A Drosophila TNF-receptor-associated factor (TRAF) binds the Ste20 kinase Misshapen and activates Jun kinase

Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified - the p21-activated protein kinase (Pak) and SPS1 families [1-3]. In contrast to Pak family members, SPS1 family me mbers do not bind and are not activated by GTP-bound p21Rac and Cdc42. We r ecently placed a member of the SPS1 family, called Misshapen (Msn), genetic ally upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein ( MAP) kinase module in Drosophila [4]. The failure to activate JNK in Drosop hila leads to embryonic lethality due to the failure of these embryos to st imulate dorsal closure [5-8]. Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an, as yet, un defined MAP kinase kinase kinase. We have identified a Drosophila TNF-recep tor-associated factor, DTRAF1, by screening for Msn-interacting proteins us ing the yeast two-hybrid system. In contrast to the mammalian TRAFs that ha ve been shown to activate JNK, DTRAF1 lacks an aminoterminal 'Ring finger' domain, and overexpression of a truncated DTRAF1, consisting of only its TR AF domain, activates JNK. We also identified another DTRAF, DTRAF2, that co ntains an amino-terminal Ring-finger domain. Msn specifically binds the TRA F domain of DTRAF1 but not that of DTRAF2. In Drosophila, DTRAF1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by i nteracting with, and activating, Msn. Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by DTRAF1. Furthermore, coexpression of Msn with DTRAF1 leads to the synergist ic activation of JNK. We have extended some of these observations to the ma mmalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals.