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Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C.

ABSTRACT: Recombinant forms of Gs alpha-1 and Gs alpha-4 were shown to act as substrates for a purified preparation of brain protein kinase C. Both forms of Gs alpha were thermally denatured during the incubation such that phosphorylation was virtually complete (greater than 90%) after 30 min. The quantity of phosphate incorporated into approximately equivalent starting amounts of the two forms of Gs alpha (4.8 pmol of Gs alpha-1 and 5.5 pmol of Gs alpha-4) at maximal phosphorylation were 0.23 +/- 0.08 pmol for Gs alpha-1 and 0.56 +/- 0.12 pmol for Gs alpha-4. Since both forms of Gs alpha were thermally denatured to the same extent after 30 min, the increased phosphorylation state of Gs alpha-4 provides evidence that Gs 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 Gs alpha-4. The guanine-nucleotide-free form of Gs 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 susceptibility of Gs alpha to phosphorylation; (ii) beta gamma-subunits, which inhibit GDP release from Gs alpha-GDP at millimolar Mg2+ concentrations, also inhibit the susceptibility of Gs alpha to phosphorylation; and (iii) guanosine 5'[beta gamma-imido]triphosphate inhibits the susceptibility of Gs alpha 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 Gs.

SUBMITTER: Pyne NJ

PROVIDER: S-EPMC1132785 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Project description:Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.

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Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C.

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