Role of endogenous regucalcin in protein tyrosine phosphatase regulation in the cloned rat hepatoma cells (H4-II-E)

Abstract

Regucalcin, a regulatory protein of Ca²⁺ signaling, is mainly present in liver cells. The role of regucalcin in hepatoma cells, however, has not been clarified. The role of endogenous regucalcin in the regulation of protein tyrosine phosphatase activity in the cloned rat hepatoma cells (H4-II-E) was investigated. Hepatoma cells were cultured for 3 days in a medium containing serum (10% fetal bovine serum). After subconfluency, the cells were used for the assay of protein phosphatase activity toward phosphotyrosine. The expression of regucalcin in hepatoma cells was detected by Western blotting using anti-regucalcin antibody. Protein tyrosine phosphatase activity was exhibited in the cytosol of hepatoma cells. The enzyme activity in the cytosol of hepatoma cells was significantly elevated by the addition of calcium chloride (10^-6-10^-4 M) in the reaction mixture. This elevation was completely blocked by the addition of trifluoperazine (TFP: 2.5 × 10^-6 M), an antagonist of calmodulin. The addition of regucalcin (10^-7 M) caused a complete inhibition of the calcium (10^-4 M) - increased enzyme activity. The presence of anti-regucalcin monoclonal antibody (25, 50, and 100 ng/ml) in the enzyme reaction mixture produced a significant increase in protein tyrosine phosphatase activity in the cytosols of hepatoma cells and normal liver cells. This increase was completely prevented by regucalcin addition. The effect of antibody (50 ng/ml) in elevating the enzyme activity was partly inhibited by vanadate (10^-4 M). Protein tyrosine phosphatase activity was significantly elevated by the culture with Bay K 8644, a Ca²⁺-channel agonist. This increase was blocked by TFP addition in the enzyme reaction mixture, and it was enhanced in the presence of anti-regucalcin antibody. The present study demonstrates that regucalcin is expressed in hepatoma cells (H4-II-E), and that the protein may have an inhibitory effect on Ca²⁺/calmodulin-dependent protein tyrosine phosphatase activity in the cells.