Conventional Protein Kinase C Isoforms Research Articles

Role of Calcium and PKC in Salivary Mucous Cell Exocrine Secretion

Fluid and exocrine secretion of mucins by salivary mucous glands is regulated predominantly by parasympathetic activation of muscarinic receptors. A direct role for subsequent putative signaling steps, phospholipase C (PLC), increased intracellular calcium ([Ca2+]i), and isoforms of protein kinase C (PKC) in mediating muscarinic exocrine secretion has not been elucidated, and these are potential therapeutic targets to enhance mucin secretion in hyposalivary patients. We found that muscarinic-induced mucin secretion by rat sublingual tubulo-acini was dependent upon PLC activation and the subsequent increase in [Ca2+]i, and further identified a transient PKC-independent component of secretion dependent upon Ca2+ release from intracellular stores, whereas sustained secretion required entry of extracellular Ca2+. Interactions among carbachol, PKC inhibitors, phorbol 12-myristate 13-acetate, and thapsigargin to modulate [Ca2+]i implicated conventional PKC isoforms in mediating sustained secretion. With increasing times during carbachol perfusion of glands, in situ, PKC-α redistributed across glandular membrane compartments and underwent a rapid and persistent accumulation near the luminal borders of mucous cells. PKC-β1 displayed transient localization near luminal borders, whereas the novel PKCs, PKC-δ or PKC-ϵ, displayed little or no redistribution in mucous cells. Collective results implicate synergistic interactions between diacylglycerol (DAG) and increasing [Ca2+]i levels to activate cPKCs in mediating sustained muscarinic-induced secretion.

Differential Protein Kinase C Isoform Regulation and Increased Constitutive Activity of Acetylcholine-Regulated Potassium Channels in Atrial Remodeling

Atrial fibrillation (AF) causes atrial-tachycardia remodeling (ATR), with enhanced constitutive acetylcholine-regulated K+ current (I(KAChC)) contributing to action potential duration shortening and AF promotion. The underlying mechanisms are unknown. To evaluate the role of protein-kinase C (PKC) isoforms in ATR-induced I(KAChC) activation. Cells from ATR-dogs (400-bpm atrial pacing for 1 week) were compared to control dog cells. In vitro tachypaced (TP; 3 Hz) canine atrial cardiomyocytes were compared to parallel 1-Hz paced cells. I(KAChC) single-channel activity was assessed in cell-attached and cell-free (inside-out) patches. Protein expression was assessed by immunoblot. In vitro TP activated I(KAChC), mimicking effects of in vivo ATR. Discrepant effects of PKC activation and inhibition between control and ATR cells suggested isoform-selective effects and altered PKC isoform distribution. Conventional PKC isoforms (cPKC; including PKCα) inhibited, whereas novel isoforms (including PKCε) enhanced, acetylcholine-regulated K+ current (I(KACh)) in inside-out patches. TP and ATR downregulated PKCα (by 33% and 37%, respectively) and caused membrane translocation of PKCε, switching PKC predominance to the stimulatory novel isoform. TP increased [Ca2+]i at 2 hours by 30%, with return to baseline at 24 hours. Buffering [Ca2+]i during TP with the cell-permeable Ca2+ chelator BAPTA-AM (1 μmol/L) or inhibiting the Ca2+-dependent protease calpain with PD150606 (20 μmol/L) prevented PKCα downregulation and TP enhancement of I(KAChC). PKCε inhibition with a cell-permeable peptide inhibitor suppressed TP/ATR-induced I(KAChC) activation, whereas cPKC inhibition enhanced I(KAChC) activity in 1-Hz cells. PKC isoforms differentially modulate I(KACh), with conventional Ca(2+)-dependent isoforms inhibiting and novel isoforms enhancing activity. ATR causes a rate-dependent PKC isoform switch, with Ca2+/calpain-dependent downregulation of inhibitory PKCα and membrane translocation of stimulatory PKCε, enhancing I(KAChC). These findings provide novel insights into mechanisms underlying I(KAChC) dysregulation in AF.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

AbstractPlatelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Unravelling the different functions of protein kinase C isoforms in platelets

Platelets tightly regulate haemostasis and arterial thrombosis. Protein kinase C (PKC) is involved in most platelet responses implicated in thrombus formation. Recent pharmacological and mouse gene knockout approaches show that the conventional PKC isoforms and the novel PKC isoforms contribute in distinct ways to these platelet responses. We hypothesize that, in platelets and other cells, the characteristic functions of PKC isoforms are established through unique activation mechanisms and unique interacting protein partners, which result in isoform-specific patterns of substrate phosphorylation. For identifying the substrate proteins in a living cell, new methodology is available and discussed.

