The specific and high affinity interaction between phosphotyrosyl- containing peptides and Src-homology 2 (SH2) domains appears to be a mechanism by which the receptor tyrosine kinases are linked to downstream signaling pathways. The function of such interaction is thought for the receptors to recruit cellular signaling molecules to form functional networks. Nck, a class II SH2- and SH3-(Src-homology 3) containing signaling molecule, is a ubiquitously expressed cellular oncogene, and a common target for varieties of cell surface receptors. Elevated expression of Nck causes cell transformation in culture and tumor formation in nude mice. Alterations (deletion, insertion, inversion, or translocation) at its chromosomal location are associated with a number of human inherited disorders and neoplasias. While the mechanism of Nck action remains to be resolved, these findings clearly indicate that Nck is involved in control of cell growth. The goal of research herein is towards elucidating the biological function and the mechanism of action of Nck. Accomplishment of this study may not only shed light on the specific function of Nck, but also the mechanism of the action of the class II SH2 and SH3 signaling molecules in general. We will be collaborating with Larry Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle), Ed Skolnik and Joseph Schlessinger (New York University Medical Center, New York) on Nck binding to CSF- l receptor, IRS-1 (insulin receptor substrate-1) and EGF receptors, respectively. The binding sites of Nck SH2 domain in these tyrosine kinase receptors/substrate will be identified in intact cells by using cell lines expressing either the wild-type or the mutant receptors/substrate. The effects of the mutations at the Nck binding sites in the receptors/substrate and interfering mutations in the SH2 domain of Nck on the signaling by the receptors will be evaluated. Anti-PY, GST-Nck and anti-Nck antibody affinity columns will be used to purify a novel p60-kDa phosphotyrosine protein which specifically mediates the interaction between Nck and EGF receptor, and function and specificity of the p60 protein will be assessed. Using purified and radiolabeled Nck SH3 domains as the probe to screen the pLox mouse embryo cDNA expression library, we have identified partial sequences of two novel genes, NS3P1 and NS3P2 (Nck SH3 binding protein 1 and 2). These proteins associate with Nck in intact cells. We will obtain the full-length cDNA of the NS3Ps and study their role in Nck-mediated cell transformation. Finally, the expression and function of Nck and NS3P genes in Nck chromosomal location (3q21-24) alteration-associated neoplastic cells will be investigated.
