Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: involvement of calmodulin binding.
The regulatory effect of regucalcin on Ca2+/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca2+ (100 microM) and calmodulin (0.30 microM). These increases were clearly inhibited by the addition of regucalcin (0.50-1.0 microM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 microg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 microM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 microM) in the presence of Ca2+ (1 and 10 microM). This increase was completely inhibited by the presence of regucalcin (0.12 microM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 microM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca2+/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin.
Omura
M
M
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka City, Japan.
Yamaguchi
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D019606:Phosphoamino Acids / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D019606:Phosphoamino Acids; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D002118:Calcium