Phosphorylation of the (Na,K)-ATPase by a plasma membrane-bound protein kinase in friend erythroleukemia cells.
A decrease in the activity of the (Na,K)-ATPase is an early and essential step in commitment of Friend virus-infected murine erythroleukemia cells to terminal erythroid differentiation. Plasma membranes from these cells were purified and shown to contain ouabain-inhibitable (Na,K)-ATPase present as approximately 0.4% of the total membrane protein. Protein kinase activity also co-purified with the plasma membrane and preferentially phosphorylated a Nonidet P-40 detergent-extractable 100,000-Da peptide. The 100,000-Da phosphopeptide migrated with the alpha subunit of dog kidney (Na,K)-ATPase when electrophoresis was carried out in the presence of sodium dodecyl sulfate in either 5 or 10% polyacrylamide gels. In two-dimensional gel electrophoresis, it separated into a series of spots between pH 5.1 and 5.4, while dog kidney alpha subunit appeared as a doublet at pH 5.3-5.4. When Nonidet P-40-solubilized plasma membranes were passed through a ouabain affinity column in the presence of Mg2+, Na+, and ATP, the 100,000-Da phosphopeptide was retained and could be eluted by ouabain. This peptide was also phosphorylated in living murine erythroleukemia cells, and proteolysis patterns of the peptide labeled in vitro, the peptide labeled in vivo, and the purified dog kidney alpha subunit using V8 protease were nearly identical. Phosphothreonine was detected in both the peptides labeled in vivo and in vitro.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D004285:Dogs; D004586:Electrophoresis; D005622:Friend murine leukemia virus; D007668:Kidney / Q000201:enzymology; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology*; D007942:Leukemia, Experimental / Q000201:enzymology; D051379:Mice; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*
D010748:Phosphopeptides; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase