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

Research output: Contribution to journalArticle

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

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Protein Kinase C-alpha
Phosphorylation
Guanine Nucleotides
GTP-Binding Proteins
Carrier Proteins
Proteins
Protein Kinase C
Substrates
Receptor Cross-Talk
Gs GTP-Binding Protein alpha Subunits
Phosphatidylinositol 4,5-Diphosphate
Guanylyl Imidodiphosphate
Adenylyl Cyclases
Hydrolysis
Brain
Chemical activation
Phosphates

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

Cite this

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title = "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.",
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",
author = "Pyne, {N J} and M Freissmuth and S Palmer",
year = "1992",
language = "English",
volume = "285 ( Pt 1)",
pages = "333--8",
journal = "Biochemical Journal",
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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. / Pyne, N J; Freissmuth, M; Palmer, S.

In: Biochemical journal, Vol. 285 ( Pt 1), 1992, p. 333-8.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C

AU - Pyne, N J

AU - Freissmuth, M

AU - Palmer, S

PY - 1992

Y1 - 1992

N2 - 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.

AB - 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.

KW - alkaloids

KW - animals

KW - autoradiography

KW - blotting, western

KW - brain

KW - electrophoresis, polyacrylamide gel

KW - GTP-binding proteins

KW - guanosine 5'-O-(3-Thiotriphosphate)

KW - kinetics

KW - phosphorylation

KW - protein Kinase C-

KW - RNA splicing

KW - rats

KW - recombinant proteins

KW - staurosporine

KW - tetradecanoylphorbol acetate

UR - http://www.biochemj.org/bj/285/bj2850333.htm

M3 - Article

VL - 285 ( Pt 1)

SP - 333

EP - 338

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -