The CD95 (Fas/APO-1) receptor is phosphorylated in vitro and in vivo and constitutively associates with several cellular proteins

Different CD95 (Fas/APO-1) isoforms and phosphorylated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylated in vivo. GST-CD95IC was phosphorylated in vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immunoprecipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35,45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not only in vitro but also in vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction. © 1996 Rapid Science Publishers.