Phosphorylation of theα subunit of the sodium channel by protein kinase C

The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases.     

Profiling the interactome of protein kinase C ζ by proteomics and bioinformatics

Background

Protein kinase C ζ (PKCζ), an isoform of the atypical protein kinase C, is a pivotal regulator in cancer. However, the molecular and cellular mechanisms whereby PKCζ regulates tumorigenesis and metastasis are still not fully understood. In this study, proteomics and bioinformatics analyses were performed to establish a protein-protein interaction (PPI) network associated with PKCζ, laying a stepping stone to further understand the diverse biological roles of PKCζ.

Methods

Protein complexes associated with PKCζ were purified by co-immunoprecipitation from breast cancer cell MDA-MB-231 and identified by LC-MS/MS. Two biological replicates and two technical replicates were analyzed. The observed proteins were filtered using the CRAPome database to eliminate the potential false positives. The proteomics identification results were combined with PPI database search to construct the interactome network. Gene ontology (GO) and pathway analysis were performed by PANTHER database and DAVID. Next, the interaction between PKCζ and protein phosphatase 2 catalytic subunit alpha (PPP2CA) was validated by co-immunoprecipitation, Western blotting and immunofluorescence. Furthermore, the TCGA database and the COSMIC database were used to analyze the expressions of these two proteins in clinical samples.

Results

The PKCζ centered PPI network containing 178 nodes and 1225 connections was built. Network analysis showed that the identified proteins were significantly associated with several key signaling pathways regulating cancer related cellular processes.

Conclusions

Through combining the proteomics and bioinformatics analyses, a PKCζ centered PPI network was constructed, providing a more complete picture regarding the biological roles of PKCζ in both cancer regulation and other aspects of cellular biology.

     

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.     

Are calcium-dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes? Studies with Ca2+/calmodulin-dependent protein kinase and protein kinase C

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Insulin promotes neuronal survival via the alternatively spliced protein kinase CδII isoform

Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.     

Modeling the roles of protein kinase Cβ and η in single-cell wound repair

Wounded cells such as Xenopus oocytes respond to damage by assembly and closure of an array of actin filaments and myosin-2 controlled by Rho GTPases, including Rho and Cdc42. Rho and Cdc42 are patterned around wounds in a characteristic manner, with active Rho concentrating in a ring-like zone inside a larger, ring-like zone of active Cdc42. How this patterning is achieved is unknown, but Rho and Cdc42 at wounds are subject to regulation by other proteins, including the protein kinases C. Specifically, Cdc42 and Rho activity are enhanced by PKCβ and inhibited by PKCη. We adapt a mathematical model of Simon and coworkers to probe the possible roles of these kinases. We show that PKCβ likely affects the magnitude of positive Rho–Abr feedback, whereas PKCη acts on Cdc42 inactivation. The model explains both qualitative and some overall quantitative features of PKC–Rho GTPase regulation. It also accounts for the previous, peculiar observation that ∼20% of cells overexpressing PKCη display zone inversions—that is, displacement of active Rho to the outside of the active Cdc42.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

Functional role of the charge at the T538 residue in the control of protein kinase Cθ

We show that protein kinase C (PKC) θ localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCθ, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCθ does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCθ molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCθ.     

Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cδ, and p38 mitogen-activated protein kinase (MAPK)

Mer tyrosine kinase (MerTK) is an integral membrane protein that is preferentially expressed by phagocytic cells, where it promotes efferocytosis and inhibits inflammatory signaling. Proteolytic cleavage of MerTK at an unidentified site leads to shedding of its soluble ectodomain (soluble MER; sMER), which can inhibit thrombosis in mice and efferocytosis in vitro. Herein, we show that MerTK is cleaved at proline 485 in murine macrophages. Site-directed deletion of 6 amino acids spanning proline 485 rendered MerTK resistant to proteolysis and suppression of efferocytosis by cleavage-inducing stimuli. LPS is a known inducer of MerTK cleavage, and the intracellular signaling pathways required for this action are unknown. LPS/TLR4-mediated generation of sMER required disintegrin and metalloproteinase ADAM17 and was independent of Myd88, instead requiring TRIF adaptor signaling. LPS-induced cleavage was suppressed by deficiency of NADPH oxidase 2 (Nox2) and PKCδ. The addition of the antioxidant N-acetyl cysteine inhibited PKCδ, and silencing of PKCδ inhibited MAPK p38, which was also required. In a mouse model of endotoxemia, we discovered that LPS induced plasma sMER, and this was suppressed by Adam17 deficiency. Thus, a TRIF-mediated pattern recognition receptor signaling cascade requires NADPH oxidase to activate PKCδ and then p38, culminating in ADAM17-mediated proteolysis of MerTK. These findings link innate pattern recognition receptor signaling to proteolytic inactivation of MerTK and generation of sMER and uncover targets to test how MerTK cleavage affects efferocytosis efficiency and inflammation resolution in vivo.     

Molecular characterization of the human protein kinase C θ * gene locus (PRKCQ)

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16?000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated.     

Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ^−/−) mice as compared with wild-type (PKCδ^+/+) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases.     

Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser₂₅₉ and Ser₄₉₈), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser₄₉₈ of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser₂₅₉ of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser₂₅₉ peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC₅₀ values; (c) recombinant human PKCδ can directly phosphorylate full length Ser₂₅₉ HDAC5 protein and HDAC5 Ser₂₅₉ peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser₇₄₄ and Ser₇₄₈ at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser₂₅₉.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock

Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory β-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.     

Prostaglandin E₂ modulates proximal tubule Na+-ATPase activity: cooperative effect between protein kinase A and protein kinase C

Previous data showed that prostaglandin E? (PGE?) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE? on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE? on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE? increased [3?S]GTPγS binding; (2) GDPβS abolished the inhibitory effect of PGE?; (3) PGE? increased PKA activity; (4) the inhibitory effect of PGE? was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE? on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE? stimulates Ca2+-independent phospholipase A? activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE? on proximal tubule sodium reabsorption.     

Identification of novel pyrrolopyrazoles as protein kinase C β II inhibitors

A novel series of pyrrolopyrazole-based protein kinase C β II inhibitors has been identified from high-throughput screening. Herein, we report our initial structure-activity relationship studies with a focus on optimizing compound ligand efficiency and physicochemical properties, which has led to potent inhibitors with good cell permeability.     

Crystal structure and allosteric activation of protein kinase C βII

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C βII was determined at 4.0 ?, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCβII was derived from small-angle X-ray scattering. Together, these results show how PKCβII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.