PHOSPHORYLATION OF THE SPLICED VARIANT FORMS OF THE RECOMBINANT STIMULATORY GUANINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN (GS-ALPHA) BY PROTEIN-KINASE-C

Recombinant forms of G(s-alpha-1) and G(s-alpha-4) were shown to act as substrates for a purified preparation of brain protein kinase C. Both forms of G(s-alpha) 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(s-alpha) (4.8 pmol of G(s-alpha-1) and 5.5 pmol of G(s-alpha-4)) at maximal phosphorylation were 0.23 +/- 0.08 pmol for G(s-alpha-1) and 0.56 +/- 0.12 pmol for G(s-alpha-4). Since both forms of G(s-alpha) were thermally denatured to the same extent after 30 min, the increased phosphorylation state of G(s-alpha-4) provides evidence that G(s-alpha-4) contains an additional phosphorylation site. Bray and co-workers [Bray, Carter, Simmons, Guo, Puckett, Kamhollz, Spiegel & 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(s-alpha-4). The guanine-nucleotide-free form of G(s-alpha) appears to be the preferred substrate for phosphorylation. This interpretation is based upon the following observations. (i) Guanosine 5'-[beta-thio]diphosphate at micromolar concentrations inhibits the susceptiblity of G(s-alpha) to phosphorylation; (ii) beta-gamma-subunits, which inhibit GDP release from G(s-alpha)-GDP at millimolar Mg2+ concentrations, also inhibit the susceptibility of G(s-alpha) to phosphorylation; and (iii) guanosine 5'[beta-gamma-imido]triphosphate inhibits the susceptiblity of G(s-alpha) to act as a substrate for phosphorylation. These studies suggest that there is potential for cross-talk between receptors which trigger Ptdlns(4,5)P2 hydrolysis and subsequently protein kinase C activation, and receptors which stimulate adenylate cyclase via G(s).