has two N-glycosylation sites (Lin et al (1992) Cell 68,
775-785), whereas ALK-5 has only one (see SEQ ID No. 9).
[0103] Binding of TGF-.beta.1 to the type I receptor is known to be
abolished by transient treatment of the cells with dithiothreitol
(DTT) (Cheifetz and Massague (1991) J. Biol. Chem. 266,
20767-20772; Wrana et al (1992) Cell 71, 1003-1014). When analyzed
by affinity cross-linking, binding of .sup.125I-TGF-.beta.1 to
ALK-5, but not to the type II receptor, was completely abolished by
DTT treatment of PAE/T.beta.R-1 cells. Affinity cross-linking
followed by immunoprecipitation by the VPN antiserum showed that
neither the ALK-5 nor the type II receptor complexes was
precipitated after DTT treatment, indicating that the VPN antiserum
reacts only with ALK-5. The data show that the VPN antiserum
recognizes a TGF-.beta. type I receptor, and that the type I and
type II receptors form a heteromeric complex.
[0104] .sup.125I-TGF-.beta.1 Binding & Affinity Crosslinking of
Transfected COS Cells
[0105] Transient expression plasmids of ALKs-1 to -6 and
T.beta.R-II were generated by subcloning into the pSV7d expression
vector or into the pcDNA I expression vector (Invitrogen).
Transient transfection of COS-1 cells and iodination of TGF-.beta.1
were carried out as described above. Crosslinking and
immunoprecipitation were performed as described for PAE cells
above.
[0106] Transfection of cDNAs for ALKs into COS-1 cells did not show
any appreciable binding of .sup.125I-TGF.beta.1, consistent with
the observation that type I receptors do not bind TGF-.beta. in the
absence of type II receptors. When the T.beta.R-II cDNA was
co-transfected with cDNAs for the different ALKs, type I
receptor-like complexes were seen, at different levels, in each
case. COS-1 cells transfected with T.beta.R-II and ALK cDNAs were
analyzed by affinity crosslinking followed by immunoprecipitation
using the DRL antisera or specific antisera against ALKs. Each one
of the ALKs bound .sup.125I-TGF-.beta.1 and was
coimmunoprecipitated with the T.beta.R-II complex using the DRL
antiserum. Comparison of the efficiency of the different ALKs to
form heteromeric complexes with T.beta.R-II, revealed that ALK-5
formed such complexes more efficiently than the other ALKs. The
size of the crosslinked complex was larger for ALK-3 than for other
ALKs, consistent with its slightly larger size.
[0107] Expression of the ALK Protein in Different Cell Types
[0108] Two different approaches were used to elucidate which ALK's
are physiological type I receptors for TGF-.beta..
[0109] Firstly, several cell lines were tested for the expression
of the ALK proteins by cross-linking followed by
immunoprecipitation using the specific antiseras against ALKs and
the TGF-.beta. type II receptor. The mink lung epithelial cell
line, Mv1Lu, is widely used to provide target cells for TGF-.beta.
action and is well characterized regarding TGF-.beta. receptors
(Laiho et al (1990) J. Biol. Chem. 265, 18518-18524; Laiho et al
(1991) J. Biol. Chem. 266, 9108-9112). Only the VPN antiserum
efficiently precipitated both type I and type II TGF-.beta.
receptors in the wild type Mv1Lu cells. The DRL antiserum also
precipitated components with the same size as those precipitated by
the VPN antiserum. A mutant cell line (R mutant) which lacks the
TGF-.beta. type I receptor and does not respond to TGF-.beta.
(Laiho et al, supra) was also investigated by cross-linking
followed by immunoprecipitation. Consistent with the results
obtained by Laiho et al (1990), supra the type III and type II
TGF-.beta. receptor complexes, but not the type I receptor complex,
were observed by affinity crosslinking. Crosslinking followed by
immunoprecipatition using the DRL antiserum revealed only the type
II receptor complex, whereas neither the type I nor type II
receptor complexes was seen using the VPN antiserum. When the cells
were metabolically labelled and subjected to immunoprecipitation
using the VPN antiserum, the 53 kDa ALK-5 protein was precipitated
in both the wild-type and R mutant Mv1Lu cells. These results
suggest that the type I receptor expressed in the R mutant is
ALK-5, which has lost the affinity for binding to TGF-.beta. after
mutation.
[0110] The type I and type II TGF-.beta. receptor complexes could
be precipitated by the VPN and DRL antisera in other cell lines,
including human foreskin fibroblasts (AG1518), human lung
adenocarcinoma cells (A549), and human oral squamous cell carcinoma
cells (HSC-2). Affinity cross-linking studies revealed multiple
TGF-.beta. type I receptor-like complexes of 70-77 kDa in these
cells. These components were less efficiently competed by excess
unlabelled TGF-.beta.1 in HSC-2 cells. Moreover, the type II
receptor complex was low or not detectable in A549 and HSC-2 cells.
Cross-linking followed by immunoprecipitation revealed that the VPN
antiserum precipitated only the 70 kDa complex among the 70-77 kDa
components. The DRL antiserum precipitated the 94 kDa type II
receptor complex as well as the 70 kDa type I receptor complex in
these cells, but not the putative type I receptor complexes of
slightly larger sizes. These results suggest that multiple type I
TGF-.beta. receptors may exist and that the 70 kDa complex
containing ALK-5 forms a heteromeric complex with the TGF-.beta.
type II receptor cloned by Lin et al (1992) Cell 68, 775-785, more
efficiently that the other species. In rat pheochromocytoma cells
(PC12) which have been reported to have no TGF-.beta. receptor
complexes by affinity cross-linking (Massague et al (1990) Ann.
N.Y. Acad. Sci. 593, 59-72), neither VPN nor DRL antisera
precipitated the TGF-.beta. receptor complexes. The antisera
against ALKs-1 to -4 and ALK6 did not efficiently immunoprecipitate
the crosslinked receptor complexes in porcine aortic endothelial
(PAE) cells or human foreskin fibroblasts.
[0111] Next, it was investigated whether ALKs could restore
responsiveness to TGF-.beta. in the R mutant of Mv1Lu cells, which
lack the ligand-binding ability of the TGF-.beta. type I receptor
but have intact type II receptor. Wild-type Mv1Lu cells and mutant
cells were transfected with ALK cDNA and were then assayed for the
production of plasminogen activator inhibitor-1 (PAI-1) which is
produced as a result of TGF-.beta. receptor activation as described
previously by Laiho et al (1991) Mol. Cell Biol. 11, 972-978.
Briefly, cells were added with or without 10 ng/ml of TGF-.beta.1
for 2 hours in serum-free MCDB 104 without methionine. Thereafter,
cultures were labelled with [.sup.35S] methionine (40 .mu.Ci/ml)
for 2 hours. The cells were removed by washing on ice once in PBS,
twice in 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 1 mM
PMSF, twice in 2 mM Tris-HCl (pH 8.0), and once in PBS.
Extracellular matrix proteins were extracted by scraping cells into
the SDS-sample buffer containing DTT, and analyzed by SDS-gel
electrophoresis followed by fluorography using Amplify. PAI-1 can
be identified as a characteristic 45kDa band (Laiho et al (1991)
Mol. Cell Biol. 11, 972-978). Wild-type Mv1Lu cells responded to
TGF-.beta. and produced PAI-1, whereas the R mutant clone did not,
even after stimulation by TGF-.beta.1. Transient transfection of
the ALK-5 cDNA into the R mutant clone led to the production of
PAI-1 in response to the stimulation by TGF-.beta.1, indicating
that the ALK-5 cDNA encodes a functional TGF-.beta. type I
receptor. In contrast, the R mutant cells that were transfected
with other ALKs did not produce PAI-1 upon the addition of
TGF-.beta.1.
[0112] Using similar approaches as those described above for the
identification of TGF-.beta.-binding ALKs, the ability of ALKs to
bind activin in the presence of ActRII was examined. COS-1 cells
were co-transfected as described above. Recombinant human activin A
was iodinated using the chloramine T method (Mathews and Vale
(1991) Cell 65, 973-982). Transfected COS-1 cells were analysed for
binding and crosslinking of .sup.125I-activin A in the presence or
absence of excess unlabelled activin A. The crosslinked complexes
were subjected to immunoprecipitation using DRL antisera or
specific ALK antisera.
[0113] All ALKs appear to bind activin A in the presence of Act
R-II. This is more clearly demonstrated by affinity cross-linking
followed by immunopreciptation. ALK-2 and ALK-4 bound
.sup.125I-activin A and were coimmunoprecipitated with ActR-II.
Other ALKs also bound .sup.125I-activin A but with a lower
efficiency compared to ALK-2 and ALK-4.
[0114] In order to investigate whether ALKs are physiological
activin type I receptors, activin responsive cells were examined
for the expression of endogenous activin type I receptors. Mv1Lu
cells, as well as the R mutant, express both type I and type II
receptors for activin, and the R mutant cells produce PAI-1 upon
the addition of activin A. Mv1Lu cells were labeled with
.sup.125I-activin A, cross-linked and immunoprecipitated by the
antisera against ActR-II or ALKs as described above.
[0115] The type I and type II receptor complexes in Mv1Lu cells
were immunoprecipitated only by the antisera against ALK-2, ALK-4
and ActR-II. Similar results were obtained using the R mutant
cells. PAE cells do not bind activin because of the lack of type II
receptors for activin, and so cells were transfected with a
chimeric receptor, to enable them to bind activin, as described
herein. A plasmid (chim A) containing the extracelluar domain and
C-terminal tail of Act R-II (amino-acids -19 to 116 and 465 to 494,
respectively (Mathews and Vale (1991) Cell, 65, 973-982)) and the
kinase domain of T.beta.R-II (amino-acids 160-543) (Lin et al
(1992) Cell, 68, 775-785) was constructed and transfected into
pcDNA/neo (Invitrogen). PAE cells were stably transfected with the
chim A plasmid by electroporation, and cells expressing the chim A
protein were established as described previously. PAE/Chim A cells
were then subjected to .sup.125I-activin A labelling crosslinking
and immunoprecipitation as described above.
[0116] Similar to Mv1Lu cells, activin type I receptor complexes in
PAE/Chim A cells were immunoprecipitated by the ALK-2 and ALK-4
antisera. These results show that both ALK-2 and ALK-4 serve as
high affinity type I receptors for activin A in these cells.
[0117] ALK-1, ALK-3 and ALK-6 bind TGF-.beta.1 and activin A in the
presence of their respective type II receptors, but the functional
consequences of the binding of the ligands remains to be
elucidated.
[0118] The experiments described supra suggested further
experiments. Specifically, it is known that TGF-.beta. family
members acts as ligands in connection with specific type I and type
II receptors, with resulting complexes interacting with members of
the Smad family. See Heldin et al., Nature 390: 465-471 (1997),
incorporated by reference. The Smad molecules are homologs of
molecules found in Drosophila ("Mad"), and C. elegans (Sma), hence,
the acronym "Smad". These are involved in signal transduction
pathways downstream of serine/threonine kinase receptors. See
Massagu et al., Trends Cell Biol. 2: 187-192 (1997). The different
members of the family have different signaling roles. Smad1, for
example, as well as Smad 2 and 3, and perhaps Smad 5, became
phosphorylated via specific type 1 serine/threonine kinase
receptors, and act in pathway restricted fashion. For example,
Xenopus Mad1 induces ventral mesoderm, in the presence of BMP. The
human Smad1 has been shown to have ventralizing activity. See Liu
et al., Nature 381: 620-623 (1996); Kretzschmer et al., Genes Dev
11: 984-995 (1997). There is also some evidence that TGF-.beta.
phosphorylates Smad1. See Lechleider et al., J. Biol. Chem. 271:
17617-17620 (1996); Yingling et al., Proc. Natl. Acad. Sci. USA 93:
8940-8944 (1996). Given what was known regarding this complex
signaling pathway, the role of ALK-1 was studied.
[0119] COS-7 cells, which do not express ALK-1, were transfected
with cDNA encoding tagged ALK-1. The tag was hemagluttinin
(hereafter "HA"), and a commercially available lipid containing
transfecting agent was used. In parallel experiments, porcine
aortic endothelial (PAE) cells were also used, because these cells
express TGF.beta. type II receptors, and ALK-5, but not ALK-1.
Hence, PAE cells were either transfected, or not. Transfection
protocols are given, supra.
[0120] The cells were then contacted with .sup.125I labelled
TGF-.beta.1, and were then contacted with ALK-1 specific antisera,
to ascertain whether cross linking had occurred. See the
experiments, supra, as well as ten Dijke et al., Science 264:
101-104 (1994), incorporated by reference. Antisera to ALK-5 were
also used.
[0121] The results indicated that the ALK-1 antiserum
immunoprecipitated complexes of the appropriate size from the
transfected COS-7 and PAE cells, but not those which were not
transfected, thereby establishing that ALK-1 is a receptor for
TGF-.beta..
[0122] This was confirmed in experiments on human umbilical vein
endothelial cells (HUVEC). These cells are known to express ALK-1
endogenously, as well as ALK-5. The ALK-5 antiserum and the ALK-1
antiserum both immunoprecipitated type I and type II receptor cross
linked complexes. The ALK-1 antiserum immunoprecipitated band
migrated slightly more slowly than the band immunprecipitated by
the ALK-5 antiserum (see, e.g., FIG. 8). This is in agreement with
the difference in size of ALK-1 and ALK-5, and it indicates that
both ALK-1 and ALK-5 bind TGF-.beta. in HUVECS.
[0123] Further, it shows that ALK-1 acts as a co-called "type I"
TGF-.beta. receptor in an endogenous, physiological setting.
[0124] Once it was determined that TGF-.beta. and ALK-1 interact,
studies were carried out to determine whether or not activation of
ALK-1 resulted in phosphorylation of Smads. To test this, COS-7
cells were transfected in the same manner described supra with
either Flag tagged Smad1, Flag tagged Smad2 or Flag tagged Smad-5
together with either a constitutively active form of ALK-1, or a
constitutively active form of ALK-5. Specifically, the variant of
ALK-1 is Q201D, and that of ALK-5 is T204D. Constitutively active
ALK-1 was used to avoid the need for an additional transfection
step. To elaborate, it is known that for the TGF-.beta. pathway to
function adequately, a complex of two, type I receptors, and two,
type II receptors must interact, so as to activate the receptors.
Constitutively active receptors, such as what was used herein, do
not require the presence of the type II receptor to function. See
Wieser et al., EMBO J 14: 2199-2208 (1995). In order to determine
if the resulting transfected cells produced phosphorylated Smads,
Smads were determined using a Flag specific antibody, which
precipitated them, and phosphorylation was determined using the
antiphosphoserine antibody of Nishimura et al., J. Biol. Chem. 273:
1872-1879 (1998). It was determined, when the data were analyzed,
that Smad1 and Smad-5 (an intracellular signalling molecule which
is structurally highly similar to Smad1) were phosphorylated
following interaction with activated ALK-1, but not following
interaction of TGF-.beta. and ALK-5. Conversely, the interaction of
TGF-.beta. and ALK-5 led to phosphorylation of Smad 2, but not Smad
1. This supports a conclusion that ALK-1 transduces signal in a
manner similar to BMPs.
[0125] FIG. 8 depicts the phosphorylation of Smad-5 following
interaction with ALK-1 but not ALK-5. Phosphorylation of both
Smad-5 and Smad1 has been shown for BMP type I receptors suggesting
ALK-1 is functionally very similar to ALK3 (BMPR-IA) and (ALK6
BMPR-IB).
[0126] Additional experiments were then carried out to study the
interaction of ALK-1 with Smad-1. Specifically, COS-7 cells were
transfected with cDNA which encoded the wild type form of the
TGF.beta. type II receptor (TBR-II), a kinase inactive form of
ALK-1, and Flag tagged Smad-1. Kinase inactive ALK-1 was used,
because the interaction of Smad-1 and receptors is known to be
transient, as once Smads are phosphorylated they dissociate from
the type I receptor. See Marcias-Silva et al., Cell 87: 1215-1224
(1996); Nakao et al., EMBO J 16: 5353-5362 (1997). Affinity
cross-linking, using .sup.125I-TGF-.beta.1, and immunoprecipitation
with Flag antibody was carried out, as discussed supra. The
expression of ALK-1 was determined using anti-HA antibody, since
the vector used to express ALK-1 effectively tagged it with HA.
[0127] The immunoprecipitating of Smad1 resulted in coprecipitation
of a cross linked TBR-II/ALK-1 complex, suggesting a direct
association of Smad1 with ALK-1.
[0128] These examples show that one can identify molecule which
inhibit, or enhance expression of a gene whose expression is
regulated by phosphorylated Smad1. To elaborate, as ALK-1 has been
identified as a key constituent of the pathway by which Smad1 is
phosphorylated, one can contact cells which express both Smad1 and
ALK-1 with a substance of interest, and then determine if the Smad1
becomes phosphorylated. The cells can be those which inherently
express both ALK-1 and Smad1, or which have been transformed or
transfected with DNA encoding one or both of these. One can
determine the phosphorylation via, e.g., the use of anti
phosphorylated serine antibodies, as discussed supra. In an
especially preferred embodiment, the assay can be carried out using
TGF-.beta., as a competing agent. The TGF-.beta., as has been
shown, does bind to ALK-1, leading to phosphorylation of Smad1.
Hence, by determining a value with TGF-.beta. alone, one can then
compare a value determined with amounts of the substance to be
tested, in the presence of TGF-.beta.. Changes in phosphorylation
levels can thus be attributed to the test substance.
[0129] In this type of system, it must be kept in mind that both
type I receptors and type II receptors must be present; however, as
indicated, supra, one can eliminate the requirement for a type II
receptor by utilizing a constitutively active form of ALK-1, such
as the form described supra. Additional approaches to inhibiting
this system will be clear to the skilled artisan. For example,
since it is known that there is interaction between Smad1 and the
ALK-1 receptor, one can test for inhibition via the use of small
molecules which inhibit the receptor/Smad interaction. Heldin et
al., supra, mention Smad6 and Smad7 as Smad1 inhibitors, albeit in
the context of a different system. Hence one can test for
inhibition, or inhibit the interaction, via adding a molecule to be
tested or for actual inhibition to a cell, wherein the molecule is
internalized by the cell, followed by assaying for phosphorylation,
via a method such as is discussed supra.
[0130] In a similar way, one can assay for inhibitors of type
I/type II receptor interaction, by testing the molecule of interest
in a system which includes both receptors, and then assaying for
phorphorylation.
[0131] Conversely, activators or agonists can also be tested for,
or utilized, following the same type of procedures.
[0132] Via using any of these systems, one can identify any gene or
genes which are activated by phosphorylated Smad1. To elaborate,
the art is very familiar with systems of expression analysis, such
as differential display PCR, subtraction hybridization, and other
systems which combine driver and testes populations of nucleic
acids, whereby transcripts which are expressed or not expressed can
be identified. By simply using an activator/inhibitor of the system
disclosed herein, on a first sample, and a second sample where none
is used, one can then carry out analysis of transcript, thereby
determining the transcripts of interest.
[0133] Also a part of the invention is the regulation of a
phosphorylation of Smad-1 or Smad-5, with inhibitors, such as
antibodies against the extracellular domain of ALK-1 or TGF-.beta.,
or enhancers, such as TGF-.beta. itself, or those portions of the
TGF-.beta. molecule which are necessary for binding. Indeed, by
appropriate truncation, one can also determine what portions of
ALK-1 are required for phosphorylation of Smad1 or Smad-5 to take
place.
[0134] The invention has been described by way of example only,
without restriction of its scope. The invention is defined by the
subject matter herein, including the claims that follow the
immediately following full Sequence Listings.
Sequence CWU 1
1
46 1 1984 DNA Homo sapiens 1 aggaaacggt ttattaggag ggagtggtgg
agctgggcca ggcaggaaga cgctggaata 60 agaaacattt ttgctccagc
ccccatccca gtcccgggag gctgccgcgc cagctgcgcc 120 gagcgagccc
ctccccggct ccagcccggt ccggggccgc gccggacccc agcccgccgt 180
ccagcgctgg cggtgcaact gcggccgcgc ggtggagggg aggtggcccc ggtccgccga
240 aggctagcgc cccgccaccc gcagagcggg cccagaggga ccatgacctt
gggctccccc 300 aggaaaggcc ttctgatgct gctgatggcc ttggtgaccc
agggagaccc tgtgaagccg 360 tctcggggcc cgctggtgac ctgcacgtgt
gagagcccac attgcaaggg gcctacctgc 420 cggggggcct ggtgcacagt
agtgctggtg cgggaggagg ggaggcaccc ccaggaacat 480 cggggctgcg
ggaacttgca cagggagctc tgcagggggc gccccaccga gttcgtcaac 540
cactactgct gcgacagcca cctctgcaac cacaacgtgt ccctggtgct ggaggccacc
600 caacctcctt cggagcagcc gggaacagat ggccagctgg ccctgatcct
gggccccgtg 660 ctggccttgc tggccctggt ggccctgggt gtcctgggcc
tgtggcatgt ccgacggagg 720 caggagaagc agcgtggcct gcacagcgag
ctgggagagt ccagtctcat cctgaaagca 780 tctgagcagg gcgacacgat
gttgggggac ctcctggaca gtgactgcac cacagggagt 840 ggctcagggc
tccccttcct ggtgcagagg acagtggcac ggcaggttgc cttggtggag 900
tgtgtgggaa aaggccgcta tggcgaagtg tggcggggct tgtggcacgg tgagagtgtg
960 gccgtcaaga tcttctcctc gagggatgaa cagtcctggt tccgggagac
tgagatctat 1020 aacacagtat tgctcagaca cgacaacatc ctaggcttca
tcgcctcaga catgacctcc 1080 cgcaactcga gcacgcagct gtggctcatc
acgcactacc acgagcacgg ctccctctac 1140 gactttctgc agagacagac
gctggagccc catctggctc tgaggctagc tgtgtccgcg 1200 gcatgcggcc
tggcgcacct gcacgtggag atcttcggta cacagggcaa accagccatt 1260
gcccaccgcg acttcaagag ccgcaatgtg ctggtcaaga gcaacctgca gtgttgcatc
1320 gccgacctgg gcctggctgt gatgcactca cagggcagcg attacctgga
catcggcaac 1380 aacccgagag tgggcaccaa gcggtacatg gcacccgagg
tgctggacga gcagatccgc 1440 acggactgct ttgagtccta caagtggact
gacatctggg cctttggcct ggtgctgtgg 1500 gagattgccc gccggaccat
cgtgaatggc atcgtggagg actatagacc acccttctat 1560 gatgtggtgc
ccaatgaccc cagctttgag gacatgaaga aggtggtgtg tgtggatcag 1620
cagaccccca ccatccctaa ccggctggct gcagacccgg tcctctcagg cctagctcag
1680 atgatgcggg agtgctggta cccaaacccc tctgcccgac tcaccgcgct
gcggatcaag 1740 aagacactac aaaaaattag caacagtcca gagaagccta
aagtgattca atagcccagg 1800 agcacctgat tcctttctgc ctgcaggggg
ctgggggggt ggggggcagt ggatggtgcc 1860 ctatctgggt agaggtagtg
tgagtgtggt gtgtgctggg gatgggcagc tgcgcctgcc 1920 tgctcggccc
ccagcccacc cagccaaaaa tacagctggg ctgaaacctg aaaaaaaaaa 1980 aaaa
1984 2 503 PRT Homo sapiens 2 Met Thr Leu Gly Ser Pro Arg Lys Gly
Leu Leu Met Leu Leu Met Ala 1 5 10 15 Leu Val Thr Gln Gly Asp Pro
Val Lys Pro Ser Arg Gly Pro Leu Val 20 25 30 Thr Cys Thr Cys Glu
Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly 35 40 45 Ala Trp Cys
Thr Val Val Leu Val Arg Glu Glu Gly Arg His Pro Gln 50 55 60 Glu
His Arg Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg 65 70
75 80 Pro Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser His Leu Cys
Asn 85 90 95 His Asn Val Ser Leu Val Leu Glu Ala Thr Gln Pro Pro
Ser Glu Gln 100 105 110 Pro Gly Thr Asp Gly Gln Leu Ala Leu Ile Leu
Gly Pro Val Leu Ala 115 120 125 Leu Leu Ala Leu Val Ala Leu Gly Val
Leu Gly Leu Trp His Val Arg 130 135 140 Arg Arg Gln Glu Lys Gln Arg
Gly Leu His Ser Glu Leu Gly Glu Ser 145 150 155 160 Ser Leu Ile Leu
Lys Ala Ser Glu Gln Gly Asp Thr Met Leu Gly Asp 165 170 175 Leu Leu
Asp Ser Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe 180 185 190
Leu Val Gln Arg Thr Val Ala Arg Gln Val Ala Leu Val Glu Cys Val 195
200 205 Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu Trp His Gly
Glu 210 215 220 Ser Val Ala Val Lys Ile Phe Ser Ser Arg Asp Glu Gln
Ser Trp Phe 225 230 235 240 Arg Glu Thr Glu Ile Tyr Asn Thr Val Leu
Leu Arg His Asp Asn Ile 245 250 255 Leu Gly Phe Ile Ala Ser Asp Met
Thr Ser Arg Asn Ser Ser Thr Gln 260 265 270 Leu Trp Leu Ile Thr His
Tyr His Glu His Gly Ser Leu Tyr Asp Phe 275 280 285 Leu Gln Arg Gln
Thr Leu Glu Pro His Leu Ala Leu Arg Leu Ala Val 290 295 300 Ser Ala
Ala Cys Gly Leu Ala His Leu His Val Glu Ile Phe Gly Thr 305 310 315
320 Gln Gly Lys Pro Ala Ile Ala His Arg Asp Phe Lys Ser Arg Asn Val
325 330 335 Leu Val Lys Ser Asn Leu Gln Cys Cys Ile Ala Asp Leu Gly
Leu Ala 340 345 350 Val Met His Ser Gln Gly Ser Asp Tyr Leu Asp Ile
Gly Asn Asn Pro 355 360 365 Arg Val Gly Thr Lys Arg Tyr Met Ala Pro
Glu Val Leu Asp Glu Gln 370 375 380 Ile Arg Thr Asp Cys Phe Glu Ser
Tyr Lys Trp Thr Asp Ile Trp Ala 385 390 395 400 Phe Gly Leu Val Leu
Trp Glu Ile Ala Arg Arg Thr Ile Val Asn Gly 405 410 415 Ile Val Glu
Asp Tyr Arg Pro Pro Phe Tyr Asp Val Val Pro Asn Asp 420 425 430 Pro
Ser Phe Glu Asp Met Lys Lys Val Val Cys Val Asp Gln Gln Thr 435 440
445 Pro Thr Ile Pro Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu
450 455 460 Ala Gln Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala
Arg Leu 465 470 475 480 Thr Ala Leu Arg Ile Lys Lys Thr Leu Gln Lys
Ile Ser Asn Ser Pro 485 490 495 Glu Lys Pro Lys Val Ile Gln 500 3
2724 DNA Homo sapiens 3 ctccgagtac cccagtgacc agagtgagag aagctctgaa
cgagggcacg cggcttgaag 60 gactgtgggc agatgtgacc aagagcctgc
attaagttgt acaatggtag atggagtgat 120 gattcttcct gtgcttatca
tgattgctct cccctcccct agtatggaag atgagaagcc 180 caaggtcaac
cccaaactct acatgtgtgt gtgtgaaggt ctctcctgcg gtaatgagga 240
ccactgtgaa ggccagcagt gcttttcctc actgagcatc aacgatggct tccacgtcta
300 ccagaaaggc tgcttccagg tttatgagca gggaaagatg acctgtaaga
ccccgccgtc 360 ccctggccaa gctgtggagt gctgccaagg ggactggtgt
aacaggaaca tcacggccca 420 gctgcccact aaaggaaaat ccttccctgg
aacacagaat ttccacttgg aggttggcct 480 cattattctc tctgtagtgt
tcgcagtatg tcttttagcc tgcctgctgg gagttgctct 540 ccgaaaattt
aaaaggcgca accaagaacg cctcaatccc cgagacgtgg agtatggcac 600
tatcgaaggg ctcatcacca ccaatgttgg agacagcact ttagcagatt tattggatca
660 ttcgtgtaca tcaggaagtg gctctggtct tccttttctg gtacaaagaa
cagtggctcg 720 ccagattaca ctgttggagt gtgtcgggaa aggcaggtat
ggtgaggtgt ggaggggcag 780 ctggcaaggg gaaaatgttg ccgtgaagat
cttctcctcc cgtgatgaga agtcatggtt 840 cagggaaacg gaattgtaca
acactgtgat gctgaggcat gaaaatatct taggtttcat 900 tgcttcagac
atgacatcaa gacactccag tacccagctg tggttaatta cacattatca 960
tgaaatggga tcgttgtacg actatcttca gcttactact ctggatacag ttagctgcct
1020 tcgaatagtg ctgtccatag ctagtggtct tgcacatttg cacatagaga
tatttgggac 1080 ccaagggaaa ccagccattg cccatcgaga tttaaagagc
aaaaatattc tggttaagaa 1140 gaatggacag tgttgcatag cagatttggg
cctggcagtc atgcattccc agagcaccaa 1200 tcagcttgat gtggggaaca
atccccgtgt gggcaccaag cgctacatgg cccccgaagt 1260 tctagatgaa
accatccagg tggattgttt cgattcttat aaaagggtcg atatttgggc 1320
ctttggactt gttttgtggg aagtggccag gcggatggtg agcaatggta tagtggagga
1380 ttacaagcca ccgttctacg atgtggttcc caatgaccca agttttgaag
atatgaggaa 1440 ggtagtctgt gtggatcaac aaaggccaaa catacccaac
agatggttct cagacccgac 1500 attaacctct ctggccaagc taatgaaaga
atgctggtat caaaatccat ccgcaagact 1560 cacagcactg cgtatcaaaa
agactttgac caaaattgat aattccctcg acaaattgaa 1620 aactgactgt
tgacattttc atagtgtcaa gaaggaagat ttgacgttgt tgtcattgtc 1680
cagctgggac ctaatgctgg cctgactggt tgtcagaatg gaatccatct gtctccctcc
1740 ccaaatggct gctttgacaa ggcagacgtc gtacccagcc atgtgttggg
gagacatcaa 1800 aaccacccta acctcgctcg atgactgtga actgggcatt
tcacgaactg ttcacactgc 1860 agagactaat gttggacaga cactgttgca
aaggtaggga ctggaggaac acagagaaat 1920 cctaaaagag atctgggcat
taagtcagtg gctttgcata gctttcacaa gtctcctaga 1980 cactccccac
gggaaactca aggaggtggt gaatttttaa tcagcaatat tgcctgtgct 2040
tctcttcttt attgcactag gaattctttg cattccttac ttgcactgtt actcttaatt
2100 ttaaagaccc aacttgccaa aatgttggct gcgtactcca ctggtctgtc
tttggataat 2160 aggaattcaa tttggcaaaa caaaatgtaa tgtcagactt
tgctgcattt tacacatgtg 2220 ctgatgttta caatgatgcc gaacattagg
aattgtttat acacaacttt gcaaattatt 2280 tattacttgt gcacttagta
gtttttacaa aactgctttg tgcatatgtt aaagcttatt 2340 tttatgtggt
cttatgattt tattacagaa atgtttttaa cactatactc taaaatggac 2400
attttctttt attatcagtt aaaatcacat tttaagtgct tcacatttgt atgtgtgtag
2460 actgtaactt tttttcagtt catatgcaga acgtatttag ccattaccca
cgtgacacca 2520 ccgaatatat tatcgattta gaagcaaaga tttcagtaga
attttagtcc tgaacgctac 2580 ggggaaaatg cattttcttc agaattatcc
attacgtgca tttaaactct gccagaaaaa 2640 aataactatt ttgttttaat
ctactttttg tatttagtag ttatttgtat aaattaaata 2700 aactgttttc
aagtcaaaaa aaaa 2724 4 509 PRT Homo sapiens 4 Met Val Asp Gly Val
Met Ile Leu Pro Val Leu Ile Met Ile Ala Leu 1 5 10 15 Pro Ser Pro
Ser Met Glu Asp Glu Lys Pro Lys Val Asn Pro Lys Leu 20 25 30 Tyr
Met Cys Val Cys Glu Gly Leu Ser Cys Gly Asn Glu Asp His Cys 35 40
45 Glu Gly Gln Gln Cys Phe Ser Ser Leu Ser Ile Asn Asp Gly Phe His
50 55 60 Val Tyr Gln Lys Gly Cys Phe Gln Val Tyr Glu Gln Gly Lys
Met Thr 65 70 75 80 Cys Lys Thr Pro Pro Ser Pro Gly Gln Ala Val Glu
Cys Cys Gln Gly 85 90 95 Asp Trp Cys Asn Arg Asn Ile Thr Ala Gln
Leu Pro Thr Lys Gly Lys 100 105 110 Ser Phe Pro Gly Thr Gln Asn Phe
His Leu Glu Val Gly Leu Ile Ile 115 120 125 Leu Ser Val Val Phe Ala
Val Cys Leu Leu Ala Cys Leu Leu Gly Val 130 135 140 Ala Leu Arg Lys
Phe Lys Arg Arg Asn Gln Glu Arg Leu Asn Pro Arg 145 150 155 160 Asp
Val Glu Tyr Gly Thr Ile Glu Gly Leu Ile Thr Thr Asn Val Gly 165 170
175 Asp Ser Thr Leu Ala Asp Leu Leu Asp His Ser Cys Thr Ser Gly Ser
180 185 190 Gly Ser Gly Leu Pro Phe Leu Val Gln Arg Thr Val Ala Arg
Gln Ile 195 200 205 Thr Leu Leu Glu Cys Val Gly Lys Gly Arg Tyr Gly
Glu Val Trp Arg 210 215 220 Gly Ser Trp Gln Gly Glu Asn Val Ala Val
Lys Ile Phe Ser Ser Arg 225 230 235 240 Asp Glu Lys Ser Trp Phe Arg
Glu Thr Glu Leu Tyr Asn Thr Val Met 245 250 255 Leu Arg His Glu Asn
Ile Leu Gly Phe Ile Ala Ser Asp Met Thr Ser 260 265 270 Arg His Ser
Ser Thr Gln Leu Trp Leu Ile Thr His Tyr His Glu Met 275 280 285 Gly
Ser Leu Tyr Asp Tyr Leu Gln Leu Thr Thr Leu Asp Thr Val Ser 290 295
300 Cys Leu Arg Ile Val Leu Ser Ile Ala Ser Gly Leu Ala His Leu His
305 310 315 320 Ile Glu Ile Phe Gly Thr Gln Gly Lys Pro Ala Ile Ala
His Arg Asp 325 330 335 Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn
Gly Gln Cys Cys Ile 340 345 350 Ala Asp Leu Gly Leu Ala Val Met His
Ser Gln Ser Thr Asn Gln Leu 355 360 365 Asp Val Gly Asn Asn Pro Arg
Val Gly Thr Lys Arg Tyr Met Ala Pro 370 375 380 Glu Val Leu Asp Glu
Thr Ile Gln Val Asp Cys Phe Asp Ser Tyr Lys 385 390 395 400 Arg Val
Asp Ile Trp Ala Phe Gly Leu Val Leu Trp Glu Val Ala Arg 405 410 415
Arg Met Val Ser Asn Gly Ile Val Glu Asp Tyr Lys Pro Pro Phe Tyr 420
425 430 Asp Val Val Pro Asn Asp Pro Ser Phe Glu Asp Met Arg Lys Val
Val 435 440 445 Cys Val Asp Gln Gln Arg Pro Asn Ile Pro Asn Arg Trp
Phe Ser Asp 450 455 460 Pro Thr Leu Thr Ser Leu Ala Lys Leu Met Lys
Glu Cys Trp Tyr Gln 465 470 475 480 Asn Pro Ser Ala Arg Leu Thr Ala
Leu Arg Ile Lys Lys Thr Leu Thr 485 490 495 Lys Ile Asp Asn Ser Leu
Asp Lys Leu Lys Thr Asp Cys 500 505 5 2932 DNA Homo sapiens 5
gctccgcgcc gagggctgga ggatgcgttc cctggggtcc ggacttatga aaatatgcat
60 cagtttaata ctgtcttgga attcatgaga tggaagcata ggtcaaagct
gtttggagaa 120 aatcagaagt acagttttat ctagccacat cttggaggag
tcgtaagaaa gcagtgggag 180 ttgaagtcat tgtcaagtgc ttgcgatctt
ttacaagaaa atctcactga atgatagtca 240 tttaaattgg tgaagtagca
agaccaatta ttaaaggtga cagtacacag gaaacattac 300 aattgaacaa
tgactcagct atacatttac atcagattat tgggagccta tttgttcatc 360
atttctcgtg ttcaaggaca gaatctggat agtatgcttc atggcactgg gatgaaatca
420 gactccgacc agaaaaagtc agaaaatgga gtaaccttag caccagagga
taccttgcct 480 tttttaaagt gctattgctc agggcactgt ccagatgatg
ctattaataa cacatgcata 540 actaatggac attgctttgc catcatagaa
gaagatgacc agggagaaac cacattagct 600 tcagggtgta tgaaatatga
aggatctgat tttcagtgca aagattctcc aaaagcccag 660 ctacgccgga
caatagaatg ttgtcggacc aatttatgta accagtattt gcaacccaca 720
ctgccccctg ttgtcatagg tccgtttttt gatggcagca ttcgatggct ggttttgctc
780 atttctatgg ctgtctgcat aattgctatg atcatcttct ccagctgctt
ttgttacaaa 840 cattattgca agagcatctc aagcagacgt cgttacaatc
gtgatttgga acaggatgaa 900 gcatttattc cagttggaga atcactaaaa
gaccttattg accagtcaca aagttctggt 960 agtgggtctg gactaccttt
attggttcag cgaactattg ccaaacagat tcagatggtc 1020 cggcaagttg
gtaaaggccg atatggagaa gtatggatgg gcaaatggcg tggcgaaaaa 1080
gtggcggtga aagtattctt taccactgaa gaagccagct ggtttcgaga aacagaaatc
1140 taccaaactg tgctaatgcg ccatgaaaac atacttggtt tcatagcggc
agacattaaa 1200 ggtacaggtt cctggactca gctctatttg attactgatt
accatgaaaa tggatctctc 1260 tatgacttcc tgaaatgtgc tacactggac
accagagccc tgcttaaatt ggcttattca 1320 gctgcctgtg gtctgtgcca
cctgcacaca gaaatttatg gcacccaagg aaagcccgca 1380 attgctcatc
gagacctaaa gagcaaaaac atcctcatca agaaaaatgg gagttgctgc 1440
attgctgacc tgggccttgc tgttaaattc aacagtgaca caaatgaagt tgatgtgccc
1500 ttgaatacca gggtgggcac caaacgctac atggctcccg aagtgctgga
cgaaagcctg 1560 aacaaaaacc acttccagcc ctacatcatg gctgacatct
acagcttcgg cctaatcatt 1620 tgggagatgg ctcgtcgttg tatcacagga
gggatcgtgg aagaatacca attgccatat 1680 tacaacatgg taccgagtga
tccgtcatac gaagatatgc gtgaggttgt gtgtgtcaaa 1740 cgtttgcggc
caattgtgtc taatcggtgg aacagtgatg aatgtctacg agcagttttg 1800
aagctaatgt cagaatgctg ggcccacaat ccagcctcca gactcacagc attgagaatt
1860 aagaagacgc ttgccaagat ggttgaatcc caagatgtaa aaatctgatg
gttaaaccat 1920 cggaggagaa actctagact gcaagaactg tttttaccca
tggcatgggt ggaattagag 1980 tggaataagg atgttaactt ggttctcaga
ctctttcttc actacgtgtt cacaggctgc 2040 taatattaaa cctttcagta
ctcttattag gatacaagct gggaacttct aaacacttca 2100 ttctttatat
atggacagct ttattttaaa tgtggttttt gatgcctttt tttaagtggg 2160
tttttatgaa ctgcatcaag acttcaatcc tgattagtgt ctccagtcaa gctctgggta
2220 ctgaattgcc tgttcataaa acggtgcttt ctgtgaaagc cttaagaaga
taaatgagcg 2280 cagcagagat ggagaaatag actttgcctt ttacctgaga
cattcagttc gtttgtattc 2340 tacctttgta aaacagccta tagatgatga
tgtgtttggg atactgctta ttttatgata 2400 gtttgtcctg tgtccttagt
gatgtgtgtg tgtctccatg cacatgcacg ccgggattcc 2460 tctgctgcca
tttgaattag aagaaaataa tttatatgca tgcacaggaa gatattggtg 2520
gccggtggtt ttgtgcttta aaaatgcaat atctgaccaa gattcgccaa tctcatacaa
2580 gccatttact ttgcaagtga gatagcttcc ccaccagctt tattttttaa
catgaaagct 2640 gatgccaagg ccaaaagaag tttaaagcat ctgtaaattt
ggactgtttt ccttcaacca 2700 ccattttttt tgtggttatt atttttgtca
cggaaagcat cctctccaaa gttggagctt 2760 ctattgccat gaaccatgct
tacaaagaaa gcacttctta ttgaagtgaa ttcctgcatt 2820 tgatagcaat
gtaagtgcct ataaccatgt tctatattct ttattctcag taacttttaa 2880
aagggaagtt atttatattt tgtgtataat gtgctttatt tgcaaatcac cc 2932 6
532 PRT Homo sapiens 6 Met Thr Gln Leu Tyr Ile Tyr Ile Arg Leu Leu
Gly Ala Tyr Leu Phe 1 5 10 15 Ile Ile Ser Arg Val Gln Gly Gln Asn
Leu Asp Ser Met Leu His Gly 20 25 30 Thr Gly Met Lys Ser Asp Ser
Asp Gln Lys Lys Ser Glu Asn Gly Val 35 40 45 Thr Leu Ala Pro Glu
Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser 50 55 60 Gly His Cys
Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly 65 70 75 80 His
Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu 85 90
95 Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp
100 105 110 Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg
Thr Asn 115 120 125 Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro
Val Val Ile Gly 130 135 140 Pro Phe Phe Asp Gly Ser Ile Arg Trp Leu
Val Leu Leu Ile Ser Met 145 150 155
160 Ala Val Cys Ile Ile Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr
165 170 175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Arg Arg Tyr Asn
Arg Asp 180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu
Ser Leu Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser
Gly Ser Gly Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile Ala Lys
Gln Ile Gln Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly Arg Tyr
Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys Val Ala
Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265 270 Arg
Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile 275 280
285 Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln
290 295 300 Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr
Asp Phe 305 310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu
Leu Lys Leu Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys His Leu
His Thr Glu Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala Ile Ala
His Arg Asp Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys Lys Asn
Gly Ser Cys Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val Lys Phe
Asn Ser Asp Thr Asn Glu Val Asp Val Pro Leu Asn Thr 385 390 395 400
Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Ser 405
410 415 Leu Asn Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr
Ser 420 425 430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg Cys Ile
Thr Gly Gly 435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn
Met Val Pro Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg Glu Val
Val Cys Val Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser Asn Arg
Trp Asn Ser Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys Leu Met
Ser Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510 Thr Ala
Leu Arg Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515 520 525
Asp Val Lys Ile 530 7 2333 DNA Homo sapiens 7 atggcggagt cggccggagc
ctcctccttc ttcccccttg ttgtcctcct gctcgccggc 60 agcggcgggt
ccgggccccg gggggtccag gctctgctgt gtgcgtgcac cagctgcctc 120
caggccaact acacgtgtga gacagatggg gcctgcatgg tttccttttt caatctggat
180 gggatggagc accatgtgcg cacctgcatc cccaaagtgg agctggtccc
tgccgggaag 240 cccttctact gcctgagctc ggaggacctg cgcaacaccc
actgctgcta cactgactac 300 tgcaacagga tcgacttgag ggtgcccagt
ggtcacctca aggagcctga gcacccgtcc 360 atgtggggcc cggtggagct
ggtaggcatc atcgccggcc cggtgttcct cctgttcctc 420 atcatcatca
ttgttttcct tgtcattaac tatcatcagc gtgtctatca caaccgccag 480
agactggaca tggaagatcc ctcatgtgag atgtgtctct ccaaagacaa gacgctccag
540 gatcttgtct acgatctctc cacctcaggg tctggctcag ggttacccct
ctttgtccag 600 cgcacagtgg cccgaaccat cgttttacaa gagattattg
gcaagggtcg gtttggggaa 660 gtatggcggg gccgctggag gggtggtgat
gtggctgtga aaatattctc ttctcgtgaa 720 gaacggtctt ggttcaggga
agcagagata taccagacgg tcatgctgcg ccatgaaaac 780 atccttggat
ttattgctgc tgacaataaa gataatggca cctggacaca gctgtggctt 840
gtttctgact atcatgagca cgggtccctg tttgattatc tgaaccggta cacagtgaca
900 attgagggga tgattaagct ggccttgtct gctgctagtg ggctggcaca
cctgcacatg 960 gagatcgtgg gcacccaagg gaagcctgga attgctcatc
gagacttaaa gtcaaagaac 1020 attctggtga agaaaaatgg catgtgtgcc
atagcagacc tgggcctggc tgtccgtcat 1080 gatgcagtca ctgacaccat
tgacattgcc ccgaatcaga gggtggggac caaacgatac 1140 atggcccctg
aagtacttga tgaaaccatt aatatgaaac actttgactc ctttaaatgt 1200
gctgatattt atgccctcgg gcttgtatat tgggagattg ctcgaagatg caattctgga
1260 ggagtccatg aagaatatca gctgccatat tacgacttag tgccctctga
cccttccatt 1320 gaggaaatgc gaaaggttgt atgtgatcag aagctgcgtc
ccaacatccc caactggtgg 1380 cagagttatg aggcactgcg ggtgatgggg
aagatgatgc gagagtgttg gtatgccaac 1440 ggcgcagccc gcctgacggc
cctgcgcatc aagaagaccc tctcccagct cagcgtgcag 1500 gaagacgtga
agatctaact gctccctctc tccacacgga gctcctggca gcgagaacta 1560
cgcacagctg ccgcgttgag cgtacgatgg aggcctacct ctcgtttctg cccagccctc
1620 tgtggccagg agccctggcc cgcaagaggg acagagcccg ggagagactc
gctcactccc 1680 atgttgggtt tgagacagac accttttcta tttacctcct
aatggcatgg agactctgag 1740 agcgaattgt gtggagaact cagtgccaca
cctcgaactg gttgtagtgg gaagtcccgc 1800 gaaacccggt gcatctggca
cgtggccagg agccatgaca ggggcgcttg ggaggggccg 1860 gaggaaccga
ggtgttgcca gtgctaagct gccctgaggg tttccttcgg ggaccagccc 1920
acagcacacc aaggtggccc ggaagaacca gaagtgcagc ccctctcaca ggcagctctg
1980 agccgcgctt tcccctcctc cctgggatgg acgctgccgg gagactgcca
gtggagacgg 2040 aatctgccgc tttgtctgtc cagccgtgtg tgcatgtgcc
gaggtgcgtc ccccgttgtg 2100 cctggttcgt gccatgccct tacacgtgcg
tgtgagtgtg tgtgtgtgtc tgtaggtgcg 2160 cacttacctg cttgagcttt
ctgtgcatgt gcaggtcggg ggtgtggtcg tcatgctgtc 2220 cgtgcttgct
ggtgcctctt ttcagtagtg agcagcatct agtttccctg gtgcccttcc 2280
ctggaggtct ctccctcccc cagagcccct catgccacag tggtactctg tgt 2333 8
505 PRT Homo sapiens 8 Met Ala Glu Ser Ala Gly Ala Ser Ser Phe Phe
Pro Leu Val Val Leu 1 5 10 15 Leu Leu Ala Gly Ser Gly Gly Ser Gly
Pro Arg Gly Val Gln Ala Leu 20 25 30 Leu Cys Ala Cys Thr Ser Cys
Leu Gln Ala Asn Tyr Thr Cys Glu Thr 35 40 45 Asp Gly Ala Cys Met
Val Ser Phe Phe Asn Leu Asp Gly Met Glu His 50 55 60 His Val Arg
Thr Cys Ile Pro Lys Val Glu Leu Val Pro Ala Gly Lys 65 70 75 80 Pro
Phe Tyr Cys Leu Ser Ser Glu Asp Leu Arg Asn Thr His Cys Cys 85 90
95 Tyr Thr Asp Tyr Cys Asn Arg Ile Asp Leu Arg Val Pro Ser Gly His
100 105 110 Leu Lys Glu Pro Glu His Pro Ser Met Trp Gly Pro Val Glu
Leu Val 115 120 125 Gly Ile Ile Ala Gly Pro Val Phe Leu Leu Phe Leu
Ile Ile Ile Ile 130 135 140 Val Phe Leu Val Ile Asn Tyr His Gln Arg
Val Tyr His Asn Arg Gln 145 150 155 160 Arg Leu Asp Met Glu Asp Pro
Ser Cys Glu Met Cys Leu Ser Lys Asp 165 170 175 Lys Thr Leu Gln Asp
Leu Val Tyr Asp Leu Ser Thr Ser Gly Ser Gly 180 185 190 Ser Gly Leu
Pro Leu Phe Val Gln Arg Thr Val Ala Arg Thr Ile Val 195 200 205 Leu
Gln Glu Ile Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg Gly 210 215
220 Arg Trp Arg Gly Gly Asp Val Ala Val Lys Ile Phe Ser Ser Arg Glu
225 230 235 240 Glu Arg Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr
Val Met Leu 245 250 255 Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala
Asp Asn Lys Asp Asn 260 265 270 Gly Thr Trp Thr Gln Leu Trp Leu Val
Ser Asp Tyr His Glu His Gly 275 280 285 Ser Leu Phe Asp Tyr Leu Asn
Arg Tyr Thr Val Thr Ile Glu Gly Met 290 295 300 Ile Lys Leu Ala Leu
Ser Ala Ala Ser Gly Leu Ala His Leu His Met 305 310 315 320 Glu Ile
