Mechanism of TRAF6 Intranuclear Translocation

Abstract

Proper cellular response to external signals requires exact spatiotemporal positioning of intracellular components. Adaptor molecules are used to assemble protein complexes to facilitate signaling pathways. We studied the interplay between two adaptor molecules, tumor necrosis factor receptor associated factor‐6 (TRAF6) and Syntenin (Syn‐1). TRAF6 transduces signaling for the TNF and the Toll‐like/Interleukin receptor families. Many studies focused on cytoplasmic TRAF6, but few report on the inhibition of TRAF6 and the mechanisms by which it enters the nucleus. We find that overexpression of Syn‐1 not only attenuates TRAF6 signaling, but causes nuclear localization in both HEK293 and RAW246.7 cells. TRAF6 is an E3‐ligase whose activity we previously reported is maintained by auto‐ubiquitination and an open conformation. Knockdown of Syn‐1 potentiates TRAF6 activation and ubiquitination. Syn‐1 exhibits reduced effect on TRAF6 mutants that cannot be ubiquitinated. This implies that Syn‐1 binds to TRAF6 in a closed state. Protein domain deletions in both TRAF6 and Syn‐1 show that the full‐length molecules are not necessary for interaction. Mutagenesis or deletion of a newly identified TRAF Interaction motif (TIM) in Syn‐1 reveals differences in how Syn‐1 affects TRAF6 signaling and localization. Syn‐1 also localizes to the nucleus when overexpressed with either wild type or a constitutively active TRAF6. Lastly, Syn‐1 binds to the transcription factor Sox4 resulting in Sox4‐Syn‐1 relocalization to the nucleus. We have observed that overexpression of Sox4 also induces TRAF6 nuclear translocation, providing a mechanism for TRAF6 entry into the nucleus.