Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

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.     

Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.     

Phorbol ester downregulates PDGFbeta receptor via PKCbeta1 in vascular smooth muscle cells

The role of protein kinase C (PKC) and their isoforms in cell growth regulation remains elusive. Here we showed that in cultured human vascular smooth muscle cells (SMC), the PKC stimulator phorbol 12-myristate 13-acetate (PMA) inhibited [(3)H]thymidine incorporation in response to the growth factor PDGF associated with downregulation of PDGFbeta (but not alpha) receptors, which was recovered to normal level after PKC was depleted. The changes in PDGFbeta receptor were inversely correlated with PKCbeta1 protein levels regulated by PMA. The downregulation of PDGFbeta receptor by PMA was fully prevented by the PKCbeta inhibitor LY379196, however, without recovery of [(3)H]thymidine incorporation to PDGF. In contrast, [(3)H]thymidine incorporation was fully recovered after depletion of PKCs. These results indicate that in human SMC PKCbeta1 mediates PDGFbeta receptor downregulation. Other PKC isoforms activated by phorbol ester also contribute to the inhibitory effects on cell growth.     

Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor

Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.     

Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation.     

Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N-methyl-D-aspartate (NMDA) receptors leads to cell death in human embryonic kidney-293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA-mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA-mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down-regulation. The region of NR2A that provides the PKC-induced enhancement of cell death was localized to a discrete segment of the C-terminus. Use of isoform-specific PKC inhibitors showed that Ca(2+)- and lipid-dependent PKC isoforms (cPKCs), specifically PKCbeta1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A-transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C-terminus of NR2A to accentuate cell death in NR1/NR2A-transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.     

Amplitude control of protein kinase C by RINCK, a novel E3 ubiquitin ligase

Protein kinase C (PKC) isozymes play a central role in cellular signaling. Levels of PKC control the amplitude of agonist-induced signaling and alterations in these levels are associated with disease states, most notably cancer, yet mechanisms that control the turnover of the protein are poorly understood. Here we identify an E3 ligase that catalyzes the ubiquitin-mediated degradation of PKC. Specifically, we identified a RING finger domain-containing protein, RINCK (for RING-finger protein that interacts with C kinase) from a yeast two-hybrid screen using the amino terminus of PKCbeta as bait. RINCK encodes a protein of 581 amino acids that contains a RING finger domain, a B-box, and two coiled-coil regions, the three domains that form the signature motif of the large family of diverse TRIM (tripartite motif) proteins. Co-immunoprecipitation studies using tsA201 cells reveal that RINCK and PKC associate with each other in cells. Studies using fragments of PKCbeta reveal that this interaction is mediated by the C1A domain of PKC. RINCK induces the ubiquitination of PKC both in vitro and in cells. Overexpression of RINCK reduces the levels of PKC in cells, whereas genetic knockdown of endogenous RINCK increases the levels of PKC. This increase was observed for all PKC isozymes examined (including conventional, novel, and atypical). The RINCK-mediated degradation of PKC occurs independently of the classic phorbol ester-mediated down-regulation: genetic depletion of RINCK had no effect on the phorbol ester-mediated down-regulation and, additionally, up-regulated the levels of isozymes that cannot bind phorbol esters. Our data reveal a novel mechanism that provides amplitude control in PKC signaling through ubiquitination catalyzed by RINCK, an E3 ligase that specifically recognizes the C1 domain of PKC isoforms.     

Lysophosphatidylcholine stimulates EGF receptor activation and mesangial cell proliferation: regulatory role of Src and PKC

Lysophosphatidylcholine (LPC), a major component of oxidized-low density lipoproteins (ox-LDL), modulates various pathobiological processes involved in vascular and glomerular diseases. Although several studies have shown increased plasma concentrations of ox-LDL as well as LPC in patients with renal disease, the role of LPC in mesangial cell proliferation and associated signaling mechanisms are not clearly understood. In this study, we have shown that LPC induced the phosphorylation of epidermal growth factor receptor (EGFR), as well as the p42/44 MAP kinases. LPC activated Src-kinase and protein kinase C (PKC), and both Src kinase inhibitor PP-2 and PKC inhibitor inhibited the activation of EGFR by LPC. LPC (5-25 microM) stimulated human mesangial cell proliferation by 4-5 fold. Preincubation of mesangial cells with the Src inhibitor (PP-2), or PKC inhibitor (bisindolylmaleimide GF109203-X), or EGF receptor kinase inhibitor (AG1478), or MEK inhibitor (PD98059) significantly inhibited LPC-mediated mesangial cell proliferation. The data suggest that LPC, by activating Src and PKC signaling pathways, stimulates EGF receptor transactivation and down-stream MAP kinase signaling resulting in mesangial hypercellularity, which is a characteristic feature of diverse renal diseases.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Interleukin 2 and a lactogen regulate proliferation and protein phosphorylation in Nb2 cells.

Cell proliferation and protein phosphorylation in response to activation of lactogenic and interleukin 2 (IL-2) receptors were studied in Nb2 cells, a rat T-lymphocyte cell line. Human growth hormone (hGH) and rat IL-2 stimulated Nb2-cell proliferation to approximately the same degree, and the actions of both mitogens were potentiated by phorbol 12-myristate 13-acetate (PMA). A monoclonal antibody specific for the rat IL-2 receptor inhibited the mitogenic actions of rat IL-2, but not those of hGH. Exposure of Nb2 cells to either mitogen for 2-3 h increased phosphorylation of an 18,600-Da protein and decreased phosphorylation of a 15,600-Da protein. PMA also inhibited phosphorylation of the latter protein, but, by itself, PMA did not stimulate phosphorylation of the 18,600-Da protein. Overall, the results suggest that hGH and IL-2 act through separate receptors to stimulate proliferation of Nb2 cells, and that some of the actions of both mitogens may be mediated, in part, through regulation of protein phosphorylation.     

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells.

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.     

Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C

Identification of selective anchoring proteins responsible for specialized localization of specific signaling proteins has led to the identification of new inhibitors of signal transduction, inhibitors of anchoring protein-ligand interactions. RACK1, the first receptor for activated C kinase identified in our lab, is a selective anchoring protein for betaII protein kinase C (betaIIPKC). We previously found that at least part of the RACK1-binding site resides in the C2 domain of betaIIPKC (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). Here we show that the V5 domain also contains part of the RACK1-binding site in betaIIPKC. In neonatal rat cardiac myocytes, the betaIIV5-3 peptide (amino acids 645-650 in betaIIPKC) selectively inhibited phorbol 12-myristate 13-acetate (PMA)-induced translocation of betaIIPKC and not betaIPKC. In addition, the betaIIV5-3 peptide inhibited cardiac myocyte hypertrophy in PMA-treated cells. Interestingly, betaIV5-3 (646-651 in betaIPKC), a selective translocation inhibitor of betaIPKC, also inhibited PMA-induced cardiac myocyte hypertrophy, demonstrating that both betaI- and betaIIPKC are essential for this cardiac function. Therefore, the betaIIV5 domain contains part of the RACK1-binding site in betaIIPKC; a peptide corresponding to this site is a selective inhibitor of betaIIPKC and, hence, enables the identification of betaIIPKC-selective functions.     

Activation of S6 kinase by repeated cycles of stretching and relaxation in rat glomerular mesangial cells. Evidence for involvement of protein kinase C.

Quiescent rat glomerular mesangial cells were exposed to repeated cycles of stretching and relaxation, and the effects on the rate of collagen production, proliferation, and S6 kinase activity were investigated. Stretch/relaxation induced increases in production of both collagen and non-collagenous proteins. Proliferation of mesangial cells was stimulated by stretch/relaxation and epidermal growth factor, but not by angiotensin II; however, administration of angiotensin II augmented stretch/relaxation-induced cell proliferation. Cytosolic S6 kinase activity was stimulated by stretch/relaxation, angiotensin II, epidermal growth factor, or phorbol 12-myristate 13-acetate. The increased S6 kinase activity was detectable within 30 min after initiation of stretch/relaxation and was blocked by either inhibitors of protein kinase C or prior down-regulation of protein kinase C following prolonged incubation with phorbol 12-myristate 13-acetate. Both translocation of protein kinase C from the cytosolic to the membrane fraction and phosphorylation of an endogenous 80-kDa protein were observed within 5 min of initiation of stretch/relaxation. These results demonstrate that in mesangial cells, mechanical factors alone can induce increases in production of collagen and non-collagenous proteins and in cell proliferation. The observation that stretch/relaxation induced stimulation of S6 kinase activity through protein kinase C-dependent mechanisms suggests that activation of protein kinase C may be a key event in initiating adaptive responses of mesangial cells to increased workload.     

Coordinated movement of RACK1 with activated betaIIPKC.

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated betaIIPKC, determines the localization and functional activity of betaIIPKC. However, the mechanism by which RACK1 localizes activated betaIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and betaIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and betaIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves betaIIPKC from one intracellular site to another.     

Protein kinase C beta1 is implicated in the regulation of neuroblastoma cell growth and proliferation.

To investigate a putative involvement of protein kinase C (PKC) isoforms in supporting neuroblastoma cell proliferation, SK-N-BE(2) neuroblastoma cells were transfected with expression vectors coding for the C2 and V5 regions from different PKC isoforms. These structures have been suggested to inhibit the activity of their corresponding PKC isoform. The PKC fragments were fused to enhanced green fluorescent protein to facilitate the detection of transfected cells. Expression of the C2 domain from a classical PKC isoform (PKCalpha), but not of C2 domains from novel PKCdelta or PKCepsilon, suppressed the number of neuroblastoma cells positive for cyclin A and bromodeoxyuridine incorporation. This indicates a role for a classical isoform in regulating proliferation of these cells. Among the V5 fragments from PKCalpha, PKCbetaI, and PKCbetaII, the PKCbetaI V5 had the most suppressive effect on proliferation markers, and this fragment also displaced PKCbetaI from the nucleus. Furthermore, a PKCbeta-specific inhibitor, LY379196, suppressed the phorbol ester- and serum-supported growth of neuroblastoma cells. There was a marked enhancement by LY379196 of the growth-suppressive and/or cytotoxic effects of paclitaxel and vincristine. These results indicate that PKCbetaI has a positive effect on the growth and proliferation of neuroblastoma cells and demonstrate that inhibition of PKCbeta may be used to enhance the effect of microtubule-interacting anticancer agents on neuroblastoma cells.     

RACK1 mediates activation of JNK by protein kinase C [corrected

Activation of the Jun-N-terminal kinase (JNK) signaling cascade by phorbol esters (TPA) or protein kinase C (PKC) is well documented, although the underlying mechanism is not known. Here, we demonstrate that the receptor for activated C kinase 1 (RACK1) serves as an adaptor for PKC-mediated JNK activation. Phosphorylation of JNK by PKC occurs on Ser129 and requires the presence of RACK1. Ser129 phosphorylation augments JNK phosphorylation by MKK4 and/or MKK7 and is required for JNK activation by TPA, TNFalpha, UV irradiation, and PKC, but not by anisomycin or MEKK1. Inhibition of RACK1 expression by siRNA attenuates JNK activation, sensitizes melanoma cells to UV-induced apoptosis, and reduces their tumorigenicity in nude mice. In finding the role of RACK1 in activation of JNK by PKC, our study also highlights the nature of crosstalk between these two signal-transduction pathways.     

Participation of phospholipase D and alpha/beta-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.     

Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein-protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best-characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle-aged animals. The levels of calcium-independent PKCδ and ε, interacting with RACK1, and related calcium-independent PKC activity were not modified by the aging process. By comparison, phorbol ester-stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium-dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.