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.     

Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.     

Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.     

Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.     

Role of atypical protein kinase C in estradiol-triggered G1/S progression of MCF-7 cells

Expression of a dominant negative atypical protein kinase C (aPKC), PKCzeta, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERalpha), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCzeta by Thr410 phosphorylation. Serine phosphorylation of PKCzeta is simultaneously induced. PKCzeta activation leads to recruitment of Ras to a multimolecular complex that also includes hERalpha, Src, PI 3-kinase, and aPKC. We propose that PKCzeta pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Enzymatic properties of a novel phorbol ester receptor/protein kinase, nPKC

A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKC epsilon, clearly distinct from the four "conventional" PKCs [Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731-741]. We purified nPKC epsilon from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKC alpha. nPKC epsilon was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKC alpha), the only PKC expressed in COS cells. The protein kinase activity of nPKC epsilon is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificities were clearly different from PKC alpha. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10-20 mM). The activation of nPKC epsilon by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKC epsilon autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKC epsilon permit clear discrimination of nPKC epsilon from conventional PKCs.     

Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase

The activity of the serine/threonine kinase c-Raf (Raf) is inhibited by increased intracellular cAMP. This is believed to require phosphorylation with the cAMP-dependent protein kinase (PKA), although the mechanism by which PKA inhibits Raf is controversial. We investigated the requirement for PKA phosphorylation using Raf mutants expressed in HEK293 or NIH 3T3 cells. Phosphopeptide mapping of (32)P-labeled Raf (WT) or a mutant lacking a putative PKA phosphorylation site (serine to alanine, S43A) confirmed that serine 43 (Ser(43)) was the major cAMP (forskolin)-stimulated phosphorylation site in vivo. Interestingly, the EGF-stimulated Raf kinase activity of the S43A mutant was inhibited by forskolin equivalently to that of the WT Raf. Forskolin also inhibited the activation of an N-terminal deletion mutant Delta5-50 Raf completely lacking this phosphorylation site. Although WT Raf was phosphorylated by PKA, phosphorylation did not inhibit Raf catalytic activity in vitro, nor did forskolin treatment inhibit the activity of an N-terminally truncated Raf protein (Raf 22W) or a full-length Raf protein (Raf-CAAX) expressed in NIH 3T3 cells. In contrast, forskolin inhibited the EGF-dependent activation of a Raf isoform (B-Raf), lacking an analogous phosphorylation site to Ser(43). Thus, these results demonstrate that PKA exerts its inhibitory effects independently of direct Raf phosphorylation and suggests instead that PKA prevents an event required for the EGF-dependent activation of Raf.     

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.     

A dominant negative protein kinase C zeta subspecies blocks NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a number of genes. NF-kappa B is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein I kappa B. Following stimulation of cells, I kappa B dissociates from NF-kappa B, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappa B activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappa B. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zeta PKC, is a required step in the activation of NF-kappa B in response to ras p21. We demonstrate here that overexpression of zeta PKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappa B into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zeta PKC dramatically inhibits the kappa B-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zeta PKC plays a decisive role in NF-kappa B regulation in mammalian cells.     

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 Ras signaling specificity by protein kinase C

Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.     

Insulin-activated protein kinase Cbeta bypasses Ras and stimulates mitogen-activated protein kinase activity and cell proliferation in muscle cells

In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Calpha (PKCalpha) and beta activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCalpha and -beta activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCbeta with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCbeta block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCbeta. Finally, blocking PKCalpha expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCalpha and -beta. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCbeta plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.     

Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase

By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.     

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.     

Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex

We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A.     

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.