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1
Muramatsu, M., K. Kaibuchi, and K. Arai. "A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester." Molecular and Cellular Biology 9, no. 2 (February 1989): 831–36. http://dx.doi.org/10.1128/mcb.9.2.831-836.1989.
Texto completo da fonteResumo:
We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
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Muramatsu, M., K. Kaibuchi, and K. Arai. "A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester." Molecular and Cellular Biology 9, no. 2 (February 1989): 831–36. http://dx.doi.org/10.1128/mcb.9.2.831.
Texto completo da fonteResumo:
We constructed mutant protein kinase C (PKC) cDNAs which expressed PKC activity in vivo in the absence of phorbol ester activation. A hybrid PKC gene, PKAC, was constructed by substituting the coding region for the N-terminal 253 amino acids of PKC alpha with the N-terminal 17 amino acids of the cyclic AMP-dependent protein kinase catalytic subunit (PKA). A truncated PKC gene, delta PKC beta, lacking the coding region for amino acid positions 6 to 159 of PKC beta was also constructed. These mutant kinase genes expressed under the control of the SR alpha promoter activated the c-fos gene enhancer in Jurkat cells and initiated maturation of Xenopus laevis oocytes. Phorbol ester binding activity was absent in both constructs but was preserved in another hybrid gene, PKCA, which was composed of the coding region for 1 to 253 amino acids of PKC alpha at the N-terminal side and the coding region for 18 to 350 amino acids of PKA at the C-terminal side. These results indicate that elimination of the regulatory domain of PKC produces constitutively active PKC that can bypass activation by the phorbol ester. delta PKC beta, in synergy with a calcium ionophore, was capable of activating the interleukin 2 promoter, indicating that cooperation of PKC-dependent and calcium-dependent pathways is necessary for activation of the interleukin 2 gene.
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Tremblay, Patricia G., and Marc-André Sirard. "Gene analysis of major signaling pathways regulated by gonadotropins in human ovarian granulosa tumor cells (KGN)†." Biology of Reproduction 103, no. 3 (May 19, 2020): 583–98. http://dx.doi.org/10.1093/biolre/ioaa079.
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Abstract The female reproductive function largely depends on timing and coordination between follicle-stimulating hormone (FSH) and luteinizing hormone. Even though it was suggested that these hormones act on granulosa cells via shared signaling pathways, mainly protein kinases A, B, and C (PKA, PKB, and PKC), there is still very little information available on how these signaling pathways are regulated by each hormone to provide such differences in gene expression throughout folliculogenesis. To obtain a global picture of the principal upstream factors involved in PKA, PKB, and PKC signaling in granulosa cells, human granulosa-like tumor cells (KGN) were treated with FSH or specific activators (forskolin, SC79, and phorbol 12-myristate 13-acetate) for each pathway to analyze gene expression with RNA-seq technology. Normalization and cutoffs (FC 1.5, P ≤ 0.05) revealed 3864 differentially expressed genes between treatments. Analysis of major upstream regulators showed that PKA is a master kinase of early cell differentiation as its activation resulted in the gene expression profile that accompanies granulosa cell differentiation. Our data also revealed that the activation of PKC in granulosa cells is also a strong differentiation signal that could control “advanced” differentiation in granulosa cells and the inflammatory cascade that occurs in the dominant follicle. According to our results, PKB activation provides support for PKA-stimulated gene expression and is also involved in granulosa cell survival throughout follicular development. Taken together, our results provide new information on PKA, PKB, and PKC signaling pathways and their roles in stimulating a follicle at the crossroad between maturation/ovulation and atresia.
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Blount, Mitsi A., Penelope Cipriani, Sara K. Redd, Ronald J. Ordas, Lauren N. Black, Diane L. Gumina, Carol A. Hoban, Janet D. Klein та Jeff M. Sands. "Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct". American Journal of Physiology-Cell Physiology 309, № 9 (1 листопада 2015): C608—C615. http://dx.doi.org/10.1152/ajpcell.00171.2014.
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Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.
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Lacroix, M., and A. Hontela. "Regulation of acute cortisol synthesis by cAMP-dependent protein kinase and protein kinase C in a teleost species, the rainbow trout (Oncorhynchus mykiss)." Journal of Endocrinology 169, no. 1 (April 1, 2001): 71–78. http://dx.doi.org/10.1677/joe.0.1690071.
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The effects of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) on acute ACTH-stimulated cortisol secretion were assessed using a specific PKA inhibitor (H-89) and a PKC activator (phorbol 12-myristate 13-acetate, PMA) in dispersed head kidney cells of rainbow trout (Oncorhynchus mykiss). To investigate the sites of action of both PKA and PKC, pregnenolone (a cortisol precursor stemmed from the rate limiting step in cortisol synthesis) and 25-OH-cholesterol (an exogenous substrate that bypasses the rate limiting step) were used as substrates, with and without ACTH stimulation. Inhibition of PKA decreased ACTH-stimulated cortisol secretion while activation of PKC had the same effect, demonstrating that PKA stimulates and PKC inhibits cortisol synthesis. Inhibition of PKA and activation of PKC had no significant effect on pregnenolone-stimulated cortisol synthesis, indicating that both PKA and PKC act upstream from the pregnenolone step. Inhibition of PKA and activation of PKC had no significant effect on basal cortisol secretion in the presence of 25-OH-cholesterol, suggesting that PKA and PKC exert their effects on the mitochondrial cholesterol translocation step. This study provided evidence for the stimulatory role of PKA and the inhibitory role of PKC in the signalling pathways leading to cortisol synthesis in teleosts.
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Itoh, Hiroyuki, Shinji Yamamura, J. Anthony Ware, Shaobin Zhuang, Shinsuke Mii, Bo Liu, and K. Craig Kent. "Differential effects of protein kinase C on human vascular smooth muscle cell proliferation and migration." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 1 (July 1, 2001): H359—H370. http://dx.doi.org/10.1152/ajpheart.2001.281.1.h359.
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Vascular smooth muscle cell (SMC) migration and proliferation contribute to intimal hyperplasia, and protein kinase C (PKC) may be required for both events. In this report, we investigated the role of PKC in proliferation and migration of SMC derived from the human saphenous vein. Activation of PKC by phorbol-12,13-dibutyrate (PDBu) or (−)-indolactam [(−)-ILV] increases SMC proliferation. Downregulation of PKC activity by prolonged incubation with phorbol ester or inhibition of PKC with chelerythrine in SMC diminished agonist-stimulated proliferation. In contrast, stimulation of PKC with PDBu or (−)-ILV inhibited basal and agonist-induced SMC chemotaxis. Moreover, downregulation of PKC or inhibition with chelerythrine accentuated migration. We postulated that the inhibitory effect of PKC on SMC chemotaxis was mediated through cAMP-dependent protein kinase (protein kinase A, PKA). In support of this hypothesis, we found that activation of PKC in SMC stimulated PKA activity. The cAMP agonist forskolin significantly inhibited SMC chemotaxis. Furthermore, the inhibitory effect of PKC on SMC chemotaxis was completely reversed by cAMP or PKA inhibitors. In search of the PKC isotype(s) underlying these differential effects of PKC in SMC, we identified eight isotypes expressed in human SMC. Only PKC-α, -βI, -δ, and -ε were eliminated by downregulation, suggesting that one or more of these four enzymes facilitate the observed phorbol ester-dependent effects of PKC in SMC. In summary, we found that PKC activation enhances proliferation but inhibits migration of human vascular SMC. These differential effect of PKC on vascular cells appears to be mediated through PKC-α, -βI, -δ, and/or -ε.
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Chen, Yongyue, Guillermo A. Altenberg, and Luis Reuss. "Mechanism of activation of Xenopus CFTR by stimulation of PKC." American Journal of Physiology-Cell Physiology 287, no. 5 (November 2004): C1256—C1263. http://dx.doi.org/10.1152/ajpcell.00229.2004.
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PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR ( XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ∼90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (∼30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.
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Hou, Lili, Lei Zhu, Min Zhang, Xingyi Zhang, Guoqing Zhang, Zhenwei Liu, Qiang Li, and Xin Zhou. "Participation of Antidiuretic Hormone (ADH) in Asthma Exacerbations Induced by Psychological Stress via PKA/PKC Signal Pathway in Airway-Related Vagal Preganglionic Neurons (AVPNs)." Cellular Physiology and Biochemistry 41, no. 6 (2017): 2230–41. http://dx.doi.org/10.1159/000475638.
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Aims: Present study was performed to examine whether ADH was implicated in psychological stress asthma and to explore the underlying molecular mechanism. Methods: We not only examined ADH levels in the cerebrospinal fluid (CSF) via radioimmunoassay, but also measured ADH receptor (ADHR) expression in airway-related vagal preganglionic neurons (AVPNs) through real-time PCR in all experimental mice. Western blotting was performed to evaluate the relationship between ADH and PKA/PKC in psychological stress asthma. Finally, the role of PKA/PKC in psychological stress asthma was analyzed. Results: Marked asthma exacerbations were noted owing to significantly elevated levels of ADH and ADHR after psychological stress induction as compared to OVA alone (asthma group). ADHR antagonists (SR-49095 or SR-121463A) dramatically lowered higher protein levels of PKAα and PKCα induced by psychological stress as compared to OVA alone, suggesting the correlation between ADH and PKA/PKC in psychological stress asthma. KT-5720 (PKA inhibitor) and Go-7874 (PKC inhibitor) further directly revealed the involvement of PKA/PKC in psychological stress asthma. Some notable changes were also noted after employing PKA and PKC inhibitors in psychological stress asthma, including reduced asthmatic inflammation (lower eosinophil peroxidase (EPO) activity, myeloperoxidase (MPO) activity, immunoglobulin E (IgE) level, and histamine release), substantial decrements in inflammatory cell counts (eosinophils and lymphocytes), and decreased cytokine secretion (IL-6, IL-10, and IFN-γ), indicating the involvement of PKA/PKC in asthma exacerbations induced by psychological stress. Conclusion: Our results strongly suggested that ADH participated in psychological stress-induced asthma exacerbations via PKA/PKC signal pathway in AVPNs.
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Wartmann, M., D. A. Jans, P. J. Parker, Y. Nagamine, B. A. Hemmings, S. Jaken, U. Eppenberger, and D. Fabbro. "Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes." Cell Regulation 2, no. 6 (June 1991): 491–502. http://dx.doi.org/10.1091/mbc.2.6.491.
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Phorbol esters, by activating protein kinase C (PKC), induce the expression of the urokinase-type plasminogen activator (uPA) gene and the proto-oncogene c-fos in LLC-PK1 (PK1) porcine kidney epithelial cells. To investigate the role of PKC in the regulation of these two 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes, the alpha-type PKC, the predominant subtype present in the PK1 cells, was overexpressed in this cell line. Two clonal PK1 derivatives overexpressing the alpha PKC 15- and 20-fold, respectively, were established. Compared with the parental and control cells, only a modest but substantially sustained (2- to 3-fold) increase in the accumulation of uPA as well as c-fos mRNAs were observed by TPA in these cells. These results indicate that the extent of induction of these genes mediated by TPA was not proportional to the amounts of alpha-type PKC stably overexpressed in these cells, suggesting that factor(s) downstream of the activation of the alpha PKC appear to be rate limiting for the induction of both TPA-inducible genes in PK1 cells.
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Wu, D., I. J. Clarke, and C. Chen. "The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs." Journal of Endocrinology 154, no. 2 (August 1997): 219–30. http://dx.doi.org/10.1677/joe.0.1540219.
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Abstract The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P<0·001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP–protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP–PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP–PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP–PKA pathway in ovine somatotrophs during the action of GRF or GHRP. Journal of Endocrinology (1997) 154, 219–230
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Dai, Yue, Larry M. Jordan, and Brent Fedirchuk. "Modulation of Transient and Persistent Inward Currents by Activation of Protein Kinase C in Spinal Ventral Neurons of the Neonatal Rat." Journal of Neurophysiology 101, no. 1 (January 2009): 112–28. http://dx.doi.org/10.1152/jn.01373.2007.
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Neuronal excitability can be regulated through modulation of voltage threshold ( Vth). Previous studies suggested that this modulation could be mediated by modulation of transient sodium currents ( IT) and/or persistent inward current (PIC). Modulation of IT and PIC through activation of protein kinase C (PKC) has previously been described as a mechanism controlling neuronal excitability. We investigated modulation of IT and PIC by PKC in neonatal rat spinal ventral neurons. In whole cell voltage clamp, activation of PKC by application of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10–30 μM) resulted in 1) a reduction of IT amplitude by 33% accompanied an increase in half-width and a decrease in the maximal rise and decay rates of the IT; 2) a reduction of PIC amplitude by 49%, with a depolarization of PIC onset by 4.5 mV. Activation of PKC caused varied effects on Vth for eliciting IT, with an unchanged Vth or depolarized Vth being the most common effects. In current-clamp recordings, PKC activation produced a small but significant depolarization (2.0 mV) of Vth for action potential generation with an increase in half-width and a decrease in amplitude and the maximal rise and decay rates of action potentials. Inclusion of PKCI19–36 (10–30 μM), a PKC inhibitor, in the recording pipette could block the OAG effects on IT and PIC. The ability of serotonin to hyperpolarize Vth was not altered by PKC activation or inhibition. This study demonstrates that activation of PKC decreases the excitability of spinal ventral neurons and that Vth can be modulated by multiple mechanisms.
