Novel Chimeric Peptide Inhibits Protein Kinase C and Induces Apoptosis in Human Immune Cells

Protein kinase C (PKC) participates in a myriad of cellular processes. Protein kinase C isoforms play different roles based on their cellular expression balance and activation. The activity of classical PKC isoforms has been shown to be crucial for immune cell population homeostasis, playing a positive role in survival and proliferation. Protein kinase C inhibitors have been used for conditions where up-regulated PKC results in a pathological state. The most commonly investigated PKC inhibitors are highly effective in inhibiting PKC function but they are relatively unspecific, some of them even inhibiting other kinase families. Protein kinase C pseudosubstrates are auto-inhibitory domains which have been used to inhibit more specifically PKC in vitro but they do not freely penetrate cells. This could be resolved by using cell-permeable PKC pseudosubstrates which would more accurately modulate cellular PKC activity and PKC-related functions in intact cells. Here we show the development of a chimeric peptide inhibitor of classical PKC isoforms, consisting of a cell permeable sequence and a pseudosubstrate sequence which was able to translocate into cells, inhibiting PKC kinase activity and PKC T-cell-specific substrate phosphorylation. We also demonstrate a dramatic reduction in T-cell proliferation at high chimeric peptide concentration; this was attributed to apoptosis induction, as demonstrated by cell shrinking, phosphatidylserine exposure and DNA fragmentation. As expected, the control peptide (pseudosubstrate) did not penetrate cells, affect cell proliferation or survival. We also show that a neoplastic T-cell line which expresses higher levels of PKC is more resistant to chimeric peptide-mediated cell death than normal cells, corroborating a PKC role in apoptosis resistance. This chimeric peptide could be useful for the specific modulation of the PKC signalling pathway in pathological conditions.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Summary Intracellular enzymes or receptors are interesting targets for the pharmacomodulation of cellular metabolism. We have previously shown that modification of relatively long peptides by a palmitoyl-lysine residue could facilitate their delivery into the cytoplasm of living cells. Several peptides containing pseudosubstrate sequences of protein kinase C (PKC) have been evaluated for their ability to modulate phosphorylation of model substrate, neuronal morphology or tumor necrosis factor secretion. In this work we have evaluated the effect of palmitoyl-modified PKC-pseudosubstrate peptides on induction of apoptosis. We have established that these peptides are able to induce apoptosis in different human cell types (primary fibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation. In contrast, control peptides (non-lipidic PKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or little effect on programmed cell death. This work highlights the pharmacological interest of lipopeptides and argues in favor of the potential role of PKC(s) in the cell death machinery. K. Thiam and E. Loing have contributed equally to this work.     

PKC theta activity maintains normal quantal size in chromaffin cells

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC θ and PKC δ, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC θ and δ were expressed in chromaffin cells, mice deficient for PKC θ, but not for PKC δ, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC θ pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC θ mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC θ, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.     

Atypical protein kinase C-zeta is essential for delayed phagocytosis of Helicobacter pylori

Phagocytosis is a rapid actin-dependent endocytic process used by macrophages and neutrophils to ingest and kill microorganisms. Perturbation of phagocytosis is central to the ability of some pathogenic microbes to cause disease, and we demonstrated previously that the ulcerogenic bacterium Helicobacter pylori (Hp) actively retards its uptake by macrophages and subsequently persists inside novel vacuoles called megasomes. Neither the receptor that mediates Hp binding nor the signaling pathways that regulate bacterial engulfment have been defined. Nevertheless, the fact that other phagocytic stimuli do not exhibit delayed phagocytosis suggests that Hp may be ingested by a unique mechanism. We now show that Hp transiently activated protein kinase C (PKC) in macrophages and that atypical PKCzeta and novel PKC(epsilon), but not conventional PKC(alpha), accumulated on forming phagosomes. Pharmacologic agents, isoform-selective pseudosubstrate peptides, and antisense oligonucleotides demonstrated that PKC(zeta) regulated local actin polymerization and bacterial engulfment, whereas other PKC isoforms did not. In contrast, opsonization of Hp with immunoglobulin G (IgG) induced rapid PKC(zeta)-independent uptake and enhanced killing of ingested bacteria. A role for atypical PKCs in phagocytosis has not been described. We conclude that Hp defines a new phagocytic pathway in macrophages that is regulated by PKC(zeta).     

Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Protein kinase C (PKC) has been implicated in a variety of cellular responses such as proliferation, differentiation, and secretion. We assessed the role of PKC in the mitogenic effects of gastrin-releasing peptide (in a small cell lung cancer (SCLC) cell line. Using antisera that specifically recognize the PKC isoforms alpha, beta, gamma, delta, and epsilon, we determined that PKC epsilon is the major isoform in the SCLC cell line NCI-N417, followed by PKC alpha and delta. In addition to the 90-kDa PKC epsilon, our anti-PKC epsilon antiserum specifically detected a 40-kDa immunoreactive protein. Treatment of the cells with either 20 nM phorbol myristate acetate or 50 nM GRP enhanced significantly the level of the 40-kDa protein in a time-dependent (1-8 h), cycloheximide-sensitive fashion. Subcellular fractionation revealed that 90% of PKC epsilon was in particulate form, while the 40-kDa immunoreactive protein was cytosolic. To test the hypothesis that the 40-kDa soluble protein represented a catalytically independent PKC epsilon fragment, cytosolic extracts were assayed for kinase activity. 45-50% of the activity was apparent in the absence of the PKC activators phosphatidylserine and diacylglycerol. This effector-independent kinase activity was further purified by affinity chromatography using a synthetic peptide corresponding to the pseudosubstrate region of PKC epsilon (ERMRPRKRQGAVRRRV) coupled to Sepharose. The partially purified protein, recognized by the anti-PKC epsilon antiserum, exhibited histone kinase activity with kinetics similar to those of the tryptically generated catalytic fragment of brain PKC epsilon. This activity was inhibited by staurosporine (IC50 = 1 x 10(-8) M) and by the pseudosubstrate inhibitor peptide (IC50 = 7.7 x 10(-8) M). The SCLC kinase and the brain PKC epsilon catalytic fragment were similar as indicated by the relative sizes of the PKC epsilon immunoreactive peptides generated with protease V8 from Staphylococcus aureus (Mr approximately 37,000, 34,000, 28,000, 26,000, and 25,000). Taken together, we conclude that a variant SCLC cell line expresses a constitutively active catalytic fragment of PKC epsilon. Regulation by 12-O-tetradecanoyl-13-acetate or GRP via de novo protein synthesis suggests a novel mechanism of control of PKC diversity with implications for small cell lung cancer and possibly other malignancies.     

Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C.

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.     

Inhibition of protein kinase C by retro-inverso pseudosubstrate analogues

A retro-inverso analogue of the pseudosubstrate sequence, Arg-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val (1), found in the regulatory domain of all protein kinase C (PKC) subspecies was synthesized. It shows to be an inhibitor (IC50 = 31 microM) of the phosphorylation, by PKC, of [Ala9.10,Lys11.12] glycogen synthase (1-12). Its analogue in which D Ala25 is replaced by D Ser is not a PKC substrate, but a more potent inhibitor, competitive with the peptidic substrate (IC50 = 5 microM, Ki = 2 microM). Both retro-inverso peptides are highly specific for PKC versus adenosine cAMP-dependent protein kinase (PKA) and are totally stable towards proteolysis by trypsin or pronase.     

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.     

PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia

Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.     

Differential recruitment of PKC isoforms in HeLa cells during redox stress

The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes.

The cytoplasmic form of protein kinase C (PKC) is inactive, probably because the pseudosubstrate region in its regulatory domain blocks the substrate-binding site in its kinase domain. Calcium ions cause a translocation to the membrane: maximum activation requires a negative lipid such as phosphatidylserine (PS) and the neutral lipid diacylglycerol (DAG) but the mechanism by which PS and DAG activate PKC is unknown. Pseudosubstrate region 19-36 of PKC-beta has six basic and one acidic amino acids and region 19-29 has five basic and no acidic amino acids. Since any binding of basic residues in the pseudosubstrate region to acidic lipids in the membrane should stabilize the active form of PKC, we studied how peptides with amino acids equivalent to residues 19-36 and 19-29 of PKC-beta bound to phospholipid vesicles. We made equilibrium dialysis, filtration, and electrophoretic mobility measurements. The fraction of bound peptide is a steep sigmoidal function of the mol fraction of negative lipid in the membrane, as predicted from a simple theoretical model that assumes the basic residues provide identical independent binding sites. The proportionality constant between the number of bound peptides/area and the concentration of peptide in the bulk aqueous phase is 1 micron for a membrane with 25% negative lipid formed in 0.1 M KCl. Equivalently, the association constant of the peptide with the membrane is 10(4) M-1, or the net binding energy is 6 kcal/mol. Thus the interaction of basic residues in the pseudosubstrate region with acidic lipids in the membrane could provide 6 kcal/mol free energy towards stabilizing the active form of PKC.     

