PKC-delta-dependent pathways contribute to PDGF-stimulated ERK1/2 activation in vascular smooth muscle

Platelet-derived growth factor (PDGF) is an important regulator of vascular smooth muscle (VSM) cell growth and migration and has been identified as a key mediator of neointima formation resulting from vascular injury. PDGF exerts its effects, in part, through activation of ERK1/2. Previously, we reported that PKC-delta, specifically compared with PKC-alpha, mediated phorbol ester- and ATP-dependent activation of ERK1/2 in VSM cells. The purpose of this study was to determine whether PKC-delta was involved in PDGF-dependent activation of ERK1/2 in VSM cells. The addition of PDGF resulted in the activation, and Src family kinase-dependent tyrosine phosphorylation, of PKC-delta. Treatment with rottlerin (0.1-10 microM), a selective PKC-delta inhibitor, or adenoviral overexpression of kinase-negative PKC-delta significantly attenuated PDGF-induced activation of ERK1/2. The effects of the PKC-delta inhibitors decreased with increasing concentrations of activator PDGF. Interestingly, treatment with Go6976 (0.1-3 microM), a selective inhibitor of cPKCs, or adenoviral overexpression of kinase-negative PKC-alpha also inhibited PDGF-stimulated ERK1/2. Furthermore, inhibition of cPKC activity with Go6976 or overexpression of kinase-negative PKC-alpha attenuated PKC-delta activation and tyrosine phosphorylation in response to PDGF. These studies indicate involvement of both PKC-delta and PKC-alpha isozymes in PDGF-stimulated signaling in VSM and suggest an unexpected role for PKC-alpha in the regulation of PKC-delta activity.     

Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using G?6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.     

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.     

Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras,protein kinase C,and protein kinase A in neuronal cells

The small GTPases Ras or Rap1 were suggested to mediate the stimulatory effect of some G protein-coupled receptors on ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G protein-coupled receptor triggers neuronal differentiation of the PC12 cell line via ERK1/2 activation, transiently activated Ras and induced the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK by PACAP, whereas Rap1 was necessary for the sustained activation phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to account for ERK activation by PACAP because 1) PACAP-elicited Rap1 GTP-loading depended only on phospholipase C, whereas maximal stimulation of ERK by PACAP also required the activity of protein kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.     

Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (Delta NPS and CAT), and dominant negative mutants of PKCs alpha, beta 1, beta 2, gamma, delta, epsilon, eta, zeta, and iota. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-alpha and PKC-epsilon were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-delta inhibited its activity. Transient transfection studies with a series of 5'-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-alpha. Deletion of the AP-1 enhancer element located at position -954 upstream from the initiation site abolished PKC-alpha-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-alpha and PKC-epsilon. The constitutively active mutants of PKC-alpha and PKC-epsilon also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-alpha or PKC-epsilon displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-alpha or PKC-epsilon in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.     

Regulation of CCK-induced amylase release by PKC-delta in rat pancreatic acinar cells

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, alpha, delta, epsilon, and zeta, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-alpha, -delta, and -epsilon, but not -zeta from soluble to membrane fraction. CCK-induced amylase release was inhibited approximately 30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-delta inhibitor, but not by G?6976, a PKC-alpha inhibitor, at concentrations from 1 to 5 microM. Neither overexpression of wild-type or dominant-negative PKC-alpha affected CCK-induced amylase release. Overexpression of PKC-delta and -epsilon enhanced amylase release, whereas only dominant-negative PKC-delta inhibited amylase release by 25%. PKC-delta overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-delta only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-delta and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-delta but had no further effect in the presence of dominant-negative PKC-delta. These results indicate that PKC-delta is the PKC isoform involved with amylase secretion.     

Cholecystokinin stimulates extracellular signal-regulated kinase through activation of the epidermal growth factor receptor,Yes, and protein kinase C. Signal amplification at the level of Raf by activation of protein kinase Cepsilon

Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.     

Opposing roles of Ras/Raf oncogenes and the MEK1/ERK signaling module in regulation of expression and adhesive function of surface transglutaminase

Tissue transglutaminase (tTG) serves as a potent and ubiquitous integrin-associated adhesion co-receptor for fibronectin on the cell surface and affects several key integrin functions. Here we report that in fibroblasts, activated H-Ras and Raf-1 oncogenes decrease biosynthesis, association with beta1 integrins, and surface expression of tTG because of down-regulation of tTG mRNA. In turn, the reduction of surface tTG inhibits adhesion of H-Ras- and Raf-1-transformed cells on fibronectin and, in particular, on its tTG-binding fragment I(6)II(1,2)I(7-9), which does not interact directly with integrins. Analysis of Ras/Raf downstream signaling with specific pharmacological inhibitors reveals that the decrease in tTG expression is mediated by the p38 MAPK, c-Jun NH2-terminal kinase, and phosphatidylinositol 3-kinase pathways. In contrast, increased activation of the ERK pathway by constitutively active MEK1 stimulates tTG mRNA expression, biosynthesis, and surface expression of tTG, whereas MEK inhibitors or dominant negative MEK1 exert an opposite effect. This modulation of surface tTG by ERK signaling alters adhesion of cells on fibronectin and its fragment that binds tTG. Furthermore, transient stimulation of ERK signaling in untransformed fibroblasts by adhesion on fibronectin or growth factors elevates tTG biosynthesis, increases complex formation with beta1 integrins, and raises surface expression of tTG. Finally, ERK activation is required for growth factor-induced redistribution of tTG on the surface of adherent fibroblasts and co-clustering of beta1 integrins and tTG at cell-matrix adhesion contacts. Together, our data indicate that down-regulation of surface tTG by Ras and Raf oncogenes contributes to adhesive deficiency of transformed fibroblasts, whereas stimulation of biosynthesis and surface expression of tTG by the MEK1/ERK module promotes and sustains cell-matrix adhesion of untransformed cells. Contrasting effects of Ras/Raf oncogenes and their immediate downstream signaling module, MEK1/ERK, on tTG expression are consistent with adhesive function of surface tTG.     

PAK1 is a novel MEK-independent raf target controlling expression of the IAP survivin in M-CSF-mediated osteoclast survival

As activation of the Ras/Raf/MEK/ERK pathway is a critical component of M-CSF-promoted osteoclast survival, determining specific mechanism by which M-CSF activates this signal transduction pathway is paramount towards advancing treatment of pathological conditions resulting in increased bone turnover. The p21 activated kinase PAK1 modulates activation of the Raf/MEK/ERK pathway by either directly activating Raf or priming MEK for activation by Raf. Therefore a role for PAK1 in M-CSF-mediated activation of the MEK/ERK pathway controlling osteoclast survival was assessed. Here we show that PAK1 is activated by M-CSF in a Ras-dependent mechanism that promotes osteoclast survival. Surprisingly, PAK1 did not modulate Raf activation or Raf-mediated MEK activation. M-CSF mediated activation of Raf was required for PAK1 activation and osteoclast survival promoted by PAK1. This survival response was MEK-independent as expression of constitutively active MEK did not rescue osteoclasts from apoptosis induced by blocking PAK1 function. Functionally, PAK1 promoted osteoclast survival by modulating expression of the IAP family member Survivin. M-CSF therefore functions to promote PAK1 activation as a novel MEK-independent Raf target to control Survivin-mediated osteoclast survival.     

PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells

Protein kinase C (PKC) isoforms play distinct roles in cellular functions. We have previously shown that ionizing radiation activates PKC isoforms (alpha, delta, epsilon, and zeta), however, isoform-specific sensitivities to radiation and its exact mechanisms in radiation mediated signal transduction are not fully understood. In this study, we showed that overexpression of PKC isoforms (alpha, delta, epsilon, and zeta) increased radiation-induced cell death in NIH3T3 cells and PKC epsilon overexpression was predominantly responsible. In addition, PKC epsilon overexpression increased ERK1/2 activation without altering other MAP-kinases such as p38 MAPK or JNK. Co-transfection of dominant negative PKC epsilon (PKC epsilon -KR) blocked both PKC epsilon -mediated ERK1/2 activation and radiation-induced cell death, while catalytically active PKC epsilon construction augmented these phenomena. When the PKC epsilon overexpressed cells were pretreated with PD98059, MEK inhibitor, radiation-induced cell death was inhibited. Co-transfection of the cells with a mutant of ERK1 or -2 (ERK1-KR or ERK2-KR) also blocked these phenomena, and co-transfection with dominant negative Ras or Raf cDNA revealed that PKC epsilon -mediated ERK1/2 activation was Ras-Raf-dependent. In conclusion, PKC epsilon -mediated ERK1/2 activation was responsible for the radiation-induced cell death.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.

The responses of human neutrophils (PMN) involve reorganization and phosphorylation of cytoskeletal components. We investigated the translocation of protein kinase C (PKC) isoforms to PMN cytoskeletal (Triton-insoluble) fractions, in conjunction with activation of the respiratory burst enzyme NADPH oxidase. In resting PMN, PKC-delta (29%) and small amounts of PKC-alpha (0.6%), but not PKC-betaII, were present in cytoskeletal fractions. Upon stimulation with the PKC agonist PMA, the levels of PKC-alpha, PKC-betaII, and PKC-delta increased in the cytoskeletal fraction, concomitant with a decrease in the noncytoskeletal (Triton-soluble) fractions. PKC-delta maximally associated with cytoskeletal fractions at 160 nM PMA and then declined, while PKC-alpha and PKC-betaII plateaued at 300 nM PMA. Translocation of PKC-delta was maximal by 2 min and sustained for at least 10 min. Translocation of PKC-alpha and PKC-betaII was biphasic, plateauing at 2-3 min and then increasing up to 10 min. Under maximal stimulation conditions, PKC isoforms were entirely cytoskeletal associated. Translocation of the NADPH oxidase component p47phox to the cytoskeletal fraction correlated with translocation of PKC-alpha and PKC-betaII, but not with translocation of PKC-delta. Oxidase activity in cytoskeletal fractions paralleled translocation of PKC-alpha, PKC-betaII, and p47phox. Stimulation with 1,2-dioctanoylglycerol resulted in little translocation of PKC isoforms or p47phox, and in minimal oxidase activity. We conclude that conventional PKC isoforms (PKC-alpha and/or PKC-betaII) may regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase. PKC-delta may modulate other PMN responses that involve cytoskeletal components.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.     

Effect of protein kinase C inhibitors on porcine oocyte activation

The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.