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Wrede, CE, LM Dickson, MK Lingohr, I. Briaud, and CJ Rhodes. "Fatty acid and phorbol ester-mediated interference of mitogenic signaling via novel protein kinase C isoforms in pancreatic beta-cells (INS-1)." Journal of Molecular Endocrinology 30, no. 3 (June 1, 2003): 271–86. http://dx.doi.org/10.1677/jme.0.0300271.
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It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Go6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Go6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.
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Garcia, Balañà, Lanuza, Tomàs, Cilleros-Mañé, Just-Borràs та Tomàs. "Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination". Cells 8, № 11 (23 жовтня 2019): 1304. http://dx.doi.org/10.3390/cells8111304.
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Background: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. Methods: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5–P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. Results: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCβI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. Conclusions: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.
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Beguin, P., A. Beggah, S. Cotecchia, and K. Geering. "Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase." American Journal of Physiology-Cell Physiology 270, no. 1 (January 1, 1996): C131—C137. http://dx.doi.org/10.1152/ajpcell.1996.270.1.c131.
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Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.
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Hu, Hui-Juan, Kathi S. Glauner, and Robert W. Gereau. "ERK Integrates PKA and PKC Signaling in Superficial Dorsal Horn Neurons. I. Modulation of A-Type K+ Currents." Journal of Neurophysiology 90, no. 3 (September 2003): 1671–79. http://dx.doi.org/10.1152/jn.00340.2003.
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The transient outward potassium currents (also known as A-type currents or IA) are important determinants of neuronal excitability. In the brain, IA is modulated by protein kinase C (PKC), protein kinase A (PKA), and extracellular signal-related kinase (ERK), three kinases that have been shown to be critical modulators of nociception. We wanted to determine the effects of these kinases on IA in superficial dorsal horn neurons. Using whole cell recordings from cultured mouse spinal cord superficial dorsal horn neurons, we found that PKC and PKA both inhibit IA in these cells, and that PKC has a tonic inhibitory action on IA. Further, we provide evidence supporting the hypothesis that PKC and PKA do not modulate IA directly, but rather act as upstream activators of ERKs, which modulate IA. These results suggest that ERKs serve as signal integrators in modulation of IA in dorsal horn neurons and that modulation of A-type potassium currents may underlie aspects of central sensitization mediated by PKC, PKA, and ERKs.
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Klein, Janet D., Christopher F. Martin, Kimilia J. Kent та Jeff M. Sands. "Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct". American Journal of Physiology-Renal Physiology 302, № 9 (1 травня 2012): F1098—F1103. http://dx.doi.org/10.1152/ajprenal.00664.2011.
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The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [32P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.
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Erclik, Mary S., та Jane Mitchell. "The role of protein kinase C-δ in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106–01 cells". American Journal of Physiology-Endocrinology and Metabolism 282, № 3 (1 березня 2002): E534—E541. http://dx.doi.org/10.1152/ajpendo.00417.2001.
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We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106–01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 μM bovine PTH-(3–34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1–34) and PTH-(3–34) (100 nM) both stimulated PKC-δ translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-δ-specific inhibitor, and a dominant negative mutant of PKC-δ were both able to significantly inhibit PTH-(1–34) and PTH-(3–34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-δ in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-α translocation from the cytosol to the membrane and inhibited ∼50% of the PTH-(1–34), forskolin, and 8-bromoadenosine 3′,5′-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-α negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-δ is the primary mediator of the effects of PTH via the PKC pathway.
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Hu, Hui-Juan, and Robert W. Gereau. "ERK Integrates PKA and PKC Signaling in Superficial Dorsal Horn Neurons. II. Modulation of Neuronal Excitability." Journal of Neurophysiology 90, no. 3 (September 2003): 1680–88. http://dx.doi.org/10.1152/jn.00341.2003.
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Protein kinases belonging to the protein kinase A (PKA), protein kinase C (PKC), and extracellular signal-related kinase (ERK) families have been identified as key players in modulating nociception at the level of the spinal cord dorsal horn, yet little is known about the effects of these kinases on membrane properties of the dorsal horn neurons. PKA, PKC, and ERK exert inhibitory effects on transient potassium currents (A-type currents or IA) in mouse superficial dorsal horn neurons ( Hu et al. 2003 ). Here we aimed to determine the effects of these kinases on action potential firing and membrane properties of these neurons to evaluate the impact of the modulation of IA (and other conductances) in these neurons. We found that activating PKC and PKA has dramatic effects on action potential firing, reflecting an increase in the excitability of superficial dorsal horn neurons. In addition, we found that inhibitors of both PKC and ERK signaling decrease the excitability of dorsal horn neurons, suggesting that these kinases exert a tonic excitation of these cells. Consistent with our findings that these kinases inhibit A-type currents, we found that PKA, PKC, and ERK act to shorten the first-spike latency after depolarization induced by current injection. In addition, activation of these kinases increases spike frequency and action potential amplitude of dorsal horn neurons. Interestingly, we found that the effects of PKA and PKC activators are blocked by inhibitors of ERK signaling, suggesting that PKA and PKC may exert their actions by activation of ERKs.
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Button, Brian, Luis Reuss, and Guillermo A. Altenberg. "Pkc-Mediated Stimulation of Amphibian Cftr Depends on a Single Phosphorylation Consensus Site. Insertion of This Site Confers Pkc Sensitivity to Human Cftr." Journal of General Physiology 117, no. 5 (April 30, 2001): 457–68. http://dx.doi.org/10.1085/jgp.117.5.457.
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Mutations of the CFTR, a phosphorylation-regulated Cl− channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.
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Yano, Kenji, Jeanette R. Bauchat, Marya B. Liimatta, David R. Clemmons, and Cunming Duan. "Down-Regulation of Protein Kinase C Inhibits Insulin-Like Growth Factor I-Induced Vascular Smooth Muscle Cell Proliferation, Migration, and Gene Expression1." Endocrinology 140, no. 10 (October 1, 1999): 4622–32. http://dx.doi.org/10.1210/endo.140.10.7035.
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Abstract Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, directed migration, differentiation, and apoptosis. The signaling mechanisms used by IGF-I to elicit these actions, however, are not well defined. In this study, we examined the role(s) of protein kinase C (PKC) in mediating the IGF-I actions in cultured porcine VSMCs. Out of the eleven known members of PKC family, PKC-α, -βI, -ε, -η, -λ,θ , and -ζ, were detectable by Western immunoblot analysis in these cells. Further analysis indicated that the subcellular distribution of several PKC isoforms is regulated by IGF-I. While IGF-I stimulated membrane translocation of PKC-η, -ε, and -ζ and regulated the cytosolic levels of PKC-βI, it had no such effect on PKC-α and -λ. To examine whether PKC activation is required for the IGF-I-regulated biological responses, phorbol myristate acetate (PMA) and GF109203X were used to down-regulate or inhibit PKC activity. Both PMA (1 μm) and GF109203X (20 μm) nearly completely suppressed the total PKC activity after a 30-min incubation (&gt; 90%), and this inhibition lasted for at least 24 h. Down-regulation or inhibition of PKC activity abolished the IGF-I-induced DNA synthesis, migration and IGFBP-5 gene expression. In contrast, the IGFBP-5 expression induced by forskolin was unaffected by PKC down-regulation or inhibition, suggesting that PKC activation is required for the IGF-regulated but not the cAMP-regulated events. Because the actions of IGF-I on DNA synthesis and IGFBP-5 gene expression in VSMCs have been shown to be mediated through the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway in porcine VSMCs, the potential role of PKC in IGF-I-induced activation of PI3 kinase and PKB/Akt were examined. Treatment with either PMA or GF109203X did not significantly affect the effects of IGF-I on PI3 kinase activation or PKB/Akt phosphorylation. These results indicated that PKC-βI, -η, -ε, and -ζ may play an essential role(s) in IGF-I regulation of VSMC migration, DNA synthesis and gene expression, and that these PKC isoforms may either act independently of the PI3 kinase pathway or act further downstream of PKB/Akt in the IGF signaling network.
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Vanoye, Carlos G., Ariel F. Castro, Thierry Pourcher, Luis Reuss, and Guillermo A. Altenberg. "Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl− currents." American Journal of Physiology-Cell Physiology 276, no. 2 (February 1, 1999): C370—C378. http://dx.doi.org/10.1152/ajpcell.1999.276.2.c370.
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Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl− currents ( I Cl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in I Cl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state I Cl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3′,5′-cyclic monophosphate reduced steady-state I Cl,swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of I Cl,swellactivation. The effects of stimulation of PKA and PKC on I Cl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state I Cl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl− channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of I Cl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects I Cl,swell.
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Rashid, Gloria, Eleanora Plotkin, Osnat Klein, Janice Green, Jacques Bernheim, and Sydney Benchetrit. "Parathyroid hormone decreases endothelial osteoprotegerin secretion: role of protein kinase A and C." American Journal of Physiology-Renal Physiology 296, no. 1 (January 2009): F60—F66. http://dx.doi.org/10.1152/ajprenal.00622.2007.
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Parathyroid hormone (PTH), which is elevated in patients with chronic renal failure, has been shown to participate in the development of vascular calcification. Previous studies have demonstrated that PTH may promote endothelial expressions of proinflammatory parameters. On the basis of these data, we evaluated whether PTH may have an impact on endothelial osteoprotegerin (OPG), a vascular-protective factor which may control vascular calcification. Endothelial cells were stimulated with 10−12 to 10−10 mol/l PTH. PKC and PKA are the main cellular pathways of PTH. Inhibitors and activators of PKC or PKA were used to determine whether these signaling pathways are involved in the control of endothelial OPG. PTH induced a decrease in OPG secretion and mRNA expression. Treatment of PTH-stimulated cells by calphostin C (PKC inhibitor) induced a further decrease in OPG secretion, while Rp-cAMP (PKA inhibitor) had no additional effect. In nonstimulated cells, a PKC activator significantly stimulated OPG secretion, while a PKA activator was associated with a decline. These effects were blunted in the presence of calphostin C and Rp-cAMP, respectively. An increase in OPG secretion induced by a PKC activator indicates that the basal OPG secretion is mediated through PKC. The decrease induced by a PKA activator, which is similar to the decrease observed with PTH, suggests that the action of PTH on OPG secretion and mRNA expression may be due to the PKA pathway.
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Sugden, D., and S. J. Rowe. "Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores." Journal of Cell Biology 119, no. 6 (December 15, 1992): 1515–21. http://dx.doi.org/10.1083/jcb.119.6.1515.
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The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.
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Hayabuchi, Y., N. B. Standen, and N. W. Davies. "Angiotensin II inhibits and alters kinetics of voltage-gated K+ channels of rat arterial smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 6 (December 1, 2001): H2480—H2489. http://dx.doi.org/10.1152/ajpheart.2001.281.6.h2480.
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The vasoconstrictor angiotensin II (ANG II) inhibits several types of K+ channels. We examined the inhibitory mechanism of ANG II on voltage-gated K+ (KV) currents ( I KV ) recorded from isolated rat arterial smooth muscle using patch-clamp techniques. Application of 100 nM ANG II accelerated the activation of I KV but also caused inactivation. These effects were abolished by the AT1 receptor antagonist losartan. The protein kinase A (PKA) inhibitor Rp-cyclic 3′,5′-hydrogen phosphothioate adenosine (100 μM) and an analog of diacylglycerol, 1,2-dioctanyoyl-rac-glycerol (2 μM), caused a significant reduction of I KV . Furthermore, the combination of 5 μM PKA inhibitor peptide 5–24 (PKA-IP) and 100 μM protein kinase C (PKC) inhibitor peptide 19–27 (PKC-IP) prevented the inhibition by ANG II, although neither alone was effective. The ANG II effect seen in the presence of PKA-IP remained during addition of the Ca2+-dependent PKC inhibitor Gö6976 (1 μM) but was abolished in the presence of 40 μM PKC-ε translocation inhibitor peptide. These results demonstrate that ANG II inhibits KVchannels through both activation of PKC-ε and inhibition of PKA.
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Xie, Guofeng, and Jean-Pierre Raufman. "Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 281, no. 4 (October 1, 2001): G1051—G1058. http://dx.doi.org/10.1152/ajpgi.2001.281.4.g1051.
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Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187 -stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.
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Mullin, James M., Jennifer A. Kampherstein, Kathleen V. Laughlin, Cheryl E. K. Clarkin, R. Daniel Miller, Zoltan Szallasi, Bechara Kachar, Alejandro Peralta Soler та Dan Rosson. "Overexpression of protein kinase C-δ increases tight junction permeability in LLC-PK1epithelia". American Journal of Physiology-Cell Physiology 275, № 2 (1 серпня 1998): C544—C554. http://dx.doi.org/10.1152/ajpcell.1998.275.2.c544.
