Protein Kinase Ce Research Articles

Invited talk: Protein kinase Ce in adipose tissue – Not merely an effector but a regulator of lipid intermediates?

Invited talk: Protein kinase Ce in adipose tissue – Not merely an effector but a regulator of lipid intermediates?

Effects of angiotensin II on protein kinase Cε and Cα expression in hepatic stellate cells

Objective To investigate the effect of angiotensinⅡon protein kinase Ce (PKCe) and protein kinase Cα (PKCα) expression in hepatic stellate cells. Methods Hepatic stellate cell (HSC)-T6 cells were treated with different concentrations of angiotensinⅡand the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium (MTT) assay. The expression of PKCe and PKCα was detected by immunofluorescence staining. PKCe and PKCα mRNA levels was detected by real time polymerase chain reaction (PCR). Results AngiotensinⅡconcentrated the proliferation of HSC-T6 cells and the level of hydroxyproline (F=25.321, 13.283, P<0.001) and showed a dose-dependent effect. With the increase of angiotensin Ⅱ concentration, PKCe significantly increased and translocated in the cell membrane; PKCα increased significantly, especially in transplanted membrane and cytoplasm (F=21.387, 19.431, P<0.01), and showed obvious dose effect. Meanwhile, Angiotensin Ⅱ increased the expression of PKCe and PKCα, and induced cell proliferation by up-regulating PKCe and PKCα mRNA levels (F=13.279, 15.174, P<0.05). Conclusions AngiotensinⅡcan up-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, increase the expression of protein kinase Ce and Cα, and promote the proliferation of hepatic stellate cells. Key words: Angiotensin Ⅱ/PD; Hepatic stellate cells/DE/EN; Protein kinase C-alpha/ME; Protein kinase C-epsilon/ME; Cell proliferation

In vitro study on the role and mechanism of interferon α-1b in regulating the inhibition of protein kinase Cε and Cα on fibrosis of hepatic stellate cells

Objective To investigate the regulation of interferon α-1b (IFNα-1b) on protein kinase Ce (PKCe) and protein kinase Cα (PKCα) which inhibit the fibrosis of hepatic stellate cells (HSC), and to explore its mechanism. Methods HSC-T6 cells were treated with different levels of IFNα-1b (100, 200, 400, 800 and 1 000 U/mL) and the proliferation of HSC-T6 cells was analyzed by methyl thiazol tetrazolium (MTT) assay. Changes of hydroxyproline level were analyzed. The expressions of PKCe and PKCα were detected by immunofluorescence staining. PKCe, PKCα, β-catenin and Survivin mRNA levels were detected by RT-PCR. PKCe, PKCα, β-catenin and Survivin protein levels were detected by Western blot. Variance analysis was conducted by using one-way ANOVA approach. Results The inhibition rates of 100, 200, 400, 800 and 1 000 U/mL IFNα-1b treatment after 24 hours of administration were (15.85±1.05)%, (36.59±1.03)%, (45.12±1.05)%, (50.00±1.01)% and (62.20±1.02)%, respectively, with statistically significant differences among groups (F=27.478, P<0.01). The 48h inhibition rates were (20.87±1.09)%, (43.96±1.08)%, (53.85±1.08)%, (64.84±1.06)% and (74.72±1.07)%, respectively, with statistically significant differences among groups (F=25.321, P<0.01). half maximal inhibitory concentration at 48 h was 343.47 U/mL. The levels of hydroxyproline in 100, 200 and 400 U/mL IFNα-1b groups were (7.48±0.28), (6.26±0.17) and (3.86±0.20) μg/mL, respectively, which were lower than that in control group (8.47±0.32) μg/mL. The differences were all statistically significant (t=4.033, 10.564 and 21.160, respective, all P<0.05). The fluorescence intensities of PKCe in 100, 200 and 400 U/mL IFNα-1b groups were all lower than that of control group. The differences were statistically significant (t=1.984, 2.457 and 7.771, respectively, all P<0.05). The fluorescence intensities of PKCα were also significantly lower than that of control group (t=9.232, 15.921 and 22.222, respectively, all P<0.01). With the increase of IFNα-1b level, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=7.020, 24.562, 45.701 and 14.241, respectively, all P<0.01). With the increase of IFNα-1b, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=9.564, 4.409, 10.036 and 6.794, respectively, all P<0.01). Conclusions IFNα-1b can down-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, reduce the expressions of PKCe, PKCα, β-catenin and Survivin, and inhibit the proliferation of HSC-T6 hepatic stellate cells. Key words: IFNα-1b; hepatic stellate cells; proliferation; molecular mechanism

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression.

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression.

Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3.

SummaryAimsSphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning.MethodsLangendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins.ResultsPre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control).ConclusionThese novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion.

Evaluation of the newly synthesized linoleic acid derivative DCP-LA as a potential anti-dementia drug

Purpose: We synthesized the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]octanoic acid (DCP-LA) with cyclopropane rings instead of cis-double bonds. Here, we introduce DCP-LA as a novel anti-dementia drug. Study section and results: DCP-LA selectively and directly activated protein kinase C-e (PKCe) to enhance the activity of presynaptic a7 acetylcholine (ACh) receptors by promoting receptor translocation to the cell surface and increasing presynaptic glutamate release. This action of DCP-LA caused a long-lasting facilitation of hippocampal synaptic transmission, which improved learning and memory impairments in animal models. Conclusion: DCP-LA could be developed as a promising anti-dementia drug, offering new hope for treating dementias including Alzheimer disease.

In Vivo NMR Studies on the Mechanism of Lipid-Induced Insulin Resistance in Humans

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and the metabolic syndrome. In this lecture I will discuss recent nuclear magnetic resonance studies that have implicated ectopic lipid deposition in liver and skeletal muscle as a causal and unifying factor in the pathogenesis of insulin resistance associated with obesity, lipodystrophy, type 2 diabetes and the metabolic syndrome. I will also present results from recent human studies demonstrating an important role of intracellular diacylglycerol, as the molecular trigger for lipid-induced insulin resistance in liver through its activation of protein kinase Ce and in muscle through its activation of protein kinase Cθ, which both result in reductions in proximal insulin signaling.

Mouse Models of the Skin: Models to Define Mechanisms of Skin Carcinogenesis

Mouse Models of the Skin: Models to Define Mechanisms of Skin Carcinogenesis

RIP140, a Janus metabolic switch involved in defense functions

RIP140, a Janus metabolic switch involved in defense functions

Effects of small interfering RNA against protein kinase Cε gene on proliferation, migration and invasion of human renal clear cell carcinoma cell line 769P

Objective To evaluate the effects of small interfering RNA (siRNA) against protein kinase Ce (PKCe) gene on the proliferation,migration and invasion of human clear cell RCC cell line 769P cells.Methods PKCe-siRNA was transfected into the 769P cells.The mRNA and protein expression of PKCe was detected by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting,respectively.The proliferation of 769P cells was measured by methyl thiazol tetrazolium (MTT) assay and colony formation assay.Cell migration and invasion was assessed by using wound-healing assay and CHEMICON cell invasion assay,respectively.Results PKCe-siRNA efficiently down-regulated the PKCe expression,which resulted in inhibition of the proliferation,migration,and invasion of clear cell RCC cell line 769P.Conclusion PKCeis may be related with proliferation,migration and invasion of 769P cells,and may be a potential therapeutic target for clear cell RCC. Key words: Protein kinase Ce; Renal clear cell carcinoma; Small interfering RNA

Allosteric modulation by protein kinase Cε leads to modified responses of EGF receptor towards tyrosine kinase inhibitors

Recently, we described a novel function of over-expressed protein kinase Cε (PKCε) as a negative allosteric modulator of EGFR signalling in several head and neck squamous carcinoma (HNSCC) cell lines. Extending this work, here we present several lines of evidence for the potency of PKCε to differently modulate the efficacy of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and lapatinib. Using the HNSCC cell line FaDu as a model, we demonstrate by co-immunoprecipitation the physical association of over-expressed PKCε with the EGFR which is stabilised by gefitinib and leads to an increase in gefitinib-induced inhibition of EGFR downstream signalling and elevated EGFR-ErbB2 heterodimerisation. Cell cycle and Western blot analysis revealed that the gefitinib-induced apoptosis was enhanced whereas the pro-apoptotic effect of lapatinib that requires another EGFR conformation was reduced by PKCε. Our findings suggest that due to elevated expression PKCε may associate with the EGFR resulting in conformational changes and different allosteric modulation of the EGFR behaviour towards TKIs. This surprising capacity indicates PKCε as a novel predictive marker protein in molecular cancer therapy with EGFR tyrosine kinase inhibitors.

