Contribution Of Protein Kinase Research Articles

The AKT isoforms 1 and 2 drive B cell fate decisions during the germinal center response.

The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2-deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1 T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2-deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.

The Contribution of Protein Kinase C to Cyclopiazonic Acid-Mediated Potentiation of Serotonin-Induced Vascular Calcium Responses

The Contribution of Protein Kinase C to Cyclopiazonic Acid-Mediated Potentiation of Serotonin-Induced Vascular Calcium Responses

Stimulation of Proglucagon Gene Expression by Human GPR119 in Enteroendocrine L-cell Line GLUTag

GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and β isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.

Protein kinase A is required for resistance to the lysosomotropic drugs quinacrine and chloroquine in the fission yeast, Schizosaccharomyces pombe

Yeast vacuoles, the functionally diverse counterparts of mammalian lysosomes, are highly dynamic organelles that undergo morphological changes during the cell cycle and in response to various cellular stresses. Here, we report results of an investigation on the contribution of protein kinase A (PKA) to vacuole integrity and dynamics in the fission yeast, Schizosaccharomyces pombe. Under normal culturing conditions, vacuoles in S. pombe wild type cells and PKA null (pka1Δ) mutants appear similar in size, morphology, and distribution. Wild type and pka1Δ cells also exhibit similar intensities of staining with quinacrine, a fluorescent lysosomotropic drug that accumulates in acidic organelles, indicating that PKA is not required for vacuole acidification in S. pombe. However, whereas quinacrine induces coalescence of vacuoles in wild type S. pombe cells, it does not induce a similar response in pka1Δ cells. Moreover, pka1Δ mutants are hypersensitive to the growth inhibitory effects of both quinacrine and the lysosomotropic toxin, chloroquine. Interestingly, we also found that the toxin rotenone, while having no obvious affect on vacuole morphology in wild type S. pombe cells, induced formation of small vacuoles aggregated at the nuclear periphery in pka1Δ cells, which are hypersensitive to growth inhibitory effects of the toxin. Our findings implicate PKA as a mediator of vacuole stress response in S. pombe.

The contribution of protein kinase C and CPI‐17 signaling pathways to hypercontractility in murine experimental colitis

Colonic smooth muscle contractility is altered in colitis, and several protein kinase pathways can mediate colonic smooth muscle contraction. In the present study, we investigated whether protein kinase C (PKC) pathways also play a role in colonic hypercontractility observed during T(H) 2 colitis in BALB/c mice. Colitis was induced in BALB/c mice by provision of 5% dextran sodium sulfate (DSS) for 7 days. Changes in smooth muscle contractility were examined using dissected circular smooth muscle preparations from the distal colon. The contribution of conventional and novel PKC isozymes to the hypercontractile response was examined with pharmacological PKC inhibitors. Western blot analyses were used to examine protein expression and phosphorylation changes. Colonic smooth muscle was associated with inflammation-induced hypercontractility and altered PKC expression. Carbachol-induced peak (phasic) and sustained (tonic) contractions were increased. Chelerythrine was the most effective PKC inhibitor of both phasic and tonic contractions. There was no general difference in the percent contribution of conventional and novel PKC isozymes toward the DSS-induced hypercontractility, but inhibition of sustained force with GF109203x was higher for inflamed muscle. The CPI-17 phosphorylation was equally suppressed in both normal and DSS conditions by Gö6976 and chelerythrine, but only for the phasic component of contraction. The outcomes suggest that both conventional and novel PKC isozymes contribute to the phasic and tonic contractile components of BALB/c colonic circular smooth muscle under normal conditions, with novel PKC isozymes having a greater contribution to the tonic contraction. However, no effect of inflammation was observed on the relative contribution of PKC and CPI-17 toward the observed hypercontractility.

Involvement of protein kinase C in IL-1β-induced expression of cyclooxygenase-2 in human gingival fibroblasts

Interleukin-1beta (IL-1beta) stimulates expression of the highly inducible enzyme cyclooxygenase-2 (COX-2) via activation of nuclear factor kappaB (NFkappaB), and consequently provokes prostaglandin E(2) (PGE(2)) synthesis, which induces inflammatory responses. In this study, the contribution of protein kinase C (PKC) to IL-1beta-induced PGE(2) synthesis in human gingival fibroblasts was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated PGE(2) release and COX-2 mRNA expression, as shown in human gingival fibroblasts stimulated by IL-1beta. However, PMA showed only a weak effect on the formation of COX-2-NFkappaB DNA-protein complex, whereas IL-1beta had a clearly stimulatory effect. In cells in which PMA-dependent PKC was down-regulated, PMA failed to induce the formation of NFkappaB DNA-protein complex and reduced the release of PMA-induced PGE(2), whereas IL-1beta stimulated the formation of COX-2-NFkappaB DNA-protein complex and PGE(2) release. The atypical PKC (aPKC) inhibitor Gö6983 clearly suppressed the formation of COX-2-NFkappaB DNA-protein complex and PGE(2) release stimulated by IL-1beta but not the inhibitor of conventional PKC (cPKC) and the novel PKC (nPKC) inhibitor Gö6976. These observations suggest that aPKC is involved in IL-1beta-induced PGE(2) synthesis, which is controlled by transcription of the COX-2 gene via the NFkappaB-dependent pathway in human gingival fibroblasts.

The contribution of protein kinase C and rho-kinase to the regulation of receptor-dependent contraction of arteries decreases with age independently of sympathetic innervation

The age-related dynamics of the activity of signaling pathways coupled with α1-adrenoreceptors and their dependence on sympathetic innervation of arterial smooth muscle has been studied. For this purpose, the effects of protein kinase C inhibitor (GF 109203X, 10−6 M and Rho-kinase inhibitor (Y27632, 10−5 M) on isometric contraction of the rat saphenous artery in response to the α1-adrenoreceptor agonist methoxamine were determined. The rats in the age of two weeks (with partially developed sympathetic innervation) had the lower vascular sensitivity to methoxamine than adult rats, but the effects of both inhibitors were more prominent. The denervation induced by excision of sympathetic ganglia in adult rats increased the arterial sensitivity to methoxamine but the sensitivity to inhibitors was unchanged. Thus, the postnatal development of the arterial smooth muscle is accompanied by diminution of the role of protein kinase C and Rho-kinase in the regulation of contraction, but these changes do not correlate with the changes in arterial sensitivity to α1-adrenergic stimulation and development of sympathetic innervation.

Altered Ca2+ handling by ryanodine receptor and Na+-Ca2+ exchange in the heart from ovariectomized rats: role of protein kinase A

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (+/-dP/dt(max)) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca(2+) homeostasis that determines the contractile function. Particular emphasis was given to Ca(2+) handling by ryanodine receptor (RyR) and Na(+)-Ca(2+) exchange (NCX). (45)Ca(2+) fluxes via the RyR, NCX, and Ca(2+)-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca(2+) by these Ca(2+) handling proteins with the overall Ca(2+) homeostasis by determining the Ca(2+) transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca(2+) handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca(2+) fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca(2+) through RyR and removal of Ca(2+) by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx.

The Contributions of Protein Kinase A and Smoothened Phosphorylation to Hedgehog Signal Transduction in Drosophila melanogaster

Protein kinase A (PKA) silences the Hedgehog (Hh) pathway in Drosophila in the absence of ligand by phosphorylating the pathway's transcriptional effector, Cubitus interruptus (Ci). Smoothened (Smo) is essential for Hh signal transduction but loses activity if three specific PKA sites or adjacent PKA-primed casein kinase 1 (CK1) sites are replaced by alanine residues. Conversely, Smo becomes constitutively active if acidic residues replace those phosphorylation sites. These observations suggest an essential positive role for PKA in responding to Hh. However, direct manipulation of PKA activity has not provided strong evidence for positive effects of PKA, with the notable exception of a robust induction of Hh target genes by PKA hyperactivity in embryos. Here we show that the latter response is mediated principally by regulatory elements other than Ci binding sites and not by altered Smo phosphorylation. Also, the failure of PKA hyperactivity to induce Hh target genes strongly through Smo phosphorylation cannot be attributed to the coincident phosphorylation of PKA sites on Ci. Finally, we show that Smo containing acidic residues at PKA and CK1 sites can be stimulated further by Hh and acts through Hh pathways that both stabilize Ci-155 and use Fused kinase activity to increase the specific activity of Ci-155.

Global Profiles of Gene Expression Induced by Adrenocorticotropin in Y1 Mouse Adrenal Cells

ACTH regulates the steroidogenic capacity, size, and structural integrity of the adrenal cortex through a series of actions involving changes in gene expression; however, only a limited number of ACTH-regulated genes have been identified, and these only partly account for the global effects of ACTH on the adrenal cortex. In this study, a National Institute on Aging 15K mouse cDNA microarray was used to identify genome-wide changes in gene expression after treatment of Y1 mouse adrenocortical cells with ACTH. ACTH affected the levels of 1275 annotated transcripts, of which 46% were up-regulated. The up-regulated transcripts were enriched for functions associated with steroid biosynthesis and metabolism; the down- regulated transcripts were enriched for functions associated with cell proliferation, nuclear transport and RNA processing, including alternative splicing. A total of 133 different transcripts, i.e. only 10% of the ACTH-affected transcripts, were represented in the categories above; most of these had not been described as ACTH-regulated previously. The contributions of protein kinase A and protein kinase C to these genome-wide effects of ACTH were evaluated in microarray experiments after treatment of Y1 cells and derivative protein kinase A-defective mutants with pharmacological probes of each pathway. Protein kinase A-dependent signaling accounted for 56% of the ACTH effect; protein kinase C-dependent signaling accounted for an additional 6%. These results indicate that ACTH affects the expression profile of Y1 adrenal cells principally through cAMP- and protein kinase A- dependent signaling. The large number of transcripts affected by ACTH anticipates a broader range of actions than previously appreciated.

Atypical Protein Kinase C Is a Novel Mediator of Dopamine-Enhanced Firing in Nucleus Accumbens Neurons

Current concepts suggest that nucleus accumbens (NAcb) dopamine mediates several motivated and addictive behaviors. Although the role of protein kinase A (PKA) and dopamine and cyclic adenosine 3',5' monophosphate-regulated phosphoprotein 32 kDa in NAcb dopamine receptor throughput has been studied extensively, the contribution of protein kinase C (PKC) to NAcb firing is poorly understood. Here we show that dopamine-mediated enhancement of spike firing in NAcb shell medium spiny neurons was prevented by the PKC inhibitor bisindolylmaleimide but not by the phospholipase C inhibitor 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione, suggesting a role for a diacylglycerol-independent atypical PKC (aPKC) isoform. In this regard, modulation of firing by dopamine was prevented by intracellular perfusion of a pseudosubstrate peptide inhibitor for aPKCs. We also provide evidence, using an in vitro kinase assay, that dopamine receptor activation increased aPKC activity in striatal membranes. Finally, direct activation of PKA with forskolin enhanced firing even during inhibition of aPKCs, suggesting that aPKCs acted upstream of PKA activation. Thus, aPKCs appear to mediate dopaminergic enhancement of spike firing in the NAcb shell, and may therefore play a critical role in NAcb- and dopamine-dependent goal-directed behaviors.

Contribution of protein kinase A and protein kinase C pathways in ultraviolet B-induced IL-8 expression by human keratinocytes

We have previously demonstrated that treatment of the human keratinocyte cell line NCTC 2544 with a UVB dose equivalent to 1 h exposure (100 mJ/cm 2) results in a significant increase of IL-8 production. In this study, we use specific inhibitors to investigate the role of both PKA- and PKC-mediated pathways in the regulation of UVB-induced IL-8 expression in NCTC 2544 cell line. We show here that the treatment of irradiated human keratinocytes with PKA inhibitors [H89 and PKA inhibitor (PKAi)] induced a significant decrease of IL-8 production at both mRNA and protein levels. However, the regulation of IL-8 production seems to be mediated via a cAMP-independent PKA pathway, since drugs known to enhance cAMP concentrations [PGE 2, cholera toxin and dibutyryl cAMP] decrease IL-8 production in irradiated cells by down-regulating NF-κB activation in response to UVB radiation. Using PMA (a potent pharmacological activator of PKC) and calphostin C (a specific PKC inhibitor), we demonstrated an up-regulation of IL-8 in NCTC 2544 cells and a down-regulation of the cytokine in UVB-irradiated cells, respectively. We also observed that in our experimental conditions, staurosporine, an inhibitor of both PKC and PMA-stimulated cellular responses, does not involve PKC inhibition in irradiated cells and significantly decreased NF-κB activity in response to UVB radiation. Finally, we concluded that a cAMP-independent PKA activation and a PKC-associated pathway are probably involved in the regulation of UVB-induced IL-8 synthesis in human keratinocytes.

