Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.
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PMID:Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. 385 14

The role of different protein kinases in the process of T cell activation has been studied using several inhibitors. The model we adopted was the activation of PBMC by monoclonal antibody OKT3. The results obtained confirm that PKC and PTK are involved. Thus, the inhibitors H-7, staurosporine, and genistein exerted a dose-dependent inhibition of CD2 up-regulation, CD25 expression, IL-2 production, and cellular proliferation. On the other hand, our data indicate that PKA is not involved since the inhibitor HA1004 was ineffective. W-7, an inhibitor of Ca(2+)-CaM protein kinases, inhibited OKT3-induced modulation of cell-surface markers and PBMC proliferation, whereas a slight increase in IL-2 release was detected at the highest dose used (20 microM). Using the MLCK inhibitor ML-9, we extended our studies to the myosin light chain kinase, which influences the organization of the cytoskeleton. ML-9-inhibited PBMC activation in terms of modulation of cell-surface markers and proliferation but stimulated IL-2 production. Similar results were obtained using the cytoskeleton disruptors demecolcine and cytochalasin B. Taken together the data described herein indicate that T cell activation is a complex event in which, aside from classical signal transduction-associated kinases PKC and PTK, at least two other kinases, Ca(2+)-CaM kinases and MLCK, seem to be involved, the latter probably through correct assembly of the cytoskeleton.
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PMID:Involvement of multiple protein kinases in CD3-mediated activation of human T lymphocytes. 790 41

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca2+. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0-2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca(2+)-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0-2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8-4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the Vmax of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphorylation site B (S828).
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PMID:Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase. 856 50

Previous work has shown that calmodulin (CaM) is constitutively phosphorylated in rat liver, probably by casein kinase II [Quadroni, M., James, P., and Carafoli, E. (1994) J. Biol. Chem. 269, 16116-16122]. A procedure is now described for the isolation of the phosphorylated forms of calmodulin (PCaM) free from CaM, since in vitro phosphorylation experiments yield a 50:50 mixture of 3-4 times phosphorylated CaM and native CaM. The activation of six target enzymes by PCaM was tested: myosin light chain kinase, 3',5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase, and CaM-kinase II. In general, the phosphorylation of CaM caused a decrease in enzyme binding affinity, increasing the Kact by 2-4-fold for MLCK, PDE, PM Ca2+-ATPase, and calcineurin. The Vmax at saturating concentrations of PCaM was less affected, with the exception of CaM-kinase II, which was only minimally activated by PCaM and NOS whose Vmax was increased 2.6 times by PCaM with respect to CaM. Phosphorylation of calmodulin had very little effect on the binding of calcium to the enzyme despite the fact that Ser 101 which is phosphorylated is located in the third calcium binding loop. CD measurements performed on CaM and PCaM indicated that phosphorylation causes a marked decrease in the alpha-helical content of the protein. Phosphorylated CaM is very prone to dephosphorylation and was thus tested as a substrate for several phosphatases. It was unaffected by calcineurin (PP2B), but was a reasonable substrate for the pleiotropic phosphatases PP1gamma and PP2A.
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PMID:Phosphorylation of calmodulin alters its potency as an activator of target enzymes. 957 70

To elucidate the apoptotic signaling pathway, we have generated a cell culture model: S2 cells stably transfected with a Drosophila cell death gene, reaper (rpr). Following rpr overexpression, caspase activation-mediated apoptotic cell death was induced in the cells. Apoptosis triggered by rpr required intracellular Ca(2+) ions and calmodulin. Furthermore, protein kinase inhibitors H-7 (a PKC, PKA, PKG, MLCK, and CKI inhibitor), calphostin C (a PKC inhibitor), or H-89 (a PKA and PKG inhibitor) completely blocked apoptosis induced by rpr, suggesting that some kind of serine/threonine protein kinase(s) act upstream of caspase in apoptotic pathway induced by rpr in S2 cells.
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PMID:Participation of intracellular Ca(2+)/calmodulin and protein kinase(s) in the pathway of apoptosis induced by a Drosophila cell death gene, reaper. 1152 81

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCmu, zeta, and gamma, AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Westem blotting and immunocytochemistry confirmed this finding. The data we present implicate kinase activities in the pathogenesis of this lesion and suggest the involvement of Sertoli cells.
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PMID:Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid. 1179 76

Protein kinase C (PKC) is a superfamily of lipid-dependent protein Ser/Thr kinases consisting of at least 10 isozymes. The present article summarizes the papers presented at the congress symposium of the 74th Annual Meeting of the Japanese Pharmacological Society, in which six special topics regarding PKC isozyme-dependent cellular functions and pathological disorders were discussed. Using a GFP-tagged PKC expression technique, each PKC subtype was suggested to vary its targeting-site in each cell in response to each stimulus and that the targeting to the specific compartment is necessary for the specific cellular responses (NS). A cardioprotective agent, JTV519, was shown to attenuate post-ischemic myocardial injury by mimicking ischemic preconditioning through specific activation of PKC delta (YK). Using an antisense technique, PKC alpha and delta/epsilon were shown to be necessary for gene expression of inducible NO synthase by interleukin-1, one of the proinflammatory cytokines, by a stimulated transactivation of NF-kappa B (TH). In canine cerebral artery, PKC delta and PKC alpha play important roles in the development and the maintenance of vasospasm induced by subarachnoid hemorrhage, respectively (SN); and stretch-induced MLC20 phosphorylation involves MLCK and PKC alpha but not PKC delta activities facilitated by inactivation of myosin phosphatase through Rho activity (KO & KN). To clarify the role of PKC isozymes in insulin resistance, the effects of insulin on glucose uptake, PKC isozyme activation and PI3K activation in rat adipocytes were shown and then platelet PKC beta activation in diabetic patients with various diabetic complications, including diabetic retinopathy, was reported (TI). These studies will promisingly open the way to a new era for the development of novel drugs controlling an isozyme-specific activity of the protein kinase C superfamily and improvement in the knowledge about the role of the protein kinase in health and disease.
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PMID:[Role of protein kinase C isozymes in cellular functions and pathological conditions]. 1186 60

Calponin (CaP), a thin filament-associated protein, plays an important role in the regulation of smooth muscle contractility. It has been known that CaP inhibits the actin-activated myosin MgATPase activity via binding to F-actin, and stimulates myosin MgATPase activity via binding to myosin. Our recent study revealed a new phenomenon that trace amount of CaP (TAC) could influence the function of different states of myosin. Our data showed that in the absence of actin, CaP, even in the concentration of 0.0001 microM, significantly increased the precipitations of 1 microM unphosphorylated myosin, Ca(2+)-CaM dependently, and independently phosphorylated myosin by MLCK, and stimulated the MgATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relative selective effect of TAC. In the presence of actin, myosin, and TAC, the increase of myosin precipitation was abolished, and no obvious changes of actin precipitations and actin-activated myosin MgATPase activities were observed implicating the highly efficiency of TAC on myosin being present in the absence of actin. Although we cannot give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present in the regulation of the function of myosin when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction between TAC and myosin can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.
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PMID:The influence of trace amount of calponin on the smooth muscle myosins in different states. 1514 57

Calponin (CaP), a thin filament-associated protein, is thought to be involved in modulating smooth muscle contractile activity, but the role and mechanism keep unknown. In this study, trace amount of calponin (TAC) was found to obviously influence myosin in different states in Ca(2+)-independent manner, suggesting a high efficient interaction between TAC and myosin. In this assay, the lowest ratio of CaP vs. myosin was 1:10,000, with the concentration of CaP 10,000-fold lower than that used previously. Myosin phosphorylation, myosin Mg(2+)-ATPase activity and protein binding activity were detected to determine the effects of TAC on the myosin in different states. The amount of precipitated myosin that bound to TAC was used as the index to determine the interaction between myosin and TAC in binding assay. Our data showed that in the absence of actin, TAC significantly increased the precipitation of unphosphorylated myosin, Ca(2+)-dependently or independently phosphorylated myosin by MLCK, and stimulated the Mg(2+)-ATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relatively selective effect of TAC. In the presence of actin, the increase of myosin precipitations was abolished, and no obvious change of actin precipitations and actin-activated myosin Mg(2+)-ATPase activities were observed implicating the high efficiency of TAC on myosin being present in the absence of actin. Although we can not give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.
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PMID:Influence of trace amount of calponin on smooth muscle myosin in different states. 1529 44

In rat erythrocytes, the regulation of Na+/Mg2+ antiport by protein kinases (PKs), protein phosphatases (PPs), intracellular Mg2+, ATP and Cl- was investigated. In untreated erythrocytes, Na+/Mg2+ antiport was slightly inhibited by the PK inhibitor staurosporine, slightly stimulated by the PP inhibitor calyculin A and strongly stimulated by vanadate. PMA stimulated Na+/Mg2+ antiport. This effect was completely inhibited by staurosporine and partially inhibited by the PKC inhibitors Ro-31-8425 and BIM I. Participation of other PKs such as PKA, the MAPK cascade, PTK, CK I, CK II, CAM II-K, PI 3-K, and MLCK was excluded by use of inhibitors. Na+/Mg2+ antiport in rat erythrocytes can thus be stimulated by PKCalpha. In non-Mg2+ -loaded erythrocytes, ATP depletion reduced Mg2+ efflux and PMA stimulation in NaCl medium. A drastic activation of Na+/Mg2+ antiport was induced by Mg2+ loading which was not further stimulated by PMA. Staurosporine, Ro-31-8425, BIM I and calyculin A did not inhibit Na+/Mg2+ antiport of Mg2+ -loaded cells. Obviously, at high [Mg2+]i Na+/Mg2+ antiport is maximally stimulated. PKCalpha or PPs are not involved in stimulation by intracellular Mg2+. ATP depletion of Mg2+ -loaded erythrocytes reduced Mg2+ efflux and the affinity of Mg2+ binding sites of the Na+/Mg2+ antiporter to Mg2+. In non-Mg2+ -loaded erythrocytes Na+/Mg2+ antiport essentially depends on Cl-. Mg2+ -loaded erythrocytes were less sensitive to the activation of Na+/Mg2+ antiport by [Cl-]i.
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PMID:Regulation of Na+/Mg2+ antiport in rat erythrocytes. 1532 47


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