H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

Epidermal growth factor receptor phosphorylates protein kinase C {delta} at Tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H(2)O(2)-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCdelta at Tyr332 in vitro. PKCdelta was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H(2)O(2)-induced tyrosine phosphorylation of PKCdelta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKCdelta at Tyr332 in the H(2)O(2)-stimulated but not in the growth-factor treated cells, and suggest that PKCdelta in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.     

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.     

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.     

Modulation of PKCdelta tyrosine phosphorylation and activity in salivary and PC-12 cells by Src kinases

Protein kinase C (PKC) delta becomes tyrosine phosphorylated in rat parotid acinar cells exposed to muscarinic and substance P receptor agonists, which initiate fluid secretion in this salivary cell. Here we examine the signaling components of PKCdelta tyrosine phosphorylation and effects of phosphorylation on PKCdelta activity. Carbachol- and substance P-promoted increases in PKCdelta tyrosine phosphorylation were blocked by inhibiting phospholipase C (PLC) but not by blocking intracellular Ca2+ concentration elevation, suggesting that diacylglycerol, rather than D-myo-inositol 1,4,5-trisphosphate production, positively modulated this phosphorylation. Stimuli-dependent increases in PKCdelta activity in parotid and PC-12 cells were blocked in vivo by inhibitors of Src tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a protein tyrosine phosphatase, reduced the enhanced PKCdelta activity. Lipid cofactors modified the tyrosine phosphorylation-dependent PKCdelta activation. Two PKCdelta regulatory sites (Thr-505 and Ser-662) were constitutively phosphorylated in unstimulated parotid cells, and these phosphorylations were not altered by stimuli that increased PKCdelta tyrosine phosphorylation. These results demonstrate that PKCdelta activity is positively modulated by tyrosine phosphorylation in parotid and PC-12 cells and suggest that PLC-dependent effects of secretagogues on salivary cells involve Src-related kinases.     

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.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.     

Activation of HGF/c-Met pathway contributes to the reactive oxygen species generation and motility of small cell lung cancer cells

Small cell lung cancer (SCLC) is a difficult disease to treat and sometimes has overexpression or mutation of c-Met receptor tyrosine kinase. The effects of c-Met/hepatocyte growth factor (c-Met/HGF, ligand for c-Met) on activation of reactive oxygen species (ROS) was determined. HGF stimulation of c-Met-overexpressing H69 SCLC cells (40 ng/ml, 15 min) resulted in an increase of ROS, measured with fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (DHE) but not in c-Met-null H446 cells. ROS was increased in juxtamembrane (JM)-mutated variants (R988C and T1010I) of c-Met compared with wild-type c-Met-expressing cells. ROS was significantly inhibited by preincubation of SCLC cells with pyrrolidine dithiocarbamate (PDTC, 100 microM) and/or SU11274 (small molecule c-Met tyrosine kinase inhibitor, 2 microM) for 3 h. PDTC and SU11274 also abrogated the HGF proliferative signal and cell motility in a cooperative fashion. H(2)O(2) treatment of SCLC cells (over 15 min) led to phosphorylation of c-Met receptor tyrosine kinase and further upregulated downstream phosphorylation of phospho-AKT, ERK1/2, and paxillin in a dose-dependent manner (125 microM to 500 microM). c-Met is an important target in lung cancer, and the pathways responsible for ROS generation together may provide novel therapeutic intervention.     

Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

Interaction between protein kinase C delta and the c-Abl tyrosine kinase in the cellular response to oxidative stress

Protein kinase C (PKC) isoforms are phosphorylated on tyrosine in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with hydrogen peroxide (H(2)O(2)) induces binding of the PKCdelta isoform and the c-Abl protein-tyrosine kinase. The results show that c-Abl phosphorylates PKCdelta in the H(2)O(2) response. We also show that PKCdelta phosphorylates and activates c-Abl in vitro. In cells, induction of c-Abl activity by H(2)O(2) is attenuated by the PKCdelta inhibitor, rottlerin, and by overexpression of the regulatory domain of PKCdelta. These findings support a functional interaction between PKCdelta and c-Abl in the cellular response to oxidative stress.     

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.     

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.     

Physical and functional interaction between protein kinase C delta and Fyn tyrosine kinase in human platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.     

Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D

Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and G?6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with G?6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.     

HGF upregulates and modifies subcellular distribution of proteins in colon cancer cell enterocytic differentiation

Hepatocyte growth factor (HGF) and its receptor, c-Met, are involved in cell transformation. To study their role in intestinal cell differentiation, we used Caco-2 colon cancer cells, which differentiate spontaneously into enterocytes during culture. Cells grown continuously in the presence of HGF reached confluence more quickly than control cells. Markers of enterocytic differentiation, such as alkaline phosphatase and sucrase-isomaltase activities, adhesion molecules, and structural proteins such as E-cadherin, villin, and F-actin were upregulated by HGF throughout the 35 days of culture, and actin fibers were reorganized. HGF also stimulated expression and tyrosine phosphorylation of c-Met and Gab-1 as well as protein kinase C (PKC)-alpha expression. PKC-alpha has been shown to be involved in intestinal differentiation. We therefore investigated the possibility that increases in PKC-alpha protein levels were responsible for the HGF-promoted events. We did this by incubating cells with G?-6976, an inhibitor of PKC-alpha and -beta1, concomitantly with HGF. This inhibitor abolished the HGF-induced increase in villin levels before, but not after, confluence. Thus HGF accelerates Caco-2 cell differentiation and stimulates the metabolic and structural events accompanying this process. These HGF-promoted events may be mediated partly by Gab-1, and the effects of HGF on villin before confluence seem to involve PKC.     

A critical role of protein kinase C delta activation loop phosphorylation in formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of p47(phox) and rapid activation of nicotinamide adenine dinucleotide phosphate oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.     

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.