title: Protein kinase activity on the cell surface of a macrophage-like cell line, J774.1 cells.
abstract: Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [gamma-32P]ATP, a transfer of [32P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg2+-dependent but cAMP-independent, and Mg2+ could be substituted for by Mn2+. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D011494:Protein Kinases