Pyne, N J and Freissmuth, M and Palmer, S (1992) Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C. Biochemical journal, 285 ( Pt . pp. 333-8. ISSN 0264-6021Full text not available in this repository. (Request a copy from the Strathclyde author)
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.
|Keywords:||alkaloids, animals, autoradiography, blotting, western, brain, electrophoresis, polyacrylamide gel, GTP-binding proteins, guanosine 5'-O-(3-Thiotriphosphate), kinetics, phosphorylation, protein Kinase C-, RNA splicing, rats, recombinant proteins, staurosporine, tetradecanoylphorbol acetate, Therapeutics. Pharmacology, Biochemistry, Cell Biology, Molecular Biology|
|Subjects:||Medicine > Therapeutics. Pharmacology|
|Department:||Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences|
|Depositing user:||Pure Administrator|
|Date Deposited:||14 Nov 2011 15:12|
|Last modified:||04 May 2016 20:20|