Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity.
We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.
Balazovich
K J
KJ
Department of Pediatrics, University of Michigan, Ann Arbor 48109.
Boxer
L A
LA
D000465:Algorithms; D001572:Benzofurans / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins; D004987:Ethers / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D015759:Ionomycin; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C / Q000097:blood*; D011955:Receptors, Drug / Q000378:metabolism; D012995:Solubility
D001572:Benzofurans; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004987:Ethers; D010703:Phorbol Esters; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D015759:Ionomycin; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2