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 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.
Department of Biochemistry, University of Saskatchewan, Sakatoon, Canada.
M D
MD
Pato
C
C
Sutherland
S J
SJ
Winder
M P
MP
Walsh
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000201:enzymology; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase