Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-07-19
2001-09-18
Wang, Andrew (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06291645
ABSTRACT:
BACKGROUND OF THE INVENTION
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 lyrosine 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
−
CD8
−
) thymocyte population was similar to normal littermates, there was a dramatic reduction in the double-positive (CD4
+
CD8
+
) thymocyte population (10-60 fold) and no detectable single positive (CD4
+
CD8
−
and CD4
−
CD8
+
) 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.
The role of Lck in TCR-mediated signaling is further supported by results from two studies in which loss of a functional Lck protein abrogated TCR-mediated signaling. In the first study, a mutant of the Jurkat leukemic T cell line, J.CaM1.6, lacking a functional Lck PTK failed to mobilize calcium, to induce tyrosine phosphoproteins, or to express activation antigens following TCR stimulation. Straus, D. and Weiss, A. (1992)
Cell
70:585-593. Reconstitution with wild-type murine Lck in this mutant restored all TCR-mediated functions. In the second study, a spontaneous variant of an IL-2-dependent cytotoxic T cell line lacking Lck also manifested a profound reduction in TCR-mediated cytolysis that was restored following Lck expression. Karnitz, L. et al. (1992)
Mol. Cell Biol.
12:4521-4530. Both mutants demonstrated comparable levels of Fyn kinase activity relative to their parental counterparts. The fact that normal levels of other Src-family PTKs in these cells are unable to compensate for the Lck deficit demonstrates that Lck plays a critical role in TCR-mediated signal transduction. Chan, A. C. et al. (1994)
Annu. Rev. Immunol.
12:555-592.
Further studies have yielded results which are consistent with the requirement for Lck in TCR-mediated signaling. Specifically, overexpression of an “activated” form of Lck(F505) in a CD4
−
negative murine T cell hybridoma resulted in enhanced antigen-induced IL-2 secretion and TCR-induced cellular tyrosine phosphoproteins. Abraham, N. et al. (1991)
Nature
350:62-66. In addition, it has been shown through further analysis of the domains within Lck that participate in TCR function that membrane localization and the SH2 domain of Lck are both required. Caron, L. et al. (1992)
Mol Cell Biol.
12:2720-2729. Mutation of the N-terminal site of myristylation (thereby preventing membrane localization of Lck(F505)) or deletion of the SH2 domain of Lck(F505) abolished the TCR-induced hyperresponsiveness as indicated by cellular tyrosine phosphorylation and antigen-induced IL-2 production. In contrast, retroviral infection of T helper hybridoma cell lines with a temperature sensitive Lck(F505) resulted in antigen-independent IL-2 production at the permissive temperature. Luo, K. and Sefton, B. M. (1992)
Mol. Cell Biol.
12:4724-4732. In this system, while deletion of the SH2 domain abrogated antigen-independent IL-2 production, deletion of the SH3 domain did not significantly alter IL-2 production. Thus, the SH2 domain may be required to interact with downstream effector molecules in propagating TCR function. Given the above-described studies, further information about the mechanisms and cellular components which regulate Lck function would offer potential new routes for modulating Lck/TCR-mediated cells signaling and lymphoid cell development and/or function.
SUMMARY OF THE INVENTION
This invention is based, at least in part, on the discovery of a family of polypeptides, designated herein as p62 polypeptides, which share at least two structural/functional properties, at least one of which is relevant to Lck function. The p62 polypeptides include, for example, an SH2 binding domain, e.g., an SH2 binding domain which binds an SH2 domain of Lck independent of phosphotyrosine and a ubiquitin binding domain.
Preferred p62 polypeptides of the present invention include several additional structural/functional domains such as a zinc finger domain, a GTPase binding domain, domains containing phosphorylation sites, a PEST domain, and an SH3 binding domain. p62 polypeptides within the scope of the invention are also characterized functionally by, for example, the ability to modulate T cell activity, e.g., T cell development/differentiation, T cell activation, lymphokine secretion; the ability to modulate B cell activity, e.g., B cell development/differentiation, B cell activation, antibody secretion; the ability to modulate ubiquitin-mediated degradation of cellular proteins; the p62 polypeptide modulates expression of cell cycle dependent kinase inhibitors, e.g., p21
cip
; the ability to bind to at least one polypeptide involved in the ras cell signaling cascade, e.g., p120-GAP; the ability to bind to GTPase; the abi
Joung Insil
Shin Jaekyoon
Strominger Jack L.
Vadlamudi Ratna K.
Dana-Farber Cancer Institute
Lahive & Cockfield LLP
Wang Andrew
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