Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-29
2002-03-12
Stucker, Jeffrey (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S091200, C435S320100, C514S04400A, C530S350000, C536S023500
Reexamination Certificate
active
06355786
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to isolated and purified proteins that modulate SHP-2 biological activity and modulate cell signaling, and to nucleic acids encoding the same. More particularly, the present invention relates to an isolated and purified transmembrane protein designated as “protein zero related” or “PZR” that binds the tyrosine phosphatase SHP-2, and an isolated and purified polynucleic acid encoding the same.
Table of Abbreviations
BSA
Bovine serum albumin
EGF
epidermal growth factor
EST
expressed sequence tags
FcyRB
an ITIM-containing hematopoietic cell protein
GC-MS
gas chromatography-mass spectroscopy
HAT
cell culture media comprising hypoxanthine,
aminopterin, and thymidine
HPLC
high pressure liquid chromatography
ITIM
immunoreceptor tyrosine-based inhibitory motif
kDa
kilodalton(s)
KIR
an ITIM-containing hematopoietic cell protein
KLH
keyhole limpet hemocyanin
LAIR
an ITIM-containing hematopoietic cell protein
Myr
myristoylation
PCR
polymerase chain reaction
PDGF
platelet-derived growth factor
PTK
protein tyrosine kinase
PTP
protein tyrosine phosphatase
PZR
protein zero related
hPZR
human PZR
hPZR1B
alternatively spliced human PZR
mPZR
mouse PZR
PZRX
intracellular domain truncated PZR
RACE
rapid amplification of cDNA ends
SH2
Src homology 2 domain
SHP-1
a protein tyrosine phosphatase
SHP-2
a protein tyrosine phosphatase
SIRP/SHPS-1
an ITIM-containing putative SHP-2 substrate
TIGR
The Institute for Genomic Research
BACKGROUND ART
Protein tyrosine phosphatases (PTPs) represent a highly diverse family of enzymes that have a pivotal role in cell proliferation, differentiation, and transformation. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991)
Science
253:401-6; Walton, K. M. and Dixon, J. E. (1993)
Annu. Rev. Biochem.
62:101-20; Hunter, T. (1995)
Cell
80:225-236. SHP-1 and SHP-2, representing a subfamily of PTPs containing SH2 domains have been extensively studied in recent years. Zhao, Z, Shen, S. H. and Fischer, E. H. (1995)
Adv. in Protein Phosphatases
9:297-317; Streuli, M. (1996)
Curr. Opinion in Cell Biol
. 183: 182-188; Scharenberg, A. M. and Kinet, J. P. (1996)
Cell
87:961-964; Tonks, N. K., & Neel, B. G. (1996)
Cell
87:365-368; Frearson, J. A. and Alexander, D. R. (1997) Bioessays 19;417427; Ulyanova, T., Blasioli, J., and Thomas, M. L. (1997)
Immunolog. Res
. 16:101-113; Byon, J. C., et al. (1997)
Proc. Soc. Exp. Biol
. &
Med
. 216:1-20; Neel, B. G. and Tonks, N. K. (1997)
Curr. Opin. Cell. Biol
. 9:193-204.
SHP-1 and SHP-2 share nearly 60% overall sequence identity and are regulated in similar manners. Nevertheless, in many systems, they have distinct physiological functions. SHP-1 has a negative role in proliferation of hematopoietic cells whereas SHP-2 is a positive transducer of growth factor signal transduction. This distinction in functions is presumably due to different physiological targets.
Recently, a number of putative substrates of SHP-1 and SHP-2 have been identified. Xiao, S., et al. (1994)
J. Biol. Chem
. 269:21244-21248; Milarski, K. L. and Saltiel, A. L. (1994)
J. Biol. Chem
. 269:21239-21243; Noguchi, T., et al. (1994)
Mol. Cell. Biol
. 14:6674-6682; Yamauchi, K., et al. (1995)
Proc. Natl. Acad. Sci. U.S.A
. 92:664-668; Yamauchi, K., et al. (1995)
J. Biol. Chem
. 270:17716-17722; Frearson, J. A., Yi, T., and Alexander, D. R. (1996)
Eur. J. Immunol
. 26:1539-1543; Valiante, N. M., et al. (1996)
J. Exp. Med
. 184:2243-2250; Carlberg, K. and Rohrschneider, L. R. (1997)
J. Biol. Chem
. 272:15943-15950; Ruff, S. J., Chen, K., and Cohen S. (1997)
J. Biol. Chem
. 272:1263-1267; Gu, H., Griffin, J. D., and Neel, B. G. (1997)
J. Biol. Chem
. 272:16421-16430; Jiao, H., et al. (1997)
Exp. Hematol
. 25:592-600. One of them, designated as SIRP or SHPS-1, has been cloned (Kharitonenkov, A., et al. (1997)
Nature
386:181-186; Fujioka, Y., et al. (1996)
Mol. Cell. Biol
. 16:6887-6899). Overexpression of catalytically inactive mutants of SHP-1 and SHP-2 resulted in the identification of several hyper-phosphorylated proteins associated with the inactive SHP-1 and/or SHP-2 (Zhao, Z., et al. (1995)
J. Biol. Chem
. 270:11765-17769; Su, L., et al. (1996)
J. Biol. Chem
. 271:10385-10390.
Although a number of putative substrates of SHP-2 have been identified, little is known at the molecular level about the signaling mechanisms of SHP-2. This lack of knowledge represents a serious deficiency in the art in view of the effects of SHP-2 as described above. Therefore, further characterization of SHP-2 signaling in vertebrates, particularly in mammals, and more particularly in humans is needed. A novel isolated and purified polypeptide having a role in SHP-2 signaling would have broad utility in view of the above-described various and multiple physiological roles of SHP-2.
SUMMARY OF THE INVENTION
The present invention contemplates an isolated and purified vertebrate protein, referred to herein as “protein zero related” or “PZR”, which plays a role in SHP-2-mediated signaling. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian PZR. Even more preferably, a polypeptide of the present invention comprises a human PZR. Even more preferably, a polypeptide of the present invention comprises the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48.
The present invention also provides an isolated and purified polynucleotide that encodes a polypeptide that plays a role in SHP-2-mediated signaling. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide designated PZR. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequence of any of SEQ ID NOs:1-8 and 17-48.
In another embodiment, the present invention provides an antibody immunoreactive with a PZR polypeptide as described above. SEQ ID NOs:1-8 and 17-48 sets forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent PZR polynucleotides and polypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the PZR polypeptide comprises human PZR. Even more preferably, the PZR polypeptide comprises the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48.
In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with a PZR as described above, the process comprising: (a) transfecting a recombinant host cell with a polynucleotide that encodes a PZR polypeptide having a SHP-2 activity-modulating function; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. SEQ ID NOs:1-8 and 17-48 set forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Preferably, the host cell is transfected with the polynucleotide of any of SEQ ID NOs:1-8 and 17-48. Even more preferably, the present invention provides an antibody prepared according to the process described above. Also contemplated by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Alternatively, the present invention provides a process of detecting a PZR polypeptide as described above, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and
Jenkins & Wilson, P.A.
Stucker Jeffrey
Vanderbilt University
Winkler Ulrike
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