Abstract
Ubiquitin ligase TRAF6, together with ubiquitin-conjugating enzyme Ubc13/Uev1, catalyzes processive assembly of unanchored K63-linked polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways. However, what domain and how it functions to enable TRAF6’s processivity are largely uncharacterized. Here, we find TRAF6 coiled-coil (CC) domain is crucial to enable its processivity. The CC domain mediates TRAF6 oligomerization to ensure efficient long polyubiquitin chain assembly. Mutating or deleting the CC domain impairs TRAF6 oligomerization and processive polyubiquitin chain assembly. Fusion of the CC domain to the E3 ubiquitin ligase CHIP/STUB1 renders the latter capable of NF-κB activation. Moreover, the CC domain, after oligomerization, interacts with Ubc13/Ub~Ubc13, which further contributes to TRAF6 processivity. Point mutations within the CC domain that weaken TRAF6 interaction with Ubc13/Ub~Ubc13 diminish TRAF6 processivity. Our results reveal that the CC oligomerization primes its interaction with Ubc13/Ub~Ubc13 to confer processivity to TRAF6 ubiquitin ligase activity.
Highlights
Ubiquitin ligase TRAF6, together with ubiquitin-conjugating enzyme Ubc13/Uev[1], catalyzes processive assembly of unanchored K63-linked polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways
TRAF6 is a prototype of RING domain-containing E3 that has been best characterized for its essential E3 activities in the interleukin-1 receptor and Toll-like receptor (IL-1R/TLR) signaling pathways[5, 6], where it works with the heterodimeric E2 Ubc13/Uev1A to catalyze polyubiquitin chain (polyUb) chain synthesis
The resulting chains, which could be either conjugated to TRAF65, 7 or unanchored derived by direct synthesis[8,9,10] or by possible en bloc shedding from conjugated TRAF6 by deubiquitinating enzymes (DUBs) with Poh1-like activity[11], activate TAK1, a heterotrimeric kinase complex consisted of TAK1 kinase subunit and TAB1 and TAB2 structural subunits[12]
Summary
Ubiquitin ligase TRAF6, together with ubiquitin-conjugating enzyme Ubc13/Uev[1], catalyzes processive assembly of unanchored K63-linked polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways. Our results reveal that the CC oligomerization primes its interaction with Ubc13/Ub~Ubc[13] to confer processivity to TRAF6 ubiquitin ligase activity. Ub~E2 coordinates with a cognate Ub ligase (E3) to ubiquitinate a protein substrate Repeating of this cascade can result in subsequent ubiquitination of Ub to form a polyubiquitin chain (polyUb)[2]. TRAF6 is a prototype of RING domain-containing E3 that has been best characterized for its essential E3 activities in the interleukin-1 receptor and Toll-like receptor (IL-1R/TLR) signaling pathways[5, 6], where it works with the heterodimeric E2 Ubc13/Uev1A to catalyze polyUb chain synthesis. Whereas these studies illustrated the importance of CC domain for TRAF6 E3 activity and signaling, a detailed biochemical understanding on how CC domain contributes to TRAF6 function, especially whether and how CC domain confers processivity to TRAF6, is lacking
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