G(I-ALPHA) AND G(I-BETA) ARE PART OF A SIGNALING COMPLEX IN BALB/C3T3 CELLS - PHOSPHORYLATION OF G(I-BETA) IN GROWTH FACTOR-ACTIVATED FIBROBLASTS

Stimulation of division of Balb/c3T3 cells by epidermal growth factor (EGF) and/or insulin is inhibited by pertussis toxin. The G-protein involvement in this response includes the growth factor receptor-induced translocation of the alpha-subunit of G(i) (G(ialpha)) to the nucleus, where G(ialpha) binds specifically to chromatin of dividing cells. This paper reports the first data of studies on the mode of interaction of tyrosine kinase growth factor receptors with G(ialpha), and the mechanism by which G(i) affects cell proliferation. When G(ialpha) was immunoprecipitated from Triton X-100 extracts of Balb/c3T3 cells, several other proteins were co-precipitated. The major proteins, of 110,000, 60,000 and 36,000 M(r) were not directly recognized by the G(ialpha) antibody, showing that G(ialpha) was in a complex with these proteins. The 36,000 M(r) protein was recognized by G(beta-common) antiserum, so confirming its identity as G(ibeta). The 36,000 M(r) protein was phosphorylated in cells activated for 20 h with platelet-derived growth factor, epidermal growth factor and insulin, but not after 3 min or 1 h of stimulation. Both G(ialpha) and G(beta-common) antibodies precipitated the phosphorylated 36,000 protein. G(ibeta) phosphorylation was similarly observed in response to activation by EGF alone for 20 h, but to a lesser extent. Phosphotyrosine antibodies also precipitated a 36,000 M(r) phosphorylated protein from growth factor-activated cells, suggesting that G(ibeta) may be phosphorylated on tyrosine. Therefore, G(ibeta) phosphorylation appears to represent a late event after activation of cells by tyrosine kinase growth factor receptors. We are currently examining the role of this event in signal transduction, particularly in relation to control of nuclear responses.