Mass spectrometric analysis of tumor necrosis factor receptor-associated factor 1 ubiquitination mediated by cellular inhibitor of apoptosis 2.
Signaling complexes formed on tumor necrosis factor receptor 2 (TNF-R2) contain adaptor proteins TNF-R-associated factors (TRAFs) 1 and 2, and cellular inhibitors of apoptosis (cIAPs) 1 and 2 which function as regulators of programmed cell death. TRAF2, cIAP1 and cIAP2 all have RING finger domains known to possess E3 ubiquitin ligase activity, implying that ubiquitination may play an important role in the TNF signaling pathway. In this report, we have shown that cIAP2 specifically mediated ubiquitination and proteasome-dependent degradation of TRAF1. To identify the sites for cIAP2-mediated ubiquitination of TRAF1, we used high pressure liquid chromatography coupled with tandem mass spectrometry. Lys185 and Lys193 of TRAF1 were found to be modified with ubiquitin chains. Mutation of Lys185 and Lys193 to Arg almost completely blocked cIAP2-mediated ubiquitination of TRAF1, indicating that they are the major, if not the only, sites of TRAF1 ubiquitination. Our data suggest that cIAP2 may regulate the turnover of TRAF1 by adding polyubiquitin chains on Lys185 or Lys193 following its recruitment to TNF-R signaling complexes.
Lee
Jong-Sik
JS
Department of Biochemistry and Protein Network Research Center, Yonsei University, Seoul, Korea.
Hong
Un-Sook
US
Lee
Tae H
TH
Yoon
Sungjoo Kim
SK
Yoon
Jong-Bok
JB
D008239:Lysine / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D009154:Mutation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D047990:TNF Receptor-Associated Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D011506:Proteins; D047990:TNF Receptor-Associated Factor 1; D025801:Ubiquitin; D008239:Lysine