Alteration of Receptor/G-protein Interaction by Putative Endogenous Protein Kinase Activity in Dictyostelium discoideum Membranes

Membranes of Dictyostelium discoideum cells were incubated under phosphorylation conditions and washed, and the effects on cAMP binding to chemotactic receptors in the absence and presence of guanosine 5’-O-(3-thiotriphosphate) (GTPγS) were investigated. Most experiments were done with adenosine 5’-O-(3-thiotriphosphate) (ATPγS), which is a good substrate for many kinases, but the product, protein phosphorothioate, is not easily hydrolyzed by phosphatases. Pretreatment of membranes under phosphorylating conditions with MgATPγS alters the site heterogeneity of the cAMP-binding forms, without a significant effect on the total number of binding sites. A similar effect was induced by GTPγS under nonphosphorylation conditions. The effects of MgATPγS were rapid (t½ = 1 min), irreversible, and not induced by Mg2+ or ATPγS alone or by magnesium adenylyl imidodiphosphate and magnesium adenylyl (β,γ-methylene)diphosphate. MgATP induced a smaller inhibition than MgATPγS, which was potentiated by the addition of exogenous cAMP-dependent protein kinase. The effect of MgATP was rapidly reversible; reversibility was reduced by the phosphatase inhibitor NaF. These results suggest that the effects of MgATPγS are mediated by an endogenous protein kinase. The major 35S-thiophosphorylated band detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a protein with Mr = 36,000. The phosphorylation of a protein with the molecular weight of the cAMP receptor (Mr = 40,000-45,000) was not observed.