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1
Amiri, Farhad, and Raul Garcia. "Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells." American Journal of Physiology-Renal Physiology 276, no. 5 (May 1, 1999): F691—F699. http://dx.doi.org/10.1152/ajprenal.1999.276.5.f691.
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It has been shown that glomerular angiotensin II (ANG II) receptors are downregulated and protein kinase C (PKC) is activated under diabetic conditions. We, therefore, investigated ANG II receptor and PKC isoform regulation in glomerular mesangial cells (MCs) under normal and elevated glucose concentrations. MCs were isolated from collagenase-treated rat glomeruli and cultured in medium containing normal or high glucose concentrations (5.5 and 25.0 mM, respectively). Competitive binding experiments were performed using the ANG II antagonists losartan and PD-123319, and PKC analysis was conducted by Western blotting. Competitive binding studies showed that the AT1 receptor was the only ANG II receptor detected on MCs grown to either subconfluence or confluence under either glucose concentration. AT1 receptor density was significantly downregulated in cells grown to confluence in high-glucose medium. Furthermore, elevated glucose concentration enhanced the presence of all MC PKC isoforms. In addition, PKCβ, PKCγ and PKCε were translocated only in cells cultured in elevated glucose concentrations following 1-min stimulation by ANG II, whereas PKCα, PKCθ, and PKCλ were translocated by ANG II only in cells grown in normal glucose. Moreover, no changes in the translocation of PKCδ, PKCι, PKCζ, and PKCμ were detected in response to ANG II stimulation under euglycemic conditions. We conclude that MCs grown in high glucose concentration show altered ANG II receptor regulation as well as PKC isoform translocation compared with cells grown in normal glucose concentration.
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Dobkin-Bekman, Masha, Liat Rahamim Ben-Navi, Boris Shterntal, Ludmila Sviridonov, Fiorenza Przedecki, Michal Naidich-Exler, Chaya Brodie, Rony Seger, and Zvi Naor. "Differential Role of PKC Isoforms in GnRH and Phorbol 12-Myristate 13-Acetate Activation of Extracellular Signal-Regulated Kinase and Jun N-Terminal Kinase." Endocrinology 151, no. 10 (September 1, 2010): 4894–907. http://dx.doi.org/10.1210/en.2010-0114.
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GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LβT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCβII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCβII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCβII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCβII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCβII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCβII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.
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Collazos, Alejandra, Barthélémy Diouf, Nathalie C. Guérineau, Corinne Quittau-Prévostel, Marion Peter, Fanny Coudane, Frédéric Hollande, and Dominique Joubert. "A Spatiotemporally Coordinated Cascade of Protein Kinase C Activation Controls Isoform-Selective Translocation." Molecular and Cellular Biology 26, no. 6 (March 15, 2006): 2247–61. http://dx.doi.org/10.1128/mcb.26.6.2247-2261.2006.
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ABSTRACT In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCβ1, PKCα, PKCε, and PKCδ, resulting in their accumulation at the entire plasma membrane (PKCβ and -δ) or selectively at the cell-cell contacts (PKCα and -ε). The duration of activation ranged from 20 s for PKCα to 20 min for PKCε. PKCα and -ε selective localization was lost in the presence of Gö6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCα localization is controlled by PKCβ1 activity and is calcium independent, while PKCε localization is dependent on PKCα activity. PKCδ was independent of the cascade linking PKCβ1, -α, and -ε. Furthermore, PKCα, but not PKCε, is involved in the TRH-induced β-catenin relocation at cell-cell contacts, suggesting that PKCε is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCβ1 activation, which in turn initiates a calcium-independent but PKCβ1 activity-dependent sequential translocation of PKCα and -ε. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.
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Bhavanasi, Dheeraj, Soochong Kim, Lawrence E. Goldfinger, and Satya P. Kunapuli. "Protein Kinase C Delta Mediates the Activation of Protein Kinase D In Platelets." Blood 116, no. 21 (November 19, 2010): 2021. http://dx.doi.org/10.1182/blood.v116.21.2021.2021.
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Abstract Abstract 2021 Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCm, PKD2 and PKCn in humans). They are part of alternate DAG receptors along with RasGRPs, Munc 13s, chimerins and DAG kinases. Members of the novel class of PKC isoforms such as PKCd, PKCh, and PKCe associate with PKD in smooth muscle cells and COS-7 cells. The mechanism of activation of PKD and the specific PKC isoforms required for its activation are not known to date. This study is aimed at investigating the pathways involved in the activation of PKD in platelets. We show that human as well as murine platelets express PKD. PKD could be activated with PAR4 agonist AYPGKF, PAR1 agonist SFLLRN and GPVI agonist convulxin. AYPGKF and SFLLRN induced PKD phosphorylation as early as 30 sec and convulxin induced PKD phosphorylation at 1 minute. PKD phosphorylation induced by AYPGKF and convulxin were sustained for 5 minutes but phosphorylation induced by SFLLRN was attenuated after 2 minutes of stimulation. AYPGKF-induced PKD phosphorylation was reduced with a calcium chelator, dimethyl BAPTA, indicating that calcium-mediated signals might play a role in activation of PKD. PKD phosphorylation in response to AYPGKF was abolished with a Gq inhibitor, YM-254890, but was not affected by Gi-coupled P2Y12 receptor antagonist ARC-69931MX, indicating that PKD phosphorylation is Gq-, but not Gi- or G 12/13-dependent. PKD phosphorylation was abolished with pan-PKC inhibitors, GF109203X and Ro31-8220, indicating that PKCs are required for PKD activation in platelets. PKD phosphorylation was significantly inhibited with a PKC delta inhibitor, rottlerin, but was not affected by the classical PKC inhibitor, Go6976, suggesting that novel PKC isoforms are important for PKD activation. In addition, 2MeSADP that fails to activate PKCd did not induce phosphorylation of PKD in platelets. Furthermore, phosphorylation of PKD induced by AYPGKF was significantly reduced in PKCd-deficient platelets compared to that of wild type platelets. Hence, we conclude that PKD is a common signaling target downstream of various agonist receptors in platelets, and Gq-mediated signals and novel PKC isoforms, in particular PKCd is required for activation of PKD. Disclosures: No relevant conflicts of interest to declare.
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Chaudhary, Divya, Diane Boschelli, Andrea Bree, Agnes Brennan, Joan Chen, Bijia Deng, Lori Fitz, et al. "Characterization of a selective Protein Kinase C theta (PKCθ) inhibitor for blocking T Cell responses (93.21)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 93.21. http://dx.doi.org/10.4049/jimmunol.182.supp.93.21.
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Abstract PKC theta (PKCθ) regulates T cell activation in lung inflammation and airway hyperresponsiveness, and in mouse models of multiple sclerosis, arthritis, and inflammatory bowel disease. We investigated inhibition of PKCθ enzyme activity by a small molecule as a strategy for modulating T cell mediated responses. Screening and lead optimization identified Compound A, a potent PKCθ inhibitor in both enzymatic and cell based assays, with low nM inhibitory activities. Compound A was highly selective (>1000 fold) over 50 kinases including representative serine/threonine, tyrosine, and phosphoinositol kinases, and the conventional and atypical PKCs, PKCβ and PKCξ. Compound A was 10 fold selective against PKCε, and 60 to 100 fold selectivity against PKCδand PKCη, respectively. We found that compound A strongly inhibited IL-2 production in antiCD3/anti-CD28 activated whole blood, using both human and mouse blood, with submicromolar potency. Based on these findings, we evaluated compound A- mediated inhibition of anti-CD3 induced IL-2 production in vivo. The results show a dose dependent inhibitory effect of compound A in this 4 hour cytokine production model. Additional cytokines inhibited by compound A in this model are IFNγ, IL-4, and TNF. IL-5 production was not reduced. We propose that compound A is a selective PKCθ inhibitor for further evaluating blockade of T cell responses in inflammation.
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Lee, Angel W. "Atypical Protein Kinase Cs Promote CSF-1-Dependent Erk Activation and Proliferation in Myeloid Cells." Blood 108, no. 11 (November 16, 2006): 4227. http://dx.doi.org/10.1182/blood.v108.11.4227.4227.
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Abstract Macrophages are integral components of the innate immune system and essential players in inflammation. Enhanced macrophage numbers underlie these pathological states. Colony stimulating factor-1 (CSF-1) is the major physiological regulator of proliferation and survival of cells of the monocyte/macrophage lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). CSF-1 and CSF-1R have emerged as drug targets in several diseases where inflammation is a critical component, e.g. breast cancer and rheumatoid arthritis. Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which of these pathways regulate proliferation and survival. Atypical PKCs (aPKCs) are implicated in cell proliferation and survival. They include the isoforms PKCζ and PKCλ/ι. Unlike the classical and novel PKCs, aPKCs are insensitive to Ca2+ and phorbol esters. In this study, we investigated the role of aPKCs in CSF-1-mediated proliferation in myeloid cells. CSF-1 is a proliferation and survival factor for 32D.R cells, a myeloid progenitor cell line transfected with the CSF-1R. Western blotting shows that PKCα, PKCδ, PKCε and PKCζ/λ/I are expressed in 32D.R. Based on studies with PKC inhibitors that have different specificities towards aPKCs (GF109203X, Ro-31-8220, Go6983 and a Myr-PKCζ peptide), maximal CSF-1-dependent proliferation in 32D.R cells appears to depend on the activity of either aPKCs or PKCε. Using phospho-specific antibodies that detect the activation state of PKCζ as well as in vitro kinase assays, we showed that CSF-1 activates aPKCs in 32D.R and bone marrow derived macrophages. In contrast, CSF-1-induced activation of PKCε was not observed. We next asked how aPKC affects CSF-1 signaling. PKCζ promotes activation of the MEK-Erk pathway in different cell types (Corbit, K.C. et al. Mol. Cell. Biol. 20, 5392). In 32D.R cells, treatment with the MEK inhibitor, U0126, reduced CSF-1-provoked proliferation by 60–70%, consistent with the inhibition observed with PKC inhibitors. Previous work from our lab showed that CSF-1 activates the Erk pathway through A-Raf and not Raf-1 (Lee and States, Mol. Cell. Biol. 18, 6779). We found that aPKC inhibitors do not affect CSF-1 induced Ras and A-Raf activity but markedly reduce MEK and Erk activity, implying that aPKC inputs into the CSF-1 Erk pathway at the level of MEK. Transient transfections with dominant-negative and constitutively active (CA) PKCζ confirmed that aPKC promotes CSF-1-induced Erk activation. aPKC inhibition does not affect CSF-1-stimulated Akt activation. To investigate the role of PKCζ in CSF-1-dependent proliferation, we established stable 32D.R mass populations overexpressing wildtype (WT) or CA PKCζ at levels 2-fold above endogenous. Comparing cells expressing CA-PKCζ to WT-PKCζ, the EC50 for CSF-1-dependent proliferation and the cell doubling time at maximal CSF-1 concentration were both reduced, consistent with a role for PKCζ in CSF-1 dependent proliferation. We will use our stable cell lines to elucidate the pathways modulated by PKCζ. Altogether, our results identify atypical PKCs as new targets of CSF-1 signaling.
7
RENDÓN-HUERTA, Erika, Guillermo MENDOZA-HERNÁNDEZ, and Martha ROBLES-FLORES. "Characterization of calreticulin as a protein interacting with protein kinase C." Biochemical Journal 344, no. 2 (November 24, 1999): 469–75. http://dx.doi.org/10.1042/bj3440469.
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A protein kinase C (PKC)-binding protein was purified to homogeneity from the Triton-insoluble fraction from rat hepatocytes homogenates. The protein was identified as the mature calreticulin chain by N-terminal amino acid sequencing and by its immunoreactivity with anti-calreticulin antibody raised against the C-terminal KDEL (single-letter code) sequence. The calculated molecular mass was 46.6 kDa but the protein migrates in SDS/PAGE as a doublet with apparent molecular masses of 60 and 55 kDa. Studies in vitro with purified calreticulin with the use of an overlay assay approach demonstrated that it binds to activated PKC isoenzymes expressed in rat hepatocytes. Phosphorylation of purified calreticulin with a PKC isoenzyme-specific immune complex kinase assay showed that it is also a very good substrate for all PKC isoforms in vitro. The treatment of intact cells with phorbol ester or with adrenaline (epinephrine) plus propranolol increased calreticulin phosphorylation, which was blocked by the pretreatment of cells with the PKC-specific inhibitor Ro 31-8220. The analysis of calreticulin immunoprecipitates from control or treated cells indicated that PKCα, PKCβ, PKCθ, PKCζ and PKCμ, but not PKCδ or PKCϵ, co-immunoprecipitated with calreticulin. Taken together, our results indicate that PKC interacts in vivo with calreticulin and suggest that they can operate in common signalling pathways.
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WEBB, Benjamin L. J., Mark A. LINDSAY, Peter J. BARNES, and Mark A. GIEMBYCZ. "Protein kinase C isoenzymes in airway smooth muscle." Biochemical Journal 324, no. 1 (May 15, 1997): 167–75. http://dx.doi.org/10.1042/bj3240167.
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The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.
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Liu, Hong, and Fan Dong. "Involvement of PKCe in the Negative Regulation of Akt Activation Stimulated by G-CSF." Blood 104, no. 11 (November 16, 2004): 2187. http://dx.doi.org/10.1182/blood.v104.11.2187.2187.
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Abstract Granulocyte colony-stimulating factor (G-CSF) supports the proliferation, differentiation and survival of myeloid cells by stimulating the activation of several signaling cascades including the serine/threonine kinase Akt pathway. Akt activation has been shown to be important for G-CSF-induced survival and granulocytic differentiation. Although significant progresses have been made in our understanding of the molecular mechanisms by which Akt is activated, much less is known about the signaling events that negatively regulate Akt activation. Interestingly, G-CSF-induced activation of Akt was completely inhibited when myeloid 32D cells transfected with the wild type G-CSF receptor were incubated with phorbol-12-myristate 13-acetate (PMA), a PKC activator. PMA-mediated inhibition of Akt activation occurred with 5 min and lasted at least 1 hour. Previously, it has been shown that a carboxyl terminally truncated G-CSF receptor (D715), whose expression is associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia (SCN), mediates significantly prolonged Akt activation. Notably, Akt activation by G-CSF in 32D cells expressing the D715 receptor mutant was rapidly downregulated by PMA treatment. The inhibitory effect of PMA on Akt activation was abolished by pretreatment of cells with the specific PKC inhibitor GF109203X, suggesting that PKC-dependent pathway negatively regulates Akt activation. Ro-31-7549, a specific inhibitor of PKCe, also abrogated PMA-mediated inhibition of Akt activation whereas rottlerin and Go6976, inhibitors of PKCd and PKC a/bI,, respectively, displayed no effect. Together, these results identified PKCe as being critically involved in PMA-mediated inhibition of Akt activation. Experiments are currently under way to determine the mechanism by which PKCe downregulates Akt activation and the role of PKCe in the regulation of cell proliferation, differentiation and survival in response to G-CSF.
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Bhavanasi, Dheeraj, Carol T. Dangelmaier, Jin Jianguo, Soochong Kim, and Satya P. Kunapuli. "Classical PKCs Regulate ADP-Induced Thromboxane Generation by Modulating Tyrosine Phosphorylation On Novel PKC Isoform Delta Through Shptp-1." Blood 120, no. 21 (November 16, 2012): 1064. http://dx.doi.org/10.1182/blood.v120.21.1064.1064.
