THE P60 TUMOR-NECROSIS-FACTOR (TNF) RECEPTOR-ASSOCIATED KINASE (TRAK)BINDS RESIDUES 344-397 WITHIN THE CYTOPLASMIC DOMAIN INVOLVED IN TNF SIGNALING

The p60 form of the tumor necrosis factor (TNF) receptor lacks motifs characteristic of tyrosine or serine/threonine protein kinases, Our re cent observations have indicated that a p60 TNF receptor-associated ki nase (p60-TRAK) from U-937 cells physically interacts with and causes the phosphorylation of the cytoplasmic domain of the TNF receptor. To define which region of the cytoplasmic domain is necessary for physica l interaction with p60-TRAK, we constructed a series of deletions (gro uped into three sets Delta 1-Delta 5, Delta 6-Delta 12, and Delta 13-D elta 16) of the p60 cytoplasmic domain, expressed them as glutathione S-transferase (GST) fusion proteins, and used them in affinity precipi tations, followed by in vitro kinase assays. Our detailed analysis ind icated that a serine-, threonine-, and proline-rich region (residues 2 43-274, Delta 2) and the N-terminal half of the cytoplasmic domain (re sidues 243-323, Delta 3) neither associated with p60-TRAK nor underwen t phosphorylation. We found that out of 222 residues (205-426) in the cytoplasmic domain, only 54 (344-397, Delta 12) were sufficient for bi nding p60-TRAK and for phosphorylation of the cytoplasmic domain. A re gion of approximately 30 residues (397-426) at the C-terminal end was found to interfere with optimal binding of the p60-TRAK activity. Thus , our results indicate that the minimal region of the cytoplasmic doma in necessary for interacting with p60-TRAK and for phosphorylation res ides within the domain previously reported to be needed for signaling the cytotoxic effect of TNF.