A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp6OC-srC kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60' and G proteins may occur with functional consequences. Preparations of pp6cc isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein a subunits (Ga) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and Ga(GDP) uncomplexed by Pr subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory Ga (Ga.) modestly increases the rate of binding of guanosine 5'-[y-[35S]thiojtriphosphate to G. as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs.Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for ppW.SrI.The oncogenes of many retroviruses encode tyrosine-specific protein kinases that are presumed to phosphorylate cellular proteins involved in the control of cell growth and differentiation. Many of these kinases belong to the src family of kinases by virtue of their structural homology with the oncogene product (pp60v-src) encoded by Rous sarcoma virus. Members of this family are cytoplasmic proteins that are myristoylated, associated with cellular membranes, and may mediate the actions of diverse cell-surface receptors (1-3).A number of lines of experimental evidence point to the existence of functional interactions between activated members of the pp6OC-src family and elements in the signal transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins). For example,. several growth factors and lymphocyte cell-surface antigens whose actions have been proposed to be mediated by members of the src subfamily (1-9) also modulate G-protein functioning (10,11) and adenylyl cyclase activity (12,13). In addition, two enzymes involved in inositol phospholipid metabolism, which is highly regulated by G proteins, may be targets of phosphorylation by activated src gene products (1,14). Furthermore, overexpression .of avian pp6OC-src in fibroblasts results in enhanced intracellular cAMP levels and adenylyl cyclase activity in response to B-adrenergic hormones, which act via the stimulatory G protein (GJ) (15,16).The molecular nature of these putative interactions has remained obscure. However, the observation that pp60c-src is a major contaminant in some highly purified G-protein preparations (17) suggests the possibility of a direct association between pp6Oc-src and G proteins. In this study, we examined the abilities of purified G proteins to serve as substrates for phosphorylation by pp60C-src. Immunoprecipitations were performed by using the rodent/avian pp60c-src-specific monoclonal antibody GD11 (27). Specifically, 20 1.l of a 1:10 dilution of GD11 ...