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)

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.
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PMID:Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function. 132 79

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, alpha-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120-270 to 500-700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca(2+)-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation-dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca(2+)-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C).
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PMID:The Ayerst Award Lecture 1990. Calcium-dependent mechanisms of regulation of smooth muscle contraction. 181 84

Simultaneous measurements of cytoplasmic Ca2+ level [( Ca2+]i) and muscle contraction in smooth muscle indicated that [Ca2+]i gradually decreases during sustained contraction. This time-dependent dissociation has been explained by the latch bridge hypothesis, positive cooperativity between phosphorylated and non-phosphorylated crossbridges, involvement of cytoskeleton phosphorylation, or connection between myosin and actin filaments by caldesmon. Furthermore, it has been found that receptor agonists induce greater contraction than high K+ for a given increase in [Ca2+]i. This stimulus-dependent dissociation may be due to the receptor agonists-induced activation of protein kinase C which in turn decreases the inhibitory effect of calponin on the actin-myosin interaction, resulting in an apparent Ca2+ sensitization. Thus, the contractions induced by receptor agonists are due not only to the increase in [Ca2+]i but also to the increase in Ca2+ sensitivity of contractile elements. Ca2+ channel blockers inhibit the increase in [Ca2+]i but not the Ca2+ sensitization, and this may be the reason why these blockers are relatively weak inhibitors of the contraction induced by receptor agonists. By contrast, cyclic AMP and cyclic GMP decrease the Ca2+ sensitivity of contractile elements in addition to their effects to decrease [Ca2+]i.
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PMID:[Calcium regulation of smooth muscle contractility]. 196 75

Calponin from chicken gizzard consists of two principal components, possibly isoforms, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cleavage with 2-nitro-5-thiocyanobenzoic acid indicated that calponin contains 2 cysteine residues. Purified fragments of 30 and 21 kDa retained the following properties of the intact protein: actin-, tropomyosin- and calmodulin-binding, and ability to inhibit the actin-activated MgATPase activity of smooth muscle myosin. Both fragments, like intact calponin, were phosphorylated by protein kinase C which inhibited their binding to actin and relieved their inhibition of the ATPase. Tryptic digestion of calponin phosphorylated by protein kinase C generated 3 phosphopeptides with the following N-terminal sequences: FASQQGMTAYGTR, GASQQGMTVYGLP, and NHSGHVQ, each possessing a single phosphoserine.
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PMID:Structural and functional characterization of calponin fragments. 215 Oct 18

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.
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PMID:Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation. 216 34

When smooth muscle calponin was incubated with protein kinase C, 1 mole of phosphate was incorporated per mole of calponin. The apparent Km value for calponin of the protein kinase was about 0.4 microM. The phosphorylation of calponin by protein kinase C was inhibited markedly by calmodulin in a calcium-dependent manner. Kinetic analysis of calmodulin-induced inhibition of calponin phosphorylation by protein kinase C revealed that calmodulin inhibited the phosphorylation in a noncompetitive fashion with calponin and the determined Ki value was 0.4 microM. These results suggest that interaction of calmodulin with calponin may play a regulatory role in the phosphorylation by protein kinase C and smooth muscle contraction.
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PMID:Modulation of smooth muscle calponin by protein kinase C and calmodulin. 222 54

Calponin is a thin filament-associated protein that has been implicated in playing an auxiliary regulatory role in smooth muscle contraction. We have used immunofluorescence and digital imaging microscopy to determine the cellular distribution of calponin in single cells freshly isolated from the ferret portal vein. In resting cells calponin is distributed throughout the cytosol, associated with filamentous structures, and is excluded from the nuclear area of the cell. The ratio of surface cortex-associated calponin to cytosol-associated calponin (R) was found to be 0.639 +/- 0.021. Upon depolarization of the cell with physiological saline solution containing 96 mM K+, the distribution of calponin did not change from that of a resting cell (R = 0.678 +/- 0.025, P = 0.369). Upon stimulation with an agonist (10 microM phenylephrine) that is known to activate protein kinase C (PKC) in these cells, the cellular distribution of calponin changed from primarily cytosolic to primarily surface cortex associated (R = 1.24 +/- 0.085, P < 0.001). This agonist-induced redistribution of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of these cells. These results suggest that the physiological function of calponin may be to mediate agonist-activated contraction via a PKC-dependent pathway.
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PMID:Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells. 752 95

Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling--sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation-dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca(2+)-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure-function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca(2+)-calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as beta-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.
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PMID:Intracellular mechanisms involved in the regulation of vascular smooth muscle tone. 758 22

Calponin is a smooth muscle-specific, thin filament-associated protein which has been implicated in the regulation of contraction via its interaction with actin and inhibition of the cross-bridge cycling rate. Calponin is phosphorylated by protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), primarily at S175, with loss of actin binding and inhibition of the actin-activated myosin MgATPase. We previously isolated calponin phosphatase from chicken gizzard smooth muscle and identified it as a type 2A protein phosphatase [Winder et al. (1992) Biochem. J. 286, 197-203]. The methods used to detect phosphatase activity in that study would additionally have detected type 1 and 2C phosphatases, but not type 2B phosphatase (Ca2+/CaM-dependent phosphatase or calcineurin). We have, therefore, examined the expression of type 2B phosphatase in smooth muscle and its ability to dephosphorylate calponin. Western blotting with polyclonal antibodies to the brain enzyme revealed the expression of type 2B phosphatase in chicken gizzard, and immunofluorescence microscopy confirmed the presence of the phosphatase in isolated smooth muscle cells (rabbit and toad stomach). The purified brain phosphatase dephosphorylated calponin (phosphorylated by PKC or CaM kinase II) in a Ca2+/CaM-dependent manner. Dephosphorylation by calcineurin restored actin-binding and actin-activated myosin MgATPase inhibition which had been reduced by PKC-catalyzed phosphorylation. We conclude that calponin dephosphorylation may be catalyzed not only by type 2A phosphatase but also by type 2B phosphatase, raising the possibility that both phosphorylation and dephosphorylation of calponin could be regulated by Ca2+/CaM.
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PMID:Dephosphorylation of calponin by type 2B protein phosphatase. 761 14

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.
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PMID:Calponin and smooth muscle regulation. 776 87


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