Val Gly Thr Gln Gly Lys Pro Gly Ile Ala His Arg Asp Leu 325 330 335
Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Met Cys Ala Ile Ala 340
345 350 Asp Leu Gly Leu Ala Val Arg His Asp Ala Val Thr Asp Thr Ile
Asp 355 360 365 Ile Ala Pro Asn Gln Arg Val Gly Thr Lys Arg Tyr Met
Ala Pro Glu 370 375 380 Val Leu Asp Glu Thr Ile Asn Met Lys His Phe
Asp Ser Phe Lys Cys 385 390 395 400 Ala Asp Ile Tyr Ala Leu Gly Leu
Val Tyr Trp Glu Ile Ala Arg Arg 405 410 415 Cys Asn Ser Gly Gly Val
His Glu Glu Tyr Gln Leu Pro Tyr Tyr Asp 420 425 430 Leu Val Pro Ser
Asp Pro Ser Ile Glu Glu Met Arg Lys Val Val Cys 435 440 445 Asp Gln
Lys Leu Arg Pro Asn Ile Pro Asn Trp Trp Gln Ser Tyr Glu 450 455 460
Ala Leu Arg Val Met Gly Lys Met Met Arg Glu Cys Trp Tyr Ala Asn 465
470 475 480 Gly Ala Ala Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu
Ser Gln 485 490 495 Leu Ser Val Gln Glu Asp Val Lys Ile 500 505 9
2308 DNA Homo sapiens 9 ggcgaggcga ggtttgctgg ggtgaggcag cggcgcggcc
gggccgggcc gggccacagg 60 cggtggcggc gggaccatgg aggcggcggt
cgctgctccg cgtccccggc tgctcctcct 120 cgtgctggcg gcggcggcgg
cggcggcggc ggcgctgctc ccgggggcga cggcgttaca 180 gtgtttctgc
cacctctgta caaaagacaa ttttacttgt gtgacagatg ggctctgctt 240
tgtctctgtc acagagacca cagacaaagt tatacacaac agcatgtgta tagctgaaat
300 tgacttaatt cctcgagata ggccgtttgt atgtgcaccc tcttcaaaaa
ctgggtctgt 360 gactacaaca tattgctgca atcaggacca ttgcaataaa
atagaacttc caactactgt 420 aaagtcatca cctggccttg gtcctgtgga
actggcagct gtcattgctg gaccagtgtg 480 cttcgtctgc atctcactca
tgttgatggt ctatatctgc cacaaccgca ctgtcattca 540 ccatcgagtg
ccaaatgaag aggacccttc attagatcgc ccttttattt cagagggtac 600
tacgttgaaa gacttaattt atgatatgac aacgtcaggt tctggctcag gtttaccatt
660 gcttgttcag agaacaattg cgagaactat tgtgttacaa gaaagcattg
gcaaaggtcg 720 atttggagaa gtttggagag gaaagtggcg gggagaagaa
gttgctgtta agatattctc 780 ctctagagaa gaacgttcgt ggttccgtga
ggcagagatt tatcaaactg taatgttacg 840 tcatgaaaac atcctgggat
ttatagcagc agacaataaa gacaatggta cttggactca 900 gctctggttg
gtgtcagatt atcatgagca tggatccctt tttgattact taaacagata 960
cacagttact gtggaaggaa tgataaaact tgctctgtcc acggcgagcg gtcttgccca
1020 tcttcacatg gagattgttg gtacccaagg aaagccagcc attgctcata
gagatttgaa 1080 atcaaagaat atcttggtaa agaagaatgg aacttgctgt
attgcagact taggactggc 1140 agtaagacat gattcagcca cagataccat
tgatattgct ccaaaccaca gagtgggaac 1200 aaaaaggtac atggcccctg
aagttctcga tgattccata aatatgaaac attttgaatc 1260 cttcaaacgt
gctgacatct atgcaatggg cttagtattc tgggaaattg ctcgacgatg 1320
ttccattggt ggaattcatg aagattacca actgccttat tatgatcttg taccttctga
1380 cccatcagtt gaagaaatga gaaaagttgt ttgtgaacag aagttaaggc
caaatatccc 1440 aaacagatgg cagagctgtg aagccttgag agtaatggct
aaaattatga gagaatgttg 1500 gtatgccaat ggagcagcta ggcttacagc
attgcggatt aagaaaacat tatcgcaact 1560 cagtcaacag gaaggcatca
aaatgtaatt ctacagcttt gcctgaactc tccttttttc 1620 ttcagatctg
ctcctgggtt ttaatttggg aggtcagttg ttctacctca ctgagaggga 1680
acagaaggat attgcttcct tttgcagcag tgtaataaag tcaattaaaa acttcccagg
1740 atttctttgg acccaggaaa cagccatgtg ggtcctttct gtgcactatg
aacgcttctt 1800 tcccaggaca gaaaatgtgt agtctacctt tattttttat
taacaaaact tgttttttaa 1860 aaagatgatt gctggtctta actttaggta
actctgctgt gctggagatc atctttaagg 1920 gcaaaggagt tggattgctg
aattacaatg aaacatgtct tattactaaa gaaagtgatt 1980 tactcctggt
tagtacattc tcagaggatt ctgaaccact agagtttcct tgattcagac 2040
tttgaatgta ctgttctata gtttttcagg atcttaaaac taacacttat aaaactctta
2100 tcttgagtct aaaaatgacc tcatatagta gtgaggaaca taattcatgc
aattgtattt 2160 tgtatactat tattgttctt tcacttattc agaacattac
atgccttcaa aatgggattg 2220 tactatacca gtaagtgcca cttctgtgtc
tttctaatgg aaatgagtag aattgctgaa 2280 agtctctatg ttaaaaccta
tagtgttt 2308 10 503 PRT Homo sapiens 10 Met Glu Ala Ala Val Ala
Ala Pro Arg Pro Arg Leu Leu Leu Leu Val 1 5 10 15 Leu Ala Ala Ala
Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr 20 25 30 Ala Leu
Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys 35 40 45
Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys 50
55 60 Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro
Arg 65 70 75 80 Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly
Ser Val Thr 85 90 95 Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn
Lys Ile Glu Leu Pro 100 105 110 Thr Thr Val Lys Ser Ser Pro Gly Leu
Gly Pro Val Glu Leu Ala Ala 115 120 125 Val Ile Ala Gly Pro Val Cys
Phe Val Cys Ile Ser Leu Met Leu Met 130 135 140 Val Tyr Ile Cys His
Asn Arg Thr Val Ile His His Arg Val Pro Asn 145 150 155 160 Glu Glu
Asp Pro Ser Leu Asp Arg Pro Phe Ile Ser Glu Gly Thr Thr 165 170 175
Leu Lys Asp Leu Ile Tyr Asp Met Thr Thr Ser Gly Ser Gly Ser Gly 180
185 190 Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Arg Thr Ile Val Leu
Gln 195 200 205 Glu Ser Ile Gly Lys Gly Arg Phe Gly Glu Val Trp Arg
Gly Lys Trp 210 215 220 Arg Gly Glu Glu Val Ala Val Lys Ile Phe Ser
Ser Arg Glu Glu Arg 225 230 235 240 Ser Trp Phe Arg Glu Ala Glu Ile
Tyr Gln Thr Val Met Leu Arg His 245 250 255 Glu Asn Ile Leu Gly Phe
Ile Ala Ala Asp Asn Lys Asp Asn Gly Thr 260 265 270 Trp Thr Gln Leu
Trp Leu Val Ser Asp Tyr His Glu His Gly Ser Leu 275 280 285 Phe Asp
Tyr Leu Asn Arg Tyr Thr Val Thr Val Glu Gly Met Ile Lys 290 295 300
Leu Ala Leu Ser Thr Ala Ser Gly Leu Ala His Leu His Met Glu Ile 305
310 315 320 Val Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu
Lys Ser 325 330 335 Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys
Ile Ala Asp Leu 340 345 350 Gly Leu Ala Val Arg His Asp Ser Ala Thr
Asp Thr Ile Asp Ile Ala 355 360 365 Pro Asn His Arg Val Gly Thr Lys
Arg Tyr Met Ala Pro Glu Val Leu 370 375 380 Asp Asp Ser Ile Asn Met
Lys His Phe Glu Ser Phe Lys Arg Ala Asp 385 390 395 400 Ile Tyr Ala
Met Gly Leu Val Phe Trp Glu Ile Ala Arg Arg Cys Ser 405 410 415 Ile
Gly Gly Ile His Glu Asp Tyr Gln Leu Pro Tyr Tyr Asp Leu Val 420 425
430 Pro Ser Asp Pro Ser Val Glu Glu Met Arg Lys Val Val Cys Glu Gln
435 440 445 Lys Leu Arg Pro Asn Ile Pro Asn Arg Trp Gln Ser Cys Glu
Ala Leu 450 455 460 Arg Val Met Ala Lys Ile Met Arg Glu Cys Trp Tyr
Ala Asn Gly Ala 465 470 475 480 Ala Arg Leu Thr Ala Leu Arg Ile Lys
Lys Thr Leu Ser Gln Leu Ser 485 490 495 Gln Gln Glu Gly Ile Lys Met
500 11 1922 DNA Mus musculus 11 gagagcacag cccttcccag tccccggagc
cgccgcgcca cgcgcgcatg atcaagacct 60 tttccccggc cccacagggc
ctctggacgt gagaccccgg ccgcctccgc aaggagaggc 120 gggggtcgag
tcgccctgtc caaaggcctc aatctaaaca atcttgattc ctgttgccgg 180
ctggcgggac cctgaatggc aggaaatctc accacatctc ttctcctatc tccaaggacc
240 atgaccttgg ggagcttcag aaggggcctt ttgatgctgt cggtggcctt
gggcctaacc 300 caggggagac ttgcgaagcc ttccaagctg gtgaactgca
cttgtgagag cccacactgc 360 aagagaccat tctgccaggg gtcatggtgc
acagtggtgc tggttcgaga gcagggcagg 420 cacccccagg tctatcgggg
ctgtgggagc ctgaaccagg agctctgctt gggacgtccc 480 acggagtttc
tgaaccatca ctgctgctat agatccttct gcaaccacaa cgtgtctctg 540
atgctggagg ccacccaaac tccttcggag gagccagaag ttgatgccca tctgcctctg
600 atcctgggtc ctgtgctggc cttgccggtc ctggtggccc tgggtgctct
gggcttgtgg 660 cgtgtccggc ggaggcagga gaagcagcgg gatttgcaca
gtgacctggg cgagtccagt 720 ctcatcctga aggcatctga acaggcagac
agcatgttgg gggacttcct ggacagcgac 780 tgtaccacgg gcagcggctc
ggggctcccc ttcttggtgc agaggacggt agctcggcag 840 gttgcgctgg
tagagtgtgt gggaaagggc cgatatggcg aggtgtggcg cggttcgtgg 900
catggcgaaa gcgtggcggt caagattttc tcctcacgag atgagcagtc ctggttccgg
960 gagacggaga tctacaacac agttctgctt agacacgaca acatcctagg
cttcatcgcc 1020 tccgacatga cttcgcggaa ctcgagcacg cagctgtggc
tcatcaccca ctaccatgaa 1080 cacggctccc tctatgactt tctgcagagg
cagacgctgg agccccagtt ggccctgagg 1140 ctagctgtgt ccccggcctg
cggcctggcg cacctacatg tggagatctt tggcactcaa 1200 ggcaaaccag
ccattgccca tcgtgacctc aagagtcgca atgtgctggt caagagtaac 1260
ttgcagtgtt gcattgcaga cctgggactg gctgtgatgc actcacaaag caacgagtac
1320 ctggatatcg gcaacacacc ccgagtgggt accaaaagat acatggcacc
cgaggtgctg 1380 gatgagcaca tccgcacaga ctgctttgag tcgtacaagt
ggacagacat ctgggccttt 1440 ggcctagtgc tatgggagat cgcccggcgg
accatcatca atggcattgt ggaggattac 1500 aggccacctt tctatgacat
ggtacccaat gaccccagtt ttgaggacat gaaaaaggtg 1560 gtgtgcgttg
accagcagac acccaccatc cctaaccggc tggctgcaga tccggtcctc 1620
tccgggctgg cccagatgat gagagagtgc tggtacccca acccctctgc tcgcctcacc
1680 gcactgcgca taaagaagac attgcagaag ctcagtcaca atccagagaa
gcccaaagtg 1740 attcactagc ccagggccac caggcttcct ctgcctaaag
tgtgtgctgg
ggaagaagac 1800 atagcctgtc tgggtagagg gagtgaagag agtgtgcacg
ctgccctgtg tgtgcctgct 1860 cagcttgctc ccagcccatc cagccaaaaa
tacagctgag ctgaaattca aaaaaaaaaa 1920 aa 1922 12 502 PRT Mus
musculus 12 Met Thr Leu Gly Ser Phe Arg Arg Gly Leu Leu Met Leu Ser
Val Ala 1 5 10 15 Leu Gly Leu Thr Gln Gly Arg Leu Ala Lys Pro Ser
Lys Leu Val Asn 20 25 30 Cys Thr Cys Glu Ser Pro His Cys Lys Arg
Pro Phe Cys Gln Gly Ser 35 40 45 Trp Cys Thr Val Val Leu Val Arg
Glu Gln Gly Arg His Pro Gln Val 50 55 60 Tyr Arg Gly Cys Gly Ser
Leu Asn Gln Glu Leu Cys Leu Gly Arg Pro 65 70 75 80 Thr Glu Phe Leu
Asn His His Cys Cys Tyr Arg Ser Phe Cys Asn His 85 90 95 Asn Val
Ser Leu Met Leu Glu Ala Thr Gln Thr Pro Ser Glu Glu Pro 100 105 110
Glu Val Asp Ala His Leu Pro Leu Ile Leu Gly Pro Val Leu Ala Leu 115
120 125 Pro Val Leu Val Ala Leu Gly Ala Leu Gly Leu Trp Arg Val Arg
Arg 130 135 140 Arg Gln Glu Lys Gln Arg Asp Leu His Ser Asp Leu Gly
Glu Ser Ser 145 150 155 160 Leu Ile Leu Lys Ala Ser Glu Gln Ala Asp
Ser Met Leu Gly Asp Phe 165 170 175 Leu Asp Ser Asp Cys Thr Thr Gly
Ser Gly Ser Gly Leu Pro Phe Leu 180 185 190 Val Gln Arg Thr Val Ala
Arg Gln Val Ala Leu Val Glu Cys Val Gly 195 200 205 Lys Gly Arg Tyr
Gly Glu Val Trp Arg Gly Ser Trp His Gly Glu Ser 210 215 220 Val Ala
Val Lys Ile Phe Ser Ser Arg Asp Glu Gln Ser Trp Phe Arg 225 230 235
240 Glu Thr Glu Ile Tyr Asn Thr Val Leu Leu Arg His Asp Asn Ile Leu
245 250 255 Gly Phe Ile Ala Ser Asp Met Thr Ser Arg Asn Ser Ser Thr
