Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased ( approximately +30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 microm) in the absence of PICK1, was strongly potentiated ( approximately +300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [(32)P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, (39)TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.     

Protein kinase C alpha controls erythropoietin receptor signaling

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent renewal divisions induced in the presence of Epo, stem cell factor, and dexamethasone.     

Protein kinase C signaling controls skeletal muscle fiber types

Slow myosin heavy chain 2 (MyHC2) gene expression in fetal avian skeletal muscle fibers is regulated by innervation and protein kinase C (PKC) activity. Fetal chick muscle fibers derived from the slow twitch medial adductor (MA) muscle express slow MyHC2 when innervated in vitro. The same pattern of slow MyHC2 regulation occurs in MA muscle fibers in which PKC activity is inhibited by staurosporine. To further test the function of PKC activity in the regulation of slow MyHC2 expression, wild-type and dominant-negative mutations of PKCalpha and PKCtheta were overexpressed in MA muscle fibers in vitro. Overexpression of wild-type PKCalpha and PKCtheta cDNAs resulted in increased PKC activities in muscle fibers and concomitant repression of slow MyHC2 expression under conditions that normally induced gene expression. Point mutations leading to single amino acid substitutions were generated in the ATP binding domains of PKCalpha and PKCtheta. Overexpression of CMVPKCalphaR368 and CMVPKCthetaR409 resulted in decreased PKC activities in transfected MA muscle fibers. Furthermore, transfection of CMVPKCalphaR368 and CMVPKCthetaR409 mutant constructs into MA muscle fibers did not repress the capacity of these fibers to express slow MyHC2 when cultured in medium containing staurosporine or when innervated. These results indicate that PKC activity represses slow MyHC2 expression and that PKC down-regulation, possibly in response to innervation, is required but not sufficient for slow MyHC2 expression.     

Protein kinase C alpha (PKC alpha): regulation and biological function

Protein kinase C alpha (PKC alpha) is a serine/threonine kinase and a member of the conventional (classical) PKCs (cPKCs), which have four conserved (C1 to C4) regions. This ubiquitously expressed PKC isotype is activated in response to many different kinds of stimuli and translocates from cytosol to the specialized cellular compartments (nucleus, focal adhesion, caveolae, etc.) where it is presumed to work. Therefore, PKC alpha has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation, motility, and inflammation. However, the responses induced by activation or overexpression of PKC alpha vary depending on the types, and sometimes conditions, of cells. For example, in some types of cells, PKC alpha is implicated in cell growth. In contrast, it may play a role in cell cycle arrest and differentiation in other types of cells. Therefore, alterations of cell responses induced by PKC alpha are not an intrinsic property of this isoform. The responses are modulated by dynamic interactions with cell-type specific factors: substrates, modulators and anchoring proteins.     

Protein kinase C isoform expression and activity in the mouse heart

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.     

Protein kinase C controls activation of the DNA integrity checkpoint

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.     

Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon)

The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.     

Protein kinase C gamma (PKC gamma): function of neuron specific isotype

The gamma isotype of protein kinase C (PKC gamma) is a member of the classical PKC (cPKC) subfamily which is activated by Ca(2+) and diacylglycerol in the presence of phosphatidylserine. Physiologically, PKC gamma is activated by a mechanism coupled with receptor-mediated breakdown of inositol phospholipid as other cPKC isotypes such as PKC alpha and PKC beta. PKC gamma is expressed solely in the brain and spinal cord and its localization is restricted to neurons, while PKC alpha and PKC beta are expressed in many tissues in addition to the brain. Within the brain, PKC gamma is the most abundant in the cerebellum, hippocampus and cerebral cortex, where the existence of neuronal plasticity has been demonstrated. Pharmacological and electrophysiological studies have shown that several neuronal functions, including long term potentiation (LTP) and long term depression (LTD), specifically require PKC gamma. Generation of mice deficient in PKC gamma provided more information regarding the physiological functions of this isotype. PKC gamma deficient mice (i) have modified long term potentiation (LTP) in hippocampus, (ii) exhibit mild deficits in spatial and contextual learning (iii) exhibit impaired motor coordination due to persistent multiple innervations of climbing fibers on Purkinje cells, (iv) show attenuation of opioid receptor activation, and (v) show decreased effects of ethanol on type A of gamma-aminobutyric acid (GABA) receptor. Furthermore, a point mutation in the PKC gamma gene may contribute to retinitis pigmentosa and Parkinsonian syndrome. This article reviews the specific functions of this neuron-specific isotype of PKC in neuronal signal transduction.     

Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation

The hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase required for replication of the HCV RNA genome. We have identified a peptide that most closely resembles a short region of the protein kinase C-related kinase 2 (PRK2) by screening of a random 12-mer peptide library displayed on the surface of the M13 bacteriophage with NS5B proteins immobilized on microwell plates. Competitive phage enzyme-linked immunosorbent assay with a synthetic peptide showed that the phage clone displaying this peptide could bind HCV RNA polymerase with a high affinity. Coimmunoprecipitation and colocalization studies demonstrated in vivo interaction of NS5B with PRK2. In vitro kinase assays demonstrated that PRK2 specifically phosphorylates NS5B by interaction with the N-terminal finger domain of NS5B (amino acids 1-187). Consistent with the in vitro NS5B-phosphorylating activity of PRK2, we detected the phosphorylated form of NS5B by metabolic cell labeling. Furthermore, HCV NS5B immunoprecipitated from HCV subgenomic replicon cells was specifically recognized by an antiphosphoserine antibody. Knock-down of the endogenous PRK2 expression using a PRK2-specific small interfering RNA inhibited HCV RNA replication. In contrast, PRK2 overexpression, which was accompanied by an increase of in the level of its active form, dramatically enhanced HCV RNA replication. Altogether, our results indicate that HCV RNA replication is regulated by NS5B phosphorylation by PRK2.     

Protein kinase C (PKC) isoforms in Drosophila

Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.     

Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.     

Protein kinase C regulation: C1 meets C-tail

In a recent issue of Cell, Leonard et?al. (2011) describe the structure of PKCβII, an AGC kinase, revealing an unanticipated intramolecular autoinhibitory interaction between its C-terminal tail and the diacylglycerol and phorbol ester binding site of its C1b domain.     

Protein kinase C controls microtubule-based traffic but not proteasomal degradation of c-Met

Upon hepatocyte growth factor stimulation, its receptor c-Met is rapidly internalized via clathrin-coated vesicles and traffics through an early endosomal compartment. We show here that c-Met accumulates progressively in perinuclear compartments, which in part include the Golgi. The c-Met content in the Golgi is principally the newly synthesized precursor form and, to a lesser extent, the internalized, recycling c-Met. By following the trafficking of c-Met inside the cell using a semi-automatic procedure and using inhibition or activation of protein kinase C (PKC) and microtubule depolymerizing agents, we show that PKC positively controls the trans-cytosolic movement of c-Met along microtubules. In parallel to its traffic, internalized c-Met is progressively degraded by a proteasome-sensitive mechanism; the lysosomal pathway does not play a substantial role. Inhibition or promotion of c-Met traffic to the perinuclear compartment does not alter the kinetics of proteasome-dependent c-Met degradation. Thus susceptibility to proteasomal degradation is not a consequence of post-endocytic traffic. The data define a PKC-controlled traffic pathway for c-Met that operates independently of its degradative pathway.     

Protein kinase C activity and isoform expression during early postnatal development of rat myocardium

Total protein kinase C (PKC) activity, its isoform expression, and concentration and fatty acid (FA) composition of diacylglycerol (DAG) were determined in the left ventricular myocardium of the rat during early postnatal development (d 2, 3, 5, 7, and 10). PKC activity measured by the incorporation of ³²P into histone IIIS decreased between d 2 and 10 in the homogenate as well as in cytosolic, membrane (100,000g), and nuclear-cytoskeletal-myofilament fractions (1000g). Likewise, the expression of PKC isoforms (α, δ, and ε) determined by immunoblotting generally declined during the period analyzed, although with a variable pattern. In the membrane and nuclear cytoskeletal myofilament fractions, PKCδ and PKCε expression decreased markedly by d 3, returning to or close to the d 2 level immediately on d 5. PKCα expression in the membrane fraction remained almost unchanged by d 7, declining thereafter. PKCδ and PKCε were associated predominantly with particulate fractions, whereas PKCα was more abundant in the cytosolic fraction. DAG concentration exhibited a significant decline by d 5, consistent with the decrease in maximal PKC activity. The unsaturation index of FA in DAG tended to decrease on d 3 owing to the lowered proportion of all polyunsaturated FA of n−6 and n−3 series. These results demonstrate that the developmental decrease in PKC activity and expression in the rat myocardium is not linear and that subcellular localization of the enzyme exhibits isoform-specific day-by-day changes during the early postnatal period. These changes are compatible with the view that PKC signaling may be involved in the control of a rapid switch of myocardial growth pattern during the first week of life.     

Protein kinase Ctheta controls Th1 cells in experimental autoimmune encephalomyelitis

Molecules that regulate encephalitogenic T cells are of interest for multiple sclerosis. In this study we show that protein kinase Ctheta (PKCtheta) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. PKCtheta-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKCtheta, but had severely diminished IFN-gamma, TNF, and IL-17 production. Increasing Ag exposure and inflammatory conditions failed to induce EAE in PKCtheta-deficient mice, showing a profound defect in the myelin oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKCtheta in the generation and effector function of autoimmune Th1 cells.     

Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry

Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk1^11A), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.     

Up-regulation of a non-kinase activity isoform of Ca(2+)/calmodulin-dependent protein kinase kinase beta1 (CaMKKbeta1) in hibernating bat brain

To improve hybrid tilapia (Oreochromis niloticusxO. aureus) survival under cold shock, the influence of diets containing various dietary lipids was investigated. Four different diets were used which consisted of 12% fish oil, 12% palmitoleic oil 12% coconut oil, and a mixture of fish oil (7%) and corn oil (5%). Our results showed that during cold shock, the proportion of saturated fatty acids in the fish steadily and significantly decreased for all of the diets, but the proportion of monounsaturated fatty acids increased. Proportions of polyenoic fatty acids initially increased then stabilized for the mixed, fish, and coconut oil diets, but did not significantly increase until day 4 for the palmitoleic oil diet. The stearoyl-CoA desaturase (SCD) activity was the lowest on day 0 and then gradually increased for all diets. At any point, the enzymatic activity of SCD was the highest for fish on the mixed and the coconut oil diet, followed by the palmitoleic oil diet, and was lowest for the fish oil diet. The expression of SCD mRNA steadily increased for all diets, but increased more substantially for the mixed diet. On day 6, the expression was the highest for fish on the mixed diet, followed by the coconut oil diet, with the lowest levels for those on the palmitoleic and fish oil diets. These results show that dietary lipids strongly affect the fatty acid composition and SCD expression in tilapia under cold shock, and cold tolerance of this species is also affected.