Insulin-induced phosphorylation of a 38 kDa DNA-binding protein in ventricular cardiomyocytes: possible implication of nuclear protein phosphatase activity.
Ventricular cardiomyocytes isolated from adult rat heart were used to analyze the effect of insulin on the phosphorylation of DNA-binding nuclear proteins and to elucidate the potential involvement of protein phosphatase-1 (PP-1) and PP-2A in this hormonal action. Cells were labelled with [33P]orthophosphate, stimulated with insulin (1.7 x 10(-7) M) and processed for the isolation of nuclei and extraction of DNA-binding proteins. Insulin was found to induce a rapid and constant increase in the serine/threonine phosphorylation of a 38 kDa DNA-binding protein, reaching 150% of control after 15 min and 180% after 150 min. Immunoprecipitation and Western blotting experiments revealed the presence of phosphorylated numatrin in the nuclear extract, however, insulin did not modify its phosphorylation state. Treatment of cardiomyocytes with okadaic acid (1 microM) resulted in a large increase (246 +/- 30%) in the phosphorylation of the 38 kDa protein. Using 32P-labelled phosphorylase as a substrate, we observed a significant inhibition of nuclear PP-1 activity to 38.5 +/- 7% (n = 3) of control after incubation of cardiomyocytes with insulin for 15 min. PP-2A, which corresponds to about 25% of total phosphatase activity, was also inhibited to the same extent. These data show the presence of an insulin-responsive 38 kDa DNA-binding phosphoprotein in the nucleus of cardiomyocytes, which is at least partly regulated by nuclear phosphatase activity. It is suggested that inhibition of nuclear PP-1 and PP-2A represents a possible mechanism of insulin signalling to the nucleus of target cells.
von Holtey
M
M
Laboratory of Molecular Cardiology, Diabetes Research Institute, Düsseldorf, Germany.
Csermely
P
P
Niggemann
J
J
Eckel
J
J
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D010750:Phosphoproteins