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)

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.
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PMID:Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. 951 39

CPI17, a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5 M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (Ki = 1.9 nM and Ki' = 5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0. 3 microM from the result of Western analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target of CPI17 in vascular smooth muscle.
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PMID:Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle. 999 Jan 34

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

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.
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PMID:Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. 1074 61

CPI-17 is a phosphorylation-dependent inhibitory protein for smooth muscle myosin phosphate. Phosphorylation at Thr(38), in vitro, by protein kinase C or Rho-kinase enhances the inhibitory potency toward myosin phosphatase. Phosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho-activated serine/threonine kinase, and its effect on smooth muscle myosin phosphatase activity were investigated. CPI-17 was phosphorylated by GST-PKN-CAT, a constitutively active GST-fusion fragment of PKN, to 1.46 mol of P/mol of CPI-17, in vitro. The K(m) value of CPI-17 for PKN was 0.96 microM. Phosphorylation of PKN dramatically increased the inhibitory effect of CPI-17 on myosin phosphatase activity. The major and inhibitory phosphorylation site was identified as Thr(38) using a point mutant of CPI-17 and a phosphorylation-state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca(2+) sensitization of smooth muscle contraction as a downstream effector of Rho and/or arachidonic acid.
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PMID:Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. 1092 61

Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.
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PMID:Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms. 1139 99

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

Contractility of smooth muscle and non-muscle microfilaments involves phosphorylation of myosin II light chain. Myosin light chain phosphatase (MLCP) is specifically inhibited by the protein kinase C-potentiated inhibitor protein of 17 kDa, called CPI-17, as part of Ca(2+) sensitization of vascular smooth muscle contraction. Phosphorylation of Thr(38) in CPI-17 enhances inhibitory potency toward MLCP over 1000-fold. In this study we mapped regions of CPI-17 required for inhibition and investigated the mechanism using deletion and point mutants. Deletion of either the N-terminal 34 residues or C-terminal 27 residues gave no change in the IC(50) of either phospho- or unphospho-CPI-17. However, further deletion to give CPI-17 proteins of 1-102, 1-89, 1-76, and 1-67, resulted in much higher IC(50) values. The results indicate there is a minimal inhibitory domain between residues 35 and 120. A single Ala substitution at Tyr(41) eliminated phosphorylation-dependent inhibition, and phospho-Thr(38) in the Y41A protein was efficiently dephosphorylated by MLCP itself. The wild type CPI-17 expressed in fibroblast-induced bundling and contraction of actomyosin filaments, whereas expression of the Y41A protein had no obvious effects. Thus, a central domain of CPI-17(35-120) including phospho-Thr(38) is necessary for recognition by myosin phosphatase and Tyr(41) arrests dephosphorylation, thereby producing inhibition.
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PMID:Defining the structural determinants and a potential mechanism for inhibition of myosin phosphatase by the protein kinase C-potentiated inhibitor protein of 17 kDa. 1151 33

1. Various smooth muscles have unique contractile characteristics, such as the degree of Ca(2+) sensitivity induced by physiological and pharmacological agents. Here we evaluated six different rabbit smooth muscle tissues for protein kinase C (PKC)-induced Ca(2+) sensitization. We also examined the expression levels of myosin light chain phosphatase (MLCP), the MLCP inhibitor phosphoprotein CPI-17, and the thin filament regulator h-calponin. 2. Immunohistochemical and Western blot analyses indicated that CPI-17 was found primarily in smooth muscle, although expression varied among different tissues. Vascular muscles contained more CPI-17 than visceral muscles, with further distinction existing between tonic and phasic subtypes. For example, the tonic femoral artery possessed approximately 8 times the cellular CPI-17 concentration of the phasic vas deferens. 3. In contrast to CPI-17 expression patterns, phasic muscles contained more MLCP myosin-targeting subunit than tonic tissues. Calponin expression was not statistically different. 4. Addition of phorbol ester to alpha-toxin-permeabilized smooth muscle caused an increase in contraction and phosphorylation of both CPI-17 and myosin light chain (MLC) at submaximal [Ca(2+)]i. These responses were several-fold greater in femoral artery as compared to vas deferens. 5. We conclude that the expression ratio of CPI-17 to MLCP correlates with the Ca(2+) sensitivities of contraction induced by a PKC activator. PKC stimulation of arterial smooth muscle with a high CPI-17 and low MLCP expression generated greater force and MLC phosphorylation than stimulation of visceral muscle with a relatively low CPI-17 and high MLCP content. This implicates CPI-17 inhibition of MLCP as an important component in modulating vascular muscle tone.
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PMID:Expression of CPI-17 and myosin phosphatase correlates with Ca(2+) sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. 1153 44

Contractility of vascular smooth muscle depends on phosphorylation of myosin light chains, and is modulated by hormonal control of myosin phosphatase activity. Signaling pathways activate kinases such as PKC or Rho-dependent kinases that phosphorylate the myosin phosphatase inhibitor protein called CPI-17. Phosphorylation of CPI-17 at Thr38 enhances its inhibitory potency 1000-fold, creating a molecular on/off switch for regulating contraction. We report the solution NMR structure of the CPI-17 inhibitory domain (residues 35-120), which retains the signature biological properties of the full-length protein. The final ensemble of 20 sets of NMR coordinates overlaid onto their mean structure with r.m.s.d. values of 0.84(+/-0.22) A for the backbone atoms. The protein forms a novel four-helix, V-shaped bundle comprised of a central anti-parallel helix pair (B/C helices) flanked by two large spiral loops formed by the N and C termini that are held together by another anti-parallel helix pair (A/D helices) stabilized by intercalated aromatic and aliphatic side-chains. Chemical shift perturbations indicated that phosphorylation of Thr38 induces a conformational change involving displacement of helix A, without significant movement of the other three helices. This conformational change seems to flex one arm of the molecule, thereby exposing new surfaces of the helix A and the nearby phosphorylation loop to form specific interactions with the catalytic site of the phosphatase. This phosphorylation-dependent conformational change offers new structural insights toward understanding the specificity of CPI-17 for myosin phosphatase and its function as a molecular switch.
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PMID:Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation. 1173 1


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