Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms

We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.     

Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network

Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and - are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.     

Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.     

Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C.

The induction of apoptosis in human keratinocytes by UV radiation involves caspase-mediated cleavage and activation of protein kinase C delta (PKCdelta). Here we examined the role of PKC activation in caspase activation and disruption of mitochondria function by UV radiation. Inhibition of PKC partially blocked UV radiation-induced cleavage of PKCdelta, pro-caspase-3, and pro-caspase-8, and the activation of these caspases. PKC inhibition also blocked the UV-induced loss of mitochondria membrane potential, but did not block the release of cytochrome c from mitochondria. Expression of the active catalytic domain of PKCdelta was sufficient to induce apoptosis and disrupt mitochondrial membrane potential, however a kinase inactive PKCdelta catalytic domain did not. Furthermore, the PKCdelta catalytic fragment generated following UV radiation localized to the mitochondria fraction, as did ectopically expressed PKCdelta catalytic domain. These results identify a functional role for PKC activation in potentiating caspase activation and disrupting mitochondrial function during UV-induced apoptosis.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Involvement of protein kinase C delta (PKCdelta) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKCdelta

Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKCdelta, we used a replication-deficient adenovirus (PKCdeltaAdV), which allowed for a tightly controlled expression of PKCdelta in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKCdeltaAdV. Overexpression of PKCdelta markedly enhanced the apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells. PKCdelta-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKCdelta-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKCdelta is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKCdelta, supporting the concept that PKCdelta plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.     

Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.

We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with G?6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death.     

Protein kinase Ctheta activity is involved in the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced signal transduction pathway leading to apoptosis in L-MAT, a human lymphoblastic T-cell line

The aromatic hydrocarbon receptor (AhR)-dependent pathway involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity has been studied extensively, but the AhR-independent molecular mechanism has not. In previous studies we found that the AhR is not expressed in L-MAT, a human lymphoblastic T-cell line. In this report, we provide the following evidence that the protein kinase C (PKC)theta activity is functionally involved in the AhR-independent signal transduction mechanism that participates in the TCDD-induced L-MAT cell apoptosis. First, only rottlerin, a novel PKC (nPKC)-selective inhibitor, blocked the apoptosis completely, in a dose-dependent manner. Second, PKCtheta was the major nPKC isoform (compared to PKCdelta) expressed in the L-MAT cell line. Third, a cell-permeable myristoylated PKCtheta pseudosubstrate peptide inhibitor also blocked the apoptosis completely, in a dose-dependent manner. Fourth, both rottlerin and myristoylated PKCtheta pseudosubstrate peptide inhibitor completely inhibited PKCtheta kinase activity in vitro at doses that effectively blocked TCDD-induced L-MAT cell apoptosis. TCDD treatment induced a time-dependent activation of nPKC kinase activity in L-MAT cells, and moreover, TCDD induced a translocation of PKCtheta from the cytosolic fraction to the particulate fraction in L-MAT cells. Finally, transient over-expression of a dominant negative PKCtheta (a kinase-dead mutant, K/R 409) in L-MAT cells conferred significant protection against TCDD-induced apoptosis.     

Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide

Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect.     

Increased membrane affinity of the C1 domain of protein kinase Cdelta compensates for the lack of involvement of its C2 domain in membrane recruitment

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 PKCdelta. We show that PKCdelta and a conventional isozyme, PKCbetaII, 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 PKCbetaII with membranes, the C2 domain of PKCdelta does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCdelta with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCdelta in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCdelta. Thus, the increased affinity of the C1 domain of PKCdelta 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.     

Protein kinase C delta is required for survival of cells expressing activated p21RAS

Inhibition of protein kinase C (PKC) activity in transformed cells and tumor cells containing activated p21(RAS) results in apoptosis. To investigate the pro-apoptotic pathway induced by the p21(RAS) oncoprotein, we first identified the specific PKC isozyme necessary to prevent apoptosis in the presence of activated p21(RAS). Dominant-negative mutants of PKC, short interfering RNA vectors, and PKC isozyme-specific chemical inhibitors directed against the PKCdelta isozyme demonstrated that PKCdelta plays a critical role in p21(RAS)-mediated apoptosis. An activating p21(RAS) mutation, or activation of the phosphatidylinositol 3-kinase (PI3K) Ras effector pathway, increased the levels of PKCdelta protein and activity in cells, whereas inhibition of p21(RAS) activity decreased the expression of the PKCdelta protein. Activation of the Akt survival pathway by oncogenic Ras required PKCdelta activity. Akt activity was dramatically decreased after PKCdelta suppression in cells containing activated p21(RAS). Conversely, constitutively activated Akt rescued cells from apoptosis induced by PKCdelta inhibition. Collectively, these findings demonstrate that p21(RAS), through its downstream effector PI3K, induces PKCdelta expression and that this increase in PKCdelta activity, acting through Akt, is required for cell survival. The p21(RAS) effector molecule responsible for the initiation of the apoptotic signal after suppression of PKCdelta activity was also determined to be PI3K. PI3K (p110(C)(AAX), where AA is aliphatic amino acid) was sufficient for induction of apoptosis after PKCdelta inhibition. Thus, the same p21(RAS) effector, PI3K, is responsible for delivering both a pro-apoptotic signal and a survival signal, the latter being mediated by PKCdelta and Akt. Selective suppression of PKCdelta activity and consequent induction of apoptosis is a potential strategy for targeting of tumor cells containing an activated p21(RAS).     

Protein kinase C delta is not activated by caspase-3 and its inhibition is sufficient to induce apoptosis in the colon cancer line, COLO 205

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.     

Protein kinase D is a downstream target of protein kinase Ctheta

Protein kinase D (PKD/PKCmu immunoprecipitated from either COS-7 cells or Jurkat T lymphocytes transiently transfected with a constitutively active mutant of PKCtheta AE (PKCthetaAE) exhibited a marked increase in basal activity. In contrast, coexpression of constitutively active mutant of PKCzeta does not induce PKD activation in both types of cells. PKCthetaAE does not induce kinase activity in immunocomplexes of PKD kinase-deficient mutants PKDK618N or PKDD733A. PKD activation in response to PKCthetaAE signaling was completely prevented by treatment with the protein kinase C (PKC) inhibitors, GF I or Ro 31-8220, or by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Our results show that PKD is a downstream target of the theta isoform of PKC in both COS-7 cells and lymphocytes. The regulation of PKD by PKCtheta reveals a new pathway in the signaling network existing between multiple members of the PKC superfamily and PKD.     

Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

The protein kinase C delta catalytic fragment targets Mcl-1 for degradation to trigger apoptosis

Proteolytic cleavage and subsequent activation of protein kinase C (PKC) delta is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCdelta catalytic fragment (PKCdelta-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCdelta is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCdelta-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCdelta-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCdelta-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCdelta-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCdelta could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCdelta. Overall our results identify Mcl-1 as an important target for PKCdelta-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli.     

Protein kinase C-theta mediates a selective T cell survival signal via phosphorylation of BAD

Protein kinase C (PKC)-activating phorbol esters protect T cells from Fas-induced apoptosis. However, the mechanism of this protective effect and the identity of the relevant PKC isoform(s) are poorly understood. Here, we show that PKCtheta plays a selective and important role in this protection. Fas triggering led to a selective caspase-3-dependent cleavage of the enzyme and proteasome-mediated degradation and inactivation of its catalytic fragment. These events preceded the onset of apoptosis. Pharmacological inhibition of PKCtheta promoted Fas-mediated apoptosis in three different types of T cells. Conversely, constitutively active PKCtheta (and, to a lesser degree, PKCepsilon) selectively protected T cells from Fas-induced apoptosis. We provide evidence that the distant Bcl-2 family member, BAD, is a PKCtheta substrate, is phosphorylated by TCR stimulation, and can mediate at least in part the anti-apoptotic effect of PKCtheta.     

Infection of glioma cells with Sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase C delta. Implications for Sindbis virus-induced apoptosis

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCdelta to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCdelta inhibitor rottlerin and a PKCdelta kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCdelta in the apoptosis induced by SVNI we used a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). PKCdelta5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCdelta. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCdelta5 but did not induce cleavage of PKCdelta or PKCdelta5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCdelta exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCdelta on specific tyrosines is required for this function.     

Dual involvement of protein kinase C delta in apoptosis induced by syndecan-2 in osteoblasts

Syndecans are proteoglycans that act as signaling molecules. Previously, we showed that syndecan-2 (SYND2) is involved in the control of osteoblastic (OB) cell apoptosis. Here, we show a novel functional interaction between SYND2 and protein kinase C delta (PKCdelta). Overexpression of SYND2 in MG63 OB cells resulted in increased PKCdelta protein level without change in PKCdelta mRNA production. In SYND2-transfected cells, the increase in PKCdelta was restricted to the cytosolic compartment, threonine 505-PKCdelta was underphosphorylated and immunoprecipitated PKCdelta showed decreased capacity to phosphorylate histone, indicating that SYND2 decreased PKCdelta activity. Inhibition of PKCdelta by Rottlerin or a dead-kinase dominant negative (DN) construct activated effector caspases and increased the number of apoptotic cells. In addition, rescue of kinase activity with a construct coding, the PKCdelta catalytic domain (CAT) reduced SYND2-induced apoptosis. This indicates that PKCdelta acts as a pro-survival kinase and that SYND2 inhibits the anti-apoptotic action of PKCdelta in OB cells. We also showed that overexpression of PKCdelta wild type (WT) induced osteoblast apoptosis. Moreover, inhibition of PKCdelta by siRNA resulted in increased apoptosis in control cells but reduced apoptosis in SYND2-overexpressing osteoblasts, indicating that SYND2 requires PKCdelta accumulation to induce apoptosis. These results show that SYND2 modulates PKCdelta actions by inhibition of the canonical allosterical activation pathway that plays an anti-apoptotic role in OB cells, and promotion of a pro-apoptotic role that may depend on PKCdelta protein level and that participates to the induction of cell death by SYND2. This establishes a functional interaction between SYND2 and PKCdelta in osteoblasts.     

Protein kinase Cepsilon binds peripherin and induces its aggregation, which is accompanied by apoptosis of neuroblastoma cells

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCepsilon induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCepsilon. PKCepsilon and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCepsilon. The interaction was specific for PKCepsilon since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCeta and -, which pulled down minute amounts. PKCepsilon interacts with vimentin through the same structures but does not induce its aggregation. When the PKCepsilon C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCepsilon through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCepsilon.