Cisplatin resistance is associated with deregulation in protein kinase C-delta

Proteolytic activation of protein kinase C (PKC)-delta has been associated with cell death induced by the DNA damaging agent cisplatin. In the present study, we have examined if PKCdelta is affected when cells acquire resistance to cisplatin. The level of PKCdelta was elevated in cisplatin-resistant HeLa (HeLa/CP) cells compared to parental HeLa cells. Prolonged cellular exposure to the PKC activator phorbol-12,13-dibutyrate (PDBu), caused downregulation of PKCdelta in HeLa cells but not in HeLa/CP cells. Treatment of HeLa cells with PDBu resulted in the translocation of PKCdelta from the cytosol to the membrane but it failed to induce PKCdelta translocation in HeLa/CP cells. PDBu, however, induced translocation and downregulation of PKCalpha in both HeLa and HeLa/CP cells. The ability of PDBu to enhance cisplatin-induced cell death was attenuated in cisplatin-resistant HeLa cells. Thus, a deregulation in PKCdelta was associated with reduced cellular sensitivity to cisplatin.     

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.     

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100 nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.     

Mutation of the hydrophobic motif in a phosphorylation-deficient mutant renders protein kinase C delta more apoptotically active

Protein kinase C delta (PKCδ) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCδ is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCδ. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCδ activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCδ (PKCδ-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCδ apoptotically more active. Furthermore, we found that the mutant PKCδ-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart.     

Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3

Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.     

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.     

The mitochondrial antiviral signaling protein, MAVS, is cleaved during apoptosis

Apoptosis of virus-infected cells is one important host strategy used to limit viral infection. Recently a member of the innate immune signaling pathway, MAVS, was localized to mitochondria, an organelle important for apoptosis regulation. Here we investigate what role MAVS may play in apoptosis. Induction of cell death led to the rapid cleavage of MAVS, resulting in its release from the outer mitochondrial membrane. This cleavage is blocked in cells incubated with proteasome or caspase inhibitors. Transfection of synthetic viral dsRNA and dsDNA also led to cleavage of MAVS, indicating that this process may be important during infection. Preventing apoptosis by over-expression of anti-apoptotic Bcl-xL blocks MAVS cleavage, placing this process downstream of caspase activation in the apoptotic program.     

The cardioprotective effect of postconditioning is mediated by ARC through inhibiting mitochondrial apoptotic pathway

Objective  Postconditioning protects the heart against ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis. However, the molecular mechanism by which postconditioning suppresses apoptosis remains to be fully understood. Apoptosis repressor with caspase recruitment domain (ARC) has been demonstrated to possess the ability to protect cardiomyocytes from apoptosis induced by ischemia/reperfusion. It is not yet clear as to whether ARC contributes to the inhibitory effect of postconditioning against cardiomyocyte apoptosis. Methods  The cultured cardiomyocytes from 1-day old male Sprague–Dawley rats were exposed to 3 h hypoxia followed by 3 h of reoxygenation. Cells were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia before 3 h of reoxygenation. Results  Hypoxia/reoxygenation led to a decrease of endogenous ARC protein levels. In contrast, postconditioning could block the reduction of endogenous ARC protein levels. Interestingly, inhibition of endogenous ARC expression by ARC antisense oligodeoxynucleotides reduced the inhibitory effect of postconditioning against apoptosis. Furthermore, our data showed that postconditioning suppressed the loss of mitochondrial membrane potential, Bax activation and the release of mitochondrial cytochrome c to cytosol. However, these inhibitory effects of postconditioning disappeared upon knockdown of endogenous ARC. Conclusion  Our data for the first time demonstrate that ARC plays an essential role in mediating the cardioprotective effect of postconditioning against apoptosis initiated by the mitochondrial pathway.     

Phosphorylation of mitochondrial phospholipid scramblase 3 by protein kinase C-delta induces its activation and facilitates mitochondrial targeting of tBid

Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.     

Gq protein-induced apoptosis is mediated by AKT kinase inhibition that leads to protein kinase C-induced c-Jun N-terminal kinase activation

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

Stimulation by glucose of cyclic AMP accumulation in mouse pancreatic islets is mediated by protein kinase C.

