Ubiquitylation by TRAF RING heterodimers: Complexity in TRAF-mediated signalling

Abstract

Ubiquitylation, one of the most abundant post-translational modification in cells, is regulated by a cascade of enzymes (E1, E2 and E3 ligases). E3 ligases, along with their partner E2 enzymes, catalyse the final step of the cascade that results in the attachment of a small regulatory protein, ubiquitin (Ub), to a substrate lysine residue. RING domain E3 ligases promote ubiquitin transfer by stabilising the Ub, which is bound to an E2 (E2~Ub conjugate), in an active conformation, primed for catalysis. As the E3 ligases confer substrate specificity, they play a vital role in regulating cell signalling. Tumour necrosis factor Receptor-Associated Factors (TRAFs) are adaptor proteins and are important for mediating both cell survival and apoptosis. Six of the seven TRAF proteins bear an N-terminal RING domain, but so far, only the E3 ligase activity of TRAF6 and its partner E2, Ubc13 has been established. The E3 ligase activity of the other RING bearing TRAFs has been proposed, but not confirmed. Studies with TRAF6 have reported that dimerisation of the RING domain and the presence of the Zn finger-1 domain, that lies C-terminal to the RING, is critical for Ub transfer. The RING dimer interface is conserved among the TRAFs that co-localise during signalling, and this study provides the first evidence of TRAF RING heterodimer formation (TRAF5-TRAF6 and TRAF2-TRAF6) and heterodimer activity. Biochemical and structural studies showed that similar to the TRAF6 RING homodimer, in the TRAF RING heterodimers, one TRAF RING monomer interacts with the E2, whereas the Zn finger-1 domain of the other TRAF monomer, stabilises the E2~ubiquitin conjugate in the primed active conformation. Although TRAF RING homo- and heterodimers form, the stability of the dimers varies, and they are difficult to fully characterise. To better evaluate RING heterodimer formation, a chemically induced dimerisation technique was established. This allowed dimer formation to be controlled and therefore, RING heterodimers to be specifically characterised. Using this technique, the activity of TRAF pairs and partner E2s was assessed using a combinatorial approach. In addition, a pilot study was undertaken to identify a system to investigate the importance of TRAF RING heterodimers for normal cellular functions. This study demonstrates that functional TRAF RING heterodimers form. The study also highlights the role of the TRAF Zn finger-1 domain during Ub transfer. Together these data suggest that both the TRAF RING homo- and heterodimers can mediate ubiquitylation, indicating a much higher level of complexity in TRAF-mediated signalling than previously anticipated.