Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells.
To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.
Grabarek
J
J
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts.
Raychowdhury
M
M
Ravid
K
K
Kent
K C
KC
Newman
P J
PJ
Ware
J A
JA
D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012313:RNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D006882:Hydroxyapatites; D007527:Isoenzymes; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D012334:RNA, Neoplasm; D012313:RNA; D017886:Durapatite; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium