Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Induction of phosphorylation of human immunodeficiency virus type 1 Nef and enhancement of CD4 downregulation by phorbol myristate acetate.

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) encodes a 27 to 34 kDa myristoylated protein that induces downregulation of CD4 from the cell surface and enhances virus infectivity. As shown by experiments on SIV-infected adult macaques, Nef is important in pathogenesis and disease progression. In vitro, protein kinase C (PKC) phosphorylates Nef, but the role of phosphorylation in the function and expression of this protein has not yet been determined. Here we show that in HIV type 1-infected cells, phosphorylation of Nef increased 8- to 12-fold after treatment with phorbol myristate acetate and phytohemagglutinin (PMA/PHA). Basal and PMA/PHA-induced phosphorylation occurred on serine residues of Nef and was independent of other HIV proteins. The PMA/PHA-induced phosphorylation of Nef was inhibited by bisindolylmaleimide I, a potent and specific inhibitor of PKC, but was unaffected by H89, an inhibitor of protein kinase A. In contrast, treatment with bisindolylmaleimide I did not affect the basal level of Nef phosphorylation, suggesting two different phosphorylation pathways. A PMA-insensitive CD4 mutant in which three serine residues in the cytoplasmic domain have been replaced by alanines was used to determine whether PMA-induced phosphorylation affects Nef-induced CD4 downregulation. In Nef-expressing cells, treatment with PMA enhanced downregulation of the CD4 serine triple mutant from the cell surface, suggesting that phosphorylation is important for Nef function.     

Regulation of phospholipase D2 activity by protein kinase C alpha

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, G? 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.     

Functional role of extracellular signal-regulated kinase activation and c-Jun induction in phorbol ester-induced promoter activation of human 12(S)-lipoxygenase gene

The functional role of mitogen-activated protein kinase (MAPK) signaling and c-Jun induction in phorbol 12-myristate 13-acetate (PMA)-induced human 12(S)-lipoxygenase gene expression was studied in human epidermoid carcinoma A431 cells. Among the family of MAPK, PMA only increased the activity of extracellular signal-regulated kinase (ERK). Treatment of cells with PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MEK), decreased the PMA-induced expression of 12(S)-lipoxygenase. Transfection of cells with Ras, Raf and ERK2 dominant negative mutants inhibited the PMA-induced promoter activation of the 12(S)-lipoxygenase gene in all cases. PMA-induced expression of c-Jun was inhibited by pretreatment with PD98059. Following treatment with PMA, the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in cells increased with time. Enhancement of binding between the c-Jun-Sp1 complex and the Sp1 oligonucleotide was observed in cells treated with PMA, suggesting the possible interaction of c-Jun-Sp1 with GC-rich binding sites in the gene promoter. These results indicate that PMA treatment induced ERK activation mainly through the Raf-MEK-ERK signaling pathway following induction of c-Jun expression, and the formation of the c-Jun-Sp1 complex. Finally, PMA activated the promoter activity of the 12(S)-lipoxygenase gene in cells overexpressing protein kinase C (PKC)delta but not PKCalpha, indicating that PKCdelta played the functional role in mediating the gene activation of 12(S)-lipoxygenase induced by PMA.     

Activation of acid sphingomyelinase by protein kinase Cdelta-mediated phosphorylation

Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.     

Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells

Both the epidermal growth factor receptor (EGFR) and protein kinase C (PKC) play important roles in glioblastoma invasive growth; however, the interaction between the EGFR and PKC is not well characterized in glioblastomas. Treatment with EGF stimulated global phosphorylation of the EGFR at Tyr(845), Tyr(992), Tyr(1068), and Tyr(1045) in glioblastoma cell lines (U-1242 MG and U-87 MG). Interestingly, phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of the EGFR only at Tyr(1068) in the two glioblastoma cell lines. Phosphorylation of the EGFR at Tyr(1068) was not detected in normal human astrocytes treated with the phorbol ester. PMA-induced phosphorylation of the EGFR at Tyr(1068) was blocked by bisindolylmaleimide (BIM), a PKC inhibitor, and rottlerin, a PKCdelta-specific inhibitor. In contrast, Go 6976, an inhibitor of classical PKC isozymes, had no effect on PMA-induced EGFR phosphorylation. Furthermore, gene silencing with PKCdelta small interfering RNA (siRNA), siRNA against c-Src, and mutant c-Src(S12C/S48A) and treatment with a c-Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) abrogated PMA-induced EGFR phosphorylation at Tyr(1068). PMA induced serine/threonine phosphorylation of Src, which was blocked by both BIM and rottlerin. Inhibition of the EGFR with AG 1478 did not significantly alter PMA-induced EGFR Tyr(1068) phosphorylation, but completely blocked EGF-induced phosphorylation of the EGFR. The effects of PMA on MAPK phosphorylation and glioblastoma cell proliferation were reduced by BIM, rottlerin, the MEK inhibitor U0126, and PKCdelta and c-Src siRNAs. Taken together, our data demonstrate that PMA transactivates the EGFR and increases cell proliferation by activating the PKCdelta/c-Src pathway in glioblastomas.     

PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype

The signaling mechanisms leading to phorbol ester myristate (PMA)-induced differentiation of HL-60 cells to the macrophagelike phenotype were investigated by using different protein kinase inhibitors. The protein kinase C inhibitor Ro 31-8220 specifically blocks PMA-induced differentiation, activation of the p42/44^ERK- and p38^RK-MAP kinase cascades and Hsp27-phosphorylation in HL-60 cells. Because Ro 31-8220 does not inhibit activation of the MAP kinase cascades by protein kinase C (PKC)-independent signals such as epidermal growth factor (EGF), heat shock, or anisomycin in these cells, only PMA-induced activation of the MAP kinases can be downstream of PKC. The MEK1 inhibitor PD 098059 and the p38^RK inhibitor SB 203580 also were used to analyze whether the PMA-induced PKC-dependent activation of MAP kinases is involved in the differentiation process. Under certain conditions, PD 098059 can completely block the PMA-induced activation of the p42^ERK as monitored by imunoprecipitation kinase assay by using the substrate myelin basic protein. SB 203580 specifically inhibits activation of p38^RK as judged by MAPKAP kinase 2 activity against the substrate Hsp27 and also blocks Hsp27 phosphorylation in the cells. In contrast, neither PD 098059 nor SB 203580 nor both inhibitors together prevent PMA-induced differentiation of the HL-60 cells to the macrophagelike phenotype. The results suggest the existence of a diversification of PMA-induced signaling in HL-60 cells downstream of PKC, leading to activation of MAP kinases that are not essential for differentiation and to phosphorylation of other, so far unidentified, targets responsible for differentiation. J. Cell. Physiol. 173:310–318, 1997. © 1997 Wiley-Liss, Inc.     

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

PKCdelta-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate.

Annexin A1 (ANX-1), a calcium-dependent, phospholipid binding protein, is known to be involved in diverse cellular processes, including regulation of cell growth and differentiation, apoptosis, and inflammation. The mitogen phorbol 12-myristate 13-acetate (PMA) induces expression and phosphorylation of ANX-1. However, the roles of ANX-1 in PMA-induced signal transduction is unknown. Here, we study the cellular localization of ANX-1 in the PMA-induced signal transduction process. We have found that PMA induces the cleavage of ANX-1 in human embryonic kidney (HEK) 293 cells, and that the cleaved form of ANX-1 translocates to the nucleus. The PMA-induced nuclear translocation of ANX-1 was inhibited by the protein kinase C (PKC)delta-specific inhibitor rottlerin, indicating that PKCdelta plays a role in nuclear translocation of the cleaved ANX-1. We propose a novel mechanism of PMA-induced translocation of ANX-1 to the nucleus that may participate in the regulation of cell proliferation and differentiation.     

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185-54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCalpha-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect.     

Signaling Pathways Involved in Dephosphorylation and Localization of the Actin-Binding Protein Cofilin in Stimulated Human Neutrophils

We have studied activation-induced dephosphorylation of proteins in human neutrophils loaded with [³²P]orthophosphate using two-dimensional gel electrophoresis and autoradiography. A major phosphoprotein of 20 kDa in resting neutrophils was markedly dephosphorylated upon activation of cells with chemotactic peptide or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Using a monoclonal anti-cofilin antibody, this phosphoprotein could be shown to be identical with cofilin, a protein implicated in actin filament remodeling. Signaling pathways leading to this dephosphorylation were further characterized. To define the role of PKC isoforms in cofilin dephosphorylation, we used different PKC inhibitors. Gö 6976 (10 μM), which inhibits preferentially PKC α and β, did not prevent PMA-induced dephosphorylation of cofilin, whereas Ro 31-8220 and CGP 41 251 (10 μM), which act also on Ca²⁺-independent PKC isoforms, almost completely suppressed this event. The lack of effect of Gö 6976 was not due to insufficient entry into the cells, as this drug suppressed PMA-induced increases in protein phosphorylation. Ca²⁺-independent PKC isoforms, rather than PKC α or β, may thus be involved in PMA-induced cofilin dephosphorylation. In contrast, Ro 31-8220 did not inhibit chemotactic peptide-induced cofilin dephosphorylation, suggesting here a PKC-independent pathway. The phosphatase inhibitor okadaic acid (1–2 μM) attenuated phosphorylation of cofilin in resting cells. This reduced level was not further attenuated by PMA. Phosphatases 1 and/or 2A may thus control cofilin phosphorylation in resting cells and contribute to PMA-induced cofilin dephosphorylation. Dephosphorylation of cofilin induced by PMA, chemotactic peptide, or okadaic acid was always accompanied by a shift of cofilin to the cell periphery into F-actin-rich areas. These findings suggest a role of cofilin in stimulus-dependent actin remodeling in motile neutrophils.     

Phorbol 12-myristate 13-acetate-induced ectodomain shedding and phosphorylation of the human meprinbeta metalloprotease

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of alpha- and beta-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin alpha-subunit. In rats and mice, the beta-subunit exists in a membrane-anchored form. In contrast, human meprinbeta is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinbeta from transfected COS-1 cells, whereas hmeprinalpha secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinbeta shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinbeta shedding. We identified tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinbeta is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinbeta is not, however, implicated in the enhanced secretion by PMA treatment.     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.     

Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes

Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-alpha, -delta, and -epsilon. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-alpha together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-alpha in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-alpha in NRVMs. WT PKC-alpha overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV beta-galactosidase (AdV betagal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-alpha-infected cells. To assess the requirement of PKC-alpha for these PMA-induced changes, AdV-DN PKC-alpha or AdV betagal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-alpha had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV betagal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV betagal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-alpha-infected NRVMs. Overexpression of DN PKC-alpha enhanced PMA-induced cell elongation. Neither WT PKC-alpha nor DN PKC-alpha affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-alpha. PMA but not WT PKC-alpha overexpression induced ERK1/2 activation. However, AdV-DN PKC-alpha partially blocked PMA-induced ERK activation. Thus PKC-alpha is necessary for certain aspects of PMA-induced NRVM hypertrophy.     

Enhancement of phorbol ester-induced protein kinase activity in human neutrophils by platelet-activating factor

We have shown that platelet-activating factor (PAF), a weak primary stimulus for neutrophil superoxide generation, synergistically enhances neutrophil oxidative responses to the tumor promoter phorbol myristate acetate (PMA). Since PMA is known to cause cytosol-to-membrane shift of calcium-activated, phospholipid-dependent protein kinase (protein kinase c, PKC) in human neutrophils, we investigated the role of PAF in modifying PMA-induced PKC activation/translocation. Protein kinase activity was measured as the incorporation of 32P from gamma-32P-ATP into histone H1 induced by enzyme in cytosolic and particulate fractions from sonicated human neutrophils. PAF did not alter the sharp decrease in cytosolic PKC activity induced by PMA. However, in the presence of PAF and PMA, total particulate protein kinase activity increased markedly over that detected in the presence of PMA alone (144 +/- 9 pmoles 32P/10(7)PMN/minute in cells treated with 20 ng/ml PMA compared to 267 +/- 24 pmoles 32P in cells exposed to PMA and 10(-6)M PAF). The increase in total particulate protein kinase activity was synergistic for the two stimuli, required the presence of cytochalasin B during stimulation, and occurred at PAF concentrations of 10(-7) M and above. Both PKC and calcium-, phospholipid-independent protein kinase activities in whole particulate fractions were augmented by PAF as were both activities in detergent-extractable particulate subfractions. PAF did not directly activate PKC obtained from control or PMA-treated neutrophils. However, the PKC-enhancing effect of PAF was inhibited in the absence of calcium during cellular stimulation. PAF also increased particulate protein kinase activity in cells simultaneously exposed to FMLP but the effect was additive for these stimuli. These results suggest that PAF enhances PMA-induced particulate PKC activity by a calcium-dependent mechanism. The enhancing effect of PAF may be directly involved in the mechanism whereby the phospholipid "primes" neutrophils for augmented oxidative responses to PMA.