Endogenous protein phosphorylation in isolated plasma membranes of AH-66 hepatoma ascites cells

The phosphorylation of endogenous membrane proteins by an endogenous protein kinase was studied in isolated plasma membranes from AH-66 hepatoma ascites cells using [gamma-32P]ATP as a precursor. The phosphorylation occurred very rapidly in the presence of 10 mM Mg2+ and reached a maximal level at 2 min. Ca2+ strongly inhibited the phosphorylation reaction and antagonized the activation produced by Mg2+. Neither cyclic AMP nor cyclic GMP had a significant effect on the phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that only membrane proteins ranging in molecular weight from 125,000 to 200,000 were heavily phosphorylated and those of less than 60,000 molecular weight were not phosphorylated at all. The protein kinase activity was readily extractable from the plasma membranes with 1 mM EDTA at pH 8.5. Among exogenous substrates, the extracted protein kinase catalyzed the phosphorylation of histone, protamine and phosvitin rather than casein. When the extracted protein kinase was subjected to chromatography on DEAE-Sepharose, a single major peak of cyclic AMP-independent protein kinase was eluted at a position quite different from those of the cytosolic protein kinases.