title: Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets.
abstract: The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.
authors.affiliation: Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
authors.forename: S L
authors.initials: SL
authors.lastname: Pelech
authors.forename: M
authors.initials: M
authors.lastname: Samiei
authors.forename: D L
authors.initials: DL
authors.lastname: Charest
authors.forename: S L
authors.initials: SL
authors.lastname: Howard
authors.forename: H
authors.initials: H
authors.lastname: Salari
mesh_terms: D000469:Alkaline Phosphatase / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000097:blood; D002451:Cell Compartmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood* / Q000276:immunology; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007976:Leupeptins; D010455:Peptides; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; C032854:leupeptin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium