Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory κBα phosphorylation and transcriptional activation of a nuclear factor-κB (NF-κB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-κB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-κB transcription factor signalling pathway.

Abbreviations used: aa, amino acid(s); AcNPV, Autographa californica nuclear polyhedrosis virus; AP-1, activator protein-1; CRE, cAMP-response element; DD, death domain; DMEM, Dulbecco's modified Eagle's medium; DR, death receptor; ECL®, enhanced chemiluminescence; ERK, extracellular-signal-regulated kinase; FBS, foetal bovine serum; Fn14-tCT, Fn14 construct encoding Fn14 with a truncated cytoplasmic tail; Fn14-WT, Fn14 construct encoding wild-type Fn14; HA, haemagglutinin; HRP, horseradish peroxidase; IκB, inhibitory κB; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor-κB; p38 MAPK, p38 mitogen-activated protein kinase; pHLZ, pHybLex/Zeo; SRE, serum-response element; TNF, tumour necrosis factor; TNFR, TNF receptor; TRAF, TNFR-associated factor; TWEAK, TNF-like weak inducer of apoptosis.

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