Acute cardioprotective effects of erythropoietin in infant rabbits are mediated by activation of protein kinases and potassium channels.

Erythropoietin is protective against cardiac ischemia, but the underlying mechanisms are unknown. We determined whether erythropoietin (0.5 - 10.0 U/ml) confers acute cardioprotection in infant rabbit hearts and the contribution of protein kinases, nitric oxide synthase and potassium channels to the underlying mechanism. Hearts from normoxic infant New Zealand White rabbits (n=8/group) were isolated and perfused in the Langendorff mode. Biventricular function was recorded under steady-state conditions prior to 30 min global no-flow ischemia and 35 min reperfusion. Administration of erythropoietin for 15 min immediately prior to ischemia resulted in a concentration-dependent increase in recovery of left and right ventricular developed pressure in rabbit hearts following myocardial ischemia and reperfusion. The optimal concentration of erythropoietin that afforded maximum recovery of developed pressure was manifest at 1.0 U/ml. Erythropoietin (1.0 U/ml) treatment resulted in phosphorylation of PKC, p38 MAP kinase and p42/44 MAP kinase. The cardioprotective effects of erythropoietin were abolished by the protein kinase inhibitors SB203580 (p38 MAP kinase), PD98059 (p42/44 MAP kinase) and chelerythrine (PKC) as well as the potassium channel blockers glibenclamide, HMR 1098, 5-HD and Paxilline. Nitrite and nitrate release from hearts before (2.3 +/- 0.9 nmol/min/g) and after (2.4 +/- 1.9 nmol/min/g) 15 min treatment with erythropoietin (1.0 U/ml) were not different. L-NAME and L-NMA did not block the cardioprotective effect of erythropoietin. We conclude the rapid activation of potassium channels and protein kinases by erythropoietin represents an important new mechanism for increasing cardioprotection.

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells.

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

Sexual dimorphism in the contribution of protein kinase c isoforms to nociception in the streptozotocin diabetic rat

The contribution of second messenger signaling, glucose level and sex hormones to sexual dimorphism in the streptozotocin model of diabetic painful peripheral neuropathy was evaluated. Streptozotocin induced elevation of blood glucose and mechanical hyperalgesia (measured by the Randall-Selitto paw-withdrawal test) were both greater in female rats. Ovariectomy abolished and estrogen implants reconstituted this sexual dimorphism; gonadectomy in males had no effect. An inhibitor of protein kinase Cϵ attenuated hyperalgesia in males and ovariectomized females, but not in normal females or in ovariectomized females with estrogen implants, whereas inhibitors of protein kinase Cδ attenuated hyperalgesia in females but not in males. Inhibitors of protein kinase A, protein kinase C (non-selective), protein kinase G and nitric oxide synthase attenuated hyperalgesia equally in both sexes. Higher blood glucose levels in diabetic females were also sex hormone dependent, and magnitude of hyperalgesia correlated with blood glucose level in diabetic male and female rats. These results demonstrate sexual dimorphism in diabetic hyperalgesia, mediated by sex hormone dependent differences in protein kinase Cϵ and protein kinase Cδ signaling and blood glucose levels and suggest that sex may be an important factor to be considered in the treatment of symptomatic diabetic neuropathy.

Bidirectional integrin alphaIIbbeta3 signalling regulating platelet adhesion under flow: contribution of protein kinase C

Platelet adhesion on von Willebrand factor (vWf) requires the co-ordinated adhesive function of glycoprotein Ib/V/IX and integrin alphaIIbbeta3. Recent evidence [Nesbitt, Kulkarni, Giuliano, Gonclaves, Dopheide, Yap, Harper, Salem and Jackson (2002) J. Biol. Chem. 277, 2965-2972] suggests that outside-in signals from both receptors play important roles in regulating platelet-adhesion dynamics under flow. In the present study, we have examined the mechanisms utilized by protein kinase C (PKC) to promote irreversible platelet adhesion on vWf. We demonstrate that PKC is primarily activated downstream of integrin alphaIIbbeta3, not glycoprotein Ib, during platelet adhesion on vWf. This integrin alphaIIbbeta3-dependent PKC activation establishes a positive-feedback loop that promotes further integrin alphaIIbbeta3 activation, calcium mobilization and firm platelet adhesion. This feedback loop appears to be most relevant at relatively low cytosolic calcium concentrations (mean Delta[Ca(2+)](i);100 nM) as artificially elevating calcium (mean Delta[Ca(2+)](i) > 500 nM) induces integrin alphaIIbbeta3 activation and irreversible platelet adhesion independent of PKC. Our studies demonstrate the existence of a complex signalling relationship operating between PKC, cytosolic calcium and integrin alphaIIbbeta3 that serves to regulate platelet-adhesion dynamics under flow. Furthermore, we have established the existence of PKC-dependent and -independent pathways regulating integrin alphaIIbbeta3 activation and stable platelet adhesion on vWf.

Contributions of protein kinase A anchoring proteins to compartmentation of cAMP signaling in the heart.

The cAMP-dependent protein kinase (PKA) transduces signals in the heart initiated by beta(1)-adrenergic, G-protein-coupled receptors after norepinephrine, sympathetic stimulation. Signaling through this pathway results in a characteristic set of cellular responses, including increases in ion fluxes and contractile strength, mobilization of energy stores, and changes in gene expression. Not all receptors that activate adenylate cyclase and increase cAMP levels, however, cause the cardiac myocyte to react in this manner. Research in the field of signal transduction over the last 25 years has addressed this issue of specificity in signaling by diffusable second messengers. PKA is in part targeted to discrete cellular locations by A-kinase anchoring proteins. Through anchoring and formation of multienzyme complexes, specific, localized signal transduction is possible. I discuss in this review recent advances in the understanding of PKA signaling complexes in the cardiac myocyte.

In rat hepatocytes, the hypertonic activation of Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is mediated by protein kinase C.

1. The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an import of extracellular Na(+) via Na(+) conductance, Na(+)-K(+)-2Cl(-) symport, and Na(+)-H(+) antiport. 2. Here, the protein kinase inhibitors staurosporine (100 nmol l(-1)) and bis-indolyl-maleimide I (400 nmol l(-1)) were used to test for a possible contribution of protein kinase C (PKC) to the hypertonic activation of these transporters in confluent primary cultures. 3. Stimulation of Na(+) conductance was monitored: (i) by use of a differential approach based on Na(+) fluxes, (ii) by means of cable analysis, and (iii) in experiments with low Na(+) pulses. All three experimental protocols in concert demonstrated a block of the activation of Na(+) conductance by staurosporine and bis-indolyl-maleimide I. 4. In addition, both compounds significantly reduced the hypertonic activation of Na(+)-K(+)-2Cl(-) symport (quantified on the basis of furosemide-sensitive (86)Rb(+) uptake) to approximately 30 %. 5. In contrast, neither staurosporine nor bis-indolyl-maleimide I had any detectable effect on the hypertonicity-induced alkalinization of cell pH via Na(+)-H(+) antiport (determined fluorometrically). 6. Staurosporine and bis-indolyl-maleimide I completely blocked the RVI of rat hepatocytes (quantified by means of confocal laser-scanning microscopy). The high efficiency of the block suggests an additional inhibitory effect of both compounds on the activity of Na(+)/K(+)-ATPase (determined as ouabain-sensitive (86)Rb(+) uptake). 7. It is concluded that the hypertonic activation of rat hepatocyte Na(+) conductance and Na(+)-K(+)-2Cl(-) symport--but not Na(+)-H(+) antiport--is probably mediated by PKC.

Activation of store-operated Ca2+ entry in RBL cells without the contribution of protein kinases.

The role of protein kinases on store-operated Ca2+ entry in rat basophilic leukaemia cells (RBL) has been studied using the whole-cell configuration of the patch-clamp technique and Ca2+ imaging with fura-2. Specific inhibitors of tyrosine kinase (lavendustin A), mitogen-activated protein (MAP) kinase (SB 203580, PD 98059), Ca2+/calmodulin-dependent kinase (CaMK, KN-62, KN-93) and protein kinase C (PKC, bisindolylmaleimide I) had no significant effect on peak current amplitude and time constant of activation. Likewise, the broad spectrum kinase blockers H-7 and staurosporine did not alter Ca2+ entry compared to control recordings. Store-mediated Ca2+ entry was unaffected if intracellular ATP was substituted by either adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) or adenylyl-imidodiphosphate (AMP-PNP). Similarly, buffering intracellular Mg2+, an essential cofactor for protein kinases, had no effect on Ca2+ influx. These results indicate that protein phosphorylation by various kinases is not required for the activation of the store-operated Ca2+ current in RBL cells.