Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl2 (10−5 and 10−4 M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 μM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10−6–10−4 M), an inhibitor of Ca2+/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca2+/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 μM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei. J. Cell. Biochem. 75:437–445, 1999. © 1999 Wiley-Liss, Inc.