Isolated DNA encoding p62 polypeptides and uses therefor

#Engagement of the T cell antigen receptor (TCR) by peptide antigen bound to the major histocompatibility complex (MHC) molecules initiates a biochemical cascade involving protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Recent biochemical and genetic evidence has implicated at least three cytoplasmic PTKs, Lck, Fyn, and ZAP-70 that are involved in the initiation of TCR signal transduction. Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592. Lck and Fyn are members of the Src-family (Cooper, J. A. (1989) "The Src Family of Protein Tyrosine Kinases" In Peptides and Protein Phosphorylation ed. Kemp, B. and Alewood, P. F. (CRC Press, Boca Raton) pp. 85-113) and ZAP-70 is a member of the Syk-family. The Src-family PTKs share a number of common structural features including: (1) an N-terminal myristylated glycine at residue 2 that permits membrane localization; (2) a unique approximately 80 amino acid N-terminal region that may dictate specific associations of the kinase; (3) an approximately 60 amino acid Src-homology 3 (SH3) domain involved in interacting with signaling molecules with proline-rich regions (reviewed in Pawson, T. et al. (1992) Cell 21:359-362); (4) an approximately 100 amino acid Src-homology 2 (SH2) domain that can specifically mediate the recruitment of tyrosine phosphoproteins (reviewed in Pawson, T. et al. (1992) Cell 21:359-362); (5) a C-terminal catalytic domain; and (6) a negative regulatory tyrosine residue C-terminal to the kinase domain. Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592.

#Lck is a 56 kDa lymphoid specific PTK that noncovalently associates with the cytoplasmic domains of CD4 and CD8 through cysteine-dependent interactions. Rudd, C. E. et al. (1988) Proc. Natl. Acad Sci. USA 85:5190-5194; Veillette, A. et al. (1988) Cell 55:301-308; Turner, J. M. et al. (1990) Cell 60:755-765; Shaw, A. S. et al. (1989) Cell 59:627-636; Shaw, A. S. et al. (1990) Mol. Cell Biol. 10:1853-1862. The extracellular domains of CD4 and CD8 serve as TCR co-receptors by binding the monomorphic regions of MHC class II or I molecules, respectively, to stabilize the interaction between T cells and antigen presenting cells. Doyle, C. et al. (1988) Nature 330:256-258; Norment, A. M. et al. (1988) Nature 336:79-81. In addition to this stabilizing function, the association of CD4 and CD8 with Lck has also suggested a potential role in signal transduction for these TCR co-receptors. Veillette, A. et al. (1989) Nature 338:257-259. Specifically, the association of Lck and CD4 has been shown to be an essential, but not the only, requirement for co-receptor function in TCR signaling. Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592.

#Further evidence, in the form of genetic studies, has been derived to demonstrate the importance of Lck in both thymocyte development and TCR-mediated cell signaling. Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592. For example, mice deficient in Lck, as a result of homologous recombination, have a pronounced arrest in thymocyte development with a 10-30 fold decrease in total thymocyte number. Molina, T. J. et al. (1992) Nature 357:161-164. Whereas the double-negative (CD4.sup.- CD8.sup.-) thymocyte population was similar to normal littermates, there was a dramatic reduction in the double-positive (CD4.sup.+ CD8.sup.+) thymocyte population (10-60 fold) and no detectable single positive (CD4.sup.+ CD8.sup.- and CD4.sup.- CD8.sup.+) thymocytes. A marked reduction also occurred in the number of peripheral T cells, though the few peripheral T cells were capable of mounting a diminished proliferative response to antibody-mediated cross-linking of the TCR. Thus, Lck appears to be critical for normal thymocyte development. Chan, A. C. et al. (1994) Annu. Rev. Immunol. 12:555-592.

#FIGS. 1A-1B is the nucleotide sequence of an approximately 2.1 kb (2083 bp) cDNA encoding a first full length human p62 polypeptide (SEQ ID NO:1).

#FIG. 2 is the predicted full length amino acid sequence (440 amino acid residues) of the human p62 polypeptide (SEQ ID NO:2) encoded by the nucleotide sequence shown in FIG. 1.

#FIGS. 3A-3B is the nucleotide sequence of an approximately 2.0 kb (1977 bp) cDNA encoding a second human p62 polypeptide (SEQ ID NO:3).

#The present invention pertains to the family of novel p62 polypeptides, or active portions thereof which are capable of, for example, modulating T or B cell development (e.g., T or B cell differentiation) and/or T or B cell activation by, for example, modulation of Lck activity. The p62 polypeptides of the invention are also capable of modulating degradation of cellular proteins, e.g., cell cycle regulatory proteins, stimulating expression of cell cycle dependent kinase inhibitors, and arresting cell cycle progression at specific boundaries, to thereby modulate cell proliferation, e.g., cell proliferation associated with tumor formation and growth. Other activities of the p62 polypeptides of the invention are described herein.

#Particularly preferred p62 polypeptides are human polypeptides. The complete nucleotide (2083 nucleotides shown in FIG. 1, SEQ ID NO:1) and amino acid sequence (440 amino acids shown in FIG. 2, SEQ ID NO:2) of a first member of the p62 polypeptide family are disclosed herein. A plasmid containing the full length nucleotide sequence encoding this first p62 polypeptide was deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209 on Dec. 19, 1995 and was assigned ATCC Accession Number 97387. This first p62 polypeptide family member is a human cytoplasmic polypeptide with a molecular weight of about 62 kD and is expressed in a variety of tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The mRNA which encodes this polypeptide includes about 2 kb. This p62 polypeptide includes several defined domains. The N-terminal 50 amino acids (amino acid residues 1-50 of the amino acid sequence of FIG. 2, SEQ ID NO:2, which are encoded by nucleotides 67-216 of the nucleotide sequence of FIG. 1, SEQ ID NO:1) of the p62 polypeptide comprise an SH2 binding domain, e.g., an SH2 binding domain which does not include phosphotyrosine. A rac GTPase binding motif appears at amino acid residues 66-82 of FIG. 2, SEQ ID NO:2 (which are encoded by nucleotides 262-312 as shown in FIG. 1, SEQ ID NO:1) of the first p62 polypeptide. The rac GTPase binding motif can be compared as follows to the proposed consensus sequence for rac GTPase set forth in Zhou et al. ((1995) J. Biol. Chem. 270:12665-12669) which also appears in human MEK5, scd1 (see also Chang et al. (1994) Cell 79:131-141), and cdc24 (see also Miyamoto et al. (1991) Biochem. Biophys. Res. Commun. 181:604-610):

#______________________________________ PROTEIN RAC GTPase CONSENSUS SEQUENCE ______________________________________ p62 66 HYRDEDGDLVAFSSDEE 82 MEK5 61 EYEDEDGDRITVRSDEE 77 scd1 786 KYVDEDGDFITITSDED 802 cdc24 696 KYQDEDGDFVVLGSDED 715 ______________________________________