The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.     

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16^INK4A and p21^Cip1, were equivalent between the genotypes, increased levels of p27^Kip1 and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27^Kip1 signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.     

Impact of cholesterol depletion on shape changes, actin reorganization, and signal transduction in neutrophil-like HL-60 cells

Stimulation of neutrophils with chemotactic peptide induces actin reorganization, formation of actin-rich protrusions, and development of polarity. Shape changes and actin polymerization can also be induced by phorbol ester-mediated direct activation of protein kinase C (PKC). We have investigated the role of cholesterol in stimulus-dependent motile events and in activation of signaling pathways in neutrophil-like differentiated HL-60 cells. Depletion of plasma membrane cholesterol using methyl-beta-cyclodextrin (MbetaCD) prevented chemotactic peptide and phorbol ester-induced shape changes and increases in cytoskeletal actin. Cholesterol depletion almost completely suppressed chemotactic peptide-mediated activation of p42/44 mitogen-activated protein kinase (MAPK). Phosphorylation of protein kinase B on Thr-308, which is indicative of activation of phosphatidylinositol 3-kinase, was in contrast only partially inhibited. Stimulus-mediated membrane recruitment of different PKC isoforms was differentially affected by treatment of cells with MbetaCD. Membrane recruitment of PKCalpha induced by chemotactic peptide or phorbol ester was suppressed, whereas that of PKCbetaII was only partially affected. Membrane association of PKCdelta was almost insensitive to cholesterol depletion. In summary, our results implicate an important role of cholesterol-containing lipid microdomains (rafts) especially in chemotactic peptide-induced activation of MAPK pathways and in chemotactic peptide- and phorbol ester-mediated activation of PKCalpha.     

Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade

As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.     

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca²⁺]_cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca²⁺]_cyt (Ca²⁺ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca²⁺ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca²⁺ signalling, we here monitor Ca²⁺ flux around the platelet by measuring net Ca²⁺ fluxes to or from the extracellular space and the intracellular Ca²⁺ stores, which act as the major sources and sinks for Ca²⁺ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na⁺ concentration ([Na⁺]_cyt), which influences platelet Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange. The intracellular store Ca²⁺ concentration ([Ca²⁺]_st) was monitored using Fluo-5N, the extracellular Ca²⁺ concentration ([Ca²⁺]_ext) was monitored using Fluo-4 whilst [Ca²⁺]_cyt and [Na⁺]_cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca²⁺ release and Ca²⁺ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca²⁺]_cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na⁺]_cyt which would be expected to reduce Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger (NCX). Thrombin-evoked rises in [Na⁺]_cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn²⁺ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca²⁺]_cyt following SERCA inhibition and either removal of extracellular Na⁺ or inhibition of Na⁺/K⁺-ATPase activity by removal of extracellular K⁺ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca²⁺]_cyt by acceleration of SERCA activity, whilst rises in [Ca²⁺]_cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na⁺/K⁺-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na⁺]_cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca²⁺ signalling.     

Activation of protein kinase C in neutrophil cytoplasts: Localization of protein substrates and possible relationship with stimulus-response coupling

Treatment of enucleated, granule-free neutrophil cytoplasts with the protein kinase C activator phorbol 12O-myristate-13-acetate (PMA) causes an increased -³²P-incorporation into a variety of polypeptides. Permeabilization of PMA-stimulated, ³²P-labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA-induced activation of generation of Superoxide anion (O⁻₂) and the phosphorylation of a cytosolic polypeptide with an apparent M_r, of 46000, whose -³²P-labeling is also enhanced by the treatment of cytoplasts with 1-oleyl-2-acetylglycerol, the Ca²⁺ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the ³²P-labeling of proteins in the 40000 M_r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O⁻₂ production by PMA.     

Intracellular pH on translocation of protein kinase C isozymes in rat pinealocytes

In rat pinealocytes, cytoplasmic alkalization causes protein kinase C (PKC) translocation, but the isozyme involved is not known. In this study, we investigated the effect of cytoplasmic alkalization on membrane-associated PKCalpha, delta, epsilon, and zeta, four isozymes present in the rat pineal gland. Treatment with NH(4)Cl, which had no effect on PKCzeta, caused a sustained increase in membrane-associated PKCalpha, delta, and epsilon that lasted for at least 60 min. The effect of NH(4)Cl on PKCalpha, delta, and epsilon was reduced by sodium propionate, an agent that counteracts the effect of NH(4)Cl on intracellular pH. Both sodium propionate and 5-(N,N-hexamethylene)amiloride (HMA), two treatments that abolished the effect of norepinephrine on cytoplasmic alkalization, also reduced norepinephrine-mediated increases in membrane-associated PKCalpha, delta, and epsilon. In contrast, these two treatments did not have an effect on the increase in membrane-associated PKC isozymes caused by 4beta-phorbol 12-myristate 13-acetate (PMA), an active phorbol ester, even though HMA was effective in abolishing PMA-mediated increases in intracellular pH. These results, apart from demonstrating that cytoplasmic alkalization by itself can cause translocation of PKCalpha, delta, and epsilon in rat pinealocytes, also indicate that the norepinephrine-stimulated cytoplasmic alkalization plays an important role in transducing signals from the adrenergic receptor to selective PKC isozymes. However, PKC translocation stimulated directly by PMA does not appear to be sensitive to changes in intracellular pH.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils

Abstract Protein kinase C (PKC) appears to have a central role in the O₂⁻ response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP₂)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling of PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O₂⁻. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.     

Syndecan‐2 regulates melanin synthesis via protein kinase C βII‐mediated tyrosinase activation

Syndecan‐2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan‐2 in melanogenesis. Syndecan‐2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA‐mediated knockdown of syndecan‐2 was associated with reduced melanin synthesis, whereas overexpression of syndecan‐2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan‐2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinase CβII. Furthermore, UVB caused increased syndecan‐2 expression, and this up‐regulation of syndecan‐2 was required for UVB‐induced melanin synthesis. Taken together, these data suggest that syndecan‐2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin‐associated diseases.     

Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca²⁺‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

CIN85 modulates the down-regulation of Fc gammaRIIa expression and function by c-Cbl in a PKC-dependent manner in human neutrophils

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Constitutive phosphorylation by protein kinase C regulates D1 dopamine receptor signaling

The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.     

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.