Protein kinase C pathway is involved in the inhibition by crocetin of vascular smooth muscle cells proliferation

Crocetin is a natural carotenoid compound isolated from Gardenia jasminoids Ellis. Our previous study showed that crocetin inhibits angiotensin II (Ang II)-induced proliferation of vascular smooth muscle cells (VSMCs). The present study investigated the involvement of the protein kinase C (PKC) pathway in the growth-inhibitory action of crocetin in VSMCs. The findings showed that PKC activity in the membrane fraction of VSMCs increased following stimulation with Ang II, which was suppressed significantly by pretreating the cells with crocetin. Inhibition of PKC activity by crocetin appeared to be associated with growth inhibition in VSMCs, because chelerythrine chloride, a specific PKC inhibitor, likewise decreased cell proliferation. PKC-a, a conventional PKC isoform, was detected in bovine aorta VSMCs by RT-PCR and western blotting analysis. Crocetin inhibited Ang II-induced membrane translocation of PKC-a, and the inhibition of crocetin on PKC activity in membrane fraction coincided with its suppression on membrane translocation of PKC-a. In addition, Ang II-induced mRNA expressions of c-fos, c-jun and c-myc were also decreased by crocetin. Taken together, the data suggest that the inhibition by crocetin of PKC activity, at least in part due to inactivation of PKC-a, and the subsequent suppression of proto-oncogene expressions might mediate its inhibitory effect on VSMCs proliferation.

Signaling Pathways Leading to Phosphorylation of Akt and GSK-3β by Activation of Cloned Human and Rat Cerebral D2and D3Receptors

Although dopamine (DA) regulates the serine/threonine kinase Akt and its downstream substrate glycogen synthase kinase-3β (GSK-3β), the direct influence of dopaminergic receptors remains poorly characterized. Short-term incubation of Chinese hamster ovary (CHO)-expressed human (h)D(₂L) and hD₃) receptors with DA (maximal effect, 5-10 min) phosphorylated Akt (Thr308 and Ser473) and GSK-3β (Ser9), actions blocked by the selective D₂ and D₃ antagonists, 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) and (3aR,9bS)-N[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl] (4-phenyl)benzamide (S33084), respectively. Similar findings were acquired with the specific D₂/D₃ receptor agonist quinelorane, which also enhanced (10 min after administration) levels of p-Akt and p-GSK-3β in rat nucleus accumbens, an action blocked by the D₂/D₃ receptor antagonist raclopride. Akt and GSK-3β phosphorylation mediated via CHO-expressed hD(₂L) and hD₃ receptors was prevented by pertussis toxin and by inhibitors of insulin-like growth factor-1 receptors as well as phosphatidylinositol 3-kinase and Src. Likewise, chelation of intracellular Ca²+ and interference with an "atypical" phorbol ester-insensitive protein kinase C (PKC) abolished recruitment of Akt and GSK-3β. Inactivation of PKCμ blocked Akt and GSK-3β phosphorylation at hD(₂L) receptors. However, blockade of conventional PKC isoforms attenuated the actions of DA at hD₃ receptors only. Furthermore, phospholipase C (PLC), calmodulin, and Akt inhibitors abolished DA-induced GSK-3β phosphorylation by hD₃ receptors, whereas phosphorylation by hD(₂L) receptors partially involved calmodulin, Akt, and extracellular signal-regulated kinase (ERK) 1/2. In conclusion, at both hD(₂L) and hD₃ receptors, DA elicited a G(i/o)- and Ca²+/calmodulin-dependent phosphorylation of Akt and GSK-3β via transactivation of insulin-like growth factor 1 receptor. However, significant differences were seen regarding the involvement of PLC, calmodulin, and ERK1/2.

Peroxynitrite causes phosphorylation of vasodilator-stimulated phosphoprotein through a PKC dependent mechanism

Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0–100 µM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine157 (Ser157) and Ser239. Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca2+ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser157, but not Ser239, which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.

