PHYSICAL AND FUNCTIONAL ASSOCIATION OF A SERINE-THREONINE PROTEIN-KINASE TO THE CYTOPLASMIC DOMAIN OF THE P80 FORM OF THE HUMAN TUMOR-NECROSIS-FACTOR RECEPTOR IN HUMAN HISTIOCYTIC LYMPHOMA U-937 CELLS

Tumor necrosis factor (TNF) binds two distinct cell surface receptors designated p60 and p80. Our previous studies indicate that a protein k inase from U-937 cells binds to and phosphorylates the p60 receptor. W hile the p80 receptor is phosphorylated in vivo, no association of a p rotein kinase has been described. We employed a fusion protein compris ing of glutathione S-transferase and the cytoplasmic domain of the p80 receptor (GST-p80CD) to identify cellular proteins that might associa te with this receptor. From S-35- and P-32-labeled cells, a protein of 59 kDa bound specifically to GST-p80CD. In vitro kinase reactions ind icated that serine/threonine protein kinase activity associated with G ST-p80CD and causes its phosphorylation. Additionally, a 59-kDa phosph oprotein was also identified after kinase reactions of proteins bound to CST-p80CD. This kinase activity required either Mg2+ or Mn2+ for op timal activity, and it phosphorylated myelin basic protein, histone H2 B, and also the cytoplasmic domain of the p60 receptor. Treatment of c ells with TNF increased the p80 receptor-associated kinase activity by 200%. In summary, our results provide evidence of a novel ligand-acti vated serine/threonine protein kinase that associates with the cytopla smic domain of the p80 receptor and causes the phosphorylation of both forms of the TNF receptor. This p80 TNF receptor associated protein a nd the associated kinase described here are referred to as p80 TRAP an d p80-TRAK, respectively.