Phosphorylation of Gz in human platelets. Selectivity and site of modification

• Published in: The Journal of biological chemistry 1991-11, Vol.266 (32), p.22051-22056
• Main Authors: LOUNSBURY, K. M, CASEY, P. J, BRASS, L. F, MANNING, D. R
• Format: Article
• Language: English
• Subjects: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate - blood; Amino Acid Sequence; Amino Acids - analysis; Biological and medical sciences; Blood Platelets - drug effects; Blood Platelets - metabolism; Cell physiology; Enzyme Activation; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - drug effects; GTP-Binding Proteins - immunology; GTP-Binding Proteins - metabolism; Humans; Immune Sera; Kinetics; Molecular and cellular biology; Molecular Sequence Data; Peptide Fragments - isolation & purification; Peptides - chemical synthesis; Peptides - immunology; Phosphorylation; Prostaglandin Endoperoxides, Synthetic - pharmacology; Protein Kinase C - blood; Recombinant Proteins - drug effects; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Signal transduction; Tetradecanoylphorbol Acetate - pharmacology; Thrombin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)54743-8

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.