Phosphorylation of pleckstrin increases proinflammatory cytokine secretion by mononuclear phagocytes in diabetes mellitus

The protein kinase C (PKC) family of intracellular enzymes plays a crucial role in signal transduction for a variety of cellular responses of mononuclear phagocytes including phagocytosis, oxidative burst, and secretion. Alterations in the activation pathways of PKC in a variety of cell types have been implicated in the pathogenesis of the complications of diabetes. In this study, we investigated the consequences of PKC activation by evaluating endogenous phosphorylation of PKC substrates with a phosphospecific PKC substrate Ab (pPKC(s)). Phosphorylation of a 40-kDa protein was significantly increased in mononuclear phagocytes from diabetics. Phosphorylation of this protein is downstream of PKC activation and its phosphorylated form was found to be associated with the membrane. Mass spectrometry analysis, immunoprecipitation, and immunoblotting experiments revealed that this 40-kDa protein is pleckstrin. We then investigated the phosphorylation and translocation of pleckstrin in response to the activation of receptor for advanced glycation end products (RAGE). The results suggest that pleckstrin is involved in RAGE signaling and advanced glycation end product (AGE)-elicited mononuclear phagocyte dysfunction. Suppression of pleckstrin expression with RNA interference silencing revealed that phosphorylation of pleckstrin is an important intermediate in the secretion and activation pathways of proinflammatory cytokines (TNF-alpha and IL-1beta) induced by RAGE activation. In summary, this study demonstrates that phosphorylation of pleckstrin is up-regulated in diabetic mononuclear phagocytes. The phosphorylation is in part due to the activation of PKC through RAGE binding, and pleckstrin is a critical molecule for proinflammatory cytokine secretion in response to elevated AGE in diabetes.     

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance‐associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.     

Increased Phosphorylation of a 17-kDa Protein Kinase C Substrate (P17) in Long-Term Potentiation

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.     

An epsilonPKC-selective inhibitor attenuates back phosphorylation of a low molecular weight protein in cardiac myocytes

We have studied epsilon PKC-mediated phosphorylation events in neonatal cardiac myocytes using back phosphorylation. 3 nM 4-beta 12-myristate-13-acetate (PMA)-intact cell treatment preferentially activates epsilon PKC in these cells (Circ. Res. 76 (1995) 654) and caused decreased 32P incorporation (back phosphorylation) into an approximately 18-kDa protein. This response required physiological levels of free Mg(2+) and short (3-5 min) incubation periods in back phosphorylation assays. Introduction of a selective epsilon PKC translocation inhibitor (epsilon V1) into these cells attenuated the 3 nM PMA-induced back phosphorylation response while translocation inhibitors to the classical PKC or deltaPKC isozymes were without effect. Pretreatment of our cells with endothelin-1 (ET1) had similar effects to 3 nM PMA albeit the magnitude of the ET1 back phosphorylation response was about one-half that of 3 nM PMA. Our results suggest that epsilon PKC phosphorylates an approximately 18-kDa protein found in the particulate cell fraction of neonatal cardiac myocytes. Epsilon PKC modulates diverse cardiac responses including contraction, ion channel functions, hypertrophy, and ischemic preconditioning. Characterization of epsilon PKC-selective phosphotransferase events may reveal novel regulatory mechanisms for this enzyme in neonatal cardiac myocytes.     

Acetylcholine-induced phosphorylation and membrane translocation of CPI-17 in bronchial smooth muscle of rats

A translocation of protein kinase C (PKC) from cytosol to plasma membrane has been reported as an association with agonist-induced Ca2+ sensitization in smooth muscle contraction. Therefore, it is possible that a downstream target of PKC, CPI-17 [PKC-potentiated inhibitory protein for heterotrimeric myosin light chain (MLC) phosphatase of 17 kDa], might also be translocated to membrane when activated. To confirm this hypothesis, cytosolic and membrane CPI-17 was measured in acetylcholine (ACh)- and high-K+ depolarization-stimulated bronchial smooth muscle of rats. An active form of CPI-17, i.e., Thr38-phosphorylated CPI-17, was also measured in cytosolic and membrane fractions. Immunoblot analyses demonstrated a translocation of CPI-17 from cytosolic to membrane fraction by ACh, but not high-K+ depolarization, stimulation in time- and concentration-dependent manners. Interestingly, phosphorylated CPI-17 was detected only in membrane fractions in the ACh-stimulated tissues. However, in the high-K+ depolarization-stimulated tissues, phosphorylated CPI-17 was not detected both in membrane and cytosolic fraction. To estimate downstream of activated CPI-17, immunoblotting for phosphorylated MLC was performed in ACh- or high-K+ depolarization-stimulated tissues. ACh- and high-K+ depolarization-induced phosphorylation of MLC was observed in its contraction-dependent manner. In conclusion, we, for the first time, suggested that CPI-17 is translocated and phosphorylated by ACh, but not high-K+ depolarization, in rat bronchial smooth muscle. ACh-induced translocation and phosphorylation of CPI-17 might be caused via the activation of muscarinic receptor.     

Selective Activation of Phospholipase Cγ1 and Distinct Protein Kinase C Subspecies in Intracellular Signaling by Hepatocyte Growth Factor/Scatter Factor in Primary Cultured Rat Neocortical Cells

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c-Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80- and 45-kDa substrates. Studies with protein kinase inhibitors and phorbol 12-myristate 13-acetate showed that protein kinase C (PKC) is activated intracellularly. The 80-kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3-kinase was enhanced, although the stimulation of brain-derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3-kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen-activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.     

Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm.

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.     

Role of Protein Kinase C (PKC) in Agonist-Induced μ-Opioid Receptor Down-Regulation

Abstract : Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express μ- and δ-opioid receptors, we investigated whether μ-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by ³H-phorbol ester and [³H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that μ-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [ d -Ala², N -Me-Phe⁴, Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [³H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

Protein kinase C delta localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity

Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.     

G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKCepsilon, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKCepsilon via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKCepsilon-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation.     

Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.     

Lymphoma protein kinase C is associated with the transmembrane glycoprotein, GP85, and may function in GP85-ankyrin binding

In this study, several complementary techniques have been used to investigate the involvement of a protein kinase C (PKC) molecule in the plasma membrane-cytoskeleton interactions that occur in mouse T-lymphoma cells. Our data indicate that the lymphoma plasma membrane contains a 78-kDa polypeptide that exists in a complex with one of the major transmembrane glycoproteins, GP85 (a wheat germ agglutinin-binding protein). This membrane-associated 78-kDa protein appears to have PKC-like properties based on the following criteria: 1) it cross-reacts with a specific antibody raised against brain PKC; 2) it has a pI of 5.6-5.8, which is similar to that of the PKC described previously in other cell types; and 3) it displays characteristic PKC enzymatic activity by phosphorylating histone H1 in a Ca2+- and phospholipid-dependent manner. Double immunocytochemical staining experiments reveal that the lymphoma PKC-like molecules translocate from the cytoplasm to the cell membrane and accumulate directly underneath receptor capped structures following addition of various ligands. Studies we have done to identify the cellular substrate(s) of the lymphoma plasma membrane-associated PKC have shown that GP85 is preferentially phosphorylated in isolated membrane preparations following addition of the PKC activator, TPA (phorbol-12-O-tetradecanoyl-phorbol 13-acetate), but not the biologically inactive TPA analogue, 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate). In addition, we have found that GP85 can be phosphorylated by purified brain protein kinase C. Analysis of the resulting phosphoamino acids indicates that phosphorylation of GP85 occurs primarily at serine residues, occurs in minor amounts (approximately 5%) at threonine residues, and does not occur at tyrosine residues. These data indicate that the lymphoma GP85 is a substrate for PKC. Furthermore, we have established that phosphorylation of GP85 by PKC enhances its binding affinity with the membrane linker molecule, ankyrin. These findings suggest that PKC-mediated phosphorylation of GP85 may be an important part of the lymphoma plasma membrane-cytoskeleton interaction.     

Regulation of cadmium-induced apoptosis by PKCδ in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCδ translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCδ cleavage to give a 41-kDa fragment, which was detected at 3–6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCδ-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCδ translocation. Cadmium chloride rapidly induced p38^MAPK activation, which was not affected by rottlerin. By contrast, the p38^MAPK inhibitor SB203580 reduced PKCδ translocation and cleavage, indicating that p38^MAPK activation precedes and regulates PKCδ activation. It is concluded that PKCδ mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP-43 in Rat Hippocampal Slices and Synaptosomes

Abstract: Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 µM), an N-methyl-d -aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates—arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)—were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 µM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.     

A protein kinase C isozyme is translocated to cytoskeletal elements on activation.

Protein kinase C (PKC)1 isozymes comprise a family of related cytosolic kinases that translocate to the cell particulate fraction on stimulation. The activated enzyme is thought to be on the plasma membrane. However, phosphorylation of protein substrates occurs throughout the cell and is inconsistent with plasma membrane localization. Using an isozyme-specific monoclonal antibody we found that, on activation, this PKC isozyme translocates to myofibrils in cardiac myocytes and to microfilaments in fibroblasts. Translocation of this activated PKC isozyme to cytoskeletal elements may explain some of the effects of PKC on cell contractility and morphology. In addition, differences in the translocation site of individual isozymes--and, therefore, phosphorylation of different substrates localized at these sites--may explain the diverse biological effects of PKC.     

Age-related alteration of PKC, a key enzyme in memory processes

Brain aging is characterized by a progressive decline of the cognitive and memory functions. It is becoming increasingly clear that protein phosphorylation and, in particular, the activity of the calcium-phospholipid-dependent protein kinase C (PKC) may be one of the fundamental cellular changes associated with memory function. PKC is a multigene family of enzymes highly expressed in brain tissues. The activation of kinase C is coupled with its translocation from the cytosol to different intracellular sites and recent studies have demonstrated the key role played by several anchoring proteins in this mechanism. PKC-phosphorylating activity appears to be impaired during senescence at brain level in a strain-dependent fashion in rodents. Whereas the levels of the various isoforms do not show age-related alterations, the enzyme translocation upon phorbol-ester treatment is deficitary among all strains investigated. Anchoring proteins may contribute to this activation deficit. We discuss also modifications of the PKC system in Alzheimer's disease that may be related to pathological alterations in neurotransmission. A better insight of the different factors controlling brain-PKC activation may be important not only for elucidating the molecular basis of neuronal transmission, but also for identifying new approaches for correcting or even preventing age-dependent changes in brain function.     

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.     

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.