Protein kinase activity associated with Fc.gamma.2a receptor of a murine macrophage like cell line, P388D1

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Abstract
The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc.gamma.2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [.gamma.-32P]ATP. The autophosphorylation of Fc.gamma.2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc.gamma.2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (> 25 mM) or KCl (> 100 mM) or by a small amount of heparin (> 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc.gamma.2a receptor used ATP as substrate with an apparent Km of 2 .mu.M as well as GTP with an apparent Km of 10 .mu.M. Prior heating (60.degree. C for 15 min) or treatment with protease (trypsin or Pronase) of Fc.gamma.2a receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. Cyclic AMP as well as dioleine and phosphatidylserine did not increase the phosphorylation of casein catalyzed by Fc.gamma.2a receptor complex. These results suggest that Fc.gamma.2a receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase. The major casein kinase active molecule associated with Fc.gamma.2a receptor was identified to be a protein of molecular weight near 37,000 by specific labeling of IgG2a-binding protein complex with the radioactive ATP analogue 5''-[(fluorosulfonyl)-benzoyl][14C]adenosine.