Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau

The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.     

Stimulus-specific differences in protein kinase C delta localization and activation mechanisms in cardiomyocytes

Protein kinase C (PKC) isoforms play key roles in the regulation of cardiac contraction, ischemic preconditioning, and hypertrophy/failure. Models of PKC activation generally focus on lipid cofactor-induced PKC translocation to membranes. This study identifies tyrosine phosphorylation as an additional mechanism that regulates PKC delta actions in cardiomyocytes. Using immunoblot analysis with antibodies to total PKC delta and PKC delta-pY(311), we demonstrate that PKC delta partitions between soluble and particulate fractions (with little Tyr(311) phosphorylation) in resting cardiomyocytes. Phorbol 12-myristate 13-acetate (PMA) promotes PKC delta translocation to membranes and phosphorylation at Tyr(311). H(2)O(2) also increases PKC delta-pY(311) in association with its release from membranes. Both PMA- and H(2)O(2)-dependent increases in PKC delta-pY(311) are mediated by Src family kinases, but they occur via different mechanisms. The H(2)O(2)-dependent increase in PKC delta-pY(311) results from Src activation and increased Src-PKC delta complex formation. The PMA-dependent increase in PKC delta-pY(311) results from a lipid cofactor-induced conformational change that renders PKC delta a better substrate for phosphorylation by precomplexed Src kinases (without Src activation). PKC delta-Y(311) phosphorylation does not grossly alter the kinetics of PMA-dependent PKC delta down-regulation. Rather, tyrosine phosphorylation regulates PKC delta kinase activity. PKC delta is recovered from the soluble fraction of H(2)O(2)-treated cardiomyocytes as a tyrosine-phosphorylated, lipid-independent enzyme with altered substrate specificity. In vitro PKC delta phosphorylation by Src also increases lipid-independent kinase activity. The magnitude of this effect varies, depending upon the substrate, suggesting that tyrosine phosphorylation fine-tunes PKC delta substrate specificity. The stimulus-specific modes for PKC delta signaling identified in this study allow for distinct PKC delta-mediated phosphorylation events and responses during growth factor stimulation and oxidant stress in cardiomyocytes.     

Regulation of hematopoietic-specific G-protein Galpha15 and Galpha16 by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein alpha subunit, Galpha(16), and its murine homologue Galpha(15), are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Galpha(15)/Galpha(16) and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Galpha(15) in COS7 cells transfected with Galpha(15) and protein kinase Calpha (PKCalpha), and phosphorylation of endogenous Galpha(16) in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Galpha(15/16) is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-beta.     

Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.     

Galpha12 and Galpha13 as key regulatory mediator in signal transduction

It is generally thought that Galpha(12) and Galpha(13)-induced responses are exclusively mediated by small G protein Rho. However, Galpha(12) and Galpha(13) elicit divergent cellular responses: phospholipase C-epsilon activation, phospholipase D activation, cytoskeletal change, oncogenic response, apoptosis, MAP kinase activation and Na/H-exchange activation. In addition to Rho activation through RhoGEF, it has been recently demonstrated that Galpha(12) and Galpha(13) interact with several proteins and regulate their activities. However, physiological importance of the interaction of Galpha(12) and Galpha(13) with these proteins has not fully established. I summarize the recent progress of Galpha(12) and Galpha(13)-mediated signaling cascade.     

Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated Galpha(13).

Although it is well established that cAMP inhibits platelet activation induced by all agonists, the thromboxane A(2) signal transduction pathway was found to be particularly sensitive to such inhibition. Therefore, we examined whether cAMP-dependent kinase mediates phosphorylation of the thromboxane A(2) receptor-G-protein complex. It was found that cAMP induces protein kinase A-dependent [gamma-(32)P]ATP labeling of solubilized membrane proteins in the region of Galpha subunits, i.e. 38-45 kDa. Moreover, ligand affinity chromatography purification of thromboxane A(2) receptor-G-protein complexes from these membranes revealed that 38-45-kDa phosphoproteins co-purify with thromboxane A(2) receptors. Immunoprecipitation of the affinity column eluate with a Galpha(13) antibody demonstrated that 8-Br-cAMP increased phosphorylation of thromboxane A(2) receptor-associated Galpha(13) by 87 +/- 27%. In separate experiments, immunopurification of Galpha(13) on microtiter wells coated with a different Galpha(13) antibody revealed that 8-Br-cAMP increased Galpha(13) phosphorylation by 53 +/- 19%. Finally, treatment of (32)P-labeled whole platelets with prostacyclin resulted in a 90 +/- 14% increase in phosphorylated Galpha(13) that was abolished by pretreatment with the adenylate cyclase inhibitor MDL-12. These results provide the first evidence that protein kinase A mediates phosphorylation of Galpha(13) both in vitro and in vivo and provides a basis for the preferential inhibition of thromboxane A(2)-mediated signaling in platelets by cAMP.     

Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.     

Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes

The Nrf2/antioxidant response element (ARE) signaling pathway plays a key role in activating cellular antioxidants, including heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO1), and glutathione. Protein kinase C (PKC) may also regulate these antioxidants, as PKC phosphorylates Nrf2 in vitro. This study examined the role of PKC in ARE-mediated gene regulation in human monocytes by curcumin, a potent inducer of the Nrf2/ARE pathway. Curcumin increased HO-1 and glutamyl cysteine ligase modulator (GCLM) expression and stimulated Nrf2 binding to the ARE. Curcumin also rapidly stimulated PKC phosphorylation and Ro-31-8220, a pan-PKC inhibitor, decreased curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Rottlerin (a PKC delta inhibitor) and PKC delta antisense oligonucleotides significantly inhibited curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Furthermore, a p38 MAP kinase inhibitor reduced GCLM and HO-1 expression and rottlerin inhibited curcumin-induced p38 phosphorylation. In summary, curcumin activates ARE-mediated gene expression in human monocytes via PKC delta, upstream of p38 and Nrf2.     

Thrombin induces nitric-oxide synthase via Galpha12/13-coupled protein kinase C-dependent I-kappaBalpha phosphorylation and JNK-mediated I-kappaBalpha degradation

An imbalance between thrombin and antithrombin III contributed to vascular hyporeactivity in sepsis, which can be attributed to excess NO production by inducible nitric-oxide synthase (iNOS). In view of the importance of the thrombin-activated coagulation pathway and excess NO as the culminating factors in vascular hyporeactivity, this study investigated the effects of thrombin on the induction of iNOS and NO production in macrophages. Thrombin induced iNOS protein in the Raw264.7 cells, which was inhibited by a thrombin inhibitor, LB30057. Thrombin increased NF-kappaB DNA binding, whose band was supershifted with anti-p65 and anti-p50 antibodies. Thrombin elicited the phosphorylation and degradation of I-kappaBalpha prior to the nuclear translocation of p65. The NF-kappaB-mediated iNOS induction was stimulated by the overexpression of activated mutants of Galpha(12/13) (Galpha(12/13)QL). Protein kinase C depletion inhibited I-kappaBalpha degradation, NF-kappaB activation, and iNOS induction by thrombin or the iNOS induction by Galpha(12/13)QL. JNK, p38 kinase, and ERK were all activated by thrombin. JNK inhibition by the stable transfection with a dominant negative mutant of JNK1 (JNK1(-)) completely suppressed the NF-kappaB-mediated iNOS induction by thrombin. Conversely, the inhibition of p38 kinase enhanced the expression of iNOS. In addition, JNK and p38 kinase oppositely controlled the NF-kappaB-mediated iNOS induction by Galpha(12/13)QL. Hence, iNOS induction by thrombin was regulated by the opposed functions of JNK and p38 kinase downstream of Galpha(12/13). In the JNK1(-) cells, thrombin did not increase either the NF-kappaB binding activity or I-kappaBalpha degradation despite I-kappaBalpha phosphorylation. These results demonstrated that thrombin induces iNOS in macrophages via Galpha(12) and Galpha(13), which leads to NF-kappaB activation involving the protein kinase C-dependent phosphorylation of I-kappaBalpha and the JNK-dependent degradation of phosphorylated I-kappaBalpha.     

Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Galpha(s) and Galpha(16) hybrid signaling

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G(s) and G(q) proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr(705) and Ser(727) phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cbeta, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Galpha(16)-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr(705) phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser(727) phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G(16)-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G(s) pathway. This postulation was confirmed by expressing constitutively active Galpha(16)QL and Galpha(s)QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Galpha(16)QL-induced STAT3 phosphorylations could be converted by the mere presence of Galpha(s)QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Galpha(16)-mediated inhibitor-sensitivity upon cicaprost induction in Galpha(s)-knocked down HEL cells again verified the pivotal role of G(s) signal. Taken together, our observations illustrate that co-stimulation of G(s) and G(q) can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.     

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).     

Cross-regulation of novel protein kinase C (PKC) isoform function in cardiomyocytes. Role of PKC epsilon in activation loop phosphorylations and PKC delta in hydrophobic motif phosphorylations

Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.     

Activation of protein kinase D3 by signaling through Rac and the alpha subunits of the heterotrimeric G proteins G12 and G13

PKD is the founding member of a novel protein kinase family that also includes PKD2 and PKD3. PKD has been the focus of most studies up to date, but little is known about the mechanisms that mediate PKD3 activation. Here, we show that addition of aluminum fluoride to COS-7 cells cotransfected with PKD3 and Galpha13 or Galpha12 induced PKD3 activation, which was associated with a transient plasma membrane translocation of cytosolic PKD3. Treatment with Clostridium difficile toxin B blocked PKD3 activation induced by either bombesin or by aluminum fluoride-stimulated Galpha12/13 but did not affect Galphaq-induced PKD3 activation. Furthermore, PKD3 immunoprecipitated from cells cotransfected with a constitutively active Rac (RacV12) exhibited a marked increase in PKD3 basal catalytic activity. In contrast, cotransfection with active Rho (RhoQ63L), Cdc42 (Cdc42Q61L), or Ras (RasV12) did not promote PKD3 activation. Expression of either COOH-terminal dominant-negative fragment of Galpha13 or dominant negative Rac (Rac N17) attenuated bombesin-induced PKD3 activation. Treatment with protein kinase C (PKC) inhibitors prevented the increase in PKD3 activity induced by RacV12 and aluminum fluoride-stimulated Galpha12/13. The catalytic activation of PKD3 in response to RacV12, alpha12/13 signaling or bombesin correlated with Ser-731/Ser-735 phosphorylation in the activation loop of this enzyme. Our results indicate that Galpha12/13 and Rac are important components in the signal transduction pathways that mediate bombesin receptor-induced PKD3 activation.     

Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation

The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha(13) and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha(13) and the PKA catalytic subunit established that PKA directly phosphorylates Galpha(13). The location of this phosphorylation site was next investigated with a new synthetic peptide (G(13)SRI(pep)) containing the PKA consensus sequence (Arg-Arg-Pro-Thr(203)) within the switch I region of Galpha(13). G(13)SRI(pep) produced a dose-dependent inhibition of PKA-mediated Galpha(13) phosphorylation. On the other hand, the Thr-phosphorylated derivative of G(13)SRI(pep) possessed no inhibitory activity, suggesting that Galpha(13) Thr(203) may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha(13)-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Galpha(13) phosphorylation stabilized coupling of Galpha(13) with platelet thromboxane A(2) receptors but destabilized coupling of Galpha(13) to its betagamma subunits. In order to determine the functional consequences of this phosphorylation on Galpha(13) signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Galpha(13) wild type (Galpha(13)-WT) or Galpha(13)-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Galpha(13) wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Galpha(13) Thr(203).     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.