Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle.
"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.
Kansai Advanced Research Center, National Institute of Information and Communications Technology, Kobe, Hyogo 651-2492 Japan. c2v@nict.go.jp
Akira
A
Yamada
Maki
M
Yoshio
Akio
A
Nakamura
Kazuhiro
K
Kohama
Kazuhiro
K
Oiwa
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D049872:Bivalvia; D029742:Caenorhabditis elegans Proteins; D019703:Calcineurin / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008274:Magnesium / Q000737:chemistry; D052998:Microcystins; D008954:Models, Biological; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000201:enzymology* / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D016559:Tacrolimus / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D052998:Microcystins; D009124:Muscle Proteins; D010456:Peptides, Cyclic; C489233:unc-22 protein, C elegans; D019319:Okadaic Acid; C078588:microcystin; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D002118:Calcium; D016559:Tacrolimus