Js. Moyers et al., IDENTIFICATION OF THE IN-VITRO PHOSPHORYLATION SITES ON G(S-ALPHA) MEDIATED BY PP60(C-SRC), Biochemical journal, 305, 1995, pp. 411-417
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.