Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2, 4-dinitrophenol (50 microM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 +/- 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 +/- 0. 2 pS. Channels had an open probability of 0.31 +/- 0.08 and opened in bursts averaging 3.55 +/- 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosol to membrane; and down-regulation of PKCalpha by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 microM) each inhibited currents. Moreover, intracellular perfusion with purified PKCalpha activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCalpha may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation.
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PMID:Metabolic stress opens K+ channels in hepatoma cells through a Ca2+- and protein kinase calpha-dependent mechanism. 866 72

Cardiac preconditioning is mediated by protein kinase C. Although endogenous calcium is a potent stimulus of protein kinase C, it remains unknown whether preischemic administration of exogenous calcium can induce protein kinase C-mediated myocardial protection against ischemia-reperfusion injury. To study this, calcium chloride was administered retrogradely through the aorta at a rate 5 nmol/min for 2 minutes to isolated perfused rat hearts 10 minutes before a 20-minute ischemia and 40-minute reperfusion insult. Calcium-mediated cardioadaptation was then linked to protein kinase C by means of the protein kinase C inhibitor chelerythrine (20 mumol.L-1.2 min-1). To determine whether exogenous calcium administration induces protein kinase C translocation and activation, immunohistochemical staining for the calcium-dependent protein kinase C isoform alpha was performed on adjacent 5 microns myocardial sections with and without calcium chloride treatment. Results indicated that preischemic calcium chloride administration improved myocardial functional recovery, as determined by enhanced developed pressure, improved coronary flow, reduced end-diastolic pressure, and decreased creatine kinase leakage during reperfusion. Beneficial effects of calcium chloride were eliminated by concurrent protein kinase C inhibition. Immunohistochemical staining for the alpha isoform of protein kinase C demonstrated that calcium chloride induces translocation of this isoform from the cytoplasm to the sarcolemma, indicating that exogenous calcium administration activates this isoform. These results suggest that calcium chloride, a safe and routinely administered agent, can induce protein kinase C-mediated cardiac preconditioning. Calcium-induced cardioadaptation to ischemia-reperfusion injury may be promising as a clinically feasible therapy before planned ischemic events such as cardiac allograft preservation and elective cardiac operations.
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PMID:Cardiac preconditioning with calcium: clinically accessible myocardial protection. 880 Jan 68

The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of 'mode 2' gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of 'mode 2' gating. This effect of PP2Ac was completely prevented by 1 microM okadaic acid. The catalytic subunit of PPI (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.
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PMID:A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells. 880 40

The gene encoding the alpha isoform of rat Ca2+/calmodulin-dependent protein kinase II was cloned, and its exon-intron organization was analyzed. The coding region of cDNA consists of 18 exons spanning more than 50 kilobase pairs. Each of the discrete functional units, such as the ATP-binding site, the autophosphorylation site responsible for Ca2+-independent activity, the calmodulin-binding site, and link structure is encoded by a single exon. The largest and smallest exons consist of 229 and 41 base pairs, respectively. All splice junction sequences flanking the introns conform to the consensus splice junction sequence and the GT-AG splice rule.
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PMID:Gene of rat Ca2+/calmodulin-dependent protein kinase II alpha isoform -- its cloning and whole structure. 891 14

Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca2+ have indirectly implicated the alpha isoform of PKC in upregulation of "late" (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of "early" differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v-Ha-ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKC alpha in regulating expression of these markers in normal and v-Ha-ras-transduced primary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKC alpha AS reduced the PKC alpha protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKC alpha, PKC delta, PKC epsilon, and PKC eta confirmed that PKC alpha AS selectively reduced the level of PKC alpha but not the other isoforms. In vitro kinase assays also revealed suppression of Ca(2+)-dependent PKC activity, which is the PKC alpha activity in this cell type, after transfection of PKC alpha AS. When PKC alpha AS-treated normal keratinocytes were stimulated to terminally differentiate with Ca2+, induction of the late differentiation markers loricrin, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS-treated controls. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, transfection of PKC alpha AS, but not the scrambled AS control, selectively downregulated PKC alpha and restored differentiation-specific expression of K1. These findings directly confirm that PKC alpha is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKC alpha contributes to the altered differentiation program of neoplastic murine keratinocytes.
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PMID:Definition by specific antisense oligonucleotides of a role for protein kinase C alpha in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes. 902 12

The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A2 (PLA2) activity in subsequently prepared MDCK-D1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA2 activity of cPLA2 immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA2 in MDCK-D1 cells. The BK-promoted increase of cPLA2 activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA2. However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKC alpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D1 cells (C. Godson, K.S. Bell, and P.A. Insel. [corrected] J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKC alpha-selective inhibitor GF109203X nor transfection of cells with PKC alpha antisense cDNA altered BK-mediated AA release. We conclude that PKC alpha is unlikely to play an important role in the regulation of cPLA2 by BK receptors in MDCK-D1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA2 activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D1 cells regulate cPLA2 via phosphorylation mediated by kinases other than ERK and PKC alpha.
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PMID:Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells. 914 65

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.
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PMID:Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line. 918 12

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.
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PMID:Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts. 924 46

We report here that amino acid analogs, which activate hsp70 promoter, are powerful transcriptional activators of human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR), an activation which was impaired when the two kappaB sites present in the LTR were mutated or deleted. Amino acid analogs also stimulated the transcription of a kappaB-controlled reporter gene. Upon treatment with amino acid analogs, the two NF-kappaB subunits (p65 and p50), which are characterized by a relatively long half-life, redistributed into the nucleus where they bound to kappaB elements. This phenomenon, which began to be detectable after 1 h of treatment, was concomitant with the degradation of the short lived inhibitory subunit IkappaB-alpha by the proteasome. However, contrasting with other NF-kappaB inducers that trigger IkappaB-alpha degradation through a phosphorylation step, amino acid analogs did not change IkappaB-alpha isoform composition. Antioxidant conditions inhibited amino acid analog stimulatory action toward NF-kappaB. This suggests that aberrant protein conformation probably generates a pro-oxidant state that is necessary for IkappaB-alpha proteolysis by the proteasome. Moreover, this activation of NF-kappaB appeared different from that mediated by endoplasmic reticulum overload as it was not inhibited by calcium chelation.
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PMID:Amino acid analogs activate NF-kappaB through redox-dependent IkappaB-alpha degradation by the proteasome without apparent IkappaB-alpha phosphorylation. Consequence on HIV-1 long terminal repeat activation. 945 29

To investigate isoform-specific roles of Ca2+/calmodulin-dependent phosphatase [calcineurin (CaN)] in ischemia-induced cell death, we raised antibodies specific to CaN A alpha and CaN A beta and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5-min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4-7 days after the induced ischemia, immunoreactivities of the CaN A alpha isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN A beta immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up-regulation of CaN A alpha in afferent fibers in CA1 and up-regulation of CaN A beta in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.
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PMID:Isoform-specific redistribution of calcineurin A alpha and A beta in the hippocampal CA1 region of gerbils after transient ischemia. 948 52


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