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. 1995 Jan 15;305(Pt 2):411–417. doi: 10.1042/bj3050411

Identification of the in vitro phosphorylation sites on Gs alpha mediated by pp60c-src.

J S Moyers 1, M E Linder 1, J D Shannon 1, S J Parsons 1
PMCID: PMC1136377  PMID: 7530445

Abstract

Overexpression of pp60c-src in mouse fibroblasts potentiates both agonist-induced signalling through beta-adrenergic receptors and cyclic AMP accumulation in response to cholera toxin [Bushman, Wilson, Luttrell, Moyers and Parsons (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7462-7466; Moyers, Bouton and Parsons (1993) Mol. Cell. Biol. 13, 2391-2400]. In reconstitution experiments in vitro, phosphorylation of Gs alpha by immune-complexed pp60c-src resulted in enhanced rates of receptor-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, 5720-5724]. These results suggest that one mechanism by which pp60c-src affects signalling through the beta-adrenergic receptor is by phosphorylation and functional alteration of the G protein. To elucidate how phosphorylation of Gs alpha might affect its function, we subjected phosphorylated, recombinant Gs alpha 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 pp60c-src, and their migrations in two-dimensional electrophoresis/t.l.c. were compared with those of tryptic phosphopeptides from intact Gs alpha. We report here that Gs alpha is phosphorylated on two residues by pp60c-src, namely, Tyr-37 and Tyr-377. Tyr-37 lies near the site of beta gamma binding in the N-terminus, within a region postulated to modulate GDP dissociation and activation by GTP [Johnson, Dhanasekaran, Gupta, Lowndes, Vaillancourt and Ruoho (1991) J. Cell Biochem. 47, 136-146], while Tyr-377 is located in the extreme C-terminus, within a region of Gs alpha important for receptor interaction [Sullivan, Miller, Masters, Beiderman, Heideman and Bourne (1987) Nature (London) 334, 712-715]. The location of these residues suggests that phosphorylation may affect the function of both of these regulatory domains.

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Selected References

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