Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: alpha, delta, epsilon, eta, and zeta isoenzymes were detected. PKC alpha associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKC delta required detergent treatment for extraction from the EDTA-EGTA-washed particulate fraction. PKC epsilon, eta, and zeta were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKC zeta, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKC zeta-immunoreactive protein (M(r) 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKC zeta with peptide epsilon (a synthetic peptide based on the pseudosubstrate domain of PKC epsilon) as substrate was 2.1 mumol P(i).min-1.(mg PKC zeta)-1 and, with peptide zeta as substrate, was 1.2 mumol P(i).min-1.(mg PKC zeta)-1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKC zeta (specific activity, 0.1-0.3 mumol P(i).min-1.mg-1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of alpha, beta, and gamma isoenzymes), were also poor substrates of PKC zeta (specific activities, 0.04 and 0.02 mumol P(i).min-1.mg-1, respectively). Chicken gizzard PKC zeta was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKC zeta are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.
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PMID:Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKC zeta. 903 90

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.
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PMID:Phosphorylation of calponin in airway smooth muscle. 903 10

To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.
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PMID:Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle. 923 49

Smooth muscle contraction is the basis of the physiological reactivity of several systems (vascular, respiratory, gastrointestinal, urogenital ...). Hyperresponsiveness of smooth muscle may also contribute to a variety of problems such as arterial hypertension, asthma and spontaneous abortion. An increase in cytoplasmic calcium concentration ([Ca2+]i) is the key event in excitation-contraction coupling in smooth muscle and the relationship linking the [Ca2+]i value to the force of contraction represents the calcium sensitivity of the contractile apparatus (CaSCA). Recently, it has become evident that CaSCA can be modified upon the action of agonists or drugs as well as in some pathophysiological situations. Such modifications induce, at a fixed [Ca2+]i value, either an increase (referred to as sensitization) or a decrease (desensitization) of the contraction force. The molecular mechanisms underlying this modulation are not yet fully elucidated. Nevertheless, recent studies have identified sites of regulation of the actomyosin interaction in smooth muscle. Sensitization primarily results from the inhibition of myosin light chain phosphatase (MLCP) by intracellular messengers such as arachidonic acid or protein kinase C. In addition, phosphorylation of thin filament-associated proteins, caldesmon and calponin, increases CaSCA. Activation of small (monomeric) G-proteins such as rho or ras is also involved. Desensitization occurs as a consequence of phosphorylation of myosin light chain kinase (MLCK) by the calcium-calmodulin activated protein kinase II, or stimulation of MLCP by cyclic GMP-activated protein kinase. In the present review, examples of physiological modulation of CaCSA as well as pharmacological and pathophysiological implications are illustrated for some smooth muscles.
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PMID:Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications. 926 58

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-epsilon. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-epsilon are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-epsilon to the surface membrane.
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PMID:Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle. 931 27

Amino acid residues 145-163 of calponin have been proposed as a putative actin-binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201] enhanced the Ca2+-induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172-His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172-His187 inhibited the binding of calponin to F-actin in a concentration-dependent manner but not the binding of caldesmon. Gly141-Gly160, including the above-mentioned putative actin-binding site, also competed with intact calponin to the same extent as Lys172-His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141-Gly160. Lys172-His187 and other tested peptides had no effect. However, Gly141-Gly160 and Lys172-His187 reduced the fluorescence intensity of pyrene-labeled F-actin with approximately equal potency. Moreover, Lys172-His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172-His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin-binding activity. These observations suggest the direct involvement of two distinct actin-binding sites, with different regulatory functions, in the interactions of calponin with actin.
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PMID:Two distinct actin-binding sites of smooth muscle calponin. 949 92

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.
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PMID:Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate. 969 7

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.
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PMID:HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery. 985 93

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebra! artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCI or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Rafl, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase C(alpha) (PKC alpha), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of mu-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCI- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg(2+)-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated in vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.
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PMID:Activation of protein kinases in canine basilar artery in vasospasm. 988 54

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin triggered by an increase in sarcoplasmic free Ca2+ concentration ([Ca2+]i). Contraction can, however, be modulated by other signal transduction pathways, one of which involves the thin filament-associated protein calponin. The h1 (basic) isoform of calponin binds to actin with high affinity and is expressed specifically in smooth muscle at a molar ratio to actin of 1:7. Calponin inhibits (i) the actin-activated MgATPase activity of smooth muscle myosin (the cross-bridge cycling rate) via its interaction with actin, (ii) the movement of actin filaments over immobilized myosin in the in vitro motility assay, and (iii) force development or shortening velocity in permeabilized smooth muscle strips and single cells. These inhibitory effects of calponin can be alleviated by protein kinase C (PKC)-catalysed phosphorylation and restored following dephosphorylation by a type 2A phosphatase. Three physiological roles of calponin can be considered based on its in vitro functional properties: (i) maintenance of relaxation at resting [Ca2+]i, (ii) energy conservation during prolonged contractions, and (iii) Ca(2+)-independent contraction mediated by phosphorylation of calponin by PKC epsilon, a Ca(2+)-independent isoenzyme of PKC.
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PMID:Regulation of smooth muscle actin-myosin interaction and force by calponin. 988 65


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