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

ABSTRACT: 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.

SUBMITTER: Winder SJ

PROVIDER: S-EPMC1133039 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Project description: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.

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

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