Abstract
We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.
Highlights
From the Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Cornell University, Zthaca, New York 14853 propriate amounts of quercetin
Wenow report that a membrane-bound protein kinase phosphorylates the p subunit of the Na’K’-ATPase in EAT. Cells and that such a phosphorylation is not observed with mouse brain microsomes
Howevsoerl, Phosphorylation assays were done at 30°C in a medium containing ubilizedNa+K+-ATPasefromeithertumor or brain were phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated a phosphatase
Summary
83704373, 1980 Printed m U.S.A. Phosphorylation of the /3 Subunit of Na’K+-ATPasein Ehrlich Ascites Tumor by a Membranebound Protein Kinase*. From the Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Cornell University, Zthaca, New York 14853 propriate amounts of quercetin. These experiments support the previously proposed modeof action of quercetin in repairing a defective or substituting for a missing regulatory subunit (2). Wenow report that a membrane-bound protein kinase phosphorylates the p subunit of the Na’K’-ATPase in EAT cells and that such a phosphorylation is not observed with mouse brain microsomes. The purified kinase from tumor cells phosphorylates purified Na’K+-ATPase from both brain and tumor and causes decreased Na’ pumping efficiency following reconstitution
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