Identification of the in vitro phosphorylation sites on G(sα) mediated by pp60(c-src)

Abstract

Overexpression of pp60(c-src) in mouse fibroblasts potentiates both agonist-induced signalling through β-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin. In reconstitution experiments in vitro, phosphorylation of G(sα) by immune-complexed pp60(c-src) resulted in enhanced rates of receptor-mediated guanosine 5'-[γ-thio]triphosphate (GTP[S]) binding and GTP hydrolysis. These results suggest that one mechanism by which pp60(c-src) affects signalling through the β-adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of G(sα) might affect its function, we subjected phosphorylated, recombinant G(sα) to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edman degradation to determine the cycle numbers at which radiolabelled phosphotyrosine was released. Candidate peptides that contained Tyr residues at the corresponding positions were synthesized, phosphorylated in vitro by pp60(c-src) and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact G(sα). We report here that G(sα) is phosphorylated on two residues by pp60(c-src), namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of βγ binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP, while Tyr-377 is located in the extreme C-terminus, within a region of G(sα) important for receptor interaction. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains.