Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats.
The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.
Ichikawa
Emiko
E
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Tsurusaki
Yoshinori
Y
Yamaguchi
Masayoshi
M
D000818:Animals; D030801:Animals, Genetically Modified; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D009206:Myocardium / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D051381:Rats; D015238:Sulfotransferases; D014268:Trifluoperazine / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D014268:Trifluoperazine; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D010749:Phosphoprotein Phosphatases; D002122:Calcium Chloride; D002118:Calcium