Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.

Weightlessness or microgravity of spaceflight induces bone loss due in part to decreased bone formation by unknown mechanisms. Due to difficulty in performing experiments in space, several ground-based simulators such as the Rotating Wall Vessel (RWV) and Random Positioning Machine (RPM) have become critical venues to continue studying space biology. However, these simulators have not been systematically compared to each other or to mechanical stimulating models. Here, we hypothesized that exposure to RWV inhibits differentiation and alters gene expression profiles of 2T3 cells, and a subset of these mechanosensitive genes behaves in a manner consistent to the RPM and opposite to the trends incurred by mechanical stimulation of mouse tibiae. Exposure of 2T3 preosteoblast cells to the RWV for 3 days inhibited alkaline phosphatase activity, a marker of differentiation, and downregulated 61 and upregulated 45 genes by more than twofold compared to static 1 g controls, as shown by microarray analysis. The microarray results were confirmed by real-time PCR and/or Western blots for seven separate genes and proteins including osteomodulin, runx2, and osteoglycin. Comparison of the RWV data to the RPM microarray study that we previously published showed 14 mechanosensitive genes that changed in the same direction. Further comparison of the RWV and RPM results to microarray data from mechanically loaded mouse tibiae reported by an independent group revealed that three genes including osteoglycin were upregulated by the loading and downregulated by our simulators. These mechanosensitive genes may provide novel insights into understanding the mechanisms regulating bone formation and potential targets for countermeasures against decreased bone formation during space flight and in pathologies associated with lack of bone formation.

Protein kinase C (PKC) is a family of closely related phospholipid-dependent protein kinases. A fully active, phospholipid-independent catalytic fragment of PKC is produced by limited proteolysis of the enzyme. The catalytic fragment allows a simplified assay system for the analysis of PKC inhibitors that interact with the catalytic domain. Recently, we reported that N-myristoylation of the synthetic peptide substrate Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) transformed a peptide that completely lacked inhibitory activity against the histone kinase reactions of PKC and its catalytic fragment into a peptide that potently inhibited both of these reactions. N-Myristoylation did not alter the potency of the peptide as a PKC substrate, and the basis for the acquisition of inhibitory activity against the catalytic fragment by N-myristoylation of the peptide remained unclear. In this report, we propose a mechanism for catalytic fragment inhibition by the N-myristoylated peptide that is based on a comparison of the inhibitory potencies of several nonphosphorylatable analogs of N-myristoyl-RKRTLRRL, a kinetic analysis of the inhibition of the histone kinase activity of the catalytic fragment by nonphosphorylatable N-myristoyl-RKRTLRRL analogs, and an analysis of the inhibitory effects of the N-myristoylated peptide series on the intrinsic ATPase activity of PKC. Our results support a mechanism in which the N-myristoylated peptides inhibit the catalytic fragment by binding to PKCfree, but not to the complex PKC-ATP, at the protein-substrate binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca 2؉ -and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca 2؉ -and PS-dependent activity of purified rat brain PKC with the same efficacy (IC 50 ‫؍‬ 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca 2؉ -and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-␣ remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/ Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-␣ was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-␣ and the tripeptide, but it was associated with an increase in the susceptibility of PKC-␣ to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca 2؉ -and PS-dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.The phospholipid-dependent isozyme family protein kinase C (PKC) 1 plays an important role in diverse physiological processes, including cell growth and differentiation, muscle contraction, and neurotransmission, and pathological developments, e.g. tumor promotion and multiple drug resistance in cancer (1-4). The PKC family consists of Ca 2ϩ -dependent, phorbol ester-activated isozymes (␣, ␤ 1 , ␤ 2 , and ␥), Ca 2ϩ -independent, phorbol ester-activated isozymes (␦, ⑀, , ⌰, and ), and Ca 2ϩ -and phorbol ester-independent isozymes ( and ). Phosphatidylserine (PS) dependence is universal among PKC isozymes (1, 5). Both stimulatory and inhibitory endogenous regulators of ...

Protein kinase C (PKC) is composed of a family of isozymes that transduce signals of certain hormones, growth factors, lectins, and neurotransmitters. This review addresses the role of PKC in the regulation of cellular proliferation and its disorders. PKC is directly activated in vivo by the second messenger diacylglycerol, a lipid produced by phospholipase C-catalyzed hydrolysis of phosphatidylinositol and polyphosphoinositides. Diacylglycerol activates PKC by reducing the enzyme's requirement for Ca2+. Phorbol ester tumor promoters and related agents potently activate PKC by a mechanism analogous to that of diacylglycerol, providing evidence that PKC activation is a critical event in tumor promotion. However, the role of PKC activation in tumor promotion is not entirely clear. For example, bryostatin is a potent PKC activator that antagonizes phorbol ester-mediated tumor promotion, and mezerein is a second-stage tumor promoter that potently activates PKC. In addition to studies concerned with tumor promotion, studies of oncogene action also indicate a role for PKC in carcinogenesis. A number of plasma membrane-associated oncogene products and related proteins are PKC substrates, and PKC activation leads to induction of the expression of oncogenes that code for nuclear proteins. PKC is implicated in human breast and colon carcinogenesis. Tumor-promoting bile acids activate PKC, and PKC expression studies in rat colonic epithelial cells and human breast cancer cells indicate a positive role for PKC in the proliferation of the cells. Altered expression of PKC in human colon and breast tumors indicates that PKC isozymes may be useful markers for these diseases.