scholarly journals Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function

1992 ◽  
Vol 286 (1) ◽  
pp. 197-203 ◽  
Author(s):  
S J Winder ◽  
M D Pato ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Chicken Gizzard ◽  
Kinase C

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.

Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKCζ

1996 ◽  
Vol 74 (1) ◽  
pp. 51-65 ◽  
Author(s):  
Odile Clément-Chomienne ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Specific Activity ◽  
Regulation Of Gene Expression ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Partial Purification ◽  
Chicken Gizzard ◽  
Kinase C

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1 and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙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 α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.

scholarly journals Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase

1993 ◽  
Vol 293 (1) ◽  
pp. 35-41 ◽  
Author(s):  
M D Pato ◽  
C Sutherland ◽  
S J Winder ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Gel Filtration ◽  
Cytosolic Fraction ◽  
Chicken Gizzard ◽  
Kinase C

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.

Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C

Parasitology ◽  
1997 ◽  
Vol 115 (2) ◽  
pp. 205-211 ◽  
Author(s):  
R. M. MARTIN ◽  
X. F. CSAR ◽  
R. B. GASSER ◽  
R. FELLEISEN ◽  
M. W. LIGHTOWLERS
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Muscle Contraction ◽  
Echinococcus Granulosus ◽  
Single Gene ◽  
Smooth Muscle Contraction ◽  
Kinase C ◽  
High Amino Acid

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 (α, β, γ and δ), 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 isoform 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.

Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle

1996 ◽  
Vol 74 (4) ◽  
pp. 485-502 ◽  
Author(s):  
Michael P. Walsh ◽  
Odile Clément-Chomienne ◽  
Jacquelyn E. Andrea ◽  
Bruce G. Allen ◽  
Arie Horowitz ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Phorbol Esters ◽  
Contractile Response ◽  
Cross Bridge ◽  
Kinase C

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca2+-independent isoenzyme of PKC. Of the two Ca2+-independent PKC isoenzymes, ε and ζ, identified in ferret aorta, PKCε is more likely to mediate the contractile response because (i) PKCε, but not PKCζ, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKCε translocates from the sarcoplasm to the sarcolemma, whereas PKCζ translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKCε, but not PKCζ, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKCε is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of α, β, and γ isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKCε but is a very poor substrate of PKCζ. A signal transduction pathway is proposed to explain Ca2+-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKCε would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (≈0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKCε is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to α1-adrenergic agonists than to K+.Key words: smooth muscle, protein kinase C, calponin.

scholarly journals Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells

1991 ◽  
Vol 277 (2) ◽  
pp. 445-450 ◽  
Author(s):  
D Y Zhao ◽  
M D Hollenberg ◽  
D L Severson
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
Kinase C ◽  
Direct Inhibitory Effect ◽  
Ic50 Values

The protein kinase C (PKC) activator phorbol 12,13-dibutyrate stimulated the phosphorylation of a 75 kDa protein (p75) in intact cultured A10 smooth-muscle cells and sonicated cell preparations; p75 was the only major substrate for endogenous PKC in sonicated A10 cells. The Ca(2+)-dependent phosphorylation of p75 in vitro was dramatically decreased in PKC-down-regulated A10 cells; however, p75 from identical sonicated cell preparations was still phosphorylated by an exogenous aortic PKC preparation. Calmodulin inhibited the phosphorylation of p75 by PKC, but not the phosphorylation of other PKC substrates (platelet P47 protein and histone). The addition of calmodulin after the phosphorylation reaction was started prevented further phosphorylation, but did not decrease the extent of phosphorylation of p75 that was reached before the addition of calmodulin. The inhibition of p75 phosphorylation was concentration-dependent, with IC50 values (concn. giving 50% inhibition) ranging from less than 0.5 to 10 micrograms of calmodulin/ml, and was Ca(2+)-dependent, requiring a free Ca2+ concentration of 10 microM or greater. These results suggest that the inhibition of the PKC-catalysed phosphorylation of p75 by calmodulin may be due to its interaction with the substrate, rather than a direct inhibitory effect on the enzyme, and that this inhibition could be regulated by intracellular Ca2+ concentration. Therefore, p75 may be a physiological link between the PKC and Ca2+/calmodulin pathways.

scholarly journals α-Tocopherol specifically inactivates cellular protein kinase C α by changing its phosphorylation state

1998 ◽  
Vol 334 (1) ◽  
pp. 243-249 ◽  
Author(s):  
Roberta RICCIARELLI ◽  
Andrea TASINATO ◽  
Sophie CLÉMENT ◽  
Nesrin K. ÖZER ◽  
Daniel BOSCOBOINIK ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Protein Phosphatase ◽  
Antioxidant Properties ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Kinase C

The mechanism of protein kinase C (PKC) regulation by α-tocopherol has been investigated in smooth-muscle cells. Treatment of rat aortic A7r5 smooth-muscle cells with α-tocopherol resulted in a time- and dose-dependent inhibition of PKC. The inhibition was not related to a direct interaction of α-tocopherol with the enzyme nor with a diminution of its expression. Western analysis demonstrated the presence of PKCα, β, δ, ε, ζ and µ isoforms in these cells. Autophosphorylation and kinase activities of the different isoforms have shown that only PKCα was inhibited by α-tocopherol. The inhibitory effects were not mimicked by β-tocopherol, an analogue of α-tocopherol with similar antioxidant properties. The inhibition of PKCα by α-tocopherol has been found to be associated with its dephosphorylation. Moreover the finding of an activation of protein phosphatase type 2A in vitro by α-tocopherol suggests that this enzyme might be responsible for the observed dephosphorylation and subsequent deactivation of PKCα. It is therefore proposed that PKCα inhibition by α-tocopherol is linked to the activation of a protein phosphatase, which in turn dephosphorylates PKCα and inhibits its activity.

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