Phosphorylation of Gz in human platelets. Selectivity and site of modification.
We have demonstrated previously that the G protein alpha subunit Gz alpha (or Gx alpha) in human platelets is subject to phosphorylation by agents that activate protein kinase C, including phorbol 12-myristate 13-acetate, thrombin, and the thromboxane A2 analog U46619. We examine here the site and selectivity of phosphorylation both in vitro using recombinant G protein alpha subunits and in situ using permeabilized and intact platelets. Protein kinase C catalyzes the rapid and nearly stoichiometric phosphorylation of recombinant Gz alpha, with the modification occurring preferentially for the GDP-bound form of the subunit. Under the same conditions, phosphorylation of recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Gs alpha-S, Gs alpha-L, and Go alpha 1 was minimal. Phosphorylation of both rGz alpha and platelet Gz alpha occurs at a serine residue near the amino terminus. This conclusion is supported by phosphoamino acid analysis and the incorporation of radiolabel from [gamma-32P]ATP into the amino-terminal CNBr peptide (residues 2-53 of the encoded protein). One of the antisera used in this study (6354, directed toward residues 24-33) recognizes only the nonphosphorylated form of Gz alpha, providing strong evidence that Ser25 or Ser27 is the site of phosphorylation. Results obtained with 6354 also suggest that phorbol ester-promoted phosphorylation of Gz alpha approaches 1 mol of phosphate per mol of subunit in permeabilized platelets.
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
K M
KM
Lounsbury
P J
PJ
Casey
L F
LF
Brass
D R
DR
Manning
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000255:Adenosine Triphosphate / Q000097:blood; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011493:Protein Kinase C / Q000097:blood; D011994:Recombinant Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D000596:Amino Acids; D007106:Immune Sera; D010446:Peptide Fragments; D010455:Peptides; D011450:Prostaglandin Endoperoxides, Synthetic; D011994:Recombinant Proteins; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013917:Thrombin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate