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)

Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (alpha, beta, gamma and delta), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoforms patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.
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PMID:Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C. 1019 Jan 76

Calponin is an actin-associated protein that appears to play an auxiliary regulatory role in the contraction of smooth muscle. We report here on the mechanisms for regulation of calponin phosphorylation in the endothelin-1-induced contraction of porcine coronary artery. Treatment of strips of porcine artery with endothelin-1 increased calponin phosphorylation and contraction in a concentration-dependent manner. The time course of the phosphorylation was biphasic, with the response to endothelin-1. The extent of phosphorylation reached a maximum within 5 min of stimulation with 10(-7)M endothelin-1 and then it declined rapidly to reach a minimum at 20 min. A potent inhibitor of protein kinase C, GF109203X, inhibited both calponin phosphorylation and contraction that were induced by endothelin-1 at 5 min, without an inhibition for myosin light chain phosphorylation. Protein phosphatase inhibitor, okadaic acid, had no effect on the extent of phosphorylation at 5 min, but it significantly inhibited the subsequent decrease in calponin phosphorylation. In contrast, in PDBu-treated strips of coronary artery, okadaic acid caused a significant steady increase of the extent of calponin phosphorylation. Our results suggest that calponin phosphorylation might be regulated by protein kinase C and okadaic acid sensitive protein phosphatases, in the endothelin-1-induced contraction of porcine coronary artery.
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PMID:Regulatory mechanisms of calponin phosphorylation in endothelin-1-induced contraction of porcine coronary artery. 1037 2

1. Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsiveness to protein kinase C (PKC) activators while intact and alpha-toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2. Western blot analyses showed that the content of the PKC alpha-isoform (PKCalpha) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17. 4. Exogenously replenishing PKCalpha alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated. 6. In in vitro experiments, CPI-17 was a much better substrate for PKCalpha than calponin, caldesmon, MLC and myosin. 7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle.
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PMID:Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. 1051 94

Calponin, an F-actin-associated protein implicated in the regulation of smooth muscle contraction, is known to be phosphorylated in vitro by protein kinase C (PKC) and Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II). Unphosphorylated calponin binds to F-actin and inhibits the actin-activated myosin ATPase activity; these properties are lost on phosphorylation. In the present study, we found that Rho-kinase phosphorylated basic calponin stoichiometrically in vitro. We identified the sites of phosphorylation of calponin by Rho-kinase as Thr-170, Ser-175, Thr-180, Thr-184, and Thr-259, and prepared antibodies that specifically recognized calponin phosphorylated at Thr-170 and Thr-184. We showed that the phosphorylation of calponin by Rho-kinase inhibited the binding of calponin to F-actin. Taken together, these results suggest that calponin is a substrate of Rho-kinase and that Rho-kinase regulates the interaction of calponin with F-actin.
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PMID:Identification of calponin as a novel substrate of Rho-kinase. 1087 72

Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation.
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PMID:A role for serine-175 in modulating the molecular conformation of calponin. 1094 74

Characteristics of supersensitivity induced by the pretreatment with AF64A, an inhibitor of choline uptake at parasympathetic nerve endings, were examined in rat iris sphincter. In preparations isolated and skinned by beta-escin after the micro injection of AF64A to eyes in vivo, the amplitude of maximum contraction in pCa 4.5 solution was increased by 180% of the control from the contralateral eyes. The Ca2+ sensitivity of the contractile system was slightly but significantly increased by AF64A injection; the half maximum contraction was obtained at pCa 5.87 and 6.05 in the control and AF64A-injected eyes, respectively. The increase in maximum contraction in AF64A injected ones was neither affected by the addition of calmodulin, GTPgammaS nor H-7. The increase in Ca2+ sensitivity by AF64A injection was not affected by calmodulin, enhanced by GTPgammaS and abolished by H-7. AF64A injection increased the total protein content only by 30% of the control. The contents of contractile proteins per iris were quantified using Western blotting with monoclonal antibodies. The contents of actin and calponin were increased by AF64A, whereas those of myosin, calmodulin and caldesmon were not affected. The results indicate that AF64A-induced enhancement of the maximum contraction is not mainly due to the increase in the contents of major contractile proteins and that the increase in Ca2+ sensitivity could be due to the mechanism in which changes in protein kinase C and/or GTP binding protein activity are involved.
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PMID:Molecular analysis of non-specific supersensitivity induced by AF64A in rat iris smooth muscle. 1098 92

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.
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PMID:Regulation of protein kinase C by the cytoskeletal protein calponin. 1100 97

SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH(2)-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH(2)-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.
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PMID:Mutagenesis analysis of human SM22: characterization of actin binding. 1105 53

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.
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PMID:Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain. 1123 12

1. Incubation of beta-escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATP gamma S under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca(2+) sensitivity of force and r-MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho-associated kinase (ROK) as it is the case in agonist-induced Ca(2+) sensitization. 2. The increase in Ca(2+) sensitivity induced by pretreatment with ATP gamma S at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with (35)S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon, calponin and CPI-17 was detected. 3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca(2+) sensitivity with an IC(50) of 1.4 microM, the ATP gamma S-induced increase in Ca(2+) sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. 4. Only staurosporine (2 microM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATP gamma S-induced Ca(2+) sensitization of force, r-MLC phosphorylation, and the (35)S incorporation into MYPT1. 5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATP gamma S also induced a staurosporine-sensitive increase in Ca(2+) sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p(21)-associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK. 6. GTP gamma S had an additive effect on ATP gamma S-induced sensitization at saturating concentrations of ATP gamma S. The additional effect of GTP gamma S was inhibited by Y 27632. 7. We conclude that treatment with ATP gamma S under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca(2+) sensitivity that is most likely to be due to thiophosphorylation of MYPT1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.
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PMID:Thiophosphorylation-induced Ca(2+) sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase. 1141 Jun 24


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