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J. Biol. Chem., Vol. 279, Issue 53, 55855-55865, December 31, 2004

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TRAF3 Forms Heterotrimers with TRAF2 and Modulates Its Ability to Mediate NF-B Activation^*

Liusheng He, Amrie C. Grammer¶, Xiaoli Wu, and Peter E. Lipsky||**

From the Flow Cytometry Section in the Office of Science and Technology and the ^¶B Cell Biology Group in the Autoimmunity Branch, ^||NIAMS, National Institutes of Health, DHHS, Bethesda, Maryland 20892

FRET experiments utilizing confocal microscopy or flow cytometry assessed homo- and heterotrimeric association of human tumor necrosis factor receptor-associated factors (TRAF) in living cells. Following transfection of HeLa cells with plasmids expressing CFP- or YFP-TRAF fusion proteins, constitutive homotypic association of TRAF2, -3, and -5 was observed, as well as heterotypic association of TRAF1-TRAF2 and TRAF3-TRAF5. A novel heterotypic association between TRAF2 and -3 was detected and confirmed by immunoprecipitation in Ramos B cells that constitutively express both TRAF2 and -3. Experiments employing deletion mutants of TRAF2 and TRAF3 revealed that this heterotypic interaction minimally involved the TRAF-C domain of TRAF3 as well as the TRAF-N domain and zinc fingers 4 and 5 of TRAF2. A novel flow cytometric FRET analysis utilizing a two-step approach to achieve linked FRET from CFP to YFP to HcRed established that TRAF2 and -3 constitutively form homo- and heterotrimers. The functional importance of TRAF2-TRAF3 heterotrimerization was demonstrated by the finding that TRAF3 inhibited spontaneous NF-B, but not AP-1, activation induced by TRAF2. Ligation of CD40 on Ramos B cells by recombinant CD154 caused TRAF2 and TRAF3 to dissociate, whereas overexpression of TRAF3 in Ramos B cells inhibited CD154-induced TRAF2-mediated activation of NF-B. Together, these results reveal a novel association between TRAF2 and TRAF3 that is mediated by unique portions of each protein and that specifically regulates activation of NF-B, but not AP-1.

Received for publication, June 29, 2004 , and in revised form, September 20, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this manuscript.

^** To whom correspondence should be addressed: NIAMS, National Institutes of Health, 9000 Rockville Pike, Bldg. 10, Rm. 9N228, Bethesda, MD 20892. Tel.: 301-496-2612; Fax: 301-402-0012; E-mail: lipskyp{at}mail.nih.gov.

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