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At least 19 recordsLinked to original sources
PubMed1988

Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II.

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.

Animals↗
PubMed1998

Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B, and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription.

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP and protein kinase A, and this induction is inhibited by insulin. Conflicting reports have suggested that insulin inhibits induction by cAMP by activating the Ras/mitogen-activated protein kinase (MAPK) pathway or by activating the phosphatidylinositol 3-kinase (PI3-kinase), but not MAPK, pathway. Insulin activated PI3-kinase phosphorylates lipids that activate protein kinase B (PKB) and Ca2+/diacylglycerol-insensitive forms of protein kinase C (PKC). We have assessed the roles of these pathways in insulin inhibition of cAMP/PKA-induced transcription of PEPCK by using dominant negative and dominant active forms of regulatory enzymes in the Ras/MAPK and PKB pathways and chemical inhibitors of PKC isoforms. Three independently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS, Ras, and MAPK, had no effect upon insulin inhibition. However, dominant active Ras prevented induction of PEPCK and also stimulated transcription mediated by Elk, a MAPK target. Insulin did not stimulate Elk-mediated transcription, indicating that insulin did not functionally activate the Ras/MAPK pathway. Inhibitors of PI3-kinase, LY294002 and wortmannin, abolished insulin inhibition of PEPCK gene transcription. However, inhibitors of PKC and mutated forms of PKB, both of which are known downstream targets of PI3-kinase, had no effect upon insulin inhibition. Dominant negative forms of PKB did not interfere with insulin inhibition and a dominant active form of PKB did not prevent induction by PKA. Phorbol ester-mediated inhibition of PEPCK transcription was blocked by bisindole maleimide and by staurosporine, but insulin-mediated inhibition was unaffected. Thus, insulin inhibition of PKA-induced PEPCK expression does not require MAPK activation but does require activation of PI3-kinase, although this signal is not transmitted through the PKB or PKC pathways.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1992

Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

Affinity Labels↗
PubMed1990

Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II.

Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junctions and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-PK II). Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 32 demonstrated that cAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1. PKC also phosphorylated seryl residues in additional peptides. CA2+/CaM-PK II phosphorylated serine and to a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228-239 based on the deduced amino acid sequence of rat connexin 32) was phosphorylated by cAMP-PK and by PKC, with kinetic properties being similar to those for other physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK II did not phosphorylate the peptide. Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated that Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by cAMP-PK or by PKC. In hepatocytes labeled with [32P]orthophosphoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyrate (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the cAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase C and cAMP dependent protein kinase.

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.

Animals↗
PubMed1985

Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates.

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.

Animals↗
PubMed1991

Ethanol has no effect on cAMP-dependent protein kinase-, protein kinase C-, or Ca(2+)-calmodulin-dependent protein kinase II-stimulated phosphorylation of highly purified substrates in vitro.

The actions of ethanol on kinase stimulated phosphorylation were examined using highly purified protein kinases and a variety of purified substrates. Ethanol (25-200 mM) failed to alter the phosphorylation of histone IIa and histone IIIs by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. Moreover, ethanol (25-200 mM) did not affect the phosphorylation of synapsin I by Ca(2+)-calmodulin-dependent protein kinase II (CAM kinase II). Finally, neither PKA nor PKC stimulated phosphorylation of the GABAA receptor (GABAA-R) was modulated by ethanol at any concentration of ethanol tested. These results suggest that ethanol, in pharmacological concentrations, has no direct actions on the ability of these kinases to catalyze the phosphorylation of specific substrate proteins. In particular, ethanol does not appear to directly influence GABAA-R phosphorylation by either PKA or PKC.

Animals↗
PubMed1999

Effect of myosin light chain kinase, protein kinase A, and protein kinase C inhibition on porcine oocyte activation.

Previous studies have shown that exposure to broad-spectrum protein kinase inhibitors results in parthenogenetic activation of metaphase II arrested porcine oocytes. The objective of this study was to determine the effect of inhibitors of myosin light chain kinase and other protein kinases on pronuclear development, dephosphorylation of a 25-kDa protein, and cortical granule exocytosis. Metaphase II arrested oocytes were obtained by in vitro maturation. Cumulus-free oocytes were cultured with specific inhibitors in modified Whitten's medium for 24 h. Treatment with inhibitors that should inhibit myosin light chain kinase--HA100 (250 microM), Wortmannin (1 microM), and a combination of Wortmannin (1 microM), KT5720 (75 nM), and Iso-H7 (50 microM)--resulted in significantly higher pronuclear development (74.0%, 18.0%, and 35.0%, respectively) than in the negative control, H7 (10 microM; 2.0-12.4% depending upon the replication). Treatment with HA100 (250 microM) resulted in the dephosphorylation of the 25-kDa protein to a 22-kDa protein in 80.0% (n = 10) of oocytes exposed. However, Wortmannin (1 microM; n = 17), KT5720 (75 nM; n = 16), and Iso-H7 (50 microM; n = 19) treatment individually and in combination (n = 19) did not result in significant (p < 0.05; n = 19) dephosphorylation over the negative control, H7 (10 microM; n = 19). HA100 treatment resulted in significant cortical granule exocytosis when evaluated by laser confocal microscopy. In addition, protein kinase assays revealed lower myosin light chain kinase activity in electroactivated oocytes (p < 0.05) and protein kinase inhibitor-treated oocytes (p < 0.05) than in negative controls, nonelectroactivated oocytes, and H7 (10 microM)-treated oocytes. Treatment with HA100 (250 microM) resulted in pronuclear formation, dephosphorylation of the 25-kDa protein, and some release of cortical granules. These observations suggest that inhibition of myosin light chain kinase, protein kinase A, and protein kinase C results in activation of porcine oocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.

Caffeic Acids↗
PubMed1996

Role of tyrosine kinases, protein kinase C, and protein kinase A in the regulation of interferon-alpha production induced by herpes simplex virus type 1.

Herpes simplex virus 1 (HSV-1) is able to induce interferon-alpha production by natural IFN-alpha-producing cells. In this study, signal transduction in this process was examined. It was found that sequestering of calcium by EGTA abolished IFN-alpha induction by HSV-infected cells. Stimulation of human PBMC by HSV-1-infected fibroblasts resulted in the production of inositol triphosphate (InsP3) and tyrosine phosphorylation of cellular proteins. The protein kinase C inhibitor, H7, and the tyrosine kinase inhibitor, herbimycin A, were able to suppress IFN-alpha gene expression as determined by IFN bioassay and RT-PCR. An IFN-alpha-specific ELISpot assay revealed that herbimycin A and H7 remarkably decreased the number of IFN-alpha-producing cells. PMA or calcium ionophore A23187 alone did not increase IFN-alpha production. However, PMA in conjugation with ionophores increased IFN-alpha production as early as 2 h. HA1004 and 2',5'-dideoxyadenosine, which are potent inhibitors of PKA pathway, had no effect on IFN-alpha production. In contrast, BrcAMP, a specific PKA activator, inhibited the IFN-alpha secretion and number of IFN-alpha-producing cells and to a lesser extent reduced the level of IFN-alpha mRNA. Our results indicate that protein kinase C, tyrosine kinases, and protein kinase A are involved in the regulation of IFN-alpha production in response to HSV-1.

Base Sequence↗
PubMed1985

Diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C) activity in bovine thyroid.

Bovine thyroid 100,000 X g supernatant contained diacylglycerol-activated, calcium/phospholipid-dependent protein kinase (protein kinase C). The protein kinase C was partially purified using ion-exchange chromatography and characterized. Substrate specificity studies revealed that the enzyme was most active when histone F1 was used as substrate. The thyroid protein kinase C was not stimulated by Ca2+ or phosphatidylserine (PS), but was stimulated by the combination of the two by 570%. Diolein stimulated the kinase by increasing its sensitivity to Ca2+. Other phospholipids could not substitute for PS and were ineffective in stimulating the protein kinase C in the absence of diolein. However, in the presence of diolein some of the other phospholipids were stimulatory albeit not to the extent of PS. Quercitin, a protein kinase C inhibitor in other systems, inhibited the thyroid enzyme in a dose-related manner. Protein kinase C could also be demonstrated using endogenous thyroid proteins as substrate. Separation of these 32P-labelled proteins by electrophoresis and subsequent autoradiography revealed that three proteins were phosphorylated by the protein kinase C of approximate molecular weights 60,000, 45,000, and less than 29,000. These results offer a possible mechanism by which Ca2+ and/or diacylglycerol effects may be mediated in thyroid.

Animals↗
PubMed1990

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C): general aspects and experimental considerations.

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) is a ubiquitous serine and threonine protein kinase that has been implicated in the regulation of a wide variety of cellular functions. Protein kinase C is tightly linked to signal transduction through phosphatidylinositol turnover, and interacts with the cAMP-protein kinase A pathway in either a "monodirectional" or "bidirectional" mode. The discovery and development of protein kinase C inhibitors and activators offer useful tools to investigate the role of the kinase in specific cellular functions. However, caution should be exercised in interpreting the results of these studies, with special attention directed at experimental design. The present review summarizes some of the general aspects of protein kinase C. Methodological considerations for planning experiments and interpretation of results are described. In addition, we present some examples for the role of protein kinase C in the pathogenesis of disease.

Calcium↗
PubMed1984

Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C).

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been found to phosphorylate and inactivate glycogen synthase. With muscle glycogen synthase as a substrate, the reaction was stimulated by Ca2+ and by phosphatidylserine. The tumor-promoting phorbol esters 12-O-tetradecanoyl phorbol 13-acetate was also a positive effector, half-maximal activation occurring at 6 nM. Phosphorylation of glycogen synthase, but not histone, was partially inhibited by glycogen, half-maximally at 0.05 mg/ml, probably via a substrate-directed mechanism. The rate of glycogen synthase phosphorylation was approximately half that for histone; the apparent Km for glycogen synthase was 0.25 mg/ml. Protein kinase C also phosphorylated casein, the preferred substrate among the individual caseins being alpha s1-casein. Glycogen synthase was phosphorylated to greater than 1 phosphate/subunit with an accompanying reduction in the -glucose-6-P/+glucose-6-P activity ratio from 0.9 to 0.5. Phosphate was introduced into serine residues in both the NH2-terminal and COOH-terminal CNBr fragments of the enzyme subunit. The two main tryptic phosphopeptides mapped in correspondence with the peptides that contain site 1a and site 2. Lesser phosphorylation in an unidentified peptide was also observed. Rabbit liver and muscle glycogen synthases were phosphorylated at similar rates by protein kinase C. The above results are compatible with a role for protein kinase C in the regulation of glycogen synthase as was suggested by a recent study of intact hepatocytes.

Animals↗
PubMed2005

PI 3-kinase, protein kinase C, and protein kinase A are involved in the trigger phase of beta1-adrenergic preconditioning.

OBJECTIVE: Using an isolated non-working rat heart model, this study investigated the mechanisms of pharmacological preconditioning (PC) induced by transient beta1-adrenoreceptor (beta1-AR) stimulation with xamoterol (XA). METHODS: After 6-hydroxydopamine (6-OHDA) pretreatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); exposed to 0.01 microM XA for 5 min with or without 10 microM atenolol (ATE), a specific antagonist of beta1-AR, followed by a 15-min XA-free perfusion before I/R (PC, ATE-PC, respectively); treated during 20 min with either phosphoinositide (PI) 3-kinase inhibitors, LY-294002 (LY, 15 microM), or wortmaninn (WO, 0.1 microM); protein kinase C (PKC) inhibitor, GF-109203X (GF, 4 nM); or protein kinase A (PKA) inhibitor, H89 (H89, 1 microM), with an infusion starting 3 min before XA (LY-PC, WO-PC, GF-PC, and H89-PC, respectively). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP), and creatine kinase (CK) release. RESULTS: XA induced an increase in the MCF after I/R (t 105 min) and a protective effect on the LVEDP, which were blocked by ATE and abolished with the different inhibitors. The transient increase in RPP following XA infusion was blocked by ATE and was not modified by the inhibitors except for H89. Recovery of RPP, measured 25 min after reperfusion, was improved by XA, blocked by ATE, and decreased with the different inhibitors. Fifteen minutes after the end of ischemia, CK release reached maximal values in all groups. XA provided significant protection whereas ATE and the four inhibitors suppressed XA-induced protection. CONCLUSION: The transient preischemic exposure to nanomolar concentrations of a beta1-AR agonist is protective against I/R. PI 3-kinase, PKC, and PKA are implicated in the trigger phase of PC. These observations were confirmed by Western blots.

Adrenergic beta-Agonists↗
PubMed1977

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and phosphodiesterases in arteries and veins of dogs. Distribution and effects of arteriovenous fistula and arterial occlusion.

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Effects of selenium compounds on phospholipid/Ca2+-dependent protein kinase (protein kinase C) system from human leukemic cells.

Selenium compounds (selenium dioxide, selenious acid, and selenic acid) were found to inhibit phospholipid/Ca2+-dependent protein kinase (protein kinase C) and the phorbol ester-stimulated phosphorylation of endogenous substrate proteins from HL60 cells. Kinetic analysis indicated that selenium dioxide (SeO2) inhibited the enzyme noncompetitively with respect to phosphatidylserine (apparent Ki, 60 microM) and Ca2+ (apparent Ki, 68 microM). The inhibitory effect of SeO2 on protein kinase C was additive to that of another inhibitor of the enzyme (alkyl-lysophospholipid) when present together. SeO2 was also equally inhibitory to myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase. It, however, affected only very slightly cyclic adenosine 3':5'-monophosphate-dependent protein kinase. It is suggested that inhibition of Ca2+-dependent reactions might be related to the anticarcinogenic property of selenium.

Animals↗
PubMed1984

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C).

Purified bovine adrenocortical cytochrome P-450scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30 degrees C, with apparent Km and Vmax of 0.14 mumol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 microM Ca2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450scc should be examined for its possible role in the regulation of adrenocortical functions.

Adrenal Cortex↗
PubMed2000

Differential role of beta(1C) and beta(1A) integrin cytoplasmic variants in modulating focal adhesion kinase, protein kinase B/AKT, and Ras/Mitogen-activated protein kinase pathways.

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.

Animals↗