Human USP14 Knockout Cell Line-Hela

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradation mechanisms that mediate the turnover of complementary pools of the intracellular proteome. Simultaneous activation of the UPS and autophagy may provide a powerful strategy for the clearance of misfolded proteins. However, it is unclear whether the UPS and autophagy can be controlled by common regulatory mechanisms. K48 deubiquitination of USP14 is known to inhibit the UPS. Here, researchers demonstrate that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, Akt-mediated activation of USP14 by phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. These studies suggest that Akt-regulated USP14 activity regulates proteasomal degradation and autophagy by controlling K48 and K63 ubiquitination, respectively. Thus, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. Inhibition of USP14 may provide a strategy to promote UPS and autophagy for the development of novel therapeutics for neurodegenerative diseases.

To test whether phosphorylation of USP14 is required for its DUB activity (K63 ubiquitination of Beclin 1), wild-type USP14 or mutant USP14 were stably expressed in Usp14 knockout cells and the effects on Beclin 1 ubiquitination were examined. As shown in Figure 1C, expression of wild-type USP14 or the USP14-DD mutant, but not the USP14-AA mutant, inhibited K63 ubiquitination of Beclin 1. Expression of wild-type USP14 inhibited Beclin 1 ubiquitination in the presence or absence of Myr-Akt expression, whereas expression of the USP14-AA mutant failed to inhibit Beclin 1 ubiquitination even in the presence of activated Akt (Figure 1D), indicating that USP14-regulated deubiquitination of Beclin 1 is dependent on Akt-mediated phosphorylation. Furthermore, even in the presence of the Akt inhibitor MK2206, the USP14-DD mutant was still able to reduce the ubiquitination level of Beclin 1, whereas wild-type USP14 lost its deubiquitination activity on Beclin 1 after Akt inhibition ( Figure 1E ). Taken together, these results suggest that Akt-mediated phosphorylation of USP14 regulates its K63 deubiquitination activity on Beclin 1.

Figure 1. Akt-mediated phosphorylation regulates the activity of USP14 DUB against Beclin 1. (A) H4 cells were serum starved overnight, and lysates were analyzed by immunoprecipitation with anti-Beclin 1, and immune complexes were analyzed by Western blot analysis with anti-ubiquitin K63-specific antibody. (B) H4 cells were infected with Myr-Akt lentiviral vector for 12 h and then serum starved for another 12 h, and lysates were analyzed by immunoprecipitation with anti-Beclin 1, and immune complexes were analyzed by A. (C) Usp14−/− H4 cells were transfected with wild-type USP14 or mutant viruses for 24 h, and lysates were analyzed by immunoprecipitation with anti-Beclin 1, and immune complexes were analyzed by Western blot analysis. (D) Usp14 knockout H4 cells were virally transfected with wild-type USP14 or USP14-AA mutants (with or without Myr-Akt as indicated) for 24 h, lysates were immunoprecipitated with anti-Beclin 1, and immune complexes were analyzed as in C. (E) Usp14 knockout H4 cells were virally transfected with wild-type USP14 or USP14-DD mutants for 18 h and then treated with or without 1 µM MK2206 for 6 h, lysates were immunoprecipitated with anti-Beclin 1, and immune complexes were analyzed as in D. (Xu, Daichao, et al. 2016)