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Abstract Abstract 1064 Introduction: Adenosine Di-phosphate (ADP) is stored in dense granules of platelets and is released upon platelet activation acting as a feedback activator by binding to G-protein coupled P2Y1 and P2Y12 receptors. ADP stimulation causes platelets to change shape, aggregate, release dense and a-granule contents and synthesize thromboxane A2 that can further act as a feedback activator potentiating platelet responses by binding to thromboxane receptor (TP). Protein kinase C is a serine threonine specific kinase that regulates multiple platelet functional responses. Specific PKC isoforms regulating platelet responses downstream of ADP receptors are not completely known. Aim: The aim of the current study is to elucidate the role of PKC isoforms in regulating ADP-induced platelet functional responses in platelets. Methods: We sought to delineate the mechanism of ADP-induced platelet responses by performing platelet aggregation (aggregometry), ATP secretion (luciferin-luciferase reaction) and thromboxane generation (ELISA kit measuring TxB2) in human or murine platelets by pre-incubating the platelets with control (DMSO) or inhibitors wherever mentioned. We also evaluated the role of PKCd to ADP-induced platelet responses by using murine platelets lacking PKCd. Background and Results: Murugappan et al have shown that PKCd was not activated downstream of ADP receptors based on the inability of ADP to cause threonine 507 phosphorylation on PKCd in platelets. However, studies from other labs have shown that PKCd can be activated when it is phosphorylated on its tyrosine residues. In the current study we show that, upon stimulation with 2MeSADP, PKCd is phosphorylated on tyrosine residue 311 in a time-dependent manner in platelets (Fig A). Also, ADP-induced thromboxane generation (Fig B) and ADP-induced thromboxane-mediated dense granule secretion were significantly inhibited in PKCd knockout murine platelets compared to those of wild type platelets. Similarly, thromboxane generation downstream of ADP receptors in human platelets pre-incubated with a PKCd inhibitor is significantly inhibited compared to control indicating a role for PKCd in mediating ADP-induced responses in platelets. Bynagari et al have shown that ADP-induced thromboxane generation is potentiated in the presence of the pan-PKC inhibitor, GF 109203X and the isoform regulating this effect is PKCe. We observed that pre-incubation of PKCe knockout murine platelets with GF 109203X further potentiated ADP-induced thromboxane generation suggesting that there are other PKC isoforms negatively regulating ADP-induced thromboxane generation. We show that this potentiating effect of thromboxane generation with GF 109203X in WT or PKCe KO murine platelets correlate with an increase in the phosphorylation of Y311 on PKCd (Fig C) suggesting that ADP-induced thromboxane generation is regulated through PKCd Y311 phosphorylation. Tyrosine phosphorylation on PKCd is mediated by Src family kinases (SFKs) as the phosphorylation is abolished with PP2, a SFK inhibitor and is only partially inhibited in Fyn knockout murine platelets suggesting that other SFKs also mediate this tyrosine phosphorylation. Surprisingly, pre-incubation of platelets with LY-333531, a classical PKC isoform (a/b) inhibitor potentiated PKCd Y311 phosphorylation (Fig D) as well as thromboxane generation (Fig E) downstream of ADP receptors suggesting a role for classical PKCs. Also, platelets pre-incubated with LY-333531 showed a decrease in the phosphorylation of SHPTP-1 (Fig F), a tyrosine phosphatase, rendering it active. The active SHPTP-1 phosphatase may dephosphorylate and activate SFKs, which can now phosphorylate PKCd on Y311 in platelets. Conclusions: In the current study, we report for the first time that the novel PKC isoform d is tyrosine phosphorylated downstream of ADP receptors through which it mediates ADP-induced thromboxane generation. We also show a novel role for classical PKC isoforms a/b in regulating tyrosine phosphorylation on novel isoform, PKCd possibly through the tyrosine phosphatase SHPTP-1 and Src family kinases in platelets. Disclosures: No relevant conflicts of interest to declare.
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Li, Luowei, Patricia S. Lorenzo, Krisztina Bogi, Peter M. Blumberg, and Stuart H. Yuspa. "Protein Kinase Cδ Targets Mitochondria, Alters Mitochondrial Membrane Potential, and Induces Apoptosis in Normal and Neoplastic Keratinocytes When Overexpressed by an Adenoviral Vector." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8547–58. http://dx.doi.org/10.1128/mcb.19.12.8547.
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ABSTRACT Inactivation of protein kinase Cδ (PKCδ) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCδ in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCδ under a cytomegalovirus promoter to overexpress PKCδ in normal and neoplastic keratinocytes. While PKCδ overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCα, PKCɛ, PKCζ, and PKCη, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCδ adenovirus. Activation of PKCδ by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCδ. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCδ translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCδ-green fluorescent protein fusion protein. Furthermore, activation of PKCδ in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCδ-overexpressing keratinocytes. These results indicate that PKCδ can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.
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Radresa, Olivier, António Guia, and Ghayath Baroudi. "Roles of PKC Isoforms in PMA-Induced Modulation of the hERG Channel (Kv11.1)." Journal of Biomolecular Screening 19, no. 6 (January 24, 2014): 890–99. http://dx.doi.org/10.1177/1087057113520227.
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Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCβ, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state ( R2 = 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.
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Lenz, Johannes C., H. Peter Reusch, Nadine Albrecht, Günter Schultz, and Michael Schaefer. "Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells." Journal of Cell Biology 159, no. 2 (October 21, 2002): 291–302. http://dx.doi.org/10.1083/jcb.200203048.
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The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKCα accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKCε was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKCε, but not of coexpressed PKCα, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKCε than for PKCα. Subsequent photolysis of caged Ca2+ immediately recruited PKCα to the membrane, and DAG-bound PKCε was displaced. At low expression levels of PKCε, PKCα concentration dependently prevented the PKCε translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation.
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Le, Man, Lada Krilov, Jianping Meng, Kelli Chapin-Kennedy, Susan Ceryak, and Bernard Bouscarel. "Bile acids stimulate PKCα autophosphorylation and activation: role in the attenuation of prostaglandin E1-induced cAMP production in human dermal fibroblasts." American Journal of Physiology-Gastrointestinal and Liver Physiology 291, no. 2 (August 2006): G275—G287. http://dx.doi.org/10.1152/ajpgi.00346.2005.
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The aim was to identify the specific PKC isoform(s) and their mechanism of activation responsible for the modulation of cAMP production by bile acids in human dermal fibroblasts. Stimulation of fibroblasts with 25–100 μM of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) led to YFP-PKCα and YFP-PKCδ translocation in 30–60 min followed by a transient 24- to 48-h downregulation of the total PKCα, PKCδ, and PKCε protein expression by 30–50%, without affecting that of PKCζ. Increased plasma membrane translocation of PKCα was associated with an increased PKCα phosphorylation, whereas increased PKCδ translocation to the perinuclear domain was associated with an increased accumulation of phospho-PKCδ Thr505 and Tyr311 in the nucleus. The PKCα specificity on the attenuation of cAMP production by CDCA was demonstrated with PKC downregulation or inhibition, as well as PKC isoform dominant-negative mutants. Under these same conditions, neither phosphatidylinositol 3-kinase, p38 MAP kinase, p42/44 MAP kinase, nor PKA inhibitors had any significant effect on the CDCA-induced cAMP production attenuation. CDCA concentrations as low as 10 μM stimulated PKCα autophosphorylation in vitro. This bile acid effect required phosphatidylserine and was completely abolished by the presence of Gö6976. CDCA at concentrations less than 50 μM enhanced the PKCα activation induced by PMA, whereas greater CDCA concentrations reduced the PMA-induced PKCα activation. CDCA alone did not affect PKCα activity in vitro. In conclusion, although CDCA and UDCA activate different PKC isoforms, PKCα plays a major role in the bile acid-induced inhibition of cAMP synthesis in fibroblasts. This study emphasizes potential consequences of increased systemic bile acid concentrations and cellular bile acid accumulation in extrahepatic tissues during cholestatic liver diseases.
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van der Merwe, Jacques Q., France Moreau, and Wallace K. MacNaughton. "Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms." American Journal of Physiology-Gastrointestinal and Liver Physiology 296, no. 6 (June 2009): G1258—G1266. http://dx.doi.org/10.1152/ajpgi.90425.2008.
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Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR2-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current ( Isc) responses to basolateral application of the selective PAR2 activating peptide, SLIGRL-NH2, were monitored as a measure of net electrogenic ion transport caused by PAR2 activation. SLIGRL-NH2 induced a transient Isc response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCβ and PLCγ following PAR2 activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCα/βI (Gö6976), and PKCδ (rottlerin), but not PKCζ (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCβI, PKCδ, and PKCε, but not PKCα or PKCζ, in membrane fractions following PAR2 activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCδ translocation. Immunoblots revealed that PAR2 activation induced phosphorylation of both cRaf and ERK1/2 via PKCδ. Inhibition of PKCβI and PI3K had only a partial effect on this response. We conclude that basolateral PAR2-induced chloride secretion involves activation of PKCβI and PKCδ via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.
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Thongon, Narongrit, La-iad Nakkrasae, Jirawan Thongbunchoo, Nateetip Krishnamra, and Narattaphol Charoenphandhu. "Enhancement of calcium transport in Caco-2 monolayer through PKCζ-dependent Cav1.3-mediated transcellular and rectifying paracellular pathways by prolactin." American Journal of Physiology-Cell Physiology 296, no. 6 (June 2009): C1373—C1382. http://dx.doi.org/10.1152/ajpcell.00053.2009.
Full textAbstract:
Previous investigations suggested that prolactin (PRL) stimulated the intestinal calcium absorption through phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and RhoA-associated coiled-coil forming kinase (ROCK) signaling pathways. However, little was known regarding its detailed mechanisms for the stimulation of transcellular and voltage-dependent paracellular calcium transport. By using Ussing chamber technique, we found that the PRL-induced increase in the transcellular calcium flux and decrease in transepithelial resistance of intestinal-like Caco-2 monolayer were not abolished by inhibitors of gene transcription and protein biosynthesis. The PRL-stimulated transcellular calcium transport was completely inhibited by the L-type calcium channel blockers (nifedipine and verapamil) and plasma membrane Ca2+-ATPase (PMCA) inhibitor (trifluoperazine) as well as small interfering RNA targeting voltage-dependent L-type calcium channel Cav1.3, but not TRPV6 or calbindin-D9k. As demonstrated by 45Ca uptake study, PI3K and PKC, but not ROCK, were essential for the PRL-enhanced apical calcium entry. In addition, PRL was unable to enhance the transcellular calcium transport after PKCζ knockdown or exposure to inhibitors of PKCζ, but not of PKCα, PKCβ, PKCε, PKCμ, or protein kinase A. Voltage-clamping experiments further showed that PRL markedly stimulated the voltage-dependent calcium transport and removed the paracellular rectification. Such PRL effects on paracellular transport were completely abolished by inhibitors of PI3K (LY-294002) and ROCK (Y-27632). It could be concluded that the PRL-stimulated transcellular calcium transport in Caco-2 monolayer was mediated by Cav1.3 and PMCA, presumably through PI3K and PKCζ pathways, while the enhanced voltage-dependent calcium transport occurred through PI3K and ROCK pathways.
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Ammoury, Rita, Roula Tahtouh, Nadine Mahfouz, Raia Doumit, Charbel Khalil, and George Hilal. "Relationship between Protein kinase C isoforms, Telomerase and Alpha- fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma." MedPharmRes 5, no. 4 (August 2, 2021): 12–26. http://dx.doi.org/10.32895/ump.mpr.5.4.3.
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Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.
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Zhang, Y. H., J. Kays, K. E. Hodgdon, T. C. Sacktor, and G. D. Nicol. "Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ." Journal of Neurophysiology 107, no. 1 (January 2012): 315–35. http://dx.doi.org/10.1152/jn.00030.2011.
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Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.
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Kunapuli, Satya P., and John C. Kostyak. "PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation." Blood 120, no. 21 (November 16, 2012): 853. http://dx.doi.org/10.1182/blood.v120.21.853.853.
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Abstract Abstract 853 Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Clément-Chomienne, Odile, and Michael P. Walsh. "Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKCζ." Biochemistry and Cell Biology 74, no. 1 (January 1, 1996): 51–65. http://dx.doi.org/10.1139/o96-006.
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The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1 and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙min−1∙mg−1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.
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Yang, Jing, Pascale H. Lane, Jennifer S. Pollock, and Pamela K. Carmines. "PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats." American Journal of Physiology-Renal Physiology 297, no. 5 (November 2009): F1220—F1228. http://dx.doi.org/10.1152/ajprenal.00314.2009.
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Type 1 diabetes (T1D) is a state of oxidative stress accompanied by PKC activation in many tissues. The primary site of O2•− production by the normal rat kidney is the medullary thick ascending limb (mTAL). We hypothesized that T1D increases O2•− production by the mTAL through a PKC-dependent mechanism involving increased expression and translocation of one or more PKC isoforms. mTAL suspensions were prepared from rats with streptozotocin-induced T1D (STZ mTALs) and from normal or sham rats (normal/sham mTALs). O2•− production by STZ mTALs was fivefold higher than normal/sham mTALs ( P < 0.05). PMA (30 min) mimicked the effect of T1D on O2•− production. Exposure to calphostin C or chelerythrine (PKC inhibitors), Gö6976 (PKCα/β inhibitor), or rottlerin (PKCδ inhibitor) decreased O2•− production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCβ inhibitors had no effect. PKC activity was increased in STZ mTALs ( P < 0.05 vs. normal/sham mTALs) and was unaltered by antioxidant exposure (tempol). PKCα protein levels were increased by 70% in STZ mTALs, with a ∼30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCβ protein levels were elevated by 29% in STZ mTALs ( P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKCδ protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKCα and PKCβ protein levels, increased PKCα translocation to the membrane, and accelerated O2•− production that is eradicated by inhibition of PKCα or PKCδ (but not PKCβ). We conclude that increased PKCα expression and activity are primarily responsible for PKC-dependent O2•− production by the mTAL during T1D.
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Xiao, Helan, Xiao-Hui Bai, Andras Kapus, Wei-Yang Lu, Alan S. Mak, and Mingyao Liu. "The Protein Kinase C Cascade Regulates Recruitment of Matrix Metalloprotease 9 to Podosomes and Its Release and Activation." Molecular and Cellular Biology 30, no. 23 (October 11, 2010): 5545–61. http://dx.doi.org/10.1128/mcb.00382-10.
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ABSTRACT Podosomes are transient cell surface structures essential for degradation of extracellular matrix during cell invasion. Protein kinase C (PKC) is involved in the regulation of podosome formation; however, the roles of individual PKC isoforms in podosome formation and proteolytic function are largely unknown. Recently, we reported that PDBu, a PKC activator, induced podosome formation in normal human bronchial epithelial cells. Here, we demonstrate that phorbol-12,13-dibutyrate (PDBu)-induced podosome formation is mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes. Interestingly, although blocking atypical PKCζ did not affect PDBu-induced podosome formation, it significantly reduced matrix degradation at podosomes. Inhibition of PKCζ reduced recruitment of matrix metalloprotease 9 (MMP-9) to podosomes and its release and activation. Downregulation of MMP-9 by small interfering RNA (siRNA) or neutralization antibody also significantly reduced matrix degradation. The regulatory effects of PKCζ on matrix degradation and recruitment of MMP-9 to podosomes were PKCζ kinase activity dependent. PDBu-induced recruitment of PKCζ and MMP-9 to podosomes was blocked by inhibition of novel PKC with rottlerin or PKCδ siRNA. Our data suggest that multiple PKC isozymes form a signaling cascade that controls podosome formation and dynamics and MMP-9 recruitment, release, and activation in a coordinated fashion.
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Song, Jaekyung Cecilia, Celina M. Hanson, Vance Tsai, Omid C. Farokhzad, Margaret Lotz, and Jeffrey B. Matthews. "Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms." American Journal of Physiology-Cell Physiology 281, no. 2 (August 1, 2001): C649—C661. http://dx.doi.org/10.1152/ajpcell.2001.281.2.c649.
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The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl− secretion (short-circuit current, I sc) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I sc and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKCε followed by the conventional PKCα and activated both isozymes by in vitro kinase assay. PKCδ was activated by PMA but did not translocate. By immunofluorescence, PKCε redistributed to the basolateral domain in response to PMA, whereas PKCα moved apically. Inhibition of I sc by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 μM) but not the conventional isoform inhibitor Gö-6976 (5 μM) or the PKCδ inhibitor rottlerin (10 μM), implicating PKCε in inhibition of Cl− secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKCα. Bryostatin-1 (100 nM) translocated PKCε and PKCα and inhibited cAMP-elicited I sc. However, unlike PMA, bryostatin-1 downregulated PKCα protein, and the decrease in TER was only transient. Carbachol (100 μM) translocated only PKCε and inhibited I sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I sc. We conclude that basolateral translocation of PKCε inhibits Cl−secretion, while apical translocation of PKCα decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.
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Schreiber, Kathy L., Louise Paquet, Bruce G. Allen, and Hansjörg Rindt. "Protein kinase C isoform expression and activity in the mouse heart." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 5 (November 1, 2001): H2062—H2071. http://dx.doi.org/10.1152/ajpheart.2001.281.5.h2062.
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The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCα and PKCβII, as well as two Ca2+-independent isoforms, PKCδ and PKCε, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCδ and PKCε were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCα in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCδ and PKCε were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.
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Alzamora, Rodrigo, Laura R. Brown, and Brian J. Harvey. "Direct Binding and Activation of Protein Kinase C Isoforms by Aldosterone and 17β-Estradiol." Molecular Endocrinology 21, no. 11 (November 1, 2007): 2637–50. http://dx.doi.org/10.1210/me.2006-0559.
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Abstract Protein kinase C (PKC) is a signal transduction protein that has been proposed to mediate rapid responses to steroid hormones. Previously, we have shown aldosterone directly activates PKCα whereas 17β-estradiol activates PKCα and PKCδ; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKCα and PKCδ involved in binding of aldosterone and 17β-estradiol, glutathione S-transferase fusion recombinant PKCα and PKCδ mutants were used to perform in vitro binding assays with [3H]aldosterone and [3H]17β-estradiol. 17β-Estradiol bound both PKCα and PKCδ but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKCα and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17β-estradiol were between 0.5–1.0 nM. Aldosterone and 17β-estradiol competed for binding to PKCα, suggesting they share the same binding site. Phorbol 12,13-dybutyrate did not compete with hormone binding; furthermore, they have an additive effect on PKC activity. EC50 for activation of PKCα and PKCδ by aldosterone and 17β-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKCα and PKCδ undergo autophosphorylation with an EC50 in the 0.5–1.0 nm range. 17β-Estradiol activated PKCα and PKCδ in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17β-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKCα and PKCδ by steroid hormones and the molecular mechanisms involved.
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KARL, Peter I., and Andras DIVALD. "Protein kinase C in cultured human placental trophoblasts: identification of isoforms and role in cAMP signalling." Biochemical Journal 320, no. 3 (December 15, 1996): 831–36. http://dx.doi.org/10.1042/bj3200831.
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The protein kinase C (PKC) superfamily is a family of serine/threonine kinases involved in the regulation of many cell functions. The objective of the present work was to identify the PKC isoenzymes present in human placental trophoblasts and to compare their relative responses to acute and chronic phorbol 12-myristate 13-acetate (PMA) treatment. In addition, the effect of PMA treatment on ligand-stimulated cAMP production was determined. In total extracts prepared from cultured or freshly purified trophoblasts, PKC isoforms α, ε and ζ were detected. Following acute treatment with PMA, PKCα and PKCε were translocated from the cytosol to the particulate fraction. Prolonged treatment (up to 36 h) with PMA resulted in the temporal down-regulation of PKCα and PKCε. PKCζ did not respond to either acute or chronic treatment with PMA. An acute 10 min treatment of the cells with PMA (10-10–10-6 M) enhanced isoprenaline- and adrenaline-stimulated cAMP production. An enhanced response was observed at all concentrations of isoprenaline tested (10-9–10-4 M), suggesting an increased capacity to respond. The acute PMA effect was evident within 2 min and near maximal by 5 min. The acute response to PMA was lost in cells where PKC was down-regulated by prior PMA treatment. Epidermal growth factor, a potential ligand for the activation of PKC in trophoblasts, enhanced isoprenaline-stimulated cAMP production. In summary, activation of PMA-responsive PKCs (PKCα or PKCε) appears to enhance ligand-stimulated cAMP production in trophoblasts. This may be a physiologically important example of ‘cross-talk’ between various signalling pathways in human placental trophoblasts.
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Pinton, Paolo, Sara Leo, Mariusz R. Wieckowski, Giulietta Di Benedetto, and Rosario Rizzuto. "Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes." Journal of Cell Biology 165, no. 2 (April 19, 2004): 223–32. http://dx.doi.org/10.1083/jcb.200311061.
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The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCα) to the increase or reduction of mitochondrial Ca2+ uptake (PKCζ and PKCβ/PKCδ, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.
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Yang, Jun-Qi, Maria Diaz-Meco, and Jorge Moscat. "Atypical protein kinase C (aPKC) isoforms ζ and λ/ι synergically control Th2 development and function (134.13)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 134.13. http://dx.doi.org/10.4049/jimmunol.184.supp.134.13.
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Abstract The protein kinase C (PKC) family consists of three subfamilies: classical, novel, and atypical PKCs. The atypical PKC (aPKC) consists of two isoforms, PKCζ and PKCλ/ι. We have recently presented in vitro, ex vivo, and in vivo genetic evidence that PKCζ is necessary for optimal activation of the IL-4 signaling cascade upstream of Jak1. Since whole-body genetic deletion of PKCλ/ι is lethal, we have used a conditional gene knockout system by loxP-Cre recombination to generated conditional PKCλ/ι KO mice to selectively delete PKCλ/ι in activated T cells. With this system, we found that PKCλ/ι was necessary for Th2 cytokine production. Upon anti-CD3/CD28 stimulation, IL-4 and IL-13 production from CD4+ T cells was significantly reduced in conditional KO mice as compared to their WT littermates. The activation of the transcription factors NFAT and NF-κB was impaired in PKCλ/ι-deficient activated T cells. Furthermore, deficiency of PKCλ/ι inhibited OVA-induced allergic airway inflammation in vivo. Recently, we have introduced the activated T cell-specific conditional PKCλ/ι deficiency into a PKCζ-/- background to generate mice with PKCζ and PKCλ/ι double knockout (DKO) in activated T cells. From these T cells, the secretion of Th2 cytokines, IL-4, IL-5 and IL-13, was more dramatically reduced and the proliferation was more significantly impaired compared to T cells with single PKCζ or PKCλ/ι deficiency. PKCλ/ι, like PKCζ, emerges as a new critical regulator of Th2 cell activation.
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Velnati, Suresh, Sara Centonze, Federico Girivetto, Daniela Capello, Ricardo M. Biondi, Alessandra Bertoni, Roberto Cantello, et al. "Identification of Key Phospholipids That Bind and Activate Atypical PKCs." Biomedicines 9, no. 1 (January 6, 2021): 45. http://dx.doi.org/10.3390/biomedicines9010045.
Full textAbstract:
PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.
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Wang, Yongyi, Min Men, Wengang Yang, Hui Zheng, and Song Xue. "MiR-31 Downregulation Protects Against Cardiac Ischemia/Reperfusion Injury by Targeting Protein Kinase C Epsilon (PKCe) Directly." Cellular Physiology and Biochemistry 36, no. 1 (2015): 179–90. http://dx.doi.org/10.1159/000374062.
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Background: Various miRNAs have been shown to participate in cardiac ischemia/reperfusion injury (I/R). miR-31 was identified as the most strikingly upregulated miRNA after acute myocardial infarction; therefore, the underlying role and mechanism of miR-31 in cardiac I/R was investigated. Methods: miR-31 expression was detected after cardiac I/R in mice. The cardioprotective effect of miR-31 downregulation was assessed in vitro and in vivo. The functional target gene and its downstream molecule were determined. Results: miR-31 expression increased after I/R. miR-31 downregulation increased cell viability and SOD activity and decreased LDH activity and MDA content in vitro. Additionally, miR-31 downregulation alleviated myocardial infarct size in vivo. PKCe was identified as the functional target gene of miR-31, and NFκB was identified as its downstream molecule that was involved in the miR-31-mediated cardioprotective effect. Conclusion: miR-31 expression increased throughout the cardiac I/R process, and miR-31 downregulation induced a cardioprotective effect via a miR-31/PKCe/NFκB-dependent pathway.
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Pan, Ting-Ting, Kay Li Neo, Li-Fang Hu, Qian Chen Yong, and Jin-Song Bian. "H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes." American Journal of Physiology-Cell Physiology 294, no. 1 (January 2008): C169—C177. http://dx.doi.org/10.1152/ajpcell.00282.2007.
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The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca2+ handling in hydrogen sulfide (H2S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H2S donor, 10−4 M) preconditioning (SP, 30 min). Intracellular Ca2+ was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCα, PKCε, and PKCδ to membrane fraction but only translocation of PKCε and PKCδ was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca2+] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca2+ removal via both accelerating uptake of Ca2+ into sarcoplasmic reticulum and enhancing Ca2+ extrusion through Na+/Ca2+ exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca2+ overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCα, PKCε, and PKCδ in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca2+ handling.
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Storz, Peter, Heike Döppler, and Alex Toker. "Protein Kinase Cδ Selectively Regulates Protein Kinase D-Dependent Activation of NF-κB in Oxidative Stress Signaling." Molecular and Cellular Biology 24, no. 7 (April 1, 2004): 2614–26. http://dx.doi.org/10.1128/mcb.24.7.2614-2626.2004.
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ABSTRACT Protein kinase D (PKD) participates in activation of the transcription factor NF-κB (nuclear factor κB) in cells exposed to oxidative stress, leading to increased cellular survival. We previously demonstrated that phosphorylation of PKD at Tyr463 in the PH (pleckstrin homology) domain is mediated by the Src-Abl pathway and that it is necessary for PKD activation and subsequent NF-κB induction. Here we show that activation of PKD in response to oxidative stress requires two sequential signaling events, i.e., phosphorylation of Tyr463 by Abl, which in turn promotes a second step, phosphorylation of the PKD activation loop (Ser738/Ser742). We show that this is mediated by PKCδ (protein kinase Cδ), a kinase that is activated by Src in response to oxidative stress. We also show that other PKCs, including PKCε and PKCζ, do not participate in PKD activation or NF-κB induction. We propose a model in which two coordinated signaling events are required for PKD activation. Tyrosine phosphorylation in the PH domain at Tyr463, mediated by the Src-Abl pathway, which in turn facilitates the phosphorylation of Ser738/Ser742 in the activation loop, mediated by the Src-PKCδ pathway. Once active, the signal is relayed to the activation of NF-κB in oxidative stress responses.
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Zhao, Chengshui, Michael Leitges, and Robert W. Gereau. "Isozyme-specific Effects of Protein Kinase C in Pain Modulation." Anesthesiology 115, no. 6 (December 1, 2011): 1261–70. http://dx.doi.org/10.1097/aln.0b013e3182390788.
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Background Protein kinase C (PKC) is a family of serine/threonine kinases that contains more than 10 isozymes. Evidence suggests that PKC may play important roles in pain modulation, but the isozyme-specific effects of PKC on different aspects of pain modulation are not fully understood. We hypothesize that different PKC isozymes play different roles in different aspects of pain modulation. Methods The nociceptive behaviors of mice with deletion of PKCα, β, γ, or δ in multiple pain models were compared with their respective wild-type littermates. Also, morphine analgesia and the development of morphine tolerance in mice with deletion of PKCγ were compared with their respective wild-type littermates. Results Thermal hyperalgesia induced by complete Freund's adjuvant injection was significantly attenuated by the deletion of PKCβ, γ, or δ, but not PKCα. Deletion of PKCγ significantly attenuated neuropathic mechanical allodynia induced by spared nerve injury, whereas deletion of PKCα enhanced this allodynia. Baseline thermal and mechanical sensitivity, nociceptive behaviors induced by formalin, mechanical allodynia induced by complete Freund's adjuvant injection, were not altered by deletion of PKCα, β, γ, or δ. Finally, morphine analgesia and the development of morphine tolerance were not altered in PKCγ-deficient mice. Conclusions PKC has isozyme-specific effects in pain modulation.
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Chaudhary, K. R., W. J. Cho, E. E. Daniel, and J. M. Seubert. "06 Epoxyeicosatrienoic acid mediated cardioprotection involves mitochondrial caveolin-1 and PKCe." Heart 97, no. 24 (November 24, 2011): e8-e8. http://dx.doi.org/10.1136/heartjnl-2011-301156.6.
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Coghlan, Matthew P., Margaret M. Chou, and Christopher L. Carpenter. "Atypical Protein Kinases Cλ and -ζ Associate with the GTP-Binding Protein Cdc42 and Mediate Stress Fiber Loss." Molecular and Cellular Biology 20, no. 8 (April 15, 2000): 2880–89. http://dx.doi.org/10.1128/mcb.20.8.2880-2889.2000.
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ABSTRACT Both the Rho family of low-molecular-weight GTP-binding proteins and protein kinases C (PKCs) mediate responses to a variety of extracellular and intracellular signals. They share many downstream targets, including remodeling of the actin cytoskeleton, activation of p70S6 kinase and c-jun N-terminal kinase (JNK), and regulation of transcription and cell proliferation. We therefore investigated whether Rho family GTP-binding proteins bind to PKCs. We found that Cdc42 associates with atypical PKCs (aPKCs) PKCζ and -λ in a GTP-dependent manner. The regulatory domain of the aPKCs mediates the interaction. Expression of activated Cdc42 results in the translocation of PKCλ from the nucleus into the cytosol, and Cdc42 and PKCλ colocalize at the plasma membrane and in the cytoplasm. Expression of activated Cdc42 leads to a loss of stress fibers, as does overexpression of either the wild type or an activated form of PKCλ. Kinase-dead PKCλ and -ζ constructs acted as dominant negatives and restored stress fibers in cells expressing the activated V12 Cdc42 mutant, indicating that Cdc42-dependent loss of stress fibers requires aPKCs. Kinase-dead PKCλ and -ζ and dominant-negative N17 Cdc42 also blocked Ras-induced loss of stress fibers, suggesting that this pathway may also be important for Ras-dependent cytoskeletal changes. N17 Rac did not block Ras-induced loss of stress fibers, nor did kinase-dead PKCλ block V12 Rac-stimulated loss of stress fibers. These results indicate that Cdc42 and Rac use different pathways to regulate stress fibers.
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Formisano, Pietro, Francesco Oriente, Francesca Fiory, Matilde Caruso, Claudia Miele, Maria Alessandra Maitan, Francesco Andreozzi, Giovanni Vigliotta, Gerolama Condorelli, and Francesco Beguinot. "Insulin-Activated Protein Kinase Cβ Bypasses Ras and Stimulates Mitogen-Activated Protein Kinase Activity and Cell Proliferation in Muscle Cells." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6323–33. http://dx.doi.org/10.1128/mcb.20.17.6323-6333.2000.
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ABSTRACT In L6 muscle cells expressing wild-type human insulin receptors (L6hIR), insulin induced protein kinase Cα (PKCα) and β activities. The expression of kinase-deficient IR mutants abolished insulin stimulation of these PKC isoforms, indicating that receptor kinase is necessary for PKC activation by insulin. In L6hIR cells, inhibition of insulin receptor substrate 1 (IRS-1) expression caused a 90% decrease in insulin-induced PKCα and -β activation and blocked insulin stimulation of mitogen-activated protein kinase (MAPK) and DNA synthesis. Blocking PKCβ with either antisense oligonucleotide or the specific inhibitor LY379196 decreased the effects of insulin on MAPK activity and DNA synthesis by >80% but did not affect epidermal growth factor (EGF)- and serum-stimulated mitogenesis. In contrast, blocking c-Ras with lovastatin or the use of the L61,S186 dominant negative Ras mutant inhibited insulin-stimulated MAPK activity and DNA synthesis by only about 30% but completely blocked the effect of EGF. PKCβ block did not affect Ras activity but almost completely inhibited insulin-induced Raf kinase activation and coprecipitation with PKCβ. Finally, blocking PKCα expression by antisense oligonucleotide constitutively increased MAPK activity and DNA synthesis, with little effect on their insulin sensitivity. We make the following conclusions. (i) The tyrosine kinase activity of the IR is necessary for insulin activation of PKCα and -β. (ii) IRS-1 phosphorylation is necessary for insulin activation of these PKCs in the L6 cells. (iii) In these cells, PKCβ plays a unique Ras-independent role in mediating insulin but not EGF or other growth factor mitogenic signals.
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Gundersen, Cameron B., Sirus A. Kohan, Qian Chen, Joseph Iagnemma, and Joy A. Umbach. "Activation of protein kinase Cη triggers cortical granule exocytosis in Xenopus oocytes." Journal of Cell Science 115, no. 6 (March 15, 2002): 1313–20. http://dx.doi.org/10.1242/jcs.115.6.1313.
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Previous work has shown that phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of protein kinase C (PKC) that mediate(s) this regulated secretory event. Because this process is initiated by lipid activators of PKC but is independent of calcium ions, we focused on the family of novel(calcium-independent) PKCs. Pharmacological investigations using Gö6976 and Gö6983 tended to exclude PKCδ, ϵ and μ as secretory triggers. Subcellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCη that colocalized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCδ and η strongly supported the conclusion that it is PKCη that initiates cortical granule exocytosis in these cells. These observations represent an important step in identifying the mechanism of secretory triggering in this system.
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Reyland, Mary E., David L. Williams, and Elizabeth K. White. "Inducible expression of protein kinase Cα suppresses steroidogenesis in Y-1 adrenocortical cells." American Journal of Physiology-Cell Physiology 275, no. 3 (September 1, 1998): C780—C789. http://dx.doi.org/10.1152/ajpcell.1998.275.3.c780.
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We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCα isoform mediates this suppression, we have developed Y-1 cell lines in which PKCα is expressed from a tetracycline-regulated promoter. Induction of PKCα expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCα expression is increased. However, expression of PKCα has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCα but not PKCε or PKCζ. These studies define for the first time the role of a specific PKC isoform (PKCα) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.
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Di Baldassarre, A., C. Di Rico, T. Bonfini, A. Iacone, M. Marchisio, S. Miscia, E. Alfani, A. R. Migliaccio, and G. Migliaccio. "Protein Kinase C (PKC) α Induces Expression of Aγ-Promoter-Controlled Genes in Cellular Models of Hemoglobin Switch." Blood 106, no. 11 (November 16, 2005): 3640. http://dx.doi.org/10.1182/blood.v106.11.3640.3640.
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Abstract PKCs are serine-threonine kinases that play an important role in many cellular functions. Almost all the 12 PKC isoforms described so far are expressed throughout erythroid differentiation of human CD34pos cells. Besides the downmodulation of PKCε expression (Bassini et al, Blood93; 1178, 1999), little is known on the role exerted by each PKC on erythroid differentiation. PKCα and PKCδ are two isoforms expressed at comparable levels (as mRNA and protein) throughout differentiation of CD36highCD235alow pro-erythroblasts into orthochromatic erythroblasts. For both proteins, the ratio between phosphorylated and total form remains constant during the differentiation of adult cells. In contrast, the ratio P-PKCα/total PKCα (but not that of P-PKCδ/total PKCδ) increases by 3-fold (with slight donor-to-donor variability) during the differentiation of CD36highCD235alow cells from cord blood. Furthermore, the protein becomes prominently localized in the nucleus of these cells. Since the most stricking difference in the differentiation of adult vs. neonatal erythroblasts is the γ/γ+β globin expression ratio (0.02–0.08 vs. 0.20–0.40 by Taqman, respectively), we hypothesized that the levels of PKCα activity might affect γ-globin expression in erythroblasts. To test this hypothesis, we used two in vitro models of HbF switching. The first model is represented by GM979 cells stably transfected with a dual luciferase (Renilla, R - Firefly, F) reporter driven by either the human γ- or β-globin promoter. The second model is represented by human erythroblasts obtained in HEMA culture. GM979 cells were transiently transfected with expression constructs encoding either the catalytic subunit (sPKCα) or the catalytic inactive (iPKCα) PKCα. The levels of expression of the γ-driven and β-driven luciferase reporters were then measured 24 hrs after transfection. Alternatively, GM979 cells were incubated with concentrations of rottlerin (30 μM) that specifically inhibit PKCα activity. sPKCα increased by 2–3-fold the expression of the luciferase driven by the γ-promoter but did not affect expression from the β promoter. Therefore, the ratio Aγ-F/(Aγ-F+2β-R) was also increased by 2–3 fold. Conversely, rottlerin inhibited (by 50%) both expression of the γ-driven luciferase and the Aγ-F/(Aγ-F+2β-R) ratio. On the other hand, transfection of the double luciferase reporter gene into adult and neonatal CD36highCD235alow cells (30–50% transfection efficiency) resulted in high levels of expression of both reporters, in a ratio consistent with the ontogenic stage of the cells [high Aγ-F/(Aγ-F+2β-R) in neonatal erythroblasts; low Aγ-F/(Aγ-F+2β-R) in adult erythroblasts]. Co-trasfection of sPKCα with this luciferase reporter in CD36highCD235alow cells (both adult and neonatal) increased by 5–6-fold the expression of the luciferase driven from the γ-promoter. Co-trasfection of iPKCα, sPKCδ and iPKCδ had no effect on the expression of the reporters in these cells. In conclusion, using several models of erythroid differentiation, we observed that increased levels of PKCα activity results in increased activity of the γ-promoter, suggesting that element(s) of the haemoglobin switching machinery may represent a specific PKCα substrate in erythroid cells.
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Jackson, Twila A., Rebecca E. Schweppe, David M. Koterwas, and Andrew P. Bradford. "Fibroblast Growth Factor Activation of the Rat PRL Promoter is Mediated by PKCδ." Molecular Endocrinology 15, no. 9 (September 1, 2001): 1517–28. http://dx.doi.org/10.1210/mend.15.9.0683.
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Abstract Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCδ is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCδ from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (α, βI, βII, andε ) did not translocate in response to fibroblast growth factors. The PKCδ subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCδ adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCδ isoform. PKC inhibitors selective for the conventional α and β isoforms or dominant negative PKCα adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCδ expression but not by an analogous dominant negative PKCα construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCδ.
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LeHoux, Jean-Guy, and Andrée Lefebvre. "Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB." Journal of Molecular Endocrinology 36, no. 1 (February 2006): 51–64. http://dx.doi.org/10.1677/jme.1.01908.
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We previously reported that H295R cells co-express three diacylglycerol (DAG)-dependent protein kinase Cs (PKCs), namely conventional (c) PKCα and novel (n) PKCε and PKCϑ. The aim of the present work was to evaluate the implication of DAG-dependent PKCs in the activation of p44/42 MAP kinase (MAPK) by angiotensin II (Ang II) and to define the role of this pathway towards CYP11B2 regulation in H295R cells. The PKC inhibitor bisindolylmaleimide 1 (Bis) inhibited Ang II-induced p44/42 MAPK phosphorylation whereas the cPKC inhibitor Gö6976 failed to do so, thus ruling out the participation of PKCα. Ang II activated nPKCε and did not affect nPKCϑ, pinpointing PKCε as the mediator of Ang II in p44/42 MAPK activation. Overexpression of wild-type ERK1 and ERK2 significantly reduced basal as well as Ang II-stimulated human -2023CYP11B2-CAT activity; conversely, the two dominant negative mutants increased them. Overexpression of constitutively active (ca) PKCsuppressed Ang II-induced -2023CYP11B2-CAT activity. Infection of H295R cells with adenoviruses (Adv) expressing caPKCε activated endogenous MEK1/2 and p44/42 MAPK. Adv-caPKCε inhibited Ang II-stimulated aldosterone synthase mRNA levels and this action was reversed by the MEK1 inhibitor, PD98059. Also, Ang II increased JunB protein levels and this effect was inhibited by PD98059 and Bis. Adv-caPKCε enhanced JunB protein levels and PD98059 attenuated the increase. JunB overexpression abolished the Ang II-induced promoter activity within -138 bp of the 5′-flanking region of CYP11B2. Collectively, these results demonstrate that PKCε inhibits CYP11B2 transcription through the p44/42 MAPK pathway and JunB in H295R cells.
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Di Marcantonio, Daniela, Jessica Vadaketh, Esteban Martinez, Prisco Mirandola, Giuliana Gobbi, Michael D. Milsom, Claudia Scholl, Stefan Fröhling, Marco Vitale, and Stephen Matthew Sykes. "Pkc Epsilon Regulates Mitochondrial Redox Biology to Support the Differentiation Blockade in Acute Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 444. http://dx.doi.org/10.1182/blood.v126.23.444.444.
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Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by the clonal expansion of myeloid progenitors that have impaired differentiation capacity. Determining the molecular machinery that regulate the survival and differentiation blockade of AML cells could serve as a foundation for designing novel therapies. PKCε is a serine-threonine kinase belonging to the subgroup of the Protein Kinase C family called Novel PKCs. Aberrant PKCε expression and activation is associated with the pathogenesis and chemotherapy resistance of many solid cancers. However, the contribution of PKCε in blood malignances such as AML is not well defined. To evaluate the role of PKCe in AML biology, we employed short hairpin RNA (shRNA)-mediated approaches to down-regulate PKCε expression in human and murine AML cell lines. We found that shRNA-mediated knockdown of PKCε significantly reduces the in vitro expansion of several human AML cell lines (MOLM-14, NOMO-1, OCI-AML3, THP-1 and U937). We also observed that blocking PKCε induces caspase-3 cleavage and increases the number of annexin V-positive cells (P<0.05), suggesting that PKCε antagonizes AML cell apoptosis. Additionally, we have also found that prior to cell death, AML cells expressing PKCε-targeting shRNAs display characteristics of myeloid differentiation. Specifically, down-regulation of PKCε results in altered expression of the myeloid differentiation transcription factors C/EBPa and PU.1 and increased expression of the mature myeloid marker CD11b (P<0.001). Moreover, upon PKCε inhibition, AML cells acquire morphological changes associated with differentiation, such as increased cytoplasmic volume, granule formation and nuclear segmentation. Interestingly, we observed similar phenotypic changes when we inhibited PKCε expression in murine AML cell lines driven by the leukemogenic fusion protein MLL-AF9 alone (MLL-AF9) or in combination with the internal tandem duplication mutation of murine Flt3 (MLL-AF9;Flt3-ITD). Specifically, we observed that PKCε down-modulation significantly reduces murine AML cell survival (P<0.001) and colony formation in methylcellulose (P<0.001) of both MLL-AF9 and MLL-AF9;Flt3-ITD cells compared to non-targeting shRNA-expressing cells. We are currently investigating how PKCε inhibition impacts AML progression in vivo using mouse models of AML driven by MLL-AF9 or MLL-AF9;Flt3-ITD. At the molecular level, we have found that PKCε is a key regulator of reactive oxygen species (ROS) biology in AML cells. Specifically, using a fluorogenic probe (CellRox) that indiscriminately detects most types of ROS, we have observed that PKCε knockdown in human AML cell lines results in increased steady-state levels of intracellular ROS compared to shRNA control cells (P<0.002). Total intracellular ROS levels are influenced by the production and clearance of distinct ROS types in various cellular compartments. To further characterize the localization and specific type(s) of ROS regulated by PKCε, we utilized four redox-sensitive GFP (roGFP) probes, which allow for direct measurement of cytoplasmic and mitochrondrial glutathione redox potential (Grx1-roGFP-Cyto and Grx1-roGFP-Mito, respectively) and hydrogen peroxide (H2O2) levels (Orp1-roGFP-Cyto and Orp1-roGFP-Mito, respectively) in live cells. Down-modulation of PKCε in NOMO-1 and OCI-AML-3 cells expressing each of these roGFP constructs resulted in a significant increase in the oxidation of Grx1-roGFP (P<.0007) and Orp1-roGFP-Mito (P<0.02) but not either of cytoplasmic constructs, suggesting that PKCε regulates the production of ROS in the mitochondria of AML cells. Since increased H2O2 production and glutathione oxidation results from increased superoxide (O2-) production in mitochondria, we next evaluated the impact of PKCε on O2- production. Using a fluorogenic probe (MitoSOX) that specifically detects O2- in live cells, we found that PKCε down-modulation increases the production of O2- in AML cells (P<0.05). Our future studies are focused on determining the precise molecular events that connect alterations in redox biology with the survival and differentiation of AML cells. Collectively, these results uncover the previously unrecognized role of PKCε as a critical regulator of mitochondrial redox biology and supporter of cell survival and impaired differentiation in AML. Disclosures No relevant conflicts of interest to declare.
