Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.     

A high-beef diet alters protein kinase C isozyme expression in rat colonic mucosa

We recently reported that a red meat (beef) diet relative to a casein-based diet increases protein kinase C (PKC) activity in rat colonic mucosa. The purpose of this study was to further elucidate the effects of a high-beef diet on colonic intracellular signal transduction by analyzing steady-state protein levels of different PKC isozymes as well as activities of the three types of sphingomyelinases. Male Wistar rats (n = 12/group) were fed AIN93G-based diets either high in beef or casein for 4 weeks. Rats fed the beef diet had significantly (P < 0.05) higher cytosolic PKC alpha and lower membrane PKC delta protein levels than rats fed the casein diet. The beef-fed rats also had alterations in subfractions of PKC zeta/lambda so that they had a significantly (P = 0.001) lower level of membrane 70 & 75 kDa fraction and a higher (P = 0.001) level of cytosolic 40 & 43 kDa fraction than rats fed the casein diet. Because protein levels analyzed with a PKC zeta-specific antibody were similar, these differences in PKC zeta/lambda were probably due to changes in PKC lambda expression. PKC beta2 levels did not differ between the dietary groups. Diet had no significant effect on the activity of acid, neutral, or alkaline sphingomyelinase. This study demonstrated that consumption of a high-beef diet is capable of modulating PKC isozyme levels in rat colon, which might be one of the mechanisms whereby red meat affects colon carcinogenesis.     

Insulin and glucose modulate protein kinase C activity in rat adipocytes

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.     

Direct evidence for protein kinase C involvement in insulin-stimulated hexose uptake.

Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.     

Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol.

The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.     

Insulin stimulates the translocation of protein kinase C in rat adipocytes

Insulin-induced changes in protein kinase C were examined in cytosol and membrane fractions of rat adipocytes enzymatically after Mono Q column chromatography and by immunoblotting. During a 5-20 min period of insulin treatment, cytosolic protein kinase C decrease by approximately 50%, whereas membrane protein kinase C increased nearly 2-fold. These findings suggest that insulin stimulates the translocation of protein kinase C in rat adipocytes.     

Comparative Analysis of PKСα and PKCζ Activities in Rat and Lamprey Erythrocytes of Different Ages

The present study aimed to find out a link between ageing of rat and lamprey erythrocytes and activity of two isoforms of protein kinase C (PKC), РKСα and РKСζ. The whole cell population was separated into fractions of different ages in Percoll density gradient. The validity of separation was confirmed by the number of immature erythrocytes, reticulocytes. PKC activity was analyzed in cytosolic and membrane cell fractions. Rat erythrocytes express both PKC isoforms, РKСα and РKСζ, whereas lamprey erythrocytes express only РKСζ. РKСα is identified as a major band at ~ 80 kDa and minor bands at ~ 55–65 kDa; РKСζ is represented by a single band at ~ 80 kDa. In young rat erythrocytes, РKСα is detected mainly in cytosolic fractions, while in membrane fractions its level is by far lower. As cells age, PKCα is translocated from the cytosol to membranes and undergoes proteolytic degradation due to repeated cycles of activation. As a result, in aged erythrocytes relative total PKCα expression (as a sum of expressions in the cytosol and membranes per total protein level) diminishes, indicating a depletion of the PKCα pool and a decline in its functional activity. In both animal species, a highest PKCζ level is observed in the cytosol of young erythrocytes. Erythrocyte ageing is accompanied by a gradual decrease in expression of free cytosolic PKCζ and concurrent increase in the level of its membrane-bound forms. However, in contrast to PKCα, PKCζ is not proteolyzed; its total level in cells and perhaps functional activity do not change throughout the erythrocyte lifespan.     

Okadaic acid activates atypical protein kinase C (zeta/lambda) in rat and 3T3/L1 adipocytes. An apparent requirement for activation of Glut4 translocation and glucose transport.

Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is known to provoke insulin-like effects on GLUT4 translocation and glucose transport, but the underlying mechanism is obscure. Presently, we found in both rat adipocytes and 3T3/L1 adipocytes that okadaic acid provoked partial insulin-like increases in glucose transport, which were inhibited by phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin and LY294002, and inhibitors of atypical protein kinase C (PKC) isoforms, zeta and lambda. Moreover, in both cell types, okadaic acid provoked increases in the activity of immunoprecipitable PKC-zeta/lambda by a PI 3-kinase-dependent mechanism. In keeping with apparent PI 3-kinase dependence of stimulatory effects of okadaic acid on glucose transport and PKC-zeta/lambda activity, okadaic acid provoked insulin-like increases in membrane PI 3-kinase activity in rat adipocytes; the mechanism for PI 3-kinase activation was uncertain, however, because it was not apparent in phosphotyrosine immunoprecipitates. Of further note, okadaic acid provoked partial insulin-like increases in the translocation of hemagglutinin antigen-tagged GLUT4 to the plasma membrane in transiently transfected rat adipocytes, and these stimulatory effects on hemagglutinin antigen-tagged GLUT4 translocation were inhibited by co-expression of kinase-inactive forms of PKC-zeta and PKC-lambda but not by a double mutant (T308A, S473A), activation-resistant form of protein kinase B. Our findings suggest that, as with insulin, PI 3-kinase-dependent atypical PKCs, zeta and lambda, are required for okadaic acid-induced increases in GLUT4 translocation and glucose transport in rat adipocytes and 3T3/L1 adipocytes.     

The rat lacrimal gland expresses the alpha isoform of PKC. Further evidence for the PMA-activated and phospholipid-independent protein kinase activity.

The molecular heterogeneity of protein kinase C (PKC) is now widely documented. In our first report, we characterized the rat lacrimal gland PKC along with a phorbol 12-myristate 13-acetate (PMA)-activated and phospholipid-independent protein kinase activity [Mauduit P., Zoukhri D. and Rossignol B. (1989) Fedn Eur. biochem. Socs Lett. 252, 5-11. In this work, we show that when the rat lacrimal gland cytosolic fraction is chromatographed on hydroxyapatite, only one peak of PKC activity can be detected. Comparison with a rat brain cytosolic fraction indicated that it is PKC-alpha which is expressed in the rat lacrimal gland. This result was confirmed by the use of polyclonal antibodies raised against rat brain PKC-alpha, beta and gamma isoforms. We also provide evidence that free arachidonic acid activates PKC, as does PMA, in a calcium and phospholipid-free system.     

Oleic acid promotes changes in the subcellular distribution of protein kinase C in isolated hepatocytes.

The effect of oleate on the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes and in perfused rat liver in the presence of physiological concentrations of serum albumin. A time- and dose-dependent translocation of PKC from the cytosol towards the membranes was observed at oleate concentrations that fell within the range of concentrations reached under several physiological conditions. Analysis of the membrane-bound isoenzymes of PKC by hydroxylapatite chromatography revealed that the beta isoenzyme was preferentially translocated to this compartment in hepatocytes incubated with oleate. Activation of PKC after incubation of hepatocytes with oleate involved at least three different effectors of the enzyme: the fatty acid itself, the diacylglycerol synthesized from oleate, and the rise in the cytosolic calcium concentration elicited by oleate. As a result of PKC activation, protein phosphorylation of intact hepatocytes in response to oleate exhibited an enhancement in the phosphate content of a protein of 82 kDa, similar to that phosphorylated in the presence of phorbol dibutyrate.     

Halothane enhances the phosphorylation of H1 histone and rat brain cytoplasmic proteins by protein kinase C

The effect of halothane, a typical volatile anesthetic, on the calcium- and phospholipid-dependent protein kinase (PKC), which is one of the key enzymes of membrane signal transduction, was examined. PKC was partially purified from the cerebral tissue of male Wistar rats. Halothane increased PKC-mediated phosphorylation of calf thymus H1 histone in the presence or absence of phorbol ester or diolein, and also increased phosphorylation of the rat brain cytosolic proteins (47 kDa and 80 kDa). A similar but slight increase in H1 histone phosphorylation was observed with isoflurane and enflurane, less lipid soluble volatile anesthetics. These findings suggest that halothane may increase PKC-mediated phosphorylation by the modification of phospholipid membrane and affect membrane signal transduction of the nerve cell under the anesthetic state.     

Glucose-induced synthesis of diacylglycerol de novo is associated with translocation (activation) of protein kinase C in rat adipocytes

Addition of glucose (5-20 mM) to rat adipocytes provoked dose-related increases in diacylglycerol, without increasing production of [3H]inositol phosphates. Cytosolic protein kinase C enzyme activity and immunoreactivity decreased within 1-5 min of 5 mM glucose addition, and further over 20 min. Membrane protein kinase C increased stoichiometrically during the first 5 min and then decreased. Higher concentrations (10 and 20 mM) of glucose provoked greater and more rapid decreases of cytosolic and membrane protein kinase C. Our findings suggest that glucose stimulates diacylglycerol production by providing substrate for phosphatidic acid synthesis de novo, and this is associated with translocative activation of protein kinase C.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

Protein kinase C-dependent antilipolysis by insulin in rat adipocytes

Recently, we have shown that protein kinase C (PKC) activated by phorbol 12-myristate 13-acetate (PMA) attenuates the beta1-adrenergic receptor (beta1-AR)-mediated lipolysis in rat adipocytes. Stimulation of cells by insulin, angiotensin II, and alpha1-AR agonist is known to cause activation of PKC. In this study, we found that lipolysis induced by the beta1-AR agonist dobutamine is decreased and is no longer inhibited by PMA in adipocytes that have been treated with 20 nM insulin for 30 min followed by washing out insulin. Such effects on lipolysis were not found after pretreatment with angiotensin II and alpha1-AR agonists. The rate of lipolysis in the insulin-treated cells was normalized by the PKCalpha- and beta-specific inhibitor G? 6976 and PKCbeta-specific inhibitor LY 333531. In the insulin-treated cells, wortmannin increased lipolysis and recovered the lipolysis-attenuating effect of PMA. Western blot analysis revealed that insulin slightly increases membrane-bound PKCalpha, betaI, and delta, and wortmannin decreases PKCbetaI, betaII, and delta in the membrane fraction. These results indicate that stimulation of insulin receptor induces a sustained activation of PKC-dependent antilipolysis in rat adipocytes.     

Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts.

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.     

Glucocorticoid-induced insulin resistance associates with activation of protein kinase C isoforms

We studied glucocorticoid-induced insulin resistance and possible role of protein kinase C (PKC). Pretreatment with dexamethasone, prednisolone and corticosterone for 60 min decreased insulin-induced [3H] 2-deoxyglucose (DOG) uptake in isolated rat adipocytes. Preincubation with Go6976, LY379196 or myristoylated PKC pseudosubstrate, conventional PKC inhibitor, but not cycloheximide or RU38486, recovered dexamethasone-induced insulin resistance. Dexamethasone activated immunoprecipitates with anti-PKC alpha, beta, and zeta antibodies. PKC zeta activity in adipocytes increased to 163%, and 264% from basal level (100%) with dexamethasone and insulin treatment, respectively. Dexamethasone provoked redistribution of both PKC beta and zeta from the cytosol to the membrane. These results indicate that dexamethasone activates both conventional and atypical PKC. However, conventional PKC is more important in glucocorticoid-induced insulin resistance.     

The role of protein kinase C in laminin-mediated neurite outgrowth

Laminin is a potent stimulator of neurite outgrowth in rat pheochromocytoma (PC12) cells. Here, we investigated the role of protein kinase C (PKC) in the mechanism of laminin-mediated neurite outgrowth in PC12 cells. Phorbol ester activators of PKC have been shown to have divergent effects on laminin-mediated neurite outgrowth. Therefore, we tested the effect of the non-phorbol PKC activator, indolactam V. At 1.0 microM indolactam V inhibited laminin-mediated neurite outgrowth by 85%. Further, the PKC inhibitor H7 blocked the inhibitory effect of indolactam V on laminin-mediated neurite outgrowth. Direct measurement of protein kinase C activity in the soluble (cytosolic) and particulate (membrane) fractions of PC12 cells showed that laminin failed to alter protein kinase C activity. These data demonstrate that PKC activation inhibits laminin-mediated neurite outgrowth and that laminin does not activate PKC in PC12 cells.     

化学诱发大鼠肝癌的亚细胞组分中两类丝氨酸蛋白激酶的动态变化

用二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌,隔周测定肝脏胞液、膜性和胞核中的蛋白激酶Ａ（ＰＫＡ）和蛋白激酶Ｃ（ＰＫＣ）的活力。发现胞液ＰＫＡ在诱癌过程中活力改变不大,胞液ＰＫＣ则逐步增高,在第１３周和２０周形成两个活力高峰。膜性ＰＫＡ和ＰＫＣ都呈双相变化,即在癌前期（１０－１４周）增加,癌形成期（１７－２０周）反而降至正常以下,胞核ＰＫＡ和ＰＫＣ也都在癌前期升至高峰,而癌形成期则低于癌前期,但仍高于正常（ＰＫＡ）或接近正常（ＰＫＣ）。因只有膜性ＰＫＣ在大鼠老化时降低,故这些变化不是鼠龄变化的结果,而是ＤＥＮ诱癌所引起,其变化机理可能与下降调节、细胞内转位或两型同工酶相反的升降变化有关。     

Direct activation of a protein kinase activity from rat lacrimal gland by PMA in a phospholipid-free system

The analysis of the cytosolic fraction from rat exorbital lacrimal gland with DEAE-cellulose ion-exchange chromatography showed the presence of a peak of protein kinase activity which was dependent on the presence of phosphatidylserine and diolein as well as calcium. This activity showed the same properties as the previously reported protein kinase C (PKC). Moreover, we have shown for the first time that this kinase or a kinase that coeluted from the column with PKC could be activated by a phorbol ester, PMA, in a phospholipid-free system, i.e. in the absence of any cofactor of PKC. These findings emphasize the need for caution in the interpretation of experimental results obtained when using phorbol esters to probe for a role of PKC in many regulatory processes.     

Inactivation of protein kinase C in rat liver during late hypoglycemic phase of sepsis

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat liver during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. Hepatic PKC was extracted and partially purified by ammonium sulfate fractionation and DEAE-cellulose chromatography. PKC activity was assayed based on the rate of incorporation of ³²p from [-³²P]ATP into histone. The results show that during early sepsis, both membrane-associated and cytosolic PKC activities remained relatively unaltered. During late sepsis, membrane-associated PKC was unaffected while cytosolic PKC activity was decreased by 19.5-34.4%. Kinetic analysis of the data on cytosolic PKC during late phase of sepsis reveals that the V_max values for ATP, histone, Ca²⁺, phosphatidylserine, and diacylglycerol were decreased by 23.4, 22.1, 19.5, 25, and 34.4%, respectively, with no changes in their K_m values. These data indicate that cytosolic PKC activity was inactivated in rat liver during late hypoglycemic phase of sepsis. Since PKC-mediated phosphorylation plays an important role in regulating hepatic glucose metabolism, an inactivation of cytosolic PKC may contribute to the development of hypoglycemia during late phase of sepsis.