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.     

Roles of tyrosine phosphorylation and cleavage of protein kinase Cdelta in its protective effect against tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.     

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues induces sustained activation of Erk1/2 via down-regulation of MKP-1: role in the apoptotic effect of etoposide

The mechanism underlying the important role of protein kinase Cdelta (PKCdelta) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCdelta in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCdelta since the phosphorylation of Erk1/2 was inhibited by a PKCdelta-KD mutant and PKCdelta small interfering RNA. We recently reported that phosphorylation of PKCdelta on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCdeltatyrosine mutants, we found that the phosphorylation of PKCdeltaon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCdelta was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCdelta. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCdelta and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCdeltaon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.     

The phosphorylation of tyrosine 332 is necessary for the caspase 3-dependent cleavage of PKCdelta and the regulation of cell apoptosis

Protein kinase C delta (PKCdelta plays a major role in the regulation of cell apoptosis and survival. PKCdelta is cleaved by caspase 3 to generate a constitutively active catalytic domain that mediates both its apoptotic and anti-apoptotic effects. The caspase cleavage site of PKCdelta in the hinge region is flanked by the two tyrosine residues, Y311 and Y332. Here, we examined the role of the phosphorylation of tyrosines 311 and 332 in the cleavage and apoptotic function of PKCdelta using the apoptotic stimuli, TRAIL and cisplatin. Tyrosine 332 was constitutively phosphorylated in the A172 and HeLa cells and was further phosphorylated by TRAIL and cisplatin. This phosphorylation was inhibited by the Src inhibitors, PP2 and SU6656, and by silencing of Src. Treatment of the A172 and HeLa cells with TRAIL induced cleavage of the WT PKCdelta and of the PKCdeltaY311F mutant, whereas a lower level of cleavage was observed in the PKCdeltaY332F mutant. Similarly, a smaller degree of cleavage of the PKCdeltaY332 mutant was observed in LNZ308 cells treated with cisplatin. Mutation of Y332F affected the apoptotic function of PKCdelta; overexpression of the PKCdeltaY332 mutant increased the apoptotic effect of TRAIL, whereas it decreased the apoptotic effect of cisplatin. Inhibition of Src decreased the cleavage of PKCdelta and modified the apoptotic responses of the cells to TRAIL and cisplatin, similar to effect of the PKCdeltaY332F mutant. These results demonstrate that the phosphorylation of tyrosine 332 by Src modulates the cleavage of PKCdelta and the sensitivity of glioma cells to TRAIL and cisplatin.     

Tyrosine 311 is phosphorylated by c-Abl and promotes the apoptotic effect of PKCdelta in glioma cells

In this study we characterized the phosphorylation of tyrosine 311 and its role in the apoptotic function of PKCdelta in glioma cells. We found that c-Abl phosphorylated PKCdelta on tyrosine 311 in response to H2O2 and that this phosphorylation contributed to the apoptotic effect of H2O2. In contrast, Src, Lyn, and Yes were not involved in the phosphorylation of tyrosine 311 by H2O2. A phosphomimetic PKCdelta mutant, in which tyrosine 311 was mutated to glutamic acid (PKCdeltaY311E), induced a large degree of cell apoptosis. Overexpression of the PKCdeltaY311E mutant induced the phosphorylation of p38 and inhibition of p38 abolished the apoptotic effect of the PKCdelta mutant. These results suggest an important role of tyrosine 311 in the apoptotic function of PKCdelta and implicate c-Abl as the kinase that phosphorylates this tyrosine.     

The localization of protein kinase Cdelta in different subcellular sites affects its proapoptotic and antiapoptotic functions and the activation of distinct downstream signaling pathways

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis and survival in diverse cellular systems. PKCdelta translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCdelta constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCdelta affects its apoptotic and survival functions. PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCdelta-ER. PKCdelta-Cyto and PKCdelta-Mito underwent cleavage, whereas no cleavage was observed in the PKCdelta-Nuc and PKCdelta-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCdelta-Cyto and PKCdelta-Mito. In contrast to the apoptotic effects of the PKCdelta-Cyto, PKCdelta-Mito, and PKCdelta-Nuc, the PKCdelta-ER protected the cells from tumor necrosis factor-related apoptosis-inducing ligand-induced and etoposide-induced apoptosis. Moreover, overexpression of a PKCdelta kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCdelta and increased tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. The localization of PKCdelta differentially affected the activation of downstream signaling pathways. PKCdelta-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCdelta-Nuc increased c-Jun NH(2)-terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCdelta-Cyto, whereas c-Jun NH(2)-terminal kinase activation mediated the apoptotic effect of PKCdelta-Nuc. Our results indicate that the subcellular localization of PKCdelta plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways.     

Tyrosine phosphorylation regulates the proteolytic activation of protein kinase Cdelta in dopaminergic neuronal cells

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 activation and PKCdelta cleavage. H(2)O(2)-induced proteolytic activation of PKC was delta mediated by the activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the proteolytic activation and proapoptotic function of the kinase in dopaminergic neuronal 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.     

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.     

Regulation of cadmium-induced apoptosis by PKCdelta in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCdelta translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCdelta cleavage to give a 41-kDa fragment, which was detected at 3-6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCdelta-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCdelta translocation. Cadmium chloride rapidly induced p38(MAPK) activation, which was not affected by rottlerin. By contrast, the p38(MAPK) inhibitor SB203580 reduced PKCdelta translocation and cleavage, indicating that p38(MAPK) activation precedes and regulates PKCdelta activation. It is concluded that PKCdelta mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

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.     

Opposing effects of protein kinase C delta and protein kinase B alpha on H(2)O(2)-induced apoptosis in CHO cells.

H(2)O(2)-induced apoptosis was enhanced in the CHO cell line overproducing protein kinase C delta (PKCdelta) as judged by DNA fragmentation. In response to the H(2)O(2) treatment, PKCdelta was tyrosine phosphorylated and recovered as a constitutively active form, but its proteolytic fragment was not generated. In contrast, H(2)O(2)-induced apoptosis was suppressed in the CHO cell line overexpressing protein kinase B alpha (PKBalpha). Consistently, phosphorylation of BAD, a pro-apoptotic protein negatively regulated by PKBalpha, was sustained in the cells overproducing PKBalpha, but was not changed in the cells overexpressing PKCdelta. In the CHO cell line overproducing both PKCdelta and PKBalpha, H(2)O(2)-induced tyrosine phosphorylation of PKCdelta was suppressed, and DNA fragmentation was diminished concomitantly. These results suggest that PKCdelta contributes to H(2)O(2)-induced apoptosis by a mechanism independent of BAD and that PKCdelta is a target of PKB for the regulation of cell survival.     

Inhibition of PKCalpha induces a PKCdelta-dependent apoptotic program in salivary epithelial cells

We have used expression of a kinase dead mutant of PKCalpha (PKCalphaKD) to explore the role of this isoform in salivary epithelial cell apoptosis. Expression of PKCalphaKD by adenovirus-mediated transduction results in a dose-dependent induction of apoptosis in salivary epithelial cells as measured by the accumulation of sub-G1 DNA, activation of caspase-3, and cleavage of PKCdelta and PKCzeta, known caspase substrates. Induction of apoptosis is accompanied by nine-fold activation of c-Jun-N-terminal kinase, and an approximately two to three-fold increase in activated mitogen-activated protein kinase (MAPK) as well as total MAPK protein. Previous studies from our laboratory have shown that PKCdelta activity is essential for the apoptotic response of salivary epithelial cells to a variety of cell toxins. To explore the contribution of PKCdelta to PKCalphaKD-induced apoptosis, salivary epithelial cells were cotransduced with PKCalphaKD and PKCdeltaKD expression vectors. Inhibition of endogenous PKCdelta blocked the ability of PKCalphaKD to induce apoptosis as indicated by cell morphology, DNA fragmentation, and caspase-3 activation, indicating that PKCdelta activity is required for the apoptotic program induced under conditions where PKCalpha is inhibited. These findings indicate that PKCalpha functions as a survival factor in salivary epithelial cells, while PKCdelta functions to regulate entry into the apoptotic pathway.     

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.     

Phosphorylation of protein kinase Cdelta on distinct tyrosine residues regulates specific cellular functions

Protein kinase Cdelta (PKCdelta) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKCdelta are involved in these two responses. Transfection of cells with PKCdelta mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKCdelta wild-type transfectant. Conversely, transfection with PKCdelta mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKCdelta wild-type transfectant. The tyrosine phosphorylation of PKCdelta and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKCdelta via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKCdelta induced by PDGF. We conclude that the tyrosine phosphorylation of PKCdelta and its association with tyrosine kinases may be an important point of divergence in PKC signaling.     

PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells

We report here that the novel protein kinase C isoform, PKCdelta, is required at or prior to the level of the mitochondria for apoptosis induced by a diverse group of cell toxins. We have used adenoviral expression of a kinase-dead (KD) mutant of PKCdelta to explore the requirement for PKCdelta in the mitochondrial-dependent apoptotic pathway. Expression of PKCdeltaKD, but not PKCalphaKD, in salivary epithelial cells resulted in a dose-dependent inhibition of apoptosis induced by etoposide, UV-irradiation, brefeldin A, and paclitaxel. DNA fragmentation was blocked up to 71% in parotid C5 cells infected with the PKCdeltaKD adenovirus, whereas caspase-3 activity was inhibited up to 65%. The activation of caspase-9-like proteases by all agents was also inhibited in parotid C5 cells expressing PKCdeltaKD. The ability of PKCdeltaKD to block the loss of mitochondrial membrane potential was similarly determined. Expression of PKCdeltaKD blocked the decrease in mitochondrial membrane potential observed in cells treated with etoposide, UV, brefeldin A, or paclitaxel in a dose-dependent manner. In contrast to the protective function of PKCdeltaKD, expression of PKCdeltaWT resulted in a potent induction of apoptosis, which could be inhibited by co-infection with PKCdeltaKD. These results suggest that PKCdelta is a common intermediate in mitochondrial-dependent apoptosis in salivary epithelial cells.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Suppression of apoptosis in the protein kinase Cdelta null mouse in vivo

Protein kinase C (PKC) delta is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used the PKCdelta(-/-) mouse to ask if loss of PKCdelta protects salivary glands against gamma-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that gamma-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater than 60% in the parotid glands of PKCdelta(-/-) mice. Primary parotid cells from PKCdelta(-/-) mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored by re-introduction of PKCdelta by adenoviral transduction. Etoposide and gamma-irradiation-induced activation of p53 is similar in primary parotid cells and parotid glands from PKCdelta(+/+) and PKCdelta(-/-) mice, indicating that PKCdelta functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal kinase is reduced in primary parotid cells from PKCdelta(-/-) cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCdelta. Similarly, c-Jun amino-terminal kinase activation is suppressed in vivo in gamma-irradiated parotid glands from PKCdelta(-/-) mice. These studies indicate an essential role for PKCdelta downstream of the p53 response and upstream of the c-Jun amino-terminal kinase activation in DNA damage-induced apoptosis in vivo and in vitro.