CD40 signalings play crucial roles in B-cell function.To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-␤ receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246 -269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230 -245 were required. Overexpression of TRAF6 activates transcription factor NFB, and its TRAF-C domain suppresses NFB activation triggered by CD40 lacking residues 246 -277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230 -245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia vi-

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The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. It also induces betaFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the betaFTZF1 promoter, thereby providing a timing mechanism for betaFTZF1 induction that is dependent on the disappearance of E75B.

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-jB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFa-induced activation of JNK but not of p38 or NF-jB. Intact RING and zinc finger domains are required for TNFa-induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFa-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in HodgkinReed-Sternberg lymphoma cells, resulting in constitutive NF-jB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13-dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF-jB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in HodgkinReed-Sternberg cells.