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 (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.
Uh
S
S
Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Van Linden
A
A
Riches
D W
DW
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology / Q000276:immunology; D002478:Cells, Cultured; D008264:Macrophages / Q000166:cytology / Q000276:immunology*; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D019319:Okadaic Acid / Q000494:pharmacology; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D015703:Antigens, CD; D010748:Phosphopeptides; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D019319:Okadaic Acid; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2