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

N J Pyne, M Freissmuth, S Palmer

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37 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)333-8
Number of pages6
JournalBiochemical journal
Volume285 ( Pt 1)
Publication statusPublished - 1992

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

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