USP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization (original) (raw)
Figure 8
The deubiquitinase activity of USP8 variants.
(A) A diagram of the USP8 protein and deletion constructs. The interaction between Smo and USP8 was determined by immunoprecipitation experiments shown in Figure S2. The activity of each USP8 construct was assessed in S2 cells by their ability to regulate the levels of Smo ubiquitination (shown below). MIT, microtubule interacting and transport domain; RHOD, Rhodanese homology domain. (B) S2 cells were transfected with Myc-Smo and the indicated USP8 constructs followed by the immunoprecipitation assay to detect Smo ubiquitination. The expression of USP8 variants was detected by a Western blot of lysates with an anti-Flag antibody (lower-middle panel). The arrow indicates a non-specific band and the asterisks indicate a possible modification of USP8C>S or USP8NT3C>S. USP8NT3C>S and USP8CT1 did not regulate Smo ubiquitination (lanes 5 and 6, top panel). (C) The effects of USP8 mutants on Smo ubiquitination were examined in S2 cells by the immunoprecipitation assay. The asterisks indicate a possible modification of USP8. Of note, both USP8C>S and USP8NT1C>S have dominant negative effects on Smo ubiquitination (lanes 3 and 4, top panel), whereas USP8NT2C>S and USP8NT3C>S do not (lanes 5 and 6, top panel). (D–E′) Wing discs expressing USP8NT3 or USP8NT3C>S by _ap_-Gal4 was immunostained for Smo and HA. The arrow and arrowhead in (D) indicate elevated Smo accumulation in A- and P-compartment cells. Notably, expression of USP8NT3C>S has no effect on Smo accumulation.