Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C

We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.     

Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation

Interactions of types I, II, and III protein kinase C (PKC) with phospholipids were investigated by following the changes in protein kinase activity and phorbol ester binding. The acidic phospholipids such as phosphatidylserine (PS), phosphatidic acid, phosphatidyl-glycerol, and cardiolipin, which are activators of PKC in the assay of protein phosphorylation, could differentially inactivate PKC I, II, and III during preincubation in the absence of divalent cation. The phospholipid-induced inactivation of PKC was concentration and time dependent and only affected the kinase activity without influencing phorbol ester binding. PKC I was the most susceptible to the phospholipid-induced inactivation, and PKC III was the least. The IC50 values of PS for PKC I, II, and III were 5, 45, and greater than 120 microM, respectively. Addition of divalent cation such as Ca2+ or Mg2+ suppressed the phospholipid-induced inactivation of PKC. In the absence of divalent cation, PKC I, II, and III all formed complexes with PS vesicles, although to a slightly different degree, as analyzed by molecule sieve chromatography. [3H]Phorbol 12,13-dibutyrate binding for PKC I, II, and III was recovered after chromatography; however, the kinase activities of all these enzymes were greatly reduced. In the presence of Ca2+, all three PKCs formed complexes with PS vesicles, and both the kinase and phorbol ester-binding activities of PKC II and III were recovered following chromatography. Under the same conditions, the phorbol ester-binding activity of PKC I was also recovered, but the kinase activity was not. The phospholipid-induced inactivation of PKC apparently results from a direct interaction of phospholipid with the catalytic domain of PKC; this interaction can be suppressed by divalent cations. In the presence of divalent cations, PS interacted preferentially with the regulatory domain of PKC and resulted in the activation of the kinase.     

p32 (gC1qBP) is a general protein kinase C (PKC)-binding protein; interaction and cellular localization of P32-PKC complexes in ray hepatocytes.

The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homogenates. The protein was identified by NH(2)-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC activators was different for each one. Whereas PKCdelta only binds to p32 in the presence of PKC activators, PKCzeta and PKCalpha increase their binding when they are in the activated form. Other PKC isoforms such as beta, epsilon, and theta bind equally well to p32 regardless of the presence of PKC activators, and PKCmu binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKCdelta) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal microscopy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKCtheta and p32 are constitutively associated in vivo, whereas PKCdelta activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.     

T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway

The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.     

Phorbol esters can persistently replace interleukin-2 (IL-2) for the growth of a human IL-2-dependent T-cell line

A selected clone from an IL-2-dependent human T-cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutylate (PDBu). Thus, a TPA(PDBu)-dependent T-cell line, designated TPA-Mat, was established from IL-2-dependent T cells. The TPA-dependency of TPA-Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA-Mat cells still showed IL-2-dependent growth. However, the TPA (PDBu)-dependent growth of TPA-Mat did not seem to be mediated by an autocrine mechanism of IL-2 or by any other growth factor production, because these factors were not detected in TPA-Mat cell supernatants. Therefore, the phorbol esters substituted for IL-2 and may be directly involved in transduction of growth signals in TPA-Mat cells. Although activity of PKC was down-regulated, messenger ribonucleic acid (mRNA) of the PKC beta-gene was detected in TPA-Mat cells cultured with PDBu. Furthermore, the growth of TPA-Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL-2-dependent TPA-Mat cells.     

Identification of a protein kinase C activating factor from murine erythroleukemia cells: characterization of the activation kinetics

A protein kinase C (PKC) activating factor (AF) has been identified in the extracellular medium of V3.17 vincristine resistant murine erythroleukemia (MEL) cells clone. The factor is a protein that stimulates the activity of PKC alpha and beta isozymes isolated from MEL cells, rat and mouse brain approximately 2 to 2.5 fold over the Vmax, respectively. AF promotes an identical activation in the presence of all the effectors but also when the amount of Ca2+ is reduced to microM concentration and in the absence of diacylglycerol (DAG). The factor shows a greater activating efficiency with PKC beta isozymes. AF binds to PKC presumably at the DAG binding site as suggested by the competition between phorbol dibutyrate and AF for binding to the kinase. Moreover, AF promotes the selective binding of PKC beta to natural or artificial membranes in the presence of microM concentrations of Ca2+. Altogether these results suggest the presence in MEL cells of a protein factor that can promote association of PKC to the membranes together with activation of the kinase, without the requirement for DAG formation. This could be visualized as a new mechanism for prolonged and selective activation of PKC.     

Downregulation of protein kinase C-γ is independent of a functional kinase domain

Prolonged activation of protein kinase C (PKC) types and β by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the γ isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-γ is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the purative ATP-binding site (Lys-380→Met-380) of the protein kinase C γ isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-γ kinase domain is not a prerequisite for downregulation of PKC activity.     

Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.     

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Ca2+-independent binding of [3H]phorbol dibutyrate to protein kinase C is supported by protamine and other polycations.