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Waidha, Kamran, Nikhil Ponnoor Anto, Divya Ram Jayaram, Avi Golan-Goldhirsh, Saravanakumar Rajendran, Etta Livneh, and Jacob Gopas. "6,6′-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity." Molecules 26, no. 9 (May 8, 2021): 2785. http://dx.doi.org/10.3390/molecules26092785.
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Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.
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Kurniawan, Rahmat. "Perancangan dan Implementasi Sistem Otentikasi OAuth 2.0 dan PKCE Berbasis Extreme Programming (XP)." Jurnal Pendidikan dan Teknologi Indonesia 2, no. 2 (February 16, 2022): 601–11. http://dx.doi.org/10.52436/1.jpti.141.
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Perusahaan XYZ adalah sebuah perusahaan swasta yang menyediakan sebuah produk Hospital Management System (HMS) yang bersifat subscription. Dimana lebih dari satu rumah sakit menggunakan produk ini dengan satu server yang terpusat. Permasalahan yang ingin diuji pada penilitian ini adalah adalah bagaimana merancang sistem otentikasi dan otorisasi untuk sistem HMS multitenant sehingga sistem tidak bisa digunakan oleh pihak yang tidak terdaftar. Tujuan penelitian ini adalah merancang dan menerapkan prosedur otentikasi dengan mekanisme otentikasi OAuth 2.0 dan PKCE pada aplikasi HMS multitentant dengan melibatkan suatu server dan client dalam melakukan proses otentikasi untuk mempermudah proses otentikasi pada tiap tenant. Pada penelitian ini akan melibatkan tiga aplikasi. Satu aplikasi sebagai middleware dimana terdapat halaman sign-in berbasis OAuth 2.0. Dan dua aplikasi lagi sebagai client dan server. Pada penelitian ini dilakukan proses pengembangan sistem menggunakan metode Extreme Programming (XP). Hasil dari penelitian ini berupa sistem login atau sistem otorisasi dan otentikasi yang secara nyata dapat memenuhi kebutuhan perusahaan XYZ. Sistem login ini memiliki kelebihan dimana mempermudah perusahaan XYZ untuk mengatur client yang terintegrasi dengan sistem ini dan bagi client mudah untuk menimplementasikannya.
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WANG, Lijun, Mark ROLFE, and Christopher G. PROUD. "Ca2+-independent protein kinase C activity is required for alpha1-adrenergic-receptor-mediated regulation of ribosomal protein S6 kinases in adult cardiomyocytes." Biochemical Journal 373, no. 2 (July 15, 2003): 603–11. http://dx.doi.org/10.1042/bj20030454.
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The α1-adrenergic agonist, phenylephrine (PE), exerts hypertrophic effects in the myocardium and activates protein synthesis. Both Ca2+-dependent protein kinase C (PKC, PKCα) and Ca2+-independent PKC isoforms (PKCδ and ε) are detectably expressed in adult rat cardiomyocytes. Stimulation of the α1-adrenergic receptor by PE results in activation of Ca2+-independent PKCs, as demonstrated by translocation of the δ and ε isoenzymes from cytosol to membrane fractions. PE also induces activation of p70 ribosomal protein S6 kinases (S6K1 and 2) in adult cardiomyocytes. We have studied the role of Ca2+-independent PKCs in the regulation of S6K activity by PE. Activation of S6K1/2 by PE was blocked by the broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) I, whereas Gö6976, a compound that only inhibits Ca2+-dependent PKCs, did not inhibit S6K activation. Rottlerin, which selectively inhibits PKCδ, also prevented PE-induced S6K activation. The isoform-specific PKC inhibitors had similar effects on the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1, a translation repressor that, like the S6Ks, lies downstream of the mammalian target of rapamycin (mTOR). Infection of cells with adenoviruses encoding dominant-negative PKCδ or ε inhibited the activation of extracellular-signal-regulated kinase (ERK) by PE, and also inhibited the activation and/or phosphorylation of S6Ks 1 and 2. The PE-induced activation of protein synthesis was abolished by BIM I and markedly attenuated by rottlerin. Our data thus suggest that Ca2+-independent PKC isoforms play an important role in coupling the α1-adrenergic receptor to mTOR signalling and protein synthesis in adult cardiomyocytes.
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Jacob, Avraham I., Miriam Horovitz-Fried, Shlomit Aga-Mizrachi, Tamar Brutman-Barazani, Hana Okhrimenko, Yehiel Zick, Chaya Brodie, and Sanford R. Sampson. "The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling." Journal of Molecular Endocrinology 44, no. 3 (December 1, 2009): 155–69. http://dx.doi.org/10.1677/jme-09-0119.
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Protein kinase C delta (PKCδ) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKCα and PKCδ regulatory and catalytic domains to elucidate which components of PKCδ are responsible for positive regulatory effects of PKCδ on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKCδ was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKCδ/α and PKCα/δ chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR–PKCδ binding was higher in cells expressing either the PKCδ/α or PKCα/δ chimera than in control cells, upregulation of IR signaling was observed only in PKCδ/α cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKCδ/α and the PKCδ/δ domains than in cells expressing the PKCα/δ domains. Basal binding of Src to PKCδ was higher in both PKCδ/α- and PKCα/δ-expressing cells compared to control. Binding of Src to IR was decreased in PKCα/δ cells but remained elevated in the PKCδ/α cells in response to insulin. Finally, insulin increased Src activity in PKCδ/α-expressing cells but decreased it in PKCα/δ-expressing cells. Thus, the regulatory domain of PKCδ via interaction with Src appears to determine the role of PKCδ as a positive regulator of IR signaling in skeletal muscle.
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Takahashi, Hideyuki, and Hideo Namiki. "Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors." Biochemical Journal 405, no. 2 (June 27, 2007): 331–40. http://dx.doi.org/10.1042/bj20070299.
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ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCα, PKCβII, PKCδ and PKCϵ, but not atypical PKCζ, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCα–GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.
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Mostafavi-Pour, Zohreh, Janet A. Askari, Scott J. Parkinson, Peter J. Parker, Tony T. C. Ng, and Martin J. Humphries. "Integrin-specific signaling pathways controlling focal adhesion formation and cell migration." Journal of Cell Biology 161, no. 1 (April 14, 2003): 155–67. http://dx.doi.org/10.1083/jcb.200210176.
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The fibronectin (FN)-binding integrins α4β1 and α5β1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of α4+/α5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with α4β1 and α5β1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, α5β1 and α4β1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; α5β1 requires a proteoglycan coreceptor (syndecan-4), and α4β1 does not. Second, adhesion via α5β1 caused an eightfold increase in protein kinase Cα (PKCα) activation, but only basal PKCα activity was observed after adhesion via α4β1. Pharmacological inhibition of PKCα and transient expression of dominant-negative PKCα, but not dominant-negative PKCδ or PKCζ constructs, suppressed focal adhesion formation and cell migration mediated by α5β1, but had no effect on α4β1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCα signaling as central to the functional differences between α4β1 and α5β1.
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Egea-Jiménez, Antonio L., Senena Corbalán-García, and Juan C. Gómez-Fernández. "The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ." Biochimica et Biophysica Acta (BBA) - Biomembranes 1838, no. 7 (July 2014): 1898–909. http://dx.doi.org/10.1016/j.bbamem.2014.04.003.
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Bharti, Ajit, Stine-Kathrein Kraeft, Mrinal Gounder, Pramod Pandey, Shengfang Jin, Zhi-Min Yuan, Susan P. Lees-Miller, et al. "Inactivation of DNA-Dependent Protein Kinase by Protein Kinase Cδ: Implications for Apoptosis." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6719–28. http://dx.doi.org/10.1128/mcb.18.11.6719.
Full textAbstract:
ABSTRACT Protein kinase Cδ (PKCδ) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCδ in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCδ (PKCδ CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCδ cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCδ associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCδ CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCδ CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCδ CF. These findings support the hypothesis that functional interactions between PKCδ and DNA-PK contribute to DNA damage-induced apoptosis.