Expression and clinical significance of novel protein kinase C ε in prostate cancer tissuss

Objective To explore the expression of novel protein kinase C e (PKCe) in normal prostate (NP) tissue, benign prostate hyperplasia(BPH), peficancerous (PC) tissue and prostate cancer (Pca), and study its correlation with the grade and stage of Pca.MethodsTen NP slides, ten BPH slides, ten PC slides and 43 Pca slides were collected from our hospital. These slides were routinely proceased and analyzed according to the requirement of immunohistochemical staining. Tumors were classified according to the 2002 TNM staging system. The grading system used in the study was based on the Gleason grade.Results We was found that the expression of PKCes in Pca (27/43) were significantly higher than those in NP(1/10), BPH (0/10) and PC (2/10) tissue, and the difference was statistically significant ( P ＜0.05 ). With regard to grade of prostate cancer, the expression of PKCe in Pca with Gleason score ≥8 group (12/13) was higher than the Gleason score 2 -4 group (4/10) and the Gleason score 5 -7 group (11/20). The difference was statistically significant (P ＜ 0.05 ). Moreover, the T3 and T4 stages had a more positive rate (10/12 & 9/10) than the T1 and T2 stages( 1/6 &7/15). There is statistically significant difference between early and advanced stage of prostate cancer ( P ＜ 0. 05 ). Furthermore, the positive expression of PKCe in prostatic carcinoma samples increased significantly in the metastasis group (9/10)compared to the non-metastasis group ( 18/33 ) ( P ＜ 0. 05 ), but the difference was not statistically significant between the concentration of prostate-specific antigen in blood serum ( P ＞ 0. 05 ).Conclusions PKCe is expressed in prostate cancer, and it correlates with the grade and stage of prostate cancer. PKCe may be related to the origin and the development of Pca, and it may be used as a prognostic factor for Pca. Key words: Prostate cancer; Protein kinase Ce ; Immunohistochemistry

The role of A1 adenosine receptor in pinacidil pretreatment-induced activation of PKCα and ε and protection of vascular reactivity and calcium sensitivity after hemorrhagic shock in rats

Objective To observe the role of adenosine receptor in the pinacidil pretreatment-induced activation of protein kinase Cα ( PKCα),protein kinase Ce (PKCe) and protection of vascular reactivity and calcium sensitivity after hemorrhagic shock in rats.Methods The changes of adenosine concentration after pinacidil pretreatment,the effects of A1,A2A,A2B and A3 adenosine receptors (A1 R,A2A R,A2BR and A3 R) inhibitors on the pinacidil pretreatment-induced protein expression and translocation of PKCα and PKCe,and the effects of A1R,A2AR,A2BR and A3R inhibitors on the pinacidil pretreatmentinduced protection of vascular reactivity and calcium sensitivity after hemorrhagic shock were measured.Results Pinacidil pretreatment implemented at 30 min before shock could increase the adenosine concentration at 2 h after shock from 7.299 to 11.626 mg/L ( P ＜ 0.01 ); Pinacidil pretreatment could promote the translocation of PKCα and PKCe from cytoplasm to membrane (P ＜0.01 ),and induce the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock,which were all inhibited by A1 R inhibitor ( P ＜ 0.01 ).Conclusion Pinacidil pretreatment could induce the activation of PKCα,PKCe and the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock in rats through A1 R. Key words: Pinacidil; Pretreatment; Vascular reactivity; Calcium sensitivity

Protein kinase (PKC)-ε와 serotonin transporter (SERT)의 C-말단과의 결합

Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of cell-cell signaling in neuronal systems. The serotonin transporter (SERT) on the plasma membrane controls the extracellular 5-HT level by reuptake of released 5-HT from the synaptic cleft, but the underlying regulation mechanism is unclear. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of SERT and found a specific interaction with protein kinase C-e (PKC-e), a PKC isotype that is characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-e bound to the tail region of SERT but not to other members of the Na?/Cl- dependent SLC6 gene family in the yeast two-hybrid assay. The C-terminal region of PKC-e is essential for interaction with SERT. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. PKC-e phosphorylated the peptide of the SERT amino (N)-terminus in vitro. These results suggest that the phosphorylation of SERT by PKC-e may regulate SERT activity in plasma membrane.

Mitochondrial translocator protein modulates metabolism and pharmacologically induced apoptosis in breast cancer cells

Dysfunctional mitochondria contribute to the onset of malignant transformation and growth. Molecules that regulate mitochondrial homeostasis are therefore the object of great attention to identify novel therapeutic strategies. The mitochondrial translocator protein (mTSPO) stands in a critical position for mitochondrial homeostasis and is involved in the physiology of breast cancer where it is overexpressed and positively associated with aggressiveness [1]. mTSPO ligands are therefore exploited for cancer imaging and chemotherapy, such as PK11195. mTSPO is associated with the voltage-dependent anion channels (VDACs), which regulate the metabolites' flux into mitochondria [2]. mTSPO expression is driven by the oncogene protein kinase Ce, suggesting a fundamental crosstalk for malignant transformation and uncontrolled proliferation. We hypothesized that mTSPO by regulating VDAC performance impinges on metabolism and pharmacologically induced cell death in breast cancer cells.

Pain TRP‐ed up by PARs

Studies of proteolytic enzymes (proteinases) like thrombin and trypsin have long been the province of cardiovascular and gastrointestinal physiologists: thrombin as the key regulator of the coagulation cascade and trypsin and other pancreatic enzymes as the means for breaking down proteins in the small intestine. Conforming to a long-standing pattern of ‘where GI physiologists tread, neuroscientists inevitably follow’, the identification of proteinase-activated receptors (PARs) on peripheral and central neurones has generated increasing interest in how they may function in the nervous system (Noorbakhsh et al. 2003; Ossovskaya & Bunnett, 2004). PARs are a four-membered family of G-protein coupled receptors (PAR1–4) whose activation by serine proteinases occurs by enzymatic cleavage at N-terminal serines to expose a ‘tethered ligand’ that binds residues in its own second extracellular domain to initiate signal transduction (Ossovskaya & Bunnett, 2004). PAR-2 has become the current favourite as a promising target in anti-inflammatory/chronic pain drug discovery programmes because it is expressed in sensory nociceptive neurones, its activation releases peptides (SP/CGRP) from afferent nerve endings in the dorsal horn and results in both thermal and mechanical hyperalgesia, and it can be activated by tryptase, which is secreted by mast cells onto adjacent neurones under inflammatory conditions or neuronal damage (Noorbakhsh et al. 2003; Ossovskaya & Bunnett, 2004). Because it is the only PAR not activated by thrombin, selective blockade of this receptor could be therapeutically useful in pain management without antithrombotic side-effects. How does PAR-2 signalling lead to thermal and mechanical hyperalgesia? A global network of research groups, headed by Nigel Bunnett, have presented a set of tour de force studies showing that PAR-2 activates multiple second messenger pathways to sensitize TRPV1 and TRPV4 receptors on nociceptive neurones and this, in turn, releases nociceptive peptides (SP and CGRP) onto centrally projecting nociceptors that can account for TRPV1-dependent thermal and TRPV4-dependent mechanical hyperalgesia (Amadesi et al. 2006; Grant et al. 2007). TRPV1 and TRPV4 receptors are calcium-permeable cationic ion channels activated by multiple sensory stimuli; of relevance here is that TRPV1 can be gated by noxious heat (> 42°C) and capsaicin, and TRPV4 by warm temperature (> 27°C), hypotonicity and shear stress (Nilius et al. 2004). Amadesi et al. (2006) found that PAR-2 colocalized with protein kinase (PK) Ce, and PKA in a subset of TRPV1-containing dorsal root neurones, that PAR-2 agonists activated these pathways, increased TRPV1-mediated calcium influx and whole-cell currents and caused thermal hyperalgesia in mice. Moreover, inhibition of PKCe and PKA prevented the PAR-2 sensitization of TRPV1 calcium transients and currents as well as PAR-2-induced thermal hyperalgesia in mice. Grant et al. (2007) found that PAR-2 also colocalized with TRPV4, SP and CGRP in dorsal root ganglia, that PAR-2 agonists enhanced TRPV4-induced calcium transients and whole-cell currents and that inhibition of phospholipase Cβ, PKA, PKC and PKD prevented the PAR-2-induced sensitization of TRPV4-induced responses. They also found that activation of TRPV4 stimulated the release of SP and CGRP from dorsal horn neurones and this release was potentiated by PAR-2 activation, similar to their previously demonstrated PAR-2 enhancement of TRPV1 release of SP and CGRP (Amadesi et al. 2004). Intraplantar injection of PAR-2 agonists caused mechanical hyperalgesia as well as sensitized pain responses to TRPV4 activation in wild-type but not in TRPV4-deleted mice. These findings provided the basis for a model outlined by Grant et al. (2007) to account for protease-induced mechanical hyperalgesia, a model that can also apply to protease-induced thermal hyperalgesia. Proteases are released in response to inflammatory stimuli where they can activate PAR-2 on nociceptive afferent nerve endings in peripheral tissue. PAR-2 couples to activation of multiple second messenger kinases (PKA, PKC, PKD) in these nerve endings to sensitize TRPV1 and TRPV4 receptors. Sensitized TRPV1 receptors now respond to non-noxious temperature while sensitized TRPV4 receptors show enhanced response to mechanical stimuli; in both cases their increased gating results in increased release of SP and CGRP from dorsal horn terminals with consequent increased transmission centrally. In terms of pathological pain states, prevention of TRPV1 and TRPV4 receptor sensitization is a more attractive therapeutic aim than inhibition of TRPV receptors per se. In this regard, it is of interest to compare results from a recent study by Alessandri-Haber et al. (2006) who found similar mechanisms of TRPV4-dependent sensitization and mechanical hyperalgesia in response to a cocktail of inflammatory mediators (bradykinin, SP, 5-HT, histamine and prostaglandin), none of which alone was sufficient to engage TRPV4-dependent hyperalgesia. PAR-2 antagonists may well pack more punch against neuropathic or inflammatory pain.

Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cε

Mice lacking protein kinase Ce (PKCe) are supersensitive to positive allosteric modulators of gamma aminobutyrate type A (GABAA) receptors. Since many of these compounds are anxiolytic, we examined whether anxiety-like behavior is altered in these mice. PKCe-null mice showed reduced anxiety-like behavior and reduced levels of the stress hormones corticosterone and adrenocorticotrophic hormone (ACTH). This was associated with increased sensitivity to neurosteroid modulators of GABAA receptors. Treatment of PKCe-null mice with the GABAA receptor antagonist bicuculline restored corticosterone levels and anxiety-like behavior to wild-type levels. These results suggest that increased GABAA receptor sensitivity to neurosteroids contributes to reduced anxiety-like behavior and stress hormone responses in PKCe-null mice. The findings also suggest PKCe as a possible therapeutic target for development of anxiolytics.

Angiotensin II inhibits rat arterial KATP channels by inhibiting steady-state protein kinase A activity and activating protein kinase Ce.

We used whole-cell patch clamp to investigate steady-state activation of ATP-sensitive K+ channels (KATP) of rat arterial smooth muscle by protein kinase A (PKA) and the pathway by which angiotensin II (Ang II) inhibits these channels. Rp-cAMPS, an inhibitor of PKA, did not affect KATP currents activated by pinacidil when the intracellular solution contained 0.1 mM ATP. However, when ATP was increased to 1.0 mM, inhibition of PKA reduced KATP current, while the phosphatase inhibitor calyculin A caused a small increase in current. Ang II (100 nM) inhibited KATP current activated by the K+ channel opener pinacidil. The degree of inhibition was greater with 1.0 mM than with 0.1 mM intracellular ATP. The effect of Ang II was abolished by the AT1 receptor antagonist losartan. The inhibition of KATP currents by Ang II was abolished by a combination of PKA inhibitor peptide 5-24 (5 microM) and PKC inhibitor peptide 19-27 (100 microM), while either alone caused only partial block of the effect. In the presence of PKA inhibitor peptide, the inhibitory effect of Ang II was unaffected by the PKC inhibitor Go 6976, which is selective for Ca2+-dependent isoforms of PKC, but was abolished by a selective peptide inhibitor of the translocation of the epsilon isoform of PKC. Our results indicate that KATP channels are activated by steady-state phosphorylation by PKA at normal intracellular ATP levels, and that Ang II inhibits the channels both through activation of PKCepsilon and inhibition of PKA.

Functional Proteomic Analysis of Protein Kinase C ε Signaling Complexes in Preconditioning.

Studies from our laboratory and others have shown that transgenic (Tg) mice expressing low levels of active protein kinase C e (PKCe) exhibit resistance to ischemic injury, a cardioprotected phenotype analogous to that observed during preconditioning. Although PKCe has been shown to activate multiple downstream targets in preconditioning, the …

Protein kinase CE mediates the inhmitory effect of epidermal growth factor on carbachol-induced chloride secretion in T 84 epithelial cells

Protein kinase CE mediates the inhmitory effect of epidermal growth factor on carbachol-induced chloride secretion in T 84 epithelial cells