The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation. Both TPA-stimulated and glucose-stimulated cyclic AMP accumulations were abolished by the omission of extracellular Ca2+. The Ca2+ ionophore A23187 did not affect cyclic AMP accumulation itself, but affected the time course of TPA-induced cyclic AMP accumulation, the effect of A23187 + TPA mimicking the time course for glucose-induced cyclic AMP accumulation. A 24 h exposure to TPA, which depletes islets of protein kinase C, abolished the effects of both TPA and glucose on cyclic AMP production. Both TPA-induced and glucose-induced cyclic AMP productions were inhibited by anti-glucagon antibody, and after pretreatment with this antibody glucose stimulation was dependent on addition of glucagon. Pretreatment of islets with TPA for 10 min potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase activity in a particulate fraction of islets. Carbamoylcholine, which is supposed to activate protein kinase C in islets, likewise stimulated cyclic AMP accumulation in islets. These observations suggest that glucose stimulates islet adenylate cyclase by activation of protein kinase C, and thereby potentiates the effect of endogenous glucagon on adenylate cyclase.     

MEKK1-induced apoptosis is mediated by Smac/Diablo release from the mitochondria

During apoptotic stimulation, the serine threonine kinase, MEKK1, is cleaved into an activated 91 kDa kinase fragment. This cleavage is mediated by caspase 3 and leads to further caspase 3 activation and apoptosis. Forced expression of the 91 kDa kinase fragment induces apoptosis through changes in membrane potential of the mitochondria mediated by permeability transition pore opening. MEKK1 activation, however, fails to release cytochrome c from the mitochondria. Herein, we determined that overexpression of MEKK1 causes mitochondrial Smac/Diablo release correlating with MEKK1-induced apoptosis. Furthermore, using siRNA that lowers Smac/Diablo expression, MEKK1-induced apoptosis was significantly reduced. Mouse embryonic fibroblast cells lacking MEKK1 expression are also resistant to etoposide-induced mitochondrial Smac/Diablo release. In contrast, etoposide-induced mitochondrial cytochrome c release was not inhibited. MEKK1 also activates the MAP kinase JNK, but MEKK1-induced mitochondrial Smac/Diablo release and apoptosis are independent of MEKK1 mediated JNK activation. Taken together, release of Smac/Diablo from the mitochondria plays a role in MEKK1-induced apoptosis.     

Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.     

Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein

Survivin is a multifunctional protein with essential roles in cell division and inhibition of apoptosis, but the molecular underpinnings of its cytoprotective properties are poorly understood. Here we show that homozygous deletion of the aryl hydrocarbon receptor-interacting protein (AIP), a survivin-associated immunophilin, causes embryonic lethality in mice by embryonic day 13.5-14, increased apoptosis of Ter119(-)/CD71(-) early erythropoietic progenitors, and loss of survivin expression in its cytosolic and mitochondrial compartments in vivo. In import assays using recombinant proteins, AIP directly mediated the import of survivin to mitochondria, thus enabling its anti-apoptotic function, whereas a survivin 1-141 mutant that does not bind AIP was not imported to mitochondria and failed to inhibit apoptosis. AIP-directed mitochondrial import of survivin did not affect cell division, was independent of the organelle transmembrane potential, did not require the chaperone Heat Shock Protein 90 (Hsp90), and was inhibited by cytosolic factor(s) present in normal cells. shRNA knockdown of the mitochondrial import receptor Tom20 abolished mitochondrial import of survivin and sensitized tumor cells to apoptosis, whereas silencing of Tom70 had no effect. Therefore, an AIP-Tom20 recognition contributes to cell survival in development and cancer by mediating the mitochondrial import of survivin.     

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.     

Induction of apoptosis in breast cancer cells by apomine is mediated by caspase and p38 mitogen activated protein kinase activation

The 1,1-bisphosphonate ester family member apomine (SR-45023A) is known to have anti-tumour activity in various cancer cell types. The aims of this study were to determine the effect of apomine on the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231, to ascertain whether any growth inhibitory effects found were due to induction of apoptosis, and to investigate the mechanism of action of apomine. Apomine caused significant growth inhibition of both cell lines after 72h of treatment. Apomine-induced growth inhibition was associated with caspase and p38 MAPK activation and DNA fragmentation. Apomine had no effect on Ras localisation, nor did addition of mevalonate to treatment media prevent apomine-induced apoptosis. We conclude that apomine induces apoptosis in breast cancer cells, an effect that is independent of oestrogen receptor status and is not via inhibition of the mevalonate pathway. Our study suggests apomine is a potential anti-neoplastic drug in breast cancer treatment.