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Aims

The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, δ and εPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.

Methods and results

Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished δPKC translocation by 3.8-fold and increased εPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of δPKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of εPKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of εPKC during IPC restored δPKC levels at the mitochondria while decreasing εPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.

Conclusion

Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, εPKC.

A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about 30 years. However, despite the wealth of information on PKC-mediated control of, T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s) and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks), cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1 → S and/or G2 → M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in T cells.

We have previously shown that domains involved in binding of protein kinase C (PKC1) isozymes to their respective anchoring proteins (RACKs2) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named ψRACK3 peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the ψRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the ψRACK sequence. We focused on εPKC ψRACK peptide, where the pseudo-εRACK sequence (ψεRACK; HDAPIGYD; corresponding to εPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to εRACK285-292) in the second amino acid. Here we show that changing the charge of the ψεRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.

Protein kinase C (PKC) family members have been implicated in numerous cellular processes. However, identifying the substrates of each PKC isozyme remains a challenge. Here, we describe a method using two dimensional (2-D) isoelectric focusing gel electrophoresis to identify substrates of delta PKC (δPKC) in MCF-7 breast carcinoma cells. We show that M2 pyruvate kinase is a substrate of δPKC, and further characterize the interaction between M2 pyruvate kinase and δPKC in MCF-7 cells by immunoprecipitation. δPKC activation in vitro or in cells did not appear to alter the enzyme activity or polymerization of M2 pyruvate kinase.

Protein kinase C (PKC) is a family of kinases that are critical in many cellular events. These enzymes are activated by lipid-derived second messengers, are dependent on binding to negatively charged phospholipids and some members also require calcium to attain full activation. The interaction with lipids and calcium activators is mediated by binding to the regulatory domains C1 and C2. In addition, many protein-protein interactions between PKC and other proteins have been described. These include interactions with adaptor proteins, substrates and cytoskeletal elements. Regulation of the interactions between PKC, small molecules and other proteins is essential for signal transduction to occur. Finally, a number of auto-inhibitory intramolecular protein-protein interactions have also been identified in PKC. This chapter focuses on mapping the sites for many of these inter and intramolecular interactions and how this information may be used to generate selective inhibitors and activators of PKC signaling.

The catalytic activity and intracellular localization of protein kinase C (PKC) are both highly regulated in vivo. This family of kinases contains conserved regulatory motifs, i.e., the C1, C2, and HR1 domains, which target PKC isoforms to specific subcellular compartments and restrict their activity spatially. Saccharomyces cerevisiae contains a single PKC isozyme, Pkc1p, which contains all of the regulatory motifs found in mammalian PKCs. Pkc1p localizes to sites of polarized growth, consistent with its main function in maintaining cell integrity. We dissected the molecular basis of Pkc1p localization by expressing each of its domains individually and in combinations as green fluorescent protein fusions. We find that the Rho1p-binding domains, HR1 and C1, are responsible for targeting Pkc1p to the bud tip and cell periphery, respectively. We demonstrate that Pkc1p activity is required for its normal localization to the bud neck, which also depends on the integrity of the septin ring. In addition, we show for the first time that yeast protein kinase C can accumulate in the nucleus, and we identify a nuclear exit signal as well as nuclear localization signals within the Pkc1p sequence. Thus, we propose that Pkc1p shuttles in and out of the nucleus and consequently has access to nuclear substrates. Surprisingly, we find that deletion of the HR1 domain results in Pkc1p localization to the mitotic spindle and that the C2 domain is responsible for this targeting. This novel nuclear and spindle localization of Pkc1p may provide a molecular explanation for previous observations that suggest a role for Pkc1p in regulating microtubule function.

We previously reported that some ATP competitive protein kinase C (PKC) inhibitors are either competitive or uncompetitive inhibitors with respect to substrate peptides. In this report, we demonstrate how the interactions between PKC and inhibitors change PKC activation kinetics. A substrate competitive inhibitor, bisindolylmaleimide I, targets activated PKC and stabilizes PKC in the activated conformation. This leads to transient activation and prolonged deactivation of PKC in the presence of bisindolylmaleimide I. In contrast, an uncompetitive substrate inhibitor, bisindolylmaleimide IV, targets quiescent PKC and stabilizes PKC in the quiescent conformation, which generates slower activation and suppressed translocation upon activation of PKC.

Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.