Gln Leu 260 265 270 Trp Leu Ile Thr His Tyr His Glu His Gly Ser Leu
Tyr Asp Phe Leu 275 280 285 Gln Arg Gln Thr Leu Glu Pro Gln Leu Ala
Leu Arg Leu Ala Val Ser 290 295 300 Pro Ala Cys Gly Leu Ala His Leu
His Val Glu Ile Phe Gly Thr Gln 305 310 315 320 Gly Lys Pro Ala Ile
Ala His Arg Asp Leu Lys Ser Arg Asn Val Leu 325 330 335 Val Lys Ser
Asn Leu Gln Cys Cys Ile Ala Asp Leu Gly Leu Ala Val 340 345 350 Met
His Ser Gln Ser Asn Glu Tyr Leu Asp Ile Gly Asn Thr Pro Arg 355 360
365 Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu His Ile
370 375 380 Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp Thr Asp Ile Trp
Ala Phe 385 390 395 400 Gly Leu Val Leu Trp Glu Ile Ala Arg Arg Thr
Ile Ile Asn Gly Ile 405 410 415 Val Glu Asp Tyr Arg Pro Pro Phe Tyr
Asp Met Val Pro Asn Asp Pro 420 425 430 Ser Phe Glu Asp Met Lys Lys
Val Val Cys Val Asp Gln Gln Thr Pro 435 440 445 Thr Ile Pro Asn Arg
Leu Ala Ala Asp Pro Val Leu Ser Gly Leu Ala 450 455 460 Gln Met Met
Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu Thr 465 470 475 480
Ala Leu Arg Ile Lys Lys Thr Leu Gln Lys Leu Ser His Asn Pro Glu 485
490 495 Lys Pro Lys Val Ile His 500 13 2070 DNA Mus musculus 13
attcatgaga tggaagcata ggtcaaagct gttcggagaa attggaacta cagttttatc
60 tagccacatc tctgagaatt ctgaagaaag cagcaggtga aagtcattgc
caagtgattt 120 tgttctgtaa ggaagcctcc ctcattcact tacaccagtg
agacagcagg accagtcatt 180 caaagggccg tgtacaggac gcgtggcaat
cagacaatga ctcagctata cacttacatc 240 agattactgg gagcctgtct
gttcatcatt tctcatgttc aagggcagaa tctagatagt 300 atgctccatg
gcactggtat gaaatcagac ttggaccaga agaagccaga aaatggagtg 360
actttagcac cagaggatac cttgcctttc ttaaagtgct attgctcagg acactgccca
420 gatgatgcta ttaataacac atgcataact aatggccatt gctttgccat
tatagaagaa 480 gatgatcagg gagaaaccac attaacttct gggtgtatga
agtatgaagg ctctgatttt 540 caatgcaagg attcaccgaa agcccagcta
cgcaggacaa tagaatgttg tcggaccaat 600 ttgtgcaacc agtatttgca
gcctacactg ccccctgttg ttataggtcc gttctttgat 660 ggcagcatcc
gatggctggt tgtgctcatt tccatggctg tctgtatagt tgctatgatc 720
atcttctcca gctgcttttg ctataagcat tattgtaaga gtatctcaag caggggtcgt
780 tacaaccgtg atttggaaca ggatgaagca tttattccag taggagaatc
attgaaagac 840 ctgattgacc agtcccaaag ctctgggagt ggatctggat
tgcctttatt ggttcagcga 900 actattgcca aacagattca gatggttcgg
caggttggta aaggccgcta tggagaagta 960 tggatgggta aatggcgtgg
tgaaaaagtg gctgtcaaag tgttttttac cactgaagaa 1020 gctagctggt
ttagagaaac agaaatctac cagacggtgt taatgcgtca tgaaaatata 1080
cttggtttta tagctgcaga cattaaaggc actggttcct ggactcagct gtatttgatt
1140 actgattacc atgaaaatgg atctctctat gacttcctga aatgtgccac
actagacacc 1200 agagccctac tcaagttagc ttattctgct gcttgtggtc
tgtgccacct ccacacagaa 1260 atttatggta cccaagggaa gcctgcaatt
gctcatcgag acctgaagag caaaaacatc 1320 cttattaaga aaaatggaag
ttgctgtatt gctgacctgg gcctagctgt taaattcaac 1380 agtgatacaa
atgaagttga catacccttg aataccaggg tgggcaccaa gcggtacatg 1440
gctccagaag tgctggatga aagcctgaat aaaaaccatt tccagcccta catcatggct
1500 gacatctata gctttggttt gatcatttgg gaaatggctc gtcgttgtat
tacaggagga 1560 atcgtggagg aatatcaatt accatattac aacatggtgc
ccagtgaccc atcctatgag 1620 gacatgcgtg aggttgtgtg tgtgaaacgc
ttgcggccaa tcgtgtctaa ccgctggaac 1680 agcgatgaat gtcttcgagc
agttttgaag ctaatgtcag aatgttgggc ccataatcca 1740 gcctccagac
tcacagcttt gagaatcaag aagacacttg caaaaatggt tgaatcccag 1800
gatgtaaaga tttgacaatt aaacaatttt gagggagaat ttagactgca agaacttctt
1860 cacccaagga atgggtggga ttagcatgga ataggatgtt gacttggttt
ccagactcct 1920 tcctctacat cttcacaggc tgctaacagt aaaccttacc
gtactctaca gaatacaaga 1980 ttggaacttg gaacttcaaa catgtcattc
tttatatatg acagctttgt tttaatgtgg 2040 ggtttttttg tttgcttttt
ttgttttgtt 2070 14 532 PRT Mus musculus 14 Met Thr Gln Leu Tyr Thr
Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe 1 5 10 15 Ile Ile Ser His
Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly 20 25 30 Thr Gly
Met Lys Ser Asp Leu Asp Gln Lys Lys Pro Glu Asn Gly Val 35 40 45
Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser 50
55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn
Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu
Thr Thr Leu 85 90 95 Thr Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp
Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala Gln Leu Arg Arg Thr
Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys Asn Gln Tyr Leu Gln
Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140 Pro Phe Phe Asp Gly
Ser Ile Arg Trp Leu Val Val Leu Ile Ser Met 145 150 155 160 Ala Val
Cys Ile Val Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr 165 170 175
Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg Tyr Asn Arg Asp 180
185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu Ser Leu Lys
Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly
Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln
Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly Arg Tyr Gly Glu Val
Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys Val Ala Val Lys Val
Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265 270 Arg Glu Thr Glu
Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile 275 280 285 Leu Gly
Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln 290 295 300
Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe 305
310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu
Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu
Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala Ile Ala His Arg Asp
Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys Lys Asn Gly Ser Cys
Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val Lys Phe Asn Ser Asp
Thr Asn Glu Val Asp Ile Pro Leu Asn Thr 385 390 395 400 Arg Val Gly
Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Ser 405 410 415 Leu
Asn Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser 420 425
430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg Cys Ile Thr Gly Gly
435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met Val Pro
Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg Glu Val Val Cys Val
Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser Asn Arg Trp Asn Ser
Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys Leu Met Ser Glu Cys
Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510 Thr Ala Leu Arg Ile
Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515 520 525 Asp Val Lys
Ile 530 15 2160 DNA Mus musculus 15 cgcggttaca tggcggagtc
ggccggagcc tcctccttct tcccccttgt tgtcctcctg 60 ctcgccggca
gcggcgggtc cgggccccgg gggatccagg ctctgctgtg tgcgtgcacc 120
agctgcctac agaccaacta cacctgtgag acagatgggg cttgcatggt ctccatcttt
180 aacctggatg gcgtggagca ccatgtacgt acctgcatcc ccaaggtgga
gctggttcct 240 gctggaaagc ccttctactg cctgagttca gaggatctgc
gcaacacaca ctgctgctat 300 attgacttct gcaacaagat tgacctcagg
gtccccagcg gacacctcaa ggagcctgcg 360 cacccctcca tgtggggccc
tgtggagctg gtcggcatca tcgccggccc cgtcttcctc 420 ctcttcctta
tcattatcat cgtcttcctg gtcatcaact atcaccagcg tgtctaccat 480
aaccgccaga ggttggacat ggaggacccc tcttgcgaga tgtgtctctc caaagacaag
540 acgctccagg atctcgtcta cgacctctcc acgtcagggt ctggctcagg
gttacccctt 600 tttgtccagc gcacagtggc ccgaaccatt gttttacaag
agattatcgg caagggccgg 660 ttcggggaag tatggcgtgg tcgctggagg
ggtggtgacg tggctgtgaa aatcttctct 720 tctcgtgaag aacggtcttg
gttccgtgaa gcagagatct accagaccgt catgctgcgc 780 catgaaaaca
tccttggctt tattgctgct gacaataaag ataatggcac ctggacccag 840
ctgtggcttg tctctgacta tcacgagcat ggctcactgt ttgattatct gaaccgctac
900 acagtgacca ttgagggaat gattaagcta gccttgtctg cagccagtgg
tttggcacac 960 ctgcatatgg agattgtggg cactcaaggg aagccgggaa
ttgctcatcg agacttgaag 1020 tcaaagaaca tcctggtgaa aaaaaatggc
atgtgtgcca ttgcagacct gggcctggct 1080 gtccgtcatg atgcggtcac
tgacaccata gacattgctc caaatcagag ggtggggacc 1140 aaacgataca
tggctcctga agtccttgac gagacaatca acatgaagca ctttgactcc 1200
ttcaaatgtg ccgacatcta tgccctcggg cttgtctact gggagattgc acgaagatgc
1260 aattctggag gagtccatga agactatcaa ctgccgtatt acgacttagt
gccctccgac 1320 ccttccattg aggagatgcg aaaggttgta tgtgaccaga
agctacggcc caatgtcccc 1380 aactggtggc agagttatga ggccttgcga
gtgatgggaa agatgatgcg ggagtgctgg 1440 tacgccaatg gtgctgcccg
tctgacagct ctgcgcatca agaagactct gtcccagcta 1500 agcgtgcagg
aagatgtgaa gatttaagct gttcctctgc ctacacaaag aacctgggca 1560
gtgaggatga ctgcagccac cgtgcaagcg tcgtggaggc ctatcctctt gtttctgccc
1620 ggccctctgg cagagccctg gcctgcaaga gggacagagc ctgggagacg
cgcgcactcc 1680 cgttgggttt gagacagaca ctttttatat ttacctcctg
atggcatgga gacctgagca 1740 aatcatgtag tcactcaatg ccacaactca
aactgcttca gtgggaagta cagagaccca 1800 gtgcattgcg tgtgcaggag
cgtgaggtgc tgggctcgcc aggagcggcc cccatacctt 1860 gtggtccact
gggctgcagg ttttcctcca gggaccagtc aactggcatc aagatattga 1920
gaggaaccgg aagtttctcc ctccttcccg tagcagtcct gagccacacc atccttctca
1980 tggacatccg gaggactgcc cctagagaca caacctgctg cctgtctgtc
cagccaagtg 2040 cgcatgtgcc gaggtgtgtc ccacattgtg cctggtctgt
gccacgcccg tgtgtgtgtg 2100 tgtgtgtgtg agtgagtgtg tgtgtgtaca
cttaacctgc ttgagcttct gtgcatgtgt 2160 16 505 PRT Mus musculus 16
Met Ala Glu Ser Ala Gly Ala Ser Ser Phe Phe Pro Leu Val Val Leu 1 5
10 15 Leu Leu Ala Gly Ser Gly Gly Ser Gly Pro Arg Gly Ile Gln Ala
Leu 20 25 30 Leu Cys Ala Cys Thr Ser Cys Leu Gln Thr Asn Tyr Thr
Cys Glu Thr 35 40 45 Asp Gly Ala Cys Met Val Ser Ile Phe Asn Leu
Asp Gly Val Glu His 50 55 60 His Val Arg Thr Cys Ile Pro Lys Val
Glu Leu Val Pro Ala Gly Lys 65 70 75 80 Pro Phe Tyr Cys Leu Ser Ser
Glu Asp Leu Arg Asn Thr His Cys Cys 85 90 95 Tyr Ile Asp Phe Cys
Asn Lys Ile Asp Leu Arg Val Pro Ser Gly His 100 105 110 Leu Lys Glu
Pro Ala His Pro Ser Met Trp Gly Pro Val Glu Leu Val 115 120 125 Gly
Ile Ile Ala Gly Pro Val Phe Leu Leu Phe Leu Ile Ile Ile Ile 130 135
140 Val Phe Leu Val Ile Asn Tyr His Gln Arg Val Tyr His Asn Arg Gln
145 150 155 160 Arg Leu Asp Met Glu Asp Pro Ser Cys Glu Met Cys Leu