Lack of constitutive activity of the free kinase domain of protein kinase C zeta. Dependence on transphosphorylation of the activation loop

Following the induction of apoptosis in mammalian cells, protein kinase C zeta (PKC zeta) is processed between the regulatory and catalytic domains by caspases, which increases its kinase activity. The catalytic domain fragments of PKC isoforms are considered to be constitutively active, because they lack the autoinhibitory amino-terminal regulatory domain, which includes a pseudosubstrate segment that plugs the active site. Phosphorylation of the activation loop at Thr(410) is known to be sufficient to activate the kinase function of full-length PKC zeta, apparently by inducing a conformational change, which displaces the amino-terminal pseudosubstrate segment from the active site. Amino acid substitutions for Thr(410) of the catalytic domain of PKC zeta (CAT zeta) essentially abolished the kinase function of ectopically expressed CAT zeta in mammalian cells. Similarly, substitution of Ala for a Phe of the docking motif for phosphoinositide-dependent kinase-1 prevented activation loop phosphorylation and abolished the kinase activity of CAT zeta. Treatment of purified CAT zeta with the catalytic subunit of protein phosphatase 1 decreased activation loop phosphorylation and kinase activity. Recombinant CAT zeta from bacteria lacked detectable kinase activity. Phosphoinositide-dependent kinase-1 phosphorylated the activation loop and activated recombinant CAT zeta from bacteria. Treatment of HeLa cells with fetal bovine serum markedly increased the phosphothreonine 410 content of CAT zeta and stimulated its kinase activity. These findings indicate that the catalytic domain of PKC zeta is intrinsically inactive and dependent on the transphosphorylation of the activation loop.     

Biochemical properties of rat protein kinase C-eta expressed in COS cells.

Using a PKC-epsilon cDNA probe a cDNA for PKC-eta has been cloned from a rat lung cDNA library. When expressed in COS cells, rat PKC-eta appeared as an 84 kDa protein. PKC-eta expressed in COS cells, was solubilized by 1% Triton X-100 and purified away from the endogenous PKC-alpha by ammonium sulphate fractionation. The activity of this PKC-eta preparation was characterized with respect to cofactor dependence and substrate specificity. Various PKC pseudosubstrate peptides are phosphorylated by PKC-eta in a phospholipid and TPA-dependent but calcium-independent manner. The polypeptide histone IIIS is a poor substrate.     

PKCeta is localized in the Golgi, ER and nuclear envelope and translocates to the nuclear envelope upon PMA activation and serum-starvation: C1b domain and the pseudosubstrate containing fragment target PKCeta to the Golgi and the nuclear envelope

Protein kinase C (PKC) represents a family of serin/threonine kinases, playing a central role in the regulation of cell growth, differentiation and transformation. These enzymes differ in their primary structure, biochemical properties, tissue distribution and subcellular localization. The specific cellular functions of PKC isoforms are largely controlled by their localization. PKCeta, a member of the novel subfamily, is expressed predominantly in epithelial tissues. However, not much is known with respect to its mechanism of activation and regulation. Our recent studies suggest its role in cell cycle control. Here we show that PKCeta is localized at the Golgi apparatus, ER and the nuclear envelope. Furthermore, using GFP-fusion proteins of the different functional domains of PKCeta we deciphered the specific structural domains of the protein responsible for its apparent localization. We show that the cysteine-rich repeat C1b is responsible for its Golgi localization, while for its presence at the ER/nuclear envelope the pseudosubstrate containing fragment coupled to the C1 domain is required. In response to short-term activation by PMA we show translocation of PKCeta to the plasma membrane and the nuclear envelope. We demonstrate that the C1b is sufficient for its translocation to the plasma membrane. Interestingly, accumulation of PKCeta at the nuclear envelope also occurred in response to serum-starvation. It should be noted that interaction of PKCeta with the cyclin E/Cdk2 complex at the perinuclear region was recently reported by us in response to serum-starvation. Thus, our studies demonstrate translocation of PKCeta to the nuclear envelope, and suggest that the spatial regulation of PKCeta could be important for its cellular functions including effects on cell cycle control and involvement in tumor promotion.     

The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions

The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.     

Oncogenic and activated wild-type ras-p21 proteins induce different isoforms of protein kinase C in mitogenic signal transduction

We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.