FIGURE 7.

USP36 competes with TrkA ubiquitination mediated by Nedd4-2. A, TrkA ubiquitination mediated by Nedd4-2 in HEK293 cells is impaired when USP36 is expressed. Lysates from HEK293 cells transfected with TrkA and/or GFP-Nedd4-2 and/or FLAG-USP36 were subjected to immunoprecipitation (IP) using TrkA antibodies. Western blotting was performed to assess TrkA ubiquitination and TrkA. The expression levels of Nedd4-2 and actin as a loading control are shown. A representative experiment is shown (n = 3). Quantification of TrkA ubiquitination levels in response to Nedd4-2 in the presence or absence of USP36 is shown (means ± S.D.; n = 3; one-tailed unpaired Student's t test). p values show the significance with respect to TrkA + Nedd4-2. B, USP36 controls TrkA levels competing the binding of Nedd4-2 and TrkA. Knockdown of USP36 increases TrkA protein levels leading to enhanced TrkA activation and signaling that results in augmented PC12 differentiation. USP36 ubiquitination and protein levels are regulated by Nedd4-2.