Abstract
Cross-linking of CD120a (p55), a receptor for tumor necrosis factor alpha (TNFalpha), initiates downstream events, including the activation of protein Ser/Thr kinases. In this report, we have characterized two protein Ser/Thr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in their phosphorylation. pp130 and pp95 were detected by co-immunoprecipitation with CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays in the presence of [gamma-(32)P]ATP. The level of phosphorylation of pp130 and pp95 was rapidly and transiently increased in response to TNFalpha in [(32)P]orthophosphate-labeled macrophages, although the level of pp130 protein associated with CD120a (p55) remained unchanged as detected by [(35)S]methionine labeling. In contrast, pp130 and pp95 were efficiently phosphorylated in in vitro kinase assays of CD120a (p55) immunoprecipitates from unstimulated cells, and the level of phosphorylation was rapidly and transiently reduced in response to TNFalpha. Both pp130 and pp95 were sensitive to dephosphorylation with purified protein phosphatase 2A, and okadaic acid, a PP1/PP2A inhibitor, mimicked the ability of TNFalpha to stimulate the phosphorylation of pp130 and pp95 in intact (32)P-labeled macrophages. Collectively, these findings suggest that pp130 and pp95 are constitutively associated with CD120a (p55) and become inducibly phosphorylated in macrophages in response to TNFalpha. We propose that the underlying mechanism of their phosphorylation may involve the inactivation of a cytoplasmic pp130/pp95 Ser/Thr phosphatase.
Highlights
That regulates many aspects of the inflammatory response, host defense, and fibrogenesis [1,2,3]
We have characterized two protein Ser/Thr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in their phosphorylation. pp130 and pp95 were detected by co-immunoprecipitation with CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays in the presence of [␥-32P]ATP
Whereas molecules such as TRADD, TNF-receptor associated factor-2, RIP, and Fas-associated death domain protein play indisputably important roles in signal transduction following ligation of CD120a (p55), other signaling proteins, including additional Ser/Thr kinases and their appropriate substrates, have been found to be present in CD120a (p55) immunoprecipitates [11] or bound by glutathione S-transferase (GST) fusion proteins containing the cytoplasmic domain of CD120a (p55) [12]
Summary
TNF␣, tumor necrosis factor-␣; pp130 and pp, phosphoproteins of 130 and 95 kDa, respectively; RIP, receptor-interacting protein; TRADD, TNF-receptor-associated death domain protein; PP1/PP2A, protein phosphatases 1 and 2A; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MEM, minimum essential medium; Pipes, 1,4-piperazinediethanesulfonic acidDTT, dithiothreitol. A similar approach employing GST fusion proteins has been used to characterize a Ser/Thr kinase activity that interacts with sequences present within the cytoplasmic domain of the lymphotoxin- receptor [14] This latter kinase appears to be specific for the lymphotoxin- receptor and does not trans-phosphorylate CD120a (p55). RIP and RIP2 are 74- and 61-kDa death domain-containing proteins bearing Ser/Thr kinase domains [15, 16] and are the only CD120a (p55)-associated kinases to be cloned their molecular weights are distinct from other receptor-asso-. We provide evidence suggestive of an indirect interaction between pp130 and pp with the cytoplasmic domain of CD120a (p55)
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