The activity of the Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), can be modulated by diacylglycerols and phorbol esters. The association of these agents with PKC is, in turn, generally understood to be dependent on Ca2+ and phospholipids. Certain substrates, e.g. protamine sulphate, are known to undergo cofactor-independent phosphorylation by PKC. We report here that, in the presence of such substrates, PKC bound 1,2-dihexanoylglycerol and phorbol dibutyrate in a Ca2+-independent manner. Histone IIIs, which is phosphorylated by PKC only in the presence of Ca2+ and phospholipid, also supported Ca2+-independent binding of 1,2-dihexanoylglycerol and phorbol dibutyrate to PKC, but to a lesser extent than did protamine. Support for Ca2+-independent binding was also exhibited by non-peptide polycations (e.g. DEAE-cellulose DE52), indicating that recognition of the catalytic site is not a prerequisite for this effect. The natural polyamines spermine and putrescine did not have this property, however. The affinity of PKC for phorbol dibutyrate and 1,2-dihexanoylglycerol was found to be unchanged by the presence of substrates or DE52. It is proposed that, in the absence of Ca2+, certain polycations favour expression of the diacylglycerol/phorbol ester binding site by stabilizing the active conformation of PKC.     

Chelerythrine is a potent and specific inhibitor of protein kinase C

The benzophenanthridine alkaloid chelerythrine is a potent, selective antagonist of the Ca++/phospholopid-dependent protein kinase (Protein kinase C: PKC) from the rat brain. Half-maximal inhibition of the kinase occurs at 0.66 microM. Chelerythrine interacted with the catalytic domain of PKC, was a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) (Ki = 0.7 microM) and a non-competitive inhibitor with respect to ATP. This effect was further evidenced by the fact that chelerythrine inhibited native PKC and its catalytic fragment identically and did not affect [3H]- phorbol 12,13 dibutyrate binding to PKC. Chelerythrine selectively inhibited PKC compared to tyrosine protein kinase, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. The potent antitumoral activity of celerythrine measured in vitro might be due at least in part to inhibition of PKC and thus suggests that PKC may be a model for rational design of antitumor drugs.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Regulatory domain of human protein kinase Cα dominantly inhibits protein kinase Cβ-I regulated growth and morphology in Saccharomyces cerevisiae

This study demonstrates that the isolated regulatory (R) domain (amino acids 1–270) of human protein kinase Cα (PKCα) is a potent inhibitor of PKCβ-I activity in a yeast expression system. The PKCα R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKCβ-I in vitro (Ki = 0.2 μM) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19–36 from PKCα. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKCα R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKCβ-I activity in vivo, in a yeast strain expressing rat PKCβ-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKCβ-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKCβ-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKCα acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes. © 1996 Wiley-Liss, Inc.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.     

Elevated levels of protein kinase C activity and alpha-isoenzyme expression in murine peritoneal B cells

Conventional murine splenic B cells are stimulated to initiate DNA synthesis by the combination of a phorbol ester protein kinase C (PKC) agonist, and a calcium ionophore; in contrast, recent work from this laboratory has shown that peritoneal B cells, enriched for the Ly-1+ B cell subset, differ in that they proliferate in response to the single signal provided by phorbol ester, acting alone. To elucidate the mechanism responsible for the abbreviated signaling requirement of peritoneal B cells, studies of intracellular Ca2+ and PKC were carried out. Measurements using the calcium sensitive dye, Indo-1, showed that base line levels of intracellular Ca2+ in peritoneal B cells were similar to those of splenic B cells, and that there was no change as a result of phorbol ester treatment. However, measurements of PKC based on the phosphorylation of histone showed enzymatic activity in peritoneal B cells to be about 60% greater than that of splenic B cells on a per microgram protein basis. Furthermore, this difference was accentuated by phorbol ester treatment, so that after 4 h, membrane and cytosol fractions from peritoneal B cells contained more than 5 times the PKC activity of the corresponding splenic B cell fractions because the down-regulation of PKC was relatively delayed in peritoneal B cells. This could not be accounted for by the onset of new PKC synthesis, but may relate to the finding that peritoneal B cells express more of the alpha-isoenzyme of PKC than splenic B cells, as shown by immunoblot analysis. Together with data from experiments using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride(H7), these results suggest that PKC activity remaining hours after phorbol ester treatment may contribute to the unusual phorbol ester responsiveness of peritoneal B cells, and indicate that B cells from separate anatomic locations differ in terms of several parameters relating to the activity and behavior of PKC.     

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm,Manduca sexta: The cytosolic enzyme

A calcium and diacylglycerol-activated and phospholipid-dependent protein kinase (protein kinase C; PKC) in the cytosol of the pupal brain of the tobacco hornworm Manduca sexta has been characterized. Two peaks of PKC activity were separated by DEAE-cellulose chromatography, both of which were dependent upon Ca²⁺, phosphatidylserine and 1,2 diolein. Maximal enzyme activity was obtained in the presence of 0.7 mM Ca²⁺ and 200 μg/ml of phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) enhanced PKC activity and calcium sensitivity markedly in the presence of phospholipids. The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate substituted for diacylglycerol in the activation of PKC. By utilizing the differential inhibition of PKC and cyclic AMP-dependent protein kinase by trifluoperazine and protein kinase inhibitor, both enzymes were measured accurately in dilute, crude cytosol preparations using the common substrate histone H1.     

The activity of a beta subtype of protein kinase C purified from nuclei of human neutrophils is enhanced by treatment with phorbol 12-myristate 13-acetate.

Two protein kinase C isoenzymes were partially purified from the nuclei of human neutrophils, and identified as beta and alpha subtypes. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) caused a 3.8-fold increase of nuclear beta PKC activity, while a minor increase of alpha PKC was observed. This selective activation of beta PKC could help to understand the molecular events involved in phorbol ester-induced cellular modifications.     

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.