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. 2017 Mar 24;7:391. doi: 10.1038/s41598-017-00149-0

Figure 4.

Figure 4

USP9X regulates RAPTOR levels and opposes its proteasomal degradation in neural progenitors. (A,B) Co-immunoprecipitation of endogenous Usp9x and Raptor from E14.5 mouse frontal cortex. (A) Anti-Usp9x antibodies precipitated Raptor protein. An interaction with pp70S6 kinase was also detected. (B) Anti-Raptor antibodies precipitated Usp9x protein. (C) USP9X over-expression increased RAPTOR protein levels. Proliferating HEK293T cells were transiently transfected with empty vector, full length USP9X (WT) or one of three USP9X variants, which do not affect enzymatic activity but are associated with human intellectual disability. Cell lysates were collected 24 hours later. Immunoblot analysis identified increased RAPTOR protein but mTOR and total S6 levels were unaffected. (D) Chemical inhibition of USP9X deubiquitylating activity leads to a rapid depletion of RAPTOR protein in neural progenitors. ReNcell VM cells were treated with 5 μM WP1130 and analysed by immunoblot. WP1130 does not deplete USP9X protein levels over 4 hours but results in almost complete loss of RAPTOR by 2 hours. pp70S6 kinase levels were not affected. Levels of another USP9X substrate MCL1 were depleted after 4 hours. (E) RAPTOR protein levels are regulated by proteasomal inhibition (epoxomicin) and inactivation of USP9X deubiquiytlating activity (WP1130) in ReNcell VM cells. Proteasomal inhibition following 4 hours exposure to 25 nM epoxomicin resulted in increased RAPTOR levels. Inhibition of USP9X DUB activity with 5 μM WP1130 for 4 hours resulted in decreased RAPTOR. Treatment of wild-type ReNcell VM cells for 4 hours with WP1130 followed by 4 hours of epoxomicin resulted in intermediate levels of RAPTOR. DMSO was used as the vehicle control for each experiment. Final concentrations of DMSO were – 0.0025% for epoxomicin; 0.00005% for WP1130; 0.00255% for WP1130 + Epoxomicin. (F) Epoxomicin treatment of 2193 and 4774 ReNcell VM cells resulted in increased RAPTOR protein levels. The addition of doxycycline (+DOX) resulted in intermediate levels of RAPTOR. Representative of two biological replicates.