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Myristoylated, alanine-rich C-kinase substrate (MARCKS) is a lipopolysaccharide-induced protein kinase C (PKC) substrate that has been proposed to regulate actin-membrane interactions, as well as actin structure at the membrane. We studied the distribution of MARCKS, the alpha isozyme of PKC (PKC alpha), and myosin I in lipopolysaccharide- treated peritoneal macrophages ingesting zymosan particles. MARCKS, PKC alpha, and myosin I colocalized with F-actin and talin in the cortical cytoplasm adjacent to forming phagocytic cups. After particle ingestion was completed, myosin I, F-actin, and talin were no longer enriched in the vicinity of the phagosome. By contrast, MARCKS and PKC alpha remained associated with the phagosome membrane until after acquisition of the lysosomal marker Lamp-1. Vinculin was not detected on phagosomes at any time point examined. Phagocytosis of zymosan was accompanied by rapid and sustained phosphorylation of MARCKS. Inhibitors of PKC reduced zymosan binding to the macrophage surface and blocked the focal accumulation of F-actin, talin, phosphotyrosine-containing proteins, MARCKS, and PKC alpha beneath attached particles. We propose that PKC- dependent phosphorylation is an early signal required for zymosan phagocytosis and that MARCKS and PKC alpha have a role in phagosome maturation. The colocalization of F-actin and MARCKS at the cytoplasmic face of the nascent phagosome reinforces the hypothesis that MARCKS regulates actin structure at the membrane. Our data also suggest that myosin I functions as a mechanical motor during particle uptake.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

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The protein kinase C (PKC) is a family of serine/threonine kinases that are key regulatory enzymes involved in growth, differentiation, cytoskeletal reorganization, tumor promotion, and migration. We investigated the functional involvement of PKC isotypes and of E-cadherin in the regulation of the locomotion of six human colon-adenocarcinoma cell lines. The different levels of the PKC α and the E-cadherin expression have predictable implications in the spontaneous locomotory activity. With the use of PKC α–specific inhibitors (safingol, Go6976) as well as the PKC δ–specific inhibitor rottlerin, we showed that only PKC α plays a major role in the regulation of tumor cell migration. The results were verified by knocking out the translation of PKC isozymes with the use of an antisense oligonucleotide strategy. After stimulation with phorbol ester we observed a translocation and a colocalization of the activated PKC α at the plasma membrane to the surrounding extracellular matrix. Furthermore, we investigated the functional involvement of E-cadherin in the locomotion with the use of a blocking antibody. A high level of PKC α expression together with a low E-cadherin expression was strongly related to a high migratory activity of the colon carcinoma cells. This correlation was independent of the differentiation grade of the tumor cell lines.

Background

Protein kinase C (PKC) signalling is often dysregulated in gastric cancer and therefore represents a potential target in cancer therapy. The Gram-negative bacterium Helicobacter pylori, which colonises the human stomach, plays a major role in the development of gastritis, peptic ulcer and gastric adenocarcinoma.

Objective

To analyse the role of PKC isozymes as mediators of H pylori-induced pathogenesis.

Methods

PKC phosphorylation was evaluated by immunoblotting and immunohistochemistry. Gene reporter assays, RT-PCR and invasion assays were performed to assess the role of PKC in the regulation of activator protein-1 (AP-1), matrix metalloproteinase-1 (MMP-1) and the invasion of H pylori-infected epithelial cells.

Results

H pylori induced phosphorylation of PKC isozymes α, δ, θ in AGS cells, which was accompanied by the phosphorylation of PKC substrates, including PKCμ and myristoylated alanine-rich C kinase substrate (MARCKS), in a CagA-independent manner. Phospholipase C, phosphatidylinositol 3-kinase and Ca2+ were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression. Invasion assays confirmed PKC involvement in H pylori-induced MMP-1 secretion. In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.

Conclusion

The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme- specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Both epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth, however, the interaction between EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of tyrosine 845, 992, 1068 and 1045 residues of the EGFR in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13- acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr 1068 residues in the two glioblastoma cell lines. The phosphorylation of EGFR at Tyr 1068 was not detected in normal human astrocytes treated with the phorbol ester. The PMA-induced phosphorylation of EGFR at Tyr 1068 was blocked by BIM, a PKC inhibitor, and rottlerin, a specific PKC δ inhibitor. In contrast Go 6976, an inhibitor of classical PKC isozymes had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKC δ siRNA, siRNA against c-Src, mutant c-Src (Ser12Cys/Ser48Ala) and treatment with a c-Src inhibitor (PP2) abrogated PMA-induced EGFR phosphorylation at Y1068. PMA induced serine/threonine phosphorylation of Src which was blocked by both BIM and rottlerin. Inhibition of EGFR with AG 1478 did not significantly alter PMA-induced EGFR (Tyr 1068) phosphorylation, but completely blocked EGF-induced phosphorylation of EGFR. The effect of PMA on mitogen activated protein kinase (MAPK) phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor UO 126, PKC δ siRNA and c-Src siRNA. Taken together, our data demonstrate that, PMA transactivates EGFR and increases cell proliferation by activating PKC δ/c-Src pathway in glioblastomas.

Background

Protein kinase C (PKC) is a family of serine/threonine kinases that contains more than 10 isozymes. Evidence suggests that PKC may play important roles in pain modulation, but the isozyme-specific effects of PKC on different aspects of pain modulation are not fully understood. We hypothesize that different PKC isozymes play different roles in different aspects of pain modulation.

Methods

The nociceptive behaviors of mice with deletion of PKC α, β, γ, or δ in multiple pain models were compared with their respective wild type littermates. Also, the morphine analgesia and the development of morphine tolerance in mice with deletion of PKC γ were compared with their respective wild type littermates.

Results

Thermal hyperalgesia induced by complete Freund’s adjuvant injection was significantly attenuated by the deletion of PKC β, γ or δ, but not PKC α. Deletion of PKC γ significantly attenuated neuropathic mechanical allodynia induced by spared nerve injury, whereas deletion of PKC α enhanced this allodynia. Baseline thermal and mechanical sensitivity, nociceptive behaviors induced by formalin, mechanical allodynia induced by complete Freund’s adjuvant injection, were not altered by deletion of PKC α, β, γ or δ. Finally, morphine analgesia and the development of morphine tolerance were not altered in PKC γ-deficient mice.

Conclusions

PKC plays isozyme-specific effects in pain modulation.

The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav−/− thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28–mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav−/− thymocytes. Vav was found to bind constitutively to PKC-θ in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.

Cardiac hypertrophy is a complex adaptive response to mechanical and neurohumoral stimuli and under continual stressor, it contributes to maladaptive responses, heart failure and death. Protein kinase C (PKC) and several other kinases play a role in the maladaptative cardiac responses, including cardiomyocyte hypertrophy, myocardial fibrosis and inflammation. Identifying specific therapies that regulate these kinases is a major focus of current research. PKC, a family of serine/threonine kinases, has emerged as potential mediators of hypertrophic stimuli associated with neurohumoral hyperactivity in heart failure. In this review, we describe the role of PKC isozymes that are involved in cardiac hypertrophy and heart failure.

The ER/Golgi protein p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β-chimaerin. C1 domains from PKC isozymes can also interact with p23/Tmp21. Our study highlights the relevance of C1 domains in protein-protein interactions in addition to their well-established lipid-binding properties.

The C1 domains in protein kinase C (PKC) isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and for mediating translocation to membranes. Previous studies revealed that the C1 domain in α- and β-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the endoplasmic reticulum/Golgi protein p23/Tmp21. Here, we found that p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β2-chimaerin. Glu227 and Leu248 in the β2-chimaerin C1 domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 domains from individual PKC isozymes differentially interact with p23/Tmp21. For PKCε, it interacts with p23/Tmp21 specifically via its C1b domain; however, this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 domain-containing proteins, and it plays an essential role in β2-chimaerin relocalization. Our study also highlights the relevance of C1 domains in protein–protein interactions in addition to their well-established lipid-binding properties.

Our laboratory has reported on a biphasic pattern of nuclear factor κB (NF-κB) activation in cultured human umbilical vein endothelial cells during infection with Rickettsia rickettsii, an obligate, intracellular bacterium, and the etiologic agent of Rocky Mountain spotted fever. Transcriptional activation of the tissue factor (TF) gene during this infection has been shown to involve NF-κB. To further understand the signal transduction events underlying these phenomena, we studied the role of protein kinase C (PKC), a ubiquitous family of phospholipid-dependent enzymes implicated in the regulation of a variety of cell signaling pathways. Two inhibitors of PKC, namely, bisindolylmaleimide I hydrochloride (BM-1) and calphostin C, which exhibit different inhibitory properties towards various isozymes of PKC, were used. Infection of cells with R. rickettsii in the presence of BM-1 (50 nM) did not significantly affect NF-κB, whereas calphostin C (2.5 μM) completely blocked the early phase of NF-κB activation. The late, sustained phase also was not affected by treatment with BM-1. Downregulation of phorbol ester-sensitive PKCs by long-term treatment with phorbol 12-myristate 13-acetate (PMA) did not inhibit NF-κB activation. Likewise, this downregulation had no effect on induction of TF activity. The activity of TF was, however, sensitive to BM-1 and calphostin C, whereas expression of TF mRNA was inhibited only by calphostin C. Overall, these results suggest the lack of involvement of classical PKC pathways in R. rickettsii-induced NF-κB activation but the possible involvement of a non-PMA-responsive PKC isoform in the posttranscriptional control of TF expression.

Studies utilizing targeted gene deletion in mice have revealed distinct roles for individual isozymes of the protein kinase C (PKC) family of enzymes in regulating sensitivity to various drugs of abuse. These changes in drug sensitivity are associated with altered patterns of drug self-administration. The purpose of this review is to summarize behavioural studies conducted on mice carrying targeted deletions of genes encoding specific PKC isozymes (namely the β, γ, δ, and ε isozymes), and to critically evaluate the possibility of using pharmacological inhibitors of specific PKC isozymes as modulators of the sensitivity to various drugs of abuse, as well as potential aids in the treatment of substance use disorders.

Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membrane-targeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca2+-triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKCδ. We show that PKCδ and a conventional isozyme, PKCβII, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKCβII with membranes, the C2 domain of PKCδ does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCδ with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCδ in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCδ. Thus, the increased affinity of the C1 domain of PKCδ allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca2+ binding to the C2 domain and DAG binding to the C1 domain for activation.

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.

Summary

SRC-3/AIB1 is a steroid receptor coactivator with potent growth promoting activity and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue rich region in SRC-3 is an important determinant for aPKC mediated phosphorylation and stabilization. The mechanism of the aPKC mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a new and potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.

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