Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.
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PMID:Roles of the cytoskeleton and of protein phosphorylation events in the osmotic stress response in eel intestinal epithelium. 1229 22

The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) were investigated with a phospho-specific antibody that recognized cofilin only when this protein was phosphorylated on ser-3. Unlike earlier studies that monitored changes in (32)P-labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 microM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide-specific phospholipase C (PI-PLC) (1.0 microM U73122). Products of PI-PLC, sn-1,2-diglyceride and inositol (1,4,5)-trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ca(2+), respectively. Treatment of neutrophils with agents that selectively activate PKC [4beta-phorbol 12-myristate 13-acetate (PMA) ] or cellular Ca(2+) (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolylmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 microM) and W-7 (50 microM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less-active analogs of these inhibitors (15 microM promethazine, 50 microM W-5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC-dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP-stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot).
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PMID:Products of phosphoinositide specific phospholipase C can trigger dephosphorylation of cofilin in chemoattractant stimulated neutrophils. 1245 91

The objectives of the present study were to investigate the role of calcium desensitization in vascular hyporeactivity, and the regulatory effects of Rho-kinase, protein kinase C (PKC), and protein kinase G (PKG) on calcium sensitivity. The vascular reactivity and calcium sensitivity with superior mesenteric artery (SMA) from hemorrhagic shock rat were observed by measuring the contraction initiated by norepinephrine (NE) and Ca2+ under depolarizing conditions (120 mmol/L K) in an isolated organ perfusion system. Angiotensin II (Ang-II) and Fasudil, the Rho-kinase agonist and inhibitor, phorbol 12-myristate 13-acetate (PMA) and staurosporine, the PKC agonist and inhibitor, 8Br-cGMP and KT-5823, the PKG agonist and inhibitor, and Calyculin A, myosin light chain phosphatase (MLCP) inhibitor were used as tool agents. The results indicated that vascular reactivity and calcium sensitivity were decreased after hemorrhagic shock. The cumulative dose-response curve of SMA to NE and Ca2+ after shock was shifted to the right. Ang-II (10 mol/L) could improve the decreased vascular reactivity by increasing the calcium sensitivity of SMA, and insulin (100 nmol/L) could further decrease the vascular reactivity by decreasing the calcium sensitivity of SMA. These results suggested that the vasculature after shock was desensitized to calcium, which played an important role in the onset of vascular hyporeactivity after shock. PMA and KT-5823 could increase the sensitivity of SMA to Ca2+ and made the cumulative dose-response curve shift to the left. In contrast, Fasudil, staurosporine, and 8Br-cGMP decreased the sensitivity of SMA to Ca2+ and made the cumulative dose-response curve of Ca2+ shift to the right. Calyculin A (10 mol/L) pretreatment further enhanced Ang-II, and PMA induced increase of calcium sensitivity, yet weakened the 8Br-cGMP-induced decrease of calcium sensitivity. Taken together, the data suggest that Rho-kinase, PKC, and PKG are involved in the regulation of calcium sensitivity of vascular smooth muscle after hemorrhagic shock, and their regulatory effects on calcium sensitivity of vasculature are possibly related to MLCP.
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PMID:The role of calcium desensitization in vascular hyporeactivity and its regulation after hemorrhagic shock in the rat. 1589 13

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.
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PMID:Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells. 1593 33

EGF inhibits carbachol-induced chloride secretion by regulating a basolateral potassium channel via phosphatidylinositol 3-kinase (PI 3-kinase) and PKCepsilon activation. Although both EGF and carbachol cause tyrosine phosphorylation of p85 of PI 3-kinase, only EGF activates the enzyme. Serine phosphorylation of p85 is thought to suppress the lipid kinase of PI 3-kinase. Our present study examined whether the differential effects of carbachol and EGF on PI 3-kinase activity correspond to varying phosphorylation of p85, and the mechanisms and consequences. T(84) colonic epithelial cells were treated with either EGF or carbachol. Cell lysates were immunoprecipitated with p85 antibody and blotted with either phosphotyrosine or phosphoserine antibodies. Protein phosphatase (PP) 1 and 2A activities were also measured. Both tyrosine and serine residues of p85 were phosphorylated by carbachol, whereas EGF induced only tyrosine phosphorylation. Moreover, EGF abolished carbachol-induced serine phosphorylation of p85 and activated PP2A without affecting PP1. Carbachol did not affect either phosphatase. Calyculin A or okadaic acid pretreatment reversed the inhibitory action of EGF on carbachol-induced chloride secretion and restored serine phosphorylation of p85. Although carbachol recruits p85, it phosphorylates both serine and tyrosine residues so that the lipid kinase of PI 3-kinase is inhibited. EGF results in p85 tyrosine phosphorylation as well as dephosphorylation of serine residues via the activation of PP2A. This explains the differential induction of PI 3-kinase enzyme activity in response to EGF and/or carbachol and has functional implications. Our data provide further insights into negative signals that regulate chloride secretion and into the molecular basis of signaling diversification in the intestinal epithelium.
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PMID:Role of protein phosphatase 2A in calcium-dependent chloride secretion by human colonic epithelial cells. 1689 52

Induction of apoptosis by the PP1/PP2A inhibitor calyculin A was inhibited if the CaMKII inhibitor KN-93 was added no later than 10 min after addition of calyculin A. The physiological relevance and mechanism of CaMKII during apoptosis, however, remains largely unclear. Here we show in MDCK and gastric parietal cells that normal transregulation of CaMKII terminates the initial burst of autophosphorylation after only 10 min. The kinetics of CaMKII involved transregulation by PP1, PP2A, PP2B and PKCalpha. Transregulation of CaMKII resulted in two kinetic phases for phosphorylation of the autoactivation site at T286/287. During the initial phase, there was a clear peak of phosphorylation that lasted 10 min. This phase was subsequently followed by a half but constant level of T286/287 phosphorylation. Calyculin A perturbed this transregulation, resulting in a hyperphosphorylated CaMKII. This effect of CA on the kinetics of CaMKII was observed in vivo as well as in vitro using isolated tubulovesicles. Calyculin A-induced hyperphosphorylation of CaMKII appears to be at least one mechanism used by cells to trigger apoptosis. Therefore, stringent limitation of CaMKII autophosphorylation at T286/287 by transregulation and prevention of hyperphosphorylation seems to restrict apoptosis.
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PMID:Stringent time-dependent transregulation of calcium calmodulin kinase II (CaMKII) is implicated in anti-apoptotic control. 1799 39


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