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The Ca2+-independent δ-isoform of protein kinase C (PKC-δ) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-δ is expressed. Western immunoblots show that the overexpressed PKC-δ is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-δ produced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12- O-tetradecanoylphorbol-13-acetate. Transepithelial electrical resistance ( R T) in cell sheets overexpressing PKC-δ was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-δ and R Twere similar to those in LLC-PK1parental cell sheets. Overexpression of PKC-δ also elicited a significant increase in transepithelial flux ofd-[14C]mannitol and a radiolabeled 2 × 106-molecular-weight dextran, suggesting with the R T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-δ overexpression results in a multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-δ results in a more disorganized arrangement of tight junctional strands. As with LLC-PK1 cell sheets treated with 12- O-tetradecanoylphorbol-13-acetate, the reduced R T, increasedd-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-δ overexpression suggest the involvement of PKC-δ in regulation of tight junctional permeability.
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Nakamura, Yuya, Masahiro Inagaki, Mayumi Tsuji, Toshihiko Gocho, Kazuaki Handa, Hitomi Hasegawa, Akihiko Yura, et al. "Linagliptin Has Wide-Ranging Anti-Inflammatory Points of Action in Human Umbilical Vein Endothelial Cells." Japanese Clinical Medicine 7 (January 2016): JCM.S39317. http://dx.doi.org/10.4137/jcm.s39317.
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Background Because of the potential anti-inflammatory effects, linagliptin, a therapeutic dipeptidyl peptidase-4 inhibitor, is used as an effective drug for diabetic patients for whom inflammation is a prognosis-related factor. We investigated the anti-inflammatory mechanism of linagliptin using seven markers. Methods We pretreated human umbilical vein endothelial cells (HUVECs), with linagliptin and lipopolysaccharide (LPS). The cytosolic fractions were evaluated for protein kinase A (PKA), protein kinase B (PKB), protein kinase C (PKC), ratio of reactive oxygen species (ROS) and Cu/Zn superoxide dismutase (SOD), activator protein 1 (AP-1), and adenosine 3′,5′-cyclic monophosphate (cAMP). Results Linagliptin increased the PKA and PKC activities and the cAMP levels in LPS-treated cells. However, it inhibited LPS-induced PKB phosphorylation, ratio of ROS and Cu/Zn SOD, and LPS-stimulated AP-1 nuclear translocation. Conclusion We reaffirmed the anti-inflammatory and antioxidant effects of linagliptin. These effects might be related to the three protein kinases. Our findings suggest that linagliptin has a wide range of anti-inflammatory effects.
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Middleton, Lisa M., and Robert D. Harvey. "PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes." American Journal of Physiology-Cell Physiology 275, no. 1 (July 1, 1998): C293—C302. http://dx.doi.org/10.1152/ajpcell.1998.275.1.c293.
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The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.
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Jiang, Quan, Mulan He, Xinyan Wang та Anderson O. L. Wong. "Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-α and -β gene expression". American Journal of Physiology-Endocrinology and Metabolism 295, № 2 (серпень 2008): E477—E490. http://dx.doi.org/10.1152/ajpendo.90386.2008.
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Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLα and SLβ mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP3)/PKC pathways and subsequent activation of the Ca2+/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLα mRNA expression. Apparently, the downstream Ca2+/CaM-dependent cascades were triggered by extracellular Ca2+ ([Ca2+]e) entry via L-type voltage-sensitive Ca2+ channels (VSCC) and Ca2+ release from IP3-sensitive intracellular Ca2+ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLβ mRNA expression, except that [Ca2+]e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLα and SLβ gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.
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Fuller, William, Jacqueline Howie, Linda M. McLatchie, Roberta J. Weber, C. James Hastie, Kerry Burness, Davor Pavlovic, and Michael J. Shattock. "FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C." American Journal of Physiology-Cell Physiology 296, no. 6 (June 2009): C1346—C1355. http://dx.doi.org/10.1152/ajpcell.00523.2008.
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FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is ∼30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 ± 0.2 pA/pF in unstimulated cells to 2.9 ± 0.1 pA/pF for PKA and 3.4 ± 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump α1-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.
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Vijayaragavan, Kausalia, Mohamed Boutjdir, and Mohamed Chahine. "Modulation of Nav1.7 and Nav1.8 Peripheral Nerve Sodium Channels by Protein Kinase A and Protein Kinase C." Journal of Neurophysiology 91, no. 4 (April 2004): 1556–69. http://dx.doi.org/10.1152/jn.00676.2003.
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Voltage-gated Na+ channels (VGSC) are transmembrane proteins that are essential for the initiation and propagation of action potentials in neuronal excitability. Because neurons express a mixture of Na+ channel isoforms and protein kinase C (PKC) isozymes, the nature of which channel is being regulated by which PKC isozyme is not known. We showed that DRG VGSC Nav1.7 (TTX-sensitive) and Nav1.8 (TTX-resistant), expressed in Xenopus oocytes were differentially regulated by protein kinase A (PKA) and PKC isozymes using the two-electrode voltage-clamp method. PKA activation resulted in a dose-dependent potentiation of Nav1.8 currents and an attenuation of Nav1.7 currents. PKA-induced increases (Nav1.8) and decreases (Nav1.7) in peak currents were not associated with shifts in voltage-dependent activation or inactivation. The PKA-mediated increase in Nav1.8 current amplitude was prevented by chloroquine, suggesting that cell trafficking may contribute to the changes in Nav1.8 current amplitudes. A dose-dependent decrease in Nav1.7 and Nav1.8 currents was observed with the PKC activators phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate. PMA induced shifts in the steady-state activation of Nav1.7 and Nav1.8 channels by 6.5 and 14 mV, respectively, in the depolarizing direction. The role of individual PKC isozymes in the regulation of Nav1.7 and Nav1.8 was determined using PKC-isozyme-specific peptide activators and inhibitors. The decrease in the Nav1.8 peak current induced by PMA was prevented by a specific ϵPKC isozyme peptide antagonist, whereas the PMA effect on Nav1.7 was prevented by ϵPKC and βIIPKC peptide inhibitors. The data showed that Nav1.7 and Nav1.8 were differentially modulated by PKA and PKC. This is the first report demonstrating a functional role for ϵPKC and βIIPKC in the regulation of Nav1.7 and Nav1.8 Na+ channels. Identification of the particular PKC isozymes(s) that mediate the regulation of Na+ channels is essential for understanding the molecular mechanism involved in neuronal ion channel regulation in normal and pathological conditions.
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Rubiś, Błazej, Sylwia Grodecka-Gazdecka, Remigiusz Lecybył, Marta Ociepa, Zygmunt Krozowski, and Wiesław H. Trzeciak. "Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells." Acta Biochimica Polonica 51, no. 4 (December 31, 2004): 919–24. https://doi.org/10.18388/abp.2004_3524.
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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.
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Greco, S., C. Storelli та S. Marsigliante. "Protein kinase C (PKC)-δ/-ε mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin". Journal of Endocrinology 188, № 1 (січень 2006): 79–89. http://dx.doi.org/10.1677/joe.1.06433.
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In this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B2 receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-α (PKC-α) and novel PKC-δ and PKC-ε; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); Gö6976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-δ and PKC-ε suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-δ/-ε, PKB/Akt and ERK1/2.
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Zhang, Q. Y., C. Hammerberg, J. J. Baldassare, P. A. Henderson, D. Burns, M. Ceska, J. J. Voorhees, and G. J. Fisher. "Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts." Journal of Immunology 149, no. 4 (August 15, 1992): 1402–8. http://dx.doi.org/10.4049/jimmunol.149.4.1402.
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Abstract Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.
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Dobado-Berrios, Pablo, Rosa Ros, Antonio Torres, Socorro García-Navarro, Mercé Jardí, Jordi Félez, Francisco Velasco, and Chari López-Pedrera. "Signal Transduction Pathways Underlying the Expression of Tissue Factor and Thrombomodulin in Promyelocytic Cells Induced to Differentiate by Retinoid Acid and Dibutyryl cAMP." Thrombosis and Haemostasis 85, no. 06 (2001): 1031–36. http://dx.doi.org/10.1055/s-0037-1615959.
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SummaryAcute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.
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Rotfeld, H., P. Hillman, D. Ickowicz, and H. Breitbart. "PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade." REPRODUCTION 147, no. 3 (March 2014): 347–56. http://dx.doi.org/10.1530/rep-13-0560.
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To enable fertilization, spermatozoa must undergo several biochemical processes in the female reproductive tract, collectively called capacitation. These processes involve protein kinase A (PKA)-dependent protein tyrosine phosphorylation including phosphatidylinositol-3-kinase (PI3K). It is not known how PKA, a serine/threonine (S/T) kinase, mediates tyrosine phosphorylation of proteins. We recently showed that inhibition of S/T phosphatase 1 (PP1) causes a significant increase in phospho-PI3K. In this study, we propose a mechanism by which PKA and PP1 mediate an increase in PI3K tyrosine phosphorylation and implicate calmodulin-dependent kinase II (CaMKII) in this process. Inhibition of sperm PP1 or PKC, stimulated CaMKII phosphorylation/activation, and inhibition of PKC enhanced PP1 phosphorylation/inactivation. Inhibition of CaMKII, using KN-93, caused significant reduction in phospho-PP1, indicating its activation. Moreover, KN-93 prevented the dephosphorylation/inactivation of PKC. We therefore suggest that CaMKII inhibits PKC, leading to PP1 inhibition and the reciprocal auto-activation of CaMKII. Thus, CaMKII can regulate its own activation by inhibiting the PKC/PP1 cascade. Inhibition of Src family kinases (SFK) caused significant inhibition of CaMKII and PP1 phosphorylation, suggesting that SFK activity results in PP1 inhibition and CaMKII activation. Activation of sperm PKA by 8Br-cAMP revealed an increase in phospho-CaMKII, which was inhibited by PKA inhibitor. Tyrosine phosphorylation of PI3K was stimulated by 8Br-cAMP and by PKC or PP1 inhibition and was abrogated by CaMKII inhibition. Furthermore, phosphorylation/activation of the tyrosine kinase Pyk2 was enhanced by PP1 inhibition, and this activation is blocked by CaMKII inhibition. Thus, PKA activates Src, which inhibits PP1, leading to CaMKII and Pyk2 activation, resulting in PI3K tyrosine phosphorylation/activation.
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Ragolia, Louis, Thomas Palaia, Enesa Paric, and John K. Maesaka. "Elevated L-PGDS activity contributes to PMA-induced apoptosis concomitant with downregulation of PI3-K." American Journal of Physiology-Cell Physiology 284, no. 1 (January 1, 2003): C119—C126. http://dx.doi.org/10.1152/ajpcell.00247.2002.
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Recently we demonstrated the induction of apoptosis by the addition of recombinant lipocalin-type prostaglandin D2 synthase (L-PGDS) to the culture medium of LLC-PK1 cells. Because protein kinase C (PKC) has been shown to be involved in the apoptotic process of various cell types, we examined the potential role of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-induced apoptosis. We report here the enzymatic activation and phosphorylation of L-PGDS in response to phorbol ester in cell culture and the direct phosphorylation of recombinant L-PGDS by PKC in vitro. Treatment of cells with PMA or L-PGDS decreased phosphatidylinositol 3-kinase (PI3-K) activity and concomitantly inhibited protein kinase B (PKB/Akt) phosphorylation, which led to the hypophosphorylation and activation of Bad. In addition, hypophosphorylation of retinoblastoma protein was also observed in response to L-PGDS-induced apoptosis. Cellular depletion of L-PGDS levels by using an antisense RNA strategy prevented PI3-K inactivation by phorbol ester and inhibited caspase-3 activation and apoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKB anti-apoptotic signaling pathways.
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KAPADIA, RINA, PETER D. YURCHENCO, and KURT AMSLER. "Binding of the Renal Epithelial Cell Line LLC-PK1 to Laminin Is Regulated by Protein Kinase C." Journal of the American Society of Nephrology 10, no. 6 (June 1999): 1214–23. http://dx.doi.org/10.1681/asn.v1061214.
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Abstract. The α6β1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of α6β1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-β1 integrin antibodies and by anti-α6 integrin antibodies. Antibodies against α2 integrin and α3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the α6β1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-β1 integrin and anti-α6 integrin antibodies. PKC activation did not alter expression of β1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the α6β1 integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface α6β1 integrin.
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Shaw, Erynn E., Philip Wood, Justyna Kulpa, Feng Hua Yang, Alastair J. Summerlee, and W. Glen Pyle. "Relaxin alters cardiac myofilament function through a PKC-dependent pathway." American Journal of Physiology-Heart and Circulatory Physiology 297, no. 1 (July 2009): H29—H36. http://dx.doi.org/10.1152/ajpheart.00482.2008.
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The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca2+ without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-δ, decreases in PKC-α and -βII, but no effect on PKC-ε. Inhibition of PKC with chelerythrine chloride or PKC-δ with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.
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Zhou, Lian, Douglas A. Baxter, and John H. Byrne. "Contribution of PKC to the maintenance of 5-HT-induced short-term facilitation at sensorimotor synapses of Aplysia." Journal of Neurophysiology 112, no. 8 (October 15, 2014): 1936–49. http://dx.doi.org/10.1152/jn.00577.2013.
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Aplysia sensorimotor synapses provide a useful model system for analyzing molecular processes that contribute to heterosynaptic plasticity. For example, previous studies demonstrated that multiple kinase cascades contribute to serotonin (5-HT)-induced short-term synaptic facilitation (STF), including protein kinase A (PKA) and protein kinase C (PKC). Moreover, the contribution of each kinase is believed to depend on the state of the synapse (e.g., depressed or nondepressed) and the time after application of 5-HT. Here, a previously unappreciated role for PKC-dependent processes was revealed to underlie the maintenance of STF at relatively nondepressed synapses. This PKC dependence was revealed when the synapse was stimulated repeatedly after application of 5-HT. The contributions of the PKA and PKC pathways were examined by blocking adenylyl cyclase-coupled 5-HT receptors with methiothepin and by blocking PKC with chelerythrine. STF was assessed 20 s after 5-HT application. The effects of PKC were consistent with enhanced mobilization of transmitter, as assessed by application of hypertonic sucrose solutions to measure the readily releasable pool of vesicles and recovery of the readily releasable pool after depletion. A computational model of transmitter release demonstrated that a PKC-dependent mobilization process was sufficient to explain the maintenance of STF at nondepressed synapses and the facilitation of depressed synapses.
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Kirton, C. Adam, and Rodger Loutzenhiser. "Alterations in basal protein kinase C activity modulate renal afferent arteriolar myogenic reactivity." American Journal of Physiology-Heart and Circulatory Physiology 275, no. 2 (August 1, 1998): H467—H475. http://dx.doi.org/10.1152/ajpheart.1998.275.2.h467.
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Myogenic vasoconstriction of the renal afferent arteriole contributes to the autoregulation of renal blood flow, glomerular filtration rate, and glomerular capillary pressure (PGC). The reactivity of the afferent arteriole to pressure and the efficiency of PGC control are subject to physiological and pathophysiological alterations, but the determinants of the myogenic response of this vessel are largely unknown. We used the in vitro perfused hydronephrotic rat kidney to investigate the role of protein kinase C (PKC) in the control of this response. Inhibition of PKC by 1 μM chelerythrine attenuated myogenic reactivity but did not affect the afferent arteriole vasoconstrictor response to KCl (35 mM)-induced depolarization. Low concentrations of phorbol ester (10 nM phorbol 12-myristate 13-acetate) and low levels of ANG II or endothelin-1 (3 pM) potentiated myogenic vasoconstriction without affecting basal afferent arteriolar diameters. These actions were blocked by 1 μM chelerythrine, suggesting a PKC-dependent mechanism. Finally, although PKC inhibition attenuated basal myogenic responses, full reactivity to pressure was restored by 1 mM 4-aminopyridine, a pharmacological inhibitor of delayed rectifier K channels, which are known to be modulated by PKC. The ability of 4-aminopyridine to circumvent the effects of PKC inhibition militates against a direct role of PKC in myogenic signaling. We interpret these observations as indicating that basal PKC activity is an important determinant of myogenic reactivity in the renal afferent arteriole. However, PKC activation does not appear to play an obligate role in myogenic signaling in this vessel. We suggest that basal PKC activity directly modulates voltage-gated K channel activity, thereby indirectly affecting myogenic reactivity.
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Rider, M. H., J. Vandamme, E. Lebeau, D. Vertommen, H. Vidal, G. G. Rousseau, J. Vandekerckhove, and L. Hue. "The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing." Biochemical Journal 285, no. 2 (July 15, 1992): 405–11. http://dx.doi.org/10.1042/bj2850405.
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Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
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Chen, Yongyue, Brian Button, Guillermo A. Altenberg, and Luis Reuss. "Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2." American Journal of Physiology-Cell Physiology 287, no. 5 (November 2004): C1436—C1444. http://dx.doi.org/10.1152/ajpcell.00045.2004.
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Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus ( X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability ( Po) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance (γ) was increased by ∼22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH2-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in Po (with a small change in γ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.
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Garcia, Neus, Maria A. Lanuza, Marta Tomàs, Víctor Cilleros-Mañé, Laia Just-Borràs, Maria Duran, Aleksandra Polishchuk, and Josep Tomàs. "PKA and PKC Balance in Synapse Elimination during Neuromuscular Junction Development." Cells 10, no. 6 (June 4, 2021): 1384. http://dx.doi.org/10.3390/cells10061384.
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During the development of the nervous system, synaptogenesis occurs in excess though only the appropriate connections consolidate. At the neuromuscular junction, competition between several motor nerve terminals results in the maturation of a single axon and the elimination of the others. The activity-dependent release of transmitter, cotransmitters, and neurotrophic factors allows the direct mutual influence between motor axon terminals through receptors such as presynaptic muscarinic ACh autoreceptors and the tropomyosin-related kinase B neurotrophin receptor. In previous studies, we investigated the synergistic and antagonistic relations between these receptors and their downstream coupling to PKA and PKC pathways and observed a metabotropic receptor-driven balance between PKA (stabilizes multinnervation) and PKC (promotes developmental axonal loss). However, how much does each kinase contribute in the developmental synapse elimination process? A detailed statistical analysis of the differences between the PKA and PKC effects in the synapse elimination could help to explore this point. The present short communication provides this analysis and results show that a similar level of PKA inhibition and PKC potentiation would be required during development to promote synapse loss.
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Karihaloo, A., K. Kato, D. A. Greene, and T. P. Thomas. "Protein kinase and Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells." American Journal of Physiology-Cell Physiology 273, no. 2 (August 1, 1997): C671—C678. http://dx.doi.org/10.1152/ajpcell.1997.273.2.c671.
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The acute regulation of inwardly directed Na(+)-myo-inositol (MI) cotransporter activity and basal and volume-sensitive MI efflux by protein kinases C (PKC) and A (PKA), cytosolic Ca2+, and phosphoinositide (PI) turnover were characterized in cultured human retinal pigment epithelial cells using 2-[3H]MI and liquid scintillation spectrometry. Kinetic analysis revealed two distinct Na(+)-MI cotransporter components differing in apparent Michaelis constant and maximal velocity. Composite Na(+)-MI cotransport activity was stimulated by PKA activation, the muscarinic agonist carbachol, and the Ca2+ ionophore A-23187 and was inhibited by PKC activation. PKC activation also increased MI efflux, but only the volume-sensitive component, whereas PKA activation increased both basal and volume-sensitive MI efflux. These studies implicate PKC as a negative modulator of MI content through Na(+)-MI cotransport inhibition and potentiation of volume-sensitive MI efflux. PKA is a positive modulator of both Na(+)-MI cotransport and basal and volume-sensitive MI efflux. Cytosolic Ca2+ release through receptor-mediated PI hydrolysis may facilitate Na(+)-MI cotransport activity.
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Matsumoto, Shigeji, Shinki Yoshida, Mizuho Ikeda, Chikako Saiki, and Mamoru Takeda. "Effects of PKC and PKA Inhibitors on the cAMP-Stimulant-Induced Enhancement of Tetrodotoxin-Resistant Na+ (Nav1.8) Currents." Open Pharmacology Journal 2, no. 1 (February 28, 2008): 17–19. http://dx.doi.org/10.2174/1874143600802010017.
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The protein kinase C (PKC) inhibitor bisindolymaleimide Ro-31-8425 (Ro-31-8425) decreases the peak tetrodotoxin- resistant (TTX-R) Na+ (Nav1.8) current in nodose ganglion (NG) neurons, and this decrease is not altered by simultaneous application of 8-bromo-cAMP (8-Br-cAMP), phorbol 12-myristate 13-acetate (PMA, a PKC activator) or forskolin (a cAMP analogue). Intracellular application of the endogenous protein kinase A (PKA) inhibitor, protein kinase inhibitor (PKI) abolishes the increase in the peak Nav1.8 current that occurs in response to the applications of 8-BrcAMP, PMA, forskolin, or prostaglandin E2 (PGE2, an adenyl cyclase activator). At a higher concentration (0.5 mM) compared with a sufficient concentration (0.01 mM) to block the cAMP-stimulant Nav1.8 current, PKI still attenuated the Ro-31-8425-induced decrease in peak Nav1.8 current. When we considered these results together, cAMP-stimulantinduced modification of Nav1.8 currents is mediated by the activation of both PKA and PKC, and PKC may be located upstream of PKA.
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Zheng, Yunhe, Chen Yang, Xiu’e Zheng, Qiangdong Guan, and Sufang Yu. "Acrylamide treatment alters the level of Ca2+ and Ca2+-related protein kinase in spinal cords of rats." Toxicology and Industrial Health 37, no. 3 (January 25, 2021): 113–23. http://dx.doi.org/10.1177/0748233720971879.
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This study aimed to analyze the neurological changes induced by acrylamide (ACR) poisoning and their underlying mechanisms within the spinal cords of male adult Wistar rats. The rats were randomly divided into three groups ( n = 9 rats per group). ACR was intraperitoneally injected to produce axonopathy according to the daily dosing schedules of 20 or 40 mg/kg/day of ACR for eight continuous weeks (three times per week). During the exposure period, body weights and gait scores were assessed, and the concentration of Ca2+ was calculated in 27 mice. Protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent protein kinase 5 (CDK5), and P35 were assessed by electrophoretic resolution and Western blotting. The contents of 3′-cyclic adenosine monophosphate (cAMP) and calmodulin (CaM) were determined using ELISA kits, and the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and PKC were determined using the commercial Signa TECTPKAassay kits. Compared with control rats, treatment with 20 and 40 mg/kg of ACR decreased body weight and increased gait scores at 8 weeks. Intracellular Ca2+ levels increased significantly in treated rats; CaM, PKC, CDK5, and P35 levels were significantly decreased; and PKA and cAMP levels remained unchanged. CaMKII, PKA, and PKC activities increased significantly. The results indicated that ACR can damage neurofilaments by affecting the contents and activities of CaM, CaMKII, PKA, cAMP, PKC, CDK5, and P35, which could result in ACR toxic neuropathy.
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Takimura, Tetsuo, Kenji Kamata, Kazuhiro Fukasawa, Hirokazu Ohsawa, Hideya Komatani, Takashi Yoshizumi, Ikuko Takahashi, Hidehito Kotani та Yoshikazu Iwasawa. "Structures of the PKC-ι kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533–551 in the C-terminal tail and their roles in ATP binding". Acta Crystallographica Section D Biological Crystallography 66, № 5 (21 квітня 2010): 577–83. http://dx.doi.org/10.1107/s0907444910005639.
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Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-θ, PKC-ι and PKC-βII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC–substrate complex has been determined. In the previously determined PKC-ι complex, residues 533–551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-ι kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 Å resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-ι are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-ι, which is different from the case of the prototypical AGC-branch kinase PKA.
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Wetsel, WC, WA Khan, I. Merchenthaler, H. Rivera, AE Halpern, HM Phung, A. Negro-Vilar, and YA Hannun. "Tissue and cellular distribution of the extended family of protein kinase C isoenzymes." Journal of Cell Biology 117, no. 1 (April 1, 1992): 121–33. http://dx.doi.org/10.1083/jcb.117.1.121.
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Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.
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Hermann-Kleiter, Natascha, Nikolaus Thuille, Christa Pfeifhofer, Thomas Gruber, Michaela Schäfer, Christof Zitt, Armin Hatzelmann, Christian Schudt, Michael Leitges та Gottfried Baier. "PKCθ and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes". Blood 107, № 12 (15 червня 2006): 4841–48. http://dx.doi.org/10.1182/blood-2005-10-4044.
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AbstractWe here investigate the crosstalk of PKC and PKA signaling during primary CD3+ T-lymphocyte activation using pharmacologic inhibitors and activators in combination with our established panel of PKC isotype–deficient mouse T cells in vitro. PKCθ and PKA inversely affect the CD3/CD28-induced IL-2 expression, whereas other PKC isotypes are dispensable in this signaling pathway. Gene ablation of PKCθ selectively results in a profound reduction of IL-2 production; however, complete abrogation of IL-2 production in these PKCθ–/– T cells was achieved only by simultaneous coactivation of the cAMP/PKA pathway in CD3+ T cells. Conversely, the reduced IL-2 production in PKC inhibitor–treated T cells can be rescued by inhibition of the cAMP/PKA pathway in wild-type but not in PKCθ–/– T cells. Mechanistically, the cAMP/PKA and PKCθ pathways converge at the level of NF-AT, as shown by DNA binding analysis. The combined increase in PKA and decrease in PKCθ activity leads to an enhanced inhibition of nuclear NF-AT translocation. This PKCθ/PKA crosstalk significantly affects neither the NF-κB, the AP-1, nor the CREB pathways. Taken together, this opposite effect between the positive PKCθ and the negative cAMP/PKA signaling pathways appears rate limiting for NF-AT transactivation and IL-2 secretion responses of CD3+ T lymphocytes.