Dephosphorylation of calponin by type 2B protein phosphatase.
Calponin is a smooth muscle-specific, thin filament-associated protein which has been implicated in the regulation of contraction via its interaction with actin and inhibition of the cross-bridge cycling rate. Calponin is phosphorylated by protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), primarily at S175, with loss of actin binding and inhibition of the actin-activated myosin MgATPase. We previously isolated calponin phosphatase from chicken gizzard smooth muscle and identified it as a type 2A protein phosphatase [Winder et al. (1992) Biochem. J. 286, 197-203]. The methods used to detect phosphatase activity in that study would additionally have detected type 1 and 2C phosphatases, but not type 2B phosphatase (Ca2+/CaM-dependent phosphatase or calcineurin). We have, therefore, examined the expression of type 2B phosphatase in smooth muscle and its ability to dephosphorylate calponin. Western blotting with polyclonal antibodies to the brain enzyme revealed the expression of type 2B phosphatase in chicken gizzard, and immunofluorescence microscopy confirmed the presence of the phosphatase in isolated smooth muscle cells (rabbit and toad stomach). The purified brain phosphatase dephosphorylated calponin (phosphorylated by PKC or CaM kinase II) in a Ca2+/CaM-dependent manner. Dephosphorylation by calcineurin restored actin-binding and actin-activated myosin MgATPase inhibition which had been reduced by PKC-catalyzed phosphorylation. We conclude that calponin dephosphorylation may be catalyzed not only by type 2A phosphatase but also by type 2B phosphatase, raising the possibility that both phosphorylation and dephosphorylation of calponin could be regulated by Ca2+/CaM.
Fraser
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ED
Department of Medical Biochemistry, University of Calgary Faculty of Medicine, Alberta, Canada.
Walsh
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MP
D000199:Actins / Q000378:metabolism; D000818:Animals; D019703:Calcineurin; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002645:Chickens; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D000096985:Calponins
D000199:Actins; D019703:Calcineurin; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009218:Myosins; D010749:Phosphoprotein Phosphatases; D000096985:Calponins