IDENTIFICATION OF THE IN-VITRO PHOSPHORYLATION SITES ON G(S-ALPHA) MEDIATED BY PP60(C-SRC)

Overexpression of pp60(c-src) in mouse fibroblasts potentiates both ag onist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttre ll, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7 466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400] . In reconstitution experiments in vitro, phosphorylation of G(s alpha ) by immune-complexed pp60(c-src) resulted in enhanced rates of recept or-mediated guanosine 5'-[gamma-thio]triphosphate (GTP[S]) binding and GTP hydrolysis [Hausdorff, Pitcher, Luttrell, Linder, Kurose, Parsons , Caron and Lefkowitz (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5720-57 24]. These results suggest that one mechanism by which pp60(c-src) aff ects signalling through the beta-adrenergic receptor is by phosphoryla tion and functional alteration of the G protein. To elucidate how phos phorylation of G(s alpha) might affect its function, we subjected phos phorylated, recombinant G(s alpha) to tryptic phosphopeptide analysis. Phosphotryptic peptides were purified by h.p.l.c. and analysed by Edm an degradation to determine the cycle numbers at which radiolabelled p hosphotyrosine was released. Candidate peptides that contained Tyr res idues at the corresponding positions were synthesized, phosphorylated in vitro by pp60(c-src), their migrations in two-dimensional electroph oresis/t.l.c. were compared with those of tryptic phosphopeptides from intact G(s alpha). We report here that G(s alpha) is phosphorylated o n two residues by pp60(c-src), namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a regio n postulated to modulate GDP dissociation and activation by GTP [Johns on, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cel l Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-ter minus, within a region of G(s alpha) important for receptor interactio n [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Na ture (London) 334, 712-715]. The location of these residues suggests t hat phosphorylation may affect the function of both of these regulator y domains.