Phosphorylation of 130-and 95-kDa substrates associated with tumor necrosis factor-alpha receptor CD120a (p55)

Cross-linking of CD120a (p55), a receptor for tumor necrosis factor (alpha (TNF alpha), initiates downstream events, including the activation of prote in Ser/Thr kinases. In this report, we have characterized two protein Ser/T hr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in thei r phosphorylation, pp130 and pp95 were detected by co-immunoprecipitation w ith CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays i n the presence of [gamma-P-32]ATP. The level of phosphorylation of pp130 an d pp95 was rapidly and transiently increased in response to TNF alpha in [P -32]orthophosphate-labeled macrophages, although the level of pp130 protein associated with CD120a (p55) remained unchanged as detected by [S-35]methi onine labeling. In contrast, pp130 and pp95 were efficiently phosphorylated in in vitro kinase assays of CD120a (p55) immunoprecipitates from unstimul ated cells, and the level of phosphorylation was rapidly and transiently re duced in response to TNF alpha. Both pp130 and pp95 were sensitive to depho sphorylation with purified protein phosphatase 2A, and okadaic acid, a PP1/ PP2A inhibitor, mimicked the ability of TNF alpha to stimulate the phosphor ylation of pp130 and pp95 in intact P-32-labeled macrophages. Collectively, these findings suggest that pp130 and pp95 are constitutively associated w ith CD120a (p55) and become inducibly phosphorylated in macrophages in resp onse to TNF alpha. We propose that the underlying mechanism of their phosph orylation may involve the inactivation of a cytoplasmic pp130/pp95 Ser/Thr phosphatase.