Involvement of protein kinase Cα in invadopodia formation in Src‐3T3 cells

Dynamic structures termed podosomes or invadopodia take the form of actin puncta or rings and are involved in extracellular matrix remodeling, adhesion, and invasion. Though various protein kinase C (PKC) isoforms are known to affect these structures, the precise role of PKCα in signaling upstream of invadopodia in transformed cells remains unclear. We have begun to analyze the role of PKCα in NIH 3T3 fibroblasts transformed with active Src (Src‐3T3 cells). PKCα colocalizes with actin and the invadopodia marker Tks5 at rosettes, and inhibition of conventional PKC isoforms interferes with PKCα localization and rosette formation. The target of PKC that is responsible for invadopodia formation is unknown; however, one possible candidate is Discs Large 1 (DLG1), which we show is a substrate of PKC. DLG1 is present at numerous actin‐based structures including invadopodia and is involved in motility and invasion. Strikingly, coexpression of DLG1 and PKCα in COS‐7 epithelial cells causes formation of actin‐based rosettes (in a cPKC‐dependent manner), suggesting that phosphorylated DLG is sufficient for establishment of these structures.This work was supported by NIH P01 DK54441 and NIH GM43154.

Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Abstract 3622Poster Board III-558Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures:No relevant conflicts of interest to declare.

Regulation of Rnd3 localization and function by protein kinase Cα-mediated phosphorylation

The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization.

Crystal-Induced Neutrophil Activation: XI. Implication and Novel Roles of Classical Protein Kinase C

Monosodium urate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathology of gouty arthritis. Furthermore, MSU crystals have recently been identified as danger signals able to induce the maturation of dendritic cells. Release of the crystals into the joint cavity promotes an acute inflammation characterized by a massive infiltration of neutrophils that leads to tissue damage. Protein kinase C (PKC) represents a family of serine/threonine kinases that play central signaling roles in multiple cellular responses. This family of kinases is divided into three subfamilies based on second messenger requirements: conventional (or classical), novel, and atypical. Despite their role in signal transduction, very little is known about the involvement of the PKC family in the inflammatory reaction induced by MSU crystals. In the present study, we show that MSU crystals activate conventional PKC isoforms, and that this activation is necessary for the MSU crystal-induced degranulation and generation of a chemotactic activity in the supernatants of MSU crystal-stimulated human neutrophils. Evidence is also obtained that the tyrosine kinase Syk is a substrate of PKC and that the PKC-mediated serine phosphorylation of Syk is necessary to its interaction with the regulatory subunit of PI3K kinases (p85) and thus to the subsequent activation of these lipid kinases. These results suggest novel means of modulating neutrophil responses (through the specific regulation of PKC) during the acute phase of MSU crystal-induced inflammation.

Knockout of the predominant conventional PKC isoform, PKCα, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake

Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50-100%, whereas both Gö compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKCalpha was estimated to account for approximately 97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCalpha knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction-induced glucose uptake were similar in muscles lacking PKCalpha and in the wild type. It can be concluded that PKCalpha, representing approximately 97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.

2008 Thomas W. Smith Memorial Lecture—Protein Kinase C α as a Novel Therapeutic Target for Treating Heart Failure

We have shown that protein kinase C (PKC) α functions as a proximal regulator of Ca 2+ handling in cardiac myocytes (Braz et al, Nat. Med. 10:248, 2004). Deletion of PKC α in the mouse resulted in augmented sarcoplasmic reticulum Ca 2+ loading, enhanced Ca 2+ transients, and augmented contractility, whereas overexpression of PKCα in the heart blunted contractility. Mechanistically, PKCα regulates Ca 2+ handling by altering inhibitor-1 phosphorylation, which suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and sarcoplasmic reticulum Ca 2+ AT-Pase 2 (SERCA2). Acute inhibition of PKCα with the pharmacologic agents Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work performing heart preparation in wild-type mice, but not in PKC α-deficient mice. Ro-32-0432 also acutely increased cardiac contractility in two different models of heart failure in vivo. Moreover, acute or chronic treatment with Ro-32-8220 in a mouse model of heart failure, due to deletion of the muscle lim protein (MLP) gene, significantly augmented cardiac contractility and restored normal pump function. Adenoviral-mediated gene therapy with a dominant negative PKCα cDNA rescued heart failure in a chronic rat model of postinfarction cardiomyopathy. Moreover, expression of dominant-negative PKCα in cardiac myocytes using a cardiac-specific transgenic system (tetracycline-regulated) also enhanced cardiac contractility and antagonized heart failure due to myocardial infarction injury. Finally, another PKCα/β inhibitor, ruboxistaurin (LY333531), antagonized heart failure after long-term pressure overload in mice. PKCα is the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Indeed, pharmacological inhibition of PKCα may serve as a novel therapeutic strategy for either enhancing cardiac contractility in the setting of severe functional deterioration or as a long-term treatment option to prevent worsening of heart failure in earlier stages.

PDGF receptor activation induces p120-catenin phosphorylation at serine 879 via a PKCα-dependent pathway

p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell–cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCα, a conventional PKC isoform implicated previously in disruption of adherens junctions.

Targeting of PED/PEA-15 Molecular Interaction with Phospholipase D1 Enhances Insulin Sensitivity in Skeletal Muscle Cells

Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) is overexpressed in several tissues of individuals affected by type 2 diabetes. In intact cells and in transgenic animal models, PED/PEA-15 overexpression impairs insulin regulation of glucose transport, and this is mediated by its interaction with the C-terminal D4 domain of phospholipase D1 (PLD1) and the consequent increase of protein kinase C-alpha activity. Here we show that interfering with the interaction of PED/PEA-15 with PLD1 in L6 skeletal muscle cells overexpressing PED/PEA-15 (L6(PED/PEA-15)) restores insulin sensitivity. Surface plasmon resonance and ELISA-like assays show that PED/PEA-15 binds in vitro the D4 domain with high affinity (K(D) = 0.37 +/- 0.13 mum), and a PED/PEA-15 peptide, spanning residues 1-24, PED-(1-24), is able to compete with the PED/PEA-15-D4 recognition. When loaded into L6(PED/PEA-15) cells and in myocytes derived from PED/PEA-15-overexpressing transgenic mice, PED-(1-24) abrogates the PED/PEA-15-PLD1 interaction and reduces protein kinase C-alpha activity to levels similar to controls. Importantly, the peptide restores insulin-stimulated glucose uptake by approximately 70%. Similar results are obtained by expression of D4 in L6(PED/PEA-15). All these findings suggest that disruption of the PED/PEA-15-PLD1 molecular interaction enhances insulin sensitivity in skeletal muscle cells and indicate that PED/PEA-15 as an important target for type 2 diabetes.

Role of PKCεin PGF2α-Stimulated MMP-2 Secretion from Human Ciliary Muscle Cells

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCbeta with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bbeta-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (k(on)(app)), but not the apparent dissociation rate (k(off)(app)), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in k(on)(app) with little effect (2-fold) on k(off)(app). Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter k(off)(app) with smaller effects on k(on)(app). We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in k(off)(app). Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.

PKC θ activity maintains normal quantal size in chromaffin cells

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC theta and PKC delta, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC theta and delta were expressed in chromaffin cells, mice deficient for PKC theta, but not for PKC delta, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC theta pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC theta mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC theta, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.

Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

The vasoconstrictor angiotensin II (Ang II) acts at G(q/11)-coupled receptors to suppress ATP-sensitive potassium (K(ATP)) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K(ATP) channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels. We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K(ATP) currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCepsilon translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K(ATP) channels by Ang II arises exclusively from the activation and translocation of PKCepsilon. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCepsilon to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K(ATP) channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCepsilon to these membrane compartments. Ang II induces the recruitment of the novel PKC isoform, PKCepsilon, to arterial smooth muscle caveolae. This translocation allows PKCepsilon access to K(ATP) channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

Acute Ethanol Administration Rapidly Increases Phosphorylation of Conventional Protein Kinase C in Specific Mammalian Brain Regions in Vivo

Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p-cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity. Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p-cPKC expression using immunohistochemistry and Western blot methods. Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p-cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p-cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p-cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKCgamma immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection. These results suggest that ethanol rapidly promotes phosphorylation of cPKC in limbic brain regions, which may underlie effects of acute ethanol on the nervous system and behavior.