Characterization of protein tyrosine phosphatase activity in rat liver microsomes: suppressive effect of endogenous regucalcin in transgenic rats.
The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of protein phosphatase activity in rat liver microsomes was investigated. Protein phosphatase activity torward phosphotyrosine, phosphoserine, and phosphothreonine was assayed in a reaction mixture containing the microsomal protein. Protein phosphatase activity toward phosphotyrosine was strong as compared with that of the enzyme activity toward phosphoserine and phosphothreonine, indicating the existence of protein tyrosine phosphatase. Protein phosphatase activity toward three phosphoaminoacids was significantly enhanced by the addition of both calcium chloride (10 micro M) and calmodulin (2.5 or 5 micro g/ml) in the reaction mixture. The presence of ethylene glycol bis (2-amino-ethylether) N, N, N', N'-tetracetic acid (EGTA; 0.1, 1 or 2 mM) or trifluoperazine (TFP; 10, 20 or 50 micro M), an antagonist of calmodulin, did not have a significant effect on protein phosphatase activity toward phosphotyrosine without calcium addition. Microsomal protein tyrosine phosphatase activity was not changed by okadaic acid (10(-6)-10(-4) M). The enzyme activity was significantly decreased by vanadate (10, 50 or 100 micro M). The addition of regucalcin (0.25 or 0.5 micro M) in the reaction mixture caused a significant inhibition of protein tyrosine phosphatase activity in liver microsomes. Western blot analysis showed a remarkable increase in regucalcin protein level in the liver microsomes of regucalcin transgenic (TG) rats. Protein tyrosine phosphatase activity was significantly suppressed in the liver microsomes of TG rats. This study demonstrates that protein tyrosine phosphatase activity is found in the liver microsomes, and that the enzyme activity is suppressed by regucalcin.
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
Yuko
Y
Fukaya
Masayoshi
M
Yamaguchi
D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; 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; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018628:Gene Dosage; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D019319:Okadaic Acid / Q000494:pharmacology; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D015238:Sulfotransferases; D014268:Trifluoperazine / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002614:Chelating Agents; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D014268:Trifluoperazine; D019000:Phosphotyrosine; D014638:Vanadates; D004533:Egtazic Acid; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D017027:Protein Tyrosine Phosphatases; D002122:Calcium Chloride; D002118:Calcium