Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (G(sα)) by protein kinase C

Abstract

Recombinant forms of G(sα 1) and G(sα 4) were shown to act as substrates for a purified preparation of brain protein kinase C. Both forms of G(sα) were thermally denatured during the incubation such that phosphorylation was virtually complete (> 90%) after 30 min. The quantity of phosphate incorporated into approximately equivalent starting amounts of the two forms of G(sα) (4.8 pmol of G(sα-1) and 5.5 pmol of G(sα-4)) at maximal phosphorylation were 0.23 ± 0.08 pmol for G(sα-1) and 0.56± 0.12 pmol for G(sα-4). Since both forms of G(sα) were thermally denatured to the same extent after 30 min, the increased phosphorylation state of G(sα-4) provides evidence that G(sα-4) contains an additional phosphorylation site. Bray and co-workers [Bray, Carter, Simmons, Guo, Puckett, Kamhollz, Spiegel and Nirenberg (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8893 -8897] proposed that an additional phosphorylation site may exist at the splice junction in G(sα-4). The guanine-nucleotide-free form of G(sα) appears to be the preferred substrate for phosphorylation. This interpretation is based upon the following observations. (i) Guanosine 5'[β-thio]diphosphate at micromolar concentrations inhibits the susceptiblity of G(sα) to phosphorylation; (ii) βγ-subunits, which inhibit GDP release from G(sα) -GDP at millimolar Mg2+ concentrations, also inhibit the susceptibility of G(sα) to phosphorylation; and (iii) guanosine 5'[βγ-imido]triphosphate inhibits the susceptiblity of G(sα) to act as a substrate for phosphorylation. These studies suggest that there is potential for cross-talk between receptors which trigger PtdIns(4,5)P2 hydrolysis and subsequently protein kinase C activation, and receptors which stimulate adenylate cyclase via G(s).