RACK1, a Receptor for Activated C Kinase and a Homolog of the β Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells

The cellular gene c-src and its viral homolog v-src (the transforming gene of Rous sarcoma virus) encode 60-kDa, cytoplasmic, membrane-associated protein-tyrosine kinases (reviewed in reference 6). For the viral protein (v-Src) or for transforming mutants of the cellular protein (c-Src or Src), a close correlation exists between elevated specific kinase activity and cell transformation. At least four factors are known to influence Src activity: (i) mutation within the coding region of the src gene, (ii) phosphorylation on Tyr 527 and Tyr 416 of Src, (iii) subcellular localization of Src and its substrates, and (iv) association of Src with other cellular proteins. Compelling evidence indicates that Src-binding proteins can regulate Src activity (reviewed in reference 6). A number of interacting proteins that upregulate Src activity have been identified. The prototype is middle T antigen (mT), the transforming protein of polyomavirus. Src complexed with mT has elevated specific activity due to dephosphorylation of Tyr 527 (5, 8, 11, 16). Activation of Src is required for the induction of mammary tumors in mT transgenic mice (24). Characterization of the Src-mT complex led to discovery of the fundamental mechanism by which the cellular Src protein is converted to a transforming protein and defined the requirement of Src for polyomavirus transformation. Thus, characterization of a single Src-binding protein contributed substantially to our understanding of both RNA and DNA tumor virology. While a number of interacting proteins that upregulate Src activity have been identified, few that downregulate Src activity have been identified. Because it is the repression of c-Src activity rather than the elevation of v-Src activity that accounts for differences in their transforming abilities (9, 29, 53, 56), it is important to search for cellular mechanisms that inactivate c-Src. Recently, caveolin, a 22-kDa integral membrane protein that is the principal structural and regulatory component of caveolae, was shown to bind Src and suppress its tyrosine kinase activity (33–35). Domains within Src kinases target the kinases to specific subcellular locations where they bind to regulatory and/or substrate proteins and are integrated into cell signaling pathways and cell cycle events (reviewed in reference 6). For example, the N-terminal unique domain (UD) confers the specificity of binding of Lck to CD4 and CD8 (64) and of Fyn to the zeta chain of the T-cell receptor (22), thus coupling intracellular tyrosine kinases to the signaling pathways of cell surface receptors. The SH3 domain binds to proline-rich motifs in proteins such as Sam68, an RNA-binding protein that binds to Src and becomes tyrosine phosphorylated during mitosis (20, 63). The SH2 domain of Src binds to tyrosine-phosphorylated proteins such as the platelet-derived growth factor (PDGF) receptor, and this interaction, which results in Src activation, is required for PDGF-induced DNA synthesis (57, 65). Thus, the UD and the SH3 and SH2 domains (UD/SH3/SH2) in Src are key binding sites for proteins that regulate Src activity and integrate Src into important signaling pathways and cell cycle events. The purpose of this study was to isolate and characterize Src-interacting proteins that potentially regulate Src activity. We focused on protein interactions that involve UD/SH3/SH2 of Src. Using the yeast two-hybrid assay to screen a human lung fibroblast cDNA library, we identified RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, as a Src SH2-binding protein. We found that the overexpression of RACK1 inhibits the activity of Src tyrosine kinases and the growth of NIH 3T3 cells. RACK1 exerts its effect on growth during the G0/G1 phase of the cell cycle.