Ser Lys Asp 165 170 175 Lys Thr Leu Gln Asp Leu Val Tyr Asp Leu Ser
Thr Ser Gly Ser Gly 180 185 190 Ser Gly Leu Pro Leu Phe Val Gln Arg
Thr Val Ala Arg Thr Ile Val 195 200 205 Leu Gln Glu Ile Ile Gly Lys
Gly Arg Phe Gly Glu Val Trp Arg Gly 210 215 220 Arg Trp Arg Gly Gly
Asp Val Ala Val Lys Ile Phe Ser Ser Arg Glu 225 230 235 240 Glu Arg
Ser Trp Phe Arg Glu Ala Glu Ile Tyr Gln Thr Val Met Leu 245 250 255
Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Asn Lys Asp Asn 260
265 270 Gly Thr Trp Thr Gln Leu Trp Leu Val Ser Asp Tyr His Glu His
Gly 275 280 285 Ser Leu Phe Asp Tyr Leu Asn Arg Tyr Thr Val Thr Ile
Glu Gly Met 290 295 300 Ile Lys Leu Ala Leu Ser Ala Ala Ser Gly Leu
Ala His Leu His Met 305 310 315 320 Glu Ile Val Gly Thr Gln Gly Lys
Pro Gly Ile Ala His Arg Asp Leu 325 330 335 Lys Ser Lys Asn Ile Leu
Val Lys Lys Asn Gly Met Cys Ala Ile Ala 340 345 350 Asp Leu Gly Leu
Ala Val Arg His Asp Ala Val Thr Asp Thr Ile Asp 355 360 365 Ile Ala
Pro Asn Gln Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu 370 375 380
Val Leu Asp Glu Thr Ile Asn Met Lys His Phe Asp Ser Phe Lys Cys 385
390 395 400 Ala Asp Ile Tyr Ala Leu Gly Leu Val Tyr Trp Glu Ile Ala
Arg Arg 405 410 415 Cys Asn Ser Gly Gly Val His Glu Asp Tyr Gln Leu
Pro Tyr Tyr Asp 420 425 430 Leu Val Pro Ser Asp Pro Ser Ile Glu Glu
Met Arg Lys Val Val Cys 435 440 445 Asp Gln Lys Leu Arg Pro Asn Val
Pro Asn Trp Trp Gln Ser Tyr Glu 450 455 460 Ala Leu Arg Val Met Gly
Lys Met Met Arg Glu Cys Trp Tyr Ala Asn 465 470 475 480 Gly Ala Ala
Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ser Gln 485 490 495 Leu
Ser Val Gln Glu Asp Val Lys Ile 500 505 17 1952 DNA Mus musculus 17
aagcggcggc agaagttgcc ggcgtggtgc tcgtagtgag ggcgcggagg acccgggacc
60 tgggaagcgg cggcgggtta acttcggctg aatcacaacc atttggcgct
gagctatgac 120 aagagagcaa acaaaaagtt aaaggagcaa cccggccata
agtgaagaga gaagtttatt 180 gataacatgc tcttacgaag ctctggaaaa
ttaaatgtgg gcaccaagaa ggaggatgga 240 gagagtacag cccccacccc
tcggcccaag atcctacgtt gtaaatgcca ccaccactgt 300 ccggaagact
cagtcaacaa tatctgcagc acagatgggt actgcttcac gatgatagaa 360
gaagatgact ctggaatgcc tgttgtcacc tctggatgtc taggactaga agggtcagat
420 tttcaatgtc gtgacactcc cattcctcat caaagaagat caattgaatg
ctgcacagaa 480 aggaatgagt gtaataaaga cctccacccc actctgcctc
ctctcaagga cagagatttt 540 gttgatgggc ccatacacca caaggccttg
cttatctctg tgactgtctg tagtttactc 600 ttggtcctca ttattttatt
ctgttacttc aggtataaaa gacaagaagc ccgacctcgg 660 tacagcattg
ggctggagca ggacgagaca tacattcctc ctggagagtc cctgagagac 720
ttgatcgagc agtctcagag ctcgggaagt ggatcaggcc tccctctgct ggtccaaagg
780 acaatagcta agcaaattca gatggtgaag cagattggaa aaggccgcta
tggcgaggtg 840 tggatgggaa agtggcgtgg agaaaaggtg gctgtgaaag
tgttcttcac cacggaggaa 900 gccagctggt tccgagagac tgagatatat
cagacggtcc tgatgcggca tgagaatatt 960 ctggggttca ttgctgcaga
tatcaaaggg actgggtcct ggactcagtt gtacctcatc 1020
acagactatc atgaaaacgg ctccctttat gactatctga aatccaccac cttagacgca
1080 aagtccatgc tgaagctagc ctactcctct gtcagcggcc tatgccattt
acacacggaa 1140 atctttagca ctcaaggcaa gccagcaatc gcccatcgag
acttgaaaag taaaaacatc 1200 ctggtgaaga aaaatggaac ttgctgcata
gcagacctgg gcttggctgt caagttcatt 1260 agtgacacaa atgaggttga
catcccaccc aacacccggg ttggcaccaa gcgctatatg 1320 cctccagaag
tgctggacga gagcttgaat agaaaccatt tccagtccta cattatggct 1380
gacatgtaca gctttggact catcctctgg gagattgcaa ggagatgtgt ttctggaggt
1440 atagtggaag aataccagct tccctatcac gacctggtgc ccagtgaccc
ttcttatgag 1500 gacatgagag aaattgtgtg catgaagaag ttacggcctt
cattccccaa tcgatggagc 1560 agtgatgagt gtctcaggca gatggggaag
cttatgacag agtgctgggc gcagaatcct 1620 gcctccaggc tgacggccct
gagagttaag aaaacccttg ccaaaatgtc agagtcccag 1680 gacattaaac
tctgacgtca gatacttgtg gacagagcaa gaatttcaca gaagcatcgt 1740
tagcccaagc cttgaacgtt agcctactgc ccagtgagtt cagactttcc tggaagagag
1800 cacggtgggc agacacagag gaacccagaa acacggattc atcatggctt
tctgaggagg 1860 agaaactgtt tgggtaactt gttcaagata tgatgcatgt
tgctttctaa gaaagccctg 1920 tattttgaat taccattttt ttataaaaaa aa 1952
18 502 PRT Mus musculus 18 Met Leu Leu Arg Ser Ser Gly Lys Leu Asn
Val Gly Thr Lys Lys Glu 1 5 10 15 Asp Gly Glu Ser Thr Ala Pro Thr
Pro Arg Pro Lys Ile Leu Arg Cys 20 25 30 Lys Cys His His His Cys
Pro Glu Asp Ser Val Asn Asn Ile Cys Ser 35 40 45 Thr Asp Gly Tyr
Cys Phe Thr Met Ile Glu Glu Asp Asp Ser Gly Met 50 55 60 Pro Val
Val Thr Ser Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 70 75 80
Cys Arg Asp Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85
90 95 Thr Glu Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro
Pro 100 105 110 Leu Lys Asp Arg Asp Phe Val Asp Gly Pro Ile His His
Lys Ala Leu 115 120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu
Val Leu Ile Ile Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln
Glu Ala Arg Pro Arg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp
Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile
Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190 Pro Leu
Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205
Gln Ile Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210
215 220 Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala
Ser 225 230 235 240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu
Met Arg His Glu 245 250 255 Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile
Lys Gly Thr Gly Ser Trp 260 265 270 Thr Gln Leu Tyr Leu Ile Thr Asp
Tyr His Glu Asn Gly Ser Leu Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr
Thr Leu Asp Ala Lys Ser Met Leu Lys Leu 290 295 300 Ala Tyr Ser Ser
Val Ser Gly Leu Cys His Leu His Thr Glu Ile Phe 305 310 315 320 Ser
Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330
335 Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly
340 345 350 Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile
Pro Pro 355 360 365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro
Glu Val Leu Asp 370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser
Tyr Ile Met Ala Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu
Trp Glu Ile Ala Arg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu
Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro
Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Met Lys Lys 435 440 445 Leu
Arg Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455
460 Gln Met Gly Lys Leu Met Thr Glu Cys Trp Ala Gln Asn Pro Ala Ser
465 470 475 480 Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys
Met Ser Glu 485 490 495 Ser Gln Asp Ile Lys Leu 500 19 28 DNA
Artificial Sequence Sense primer, extracellular domain. 19
gcggatcctg ttgtgaaggn aatatgtg 28 20 24 DNA Artificial Sequence
Sense primer, kinase domain II 20 gcgatccgtc gcagtcaaaa tttt 24 21
26 DNA Artificial Sequence Sense Primer, Kinase domain VIB 21
gcggatccgc gatatattaa aagcaa 26 22 20 DNA Artificial sequence
Anti-sense primer, Kinase Domain VIB 22 cggaattctg gtgccatata 20 23
37 DNA Artificial Sequence Oligonucleotide probe 23 attcaagggc
acatcaactt catttgtgtc actgttg 37 24 26 DNA Artificial Sequence 5'
Oligonucleotide primer 24 gcggatccac catggcggag tcggcc 26 25 20 DNA
Artificial sequence 3' Oligonucleotide primer 25 aacaccgggc
cggcgatgat 20 26 6 PRT Artificial Sequence Consensus sequence in
Subdomain I 26 Gly Xaa Gly Xaa Xaa Gly 1 5 27 6 PRT Homo sapiens 27
Asp Phe Lys Ser Arg Asn 1 5 28 6 PRT Homo sapiens 28 Asp Leu Lys
Ser Lys Asn 1 5 29 6 PRT Homo sapiens 29 Gly Thr Lys Arg Tyr Met 1
5 30 182 PRT Homo sapiens 30 Leu Asp Thr Leu Val Gly Lys Gly Arg
Phe Ala Glu Val Tyr Lys Ala 1 5 10 15 Lys Leu Lys Gln Asn Thr Ser
Glu Gln Phe Glu Thr Val Ala Val Lys 20 25 30 Ile Phe Pro Tyr Asp
His Tyr Ala Ser Trp Lys Asp Arg Lys Asp Ile 35 40 45 Phe Ser Asp
Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr 50 55 60 Ala
Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr 65 70
75 80 Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His
Val 85 90 95 Ile Ser Trp Glu Asp Leu Arg Asn Val Gly Ser Ser Leu
Ala Arg Gly 100 105 110 Leu Ser His Leu His Ser Asp His Thr Pro Cys
Gly Arg Pro Lys Met 115 120 125 Pro Ile Val His Arg Asp Leu Lys Ser
Ser Asn Ile Leu Val Lys Asn 130 135 140 Asp Leu Thr Cys Cys Leu Cys
Asp Phe Gly Leu Ser Leu Arg Leu Gly 145 150 155 160 Pro Tyr Ser Ser
Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr 165 170 175 Ala Arg
Tyr Met Ala Pro 180 31 176 PRT Mus musculus 31 Leu Leu Glu Ile Lys
Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala 1 5 10 15 Gln Leu Met
Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp 20 25 30 Lys
Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met 35 40
45 Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser
50 55 60 Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp
Lys Gly 65 70 75 80 Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr
Trp Asn Glu Leu 85 90 95 Cys His Val Ala Glu Thr Met Ser Arg Gly
Leu Ser Tyr Leu His Glu 100 105 110 Asp Val Pro Trp Cys Arg Gly Glu
Gly His Lys Pro Ser Ile Ala His 115 120 125 Arg Asp Phe Lys Ser Lys
Asn Val Leu Leu Lys Ser Asp Leu Thr Ala 130 135 140 Val Leu Ala Asp
Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro 145 150 155 160 Pro
Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro 165 170
175 32 175 PRT Mus musculus 32 Leu Leu Glu Val Lys Ala Arg Gly Arg
Phe Gly Cys Val Trp Lys Ala 1 5 10 15 Gln Leu Leu Asn Glu Tyr Val
Ala Val Lys Ile Phe Pro Ile Gln Asp 20 25 30 Lys Gln Ser Trp Gln
Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly Met 35 40 45 Lys His Glu
Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr 50 55 60 Ser
Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly 65 70
75 80 Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu
Leu 85 90 95 Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr
Leu His Glu 100 105 110 Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro
Ala Ile Ser His Arg 115 120 125 Asp Ile Lys Ser Lys Asn Val Leu Leu
Lys Asn Asn Leu Thr Ala Cys 130 135 140 Ile Ala Asp Phe Gly Leu Ala
Leu Lys Phe Glu Ala Gly Lys Ser Ala 145 150 155 160 Gly Asp Thr His
Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro 165 170 175 33 178 PRT
Caenorhabditis elegans 33 Leu Thr Gly Arg Val Gly Ser Gly Arg Phe
Gly Asn Val Ser Arg Gly 1 5 10 15 Asp Tyr Arg Gly Glu Ala Val Ala
Val Lys Val Phe Asn Ala Leu Asp 20 25 30 Glu Pro Ala Phe His Lys
Glu Thr Glu Ile Phe Glu Thr Arg Met Leu 35 40 45 Arg His Pro Asn
Val Leu Arg Tyr Ile Gly Ser Asp Arg Val Asp Thr 50 55 60 Gly Phe
Val Thr Glu Leu Trp Leu Val Thr Glu Tyr His Pro Ser Gly 65 70 75 80
Ser Leu His Asp Phe Leu Leu Glu Asn Thr Val Asn Ile Glu Thr Tyr 85
90 95 Tyr Asn Leu Met Arg Ser Thr Ala Ser Gly Leu Ala Phe Leu His
Asn 100 105 110 Gln Ile Gly Gly Ser Lys Glu Ser Asn Lys Pro Ala Met
Ala His Arg 115 120 125 Asp Ile Lys Ser Lys Asn Ile Met Val Lys Asn
Asp Leu Thr Cys Ala 130 135 140 Ile Gly Asp Leu Gly Leu Ser Leu Ser
Lys Pro Glu Asp Ala Ala Ser 145 150 155 160 Asp Ile Ile Ala Asn Glu
Asn Tyr Lys Cys Gly Thr Val Arg Tyr Leu 165 170 175 Ala Pro 34 513
PRT Mus musculus 34 Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe
Leu Ile Ser Cys 1 5 10 15 Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu
Thr Gln Glu Cys Leu Phe 20 25 30 Phe Asn Ala Asn Trp Glu Lys Asp
Arg Thr Asn Gln Thr Gly Val Glu 35 40 45 Pro Cys Tyr Gly Asp Lys
Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50 55 60 Lys Asn Ile Ser
Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu 65 70 75 80 Asp Asp
Ile Asn Cys Tyr Asp Arg Thr Asp Cys Val Glu Lys Lys Asp 85 90 95
Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 100
105 110 Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser
Asn 115 120 125 Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu
Tyr Ser Leu 130 135 140 Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile
Cys Ala Phe Trp Val 145 150 155 160 Tyr Arg His His Lys Met Ala Tyr
Pro Pro Val Leu Val Pro Thr Gln 165 170 175 Asp Pro Gly Pro Pro Pro
Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu 180 185 190 Gln Leu Leu Glu
Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys 195 200 205 Ala Gln
Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln 210 215 220
Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly 225
230 235 240 Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys
Arg Gly 245 250 255 Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala
Phe His Glu Lys 260 265 270 Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn
Val Val Ser Trp Asn Glu 275 280 285 Leu Cys His Ile Ala Glu Thr Met
Ala Arg Gly Leu Ala Tyr Leu His 290 295 300 Glu Asp Ile Pro Gly Leu
Lys Asp Gly His Lys Pro Ala Ile Ser His 305 310 315 320 Arg Asp Ile
Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala 325 330 335 Cys
Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser 340 345
350 Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro
355 360 365 Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe
Leu Arg 370 375 380 Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu
Leu Ala Ser Arg 385 390 395 400 Cys Thr Ala Ala Asp Gly Pro Val Asp
Glu Tyr Met Leu Pro Phe Glu 405 410 415 Glu Glu Ile Gly Gln His Pro
Ser Leu Glu Asp Met Gln Glu Val Val 420 425 430 Val His Lys Lys Lys
Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His 435 440 445 Ala Gly Met
Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His 450 455 460 Asp
Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr 465 470
475 480 Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val
Thr 485 490 495 Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys
Glu Ser Ser 500 505 510 Leu 35 536 PRT Mus musculus 35 Met Thr Ala
Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala
Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25
30 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg
35 40 45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser
Trp Arg 50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly
Cys Trp Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys
Val Ala Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys
Glu Gly Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu
Pro Gly Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala
Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile
Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr 145 150 155
160 Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Val Arg
165 170 175 Gln Cys Gln Arg Trp Ala Gly Arg Arg Asp Gly Cys Ala Asp
Ser Phe 180 185 190 Lys Pro Leu Pro Phe Gln Asp Pro Gly Pro Pro Pro
Pro Ser Pro Leu 195 200 205 Val Gly Leu Lys Pro Leu Gln Leu Leu Glu
Ile Lys Ala Arg Gly Arg 210 215 220 Phe Gly Cys Val Trp Lys Ala Gln
Leu Met Asn Asp Phe Val Ala Val 225 230 235 240 Lys Ile Phe Pro Leu
Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu 245 250 255 Ile Phe Ser
Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile 260 265 270 Ala
Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile 275 280
285 Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn
290 295 300 Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met
Ser Arg 305 310 315 320 Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp
Cys Arg Gly Glu Gly 325 330 335 His Lys Pro Ser Ile Ala His Arg Asp
Phe Lys Ser Lys Asn Val Leu 340 345 350 Leu Lys Ser Asp Leu Thr Ala
Val Leu Ala Asp Phe Gly Leu Ala Val 355 360 365
Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly 370
375 380 Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn
Phe 385 390 395 400 Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala
Met Gly Leu Val 405 410 415 Leu Trp Glu Leu Val Ser Arg Cys Lys Ala
Ala Asp Gly Pro Val Asp 420 425 430 Glu Tyr Met Leu Pro Phe Glu Glu
Glu Ile Gly Gln His Pro Ser Leu 435 440 445 Glu Glu Leu Gln Glu Val
Val Val His Lys Lys Met Arg Pro Thr Ile 450 455 460 Lys Asp His Trp
Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr 465 470 475 480 Ile
Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly 485 490
495 Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr
500 505 510 Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn
Val Asp 515 520 525 Leu Leu Pro Lys Glu Ser Ser Ile 530 535 36 567
PRT Homo sapiens 36 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu
His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro
His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp
Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met
Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu
Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95
Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100
105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys
Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp
Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr
Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr
Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val
Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys
Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met
Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220
Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225
230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg
Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser
Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu
Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp
Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala
Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu
Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu
Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345
350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys
355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser
Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys
Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser
Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg
Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu
Asn Ala Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala
Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly
Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470
475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg
Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly
Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His
Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg
Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser
Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn
Thr Thr Lys 565 37 97 PRT Caenorhabditis elegans 37 Cys His Cys Ser
Arg Glu Val Gly Cys Asn Ala Arg Thr Thr Gly Trp 1 5 10 15 Val Pro
Gly Ile Glu Phe Leu Asn Glu Thr Asp Arg Ser Phe Tyr Glu 20 25 30
Asn Thr Cys Tyr Thr Asp Gly Ser Cys Tyr Gln Ser Ala Arg Pro Ser 35
40 45 Pro Glu Ile Ser His Phe Gly Cys Met Asp Glu Lys Ser Val Thr
Asp 50 55 60 Glu Thr Glu Phe His Asp Thr Ala Ala Lys Val Cys Thr
Asn Asn Thr 65 70 75 80 Lys Asp Pro His Ala Thr Val Trp Ile Cys Cys
Asp Lys Gly Asn Phe 85 90 95 Cys 38 6 PRT Artificial Sequence
Serine/threonine kinase consensus 38 Asp Leu Lys Pro Glu Asn 1 5 39
6 PRT Artificial Sequence Tyrosine kinase consensus 39 Asp Leu Ala
Ala Arg Asn 1 5 40 6 PRT Artificial Sequence Act R-II motif 40 Asp
Ile Lys Ser Lys Asn 1 5 41 6 PRT Artificial Sequence Act R-IIB
motif 41 Asp Phe Lys Ser Lys Asn 1 5 42 6 PRT Artificial Sequence
TaR-II motif 42 Asp Leu Lys Ser Ser Asn 1 5 43 6 PRT Artificial
Sequence Artificial Peptide 43 Gly Xaa Xaa Xaa Xaa Xaa 1 5 44 6 PRT
Artificial Sequence Synthetic peptide 44 Xaa Pro Xaa Xaa Trp Xaa 1
5 45 6 PRT Homo sapiens 45 Gly Thr Arg Arg Tyr Met 1 5 46 6 PRT
Homo sapiens 46 Gly Thr Ala Arg Tyr Met 1 5
* * * * *