A tyrosine-specific protein kinase from ehrlich ascites tumor cells

Abstract A protein tyrosine kinase that phosphorylates both α and β subunits of inactivated (Na + ,K + )-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn 2+ , Mg 2+ , or Fe 2+ but was inhibited by Cu 2+ or Zn 2+ . The purified enzyme phosphorylated the β subunit about five times faster than the α subunit of the (Na + ,K + )-ATPase. The random polymer poly(Glu 80 Tyr 20 ) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na + ,K + )-ATPase was preferentially phosphorylated on the a subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na + ,K + )-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an M r of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu 80 Tyr 20 ) was partially purified from the same membrane.