SMOOTH-MUSCLE CALDESMON PHOSPHATASE IS SMP-I, A TYPE-2A PROTEIN PHOSPHATASE

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 reco vered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fracti onation of the cytosolic fraction revealed a single major peak of phos phatase activity which co-eluted with calponin phosphatase [Winder, Pa to and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphat ase. Further purification of caldesmon phosphatase was achieved by seq uential chromatography on columns of DEAE-Sephacel, omega-amino-octyl- agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sephar ose. A single peak of caldesmon phosphatase activity was detected at e ach 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 subuni t. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Sa fer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winde r 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 o f 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 su bstrate of SMP-I than are any of the three phosphorylated forms of cal desmon. We therefore suggest that caldesmon phosphorylation in vivo ca n be maintained after kinase inactivation due to slow dephosphorylatio n by SMP-I, whereas calponin and myosin are rapidly dephosphorylated b y SMP-I and SMP-III/SMP-IV respectively. This may have important funct ional consequences in terms of the contractile properties of smooth mu scle.