Activation and auto‐destruction of the ubiquitin ligase Itch

Abstract

Ubiquitin ligases of the Nedd4 family are maintained inactive by intramolecular interactions (1). We reported that, like Nedd4, the closely related ubiquitin ligase Itch strongly binds to the endosomal protein LITAF through its WW domains, that recognize two PPXY motifs in the N‐terminal half of LITAF. Unlike Nedd4, Itch binding to LITAF brings the ligase to the lysosomal compartments. We thus postulated that LITAF overexpression might bring Itch to the lysosome, where it would access different substrates and target them for degradation (2). Concomitantly, LITAF binding to Itch may increase Itch ligase activity, as other PPXY motif proteins such as NDFIP and LRAD3 (3,4). Here, we set up to determine if Itch ubiquitin ligase activity is indeed up regulated by LITAF binding, and if Itch most common substrate degradation is increased in response to LITAF binding to Itch. We find that Itch, WWP1, WWP2 and Nedd4 all bind to LITAF, and that the four ligases autoubiquitylation is greatly increased upon LITAF binding, albeit to different levels. Both LITAF overexpression and addition of recombinant LITAF to crude cell lisates lead to rapid degradation of Itch, and, to a lesser extent, Nedd4. Itch substrates cJun and Endophilin degradation was also increased. In vitro ubiquitylation assays show that Itch autoubiquitylation is enhanced in the presence of LITAF, but substrate ubiquitylation was not significantly increased. In conclusion, LITAF binding strongly activate Itch autoubiquitylation, as well as that of other ligases of the Nedd4 family. The consequence of this activation is Itch proteasomal degradation, with little effect on its substrates ubiquitylation. LITAF is thus likely to be a negative regulator of Itch in cells where it is strongly expressed.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.