Fig 2: siRNA depletion of BRAP, but not USP15, stimulates MEK phosphorylation in non-starved cells stimulated with a short pulse of EGF. A, HeLa cells were treated for 72 h with siRNA targeting KSR1, BRAP, USP4, or USP15 (oligo #17) and serum-starved for the last 16 h before stimulation with EGF (1 ng/ml) for 5 min. Knockdown efficiency as well as MEK and ERK phosphorylation were assessed by Western blotting (IB). B, cells were treated as in A but were not serum-starved. Graphs show results from four biological replicates (error bars, S.E., two-tailed paired t test compared with control; BRAP, p < 0.05; USP15: p < 0.01).

Fig 3: USP15 regulates CRAF expression levels. A, samples shown in Fig. 4A were probed with the indicated antibodies. Quantitation shows the mean of three biological replicates (error bars, S.E.; two-tailed paired t test compared with control; USP15-1, p < 0.05; USP15-17, p < 0.025). IB, immunoblot. B, CRAF was immunoprecipitated (IP) from lysates shown in Fig. 4B and probed with anti-ubiquitin antibodies (Ab). C, samples from epomoxicin (Epo) and vehicle-treated cells (Fig. 4C) were probed with anti-CRAF antibodies. D, HeLa cells were treated with control reagents or USP15 siRNA (Oligo #17) for 66 h before cycloheximide (CHX; 100 μg/ml) treatment. Cells were lysed at indicated time points in Nonidet P-40 lysis buffer, and lysates were probed with anti-CRAF antibody. E, HEK293T cells were treated with control reagents or USP15 siRNA (Oligo#1) for 24 h before transfection with myc-CRAF or an empty vector for another 48 h. Cell lysates were probed with anti-USP15 and anti-CRAF antibodies. Endogenous CRAF levels are only visible in the darker exposure.

Fig 4: Interactions between three closely related DUBs and a subset of RING E3 ligases. A, shown is a schematic representation of USP15, -4, and -11 domain structures. % indicates identity at amino acid level. B, shown is a summary of interactions between USP4, USP15, and USP11 (baits) and RING E3 ligases (preys) observed in a directed DUB:RING E3 ligase Y2H mating screen. Yeast diploids were scored after 5 days for growth using HIS3 and ADE2 reporters. The relative level of colony growth (+, ++, +++) is indicated by line thickness. Note that USP15 shares interactors with both USP4 and USP11. BRAP exclusively interacts with USP4 and USP15 among 55 DUBs tested in this screen. C, shown is a summary of the fragment-based mapping of the interaction between USP15 and BRAP described in Table 1.

Fig 5: USP15 is a positive regulator of MAP kinase signaling. USP15, but not USP4 depletion inhibits MEK and MAPK activation. A, HeLa cells were treated >72 h with 40 nm siRNA oligos against BRAP, USP4, USP15, USP4, and USP15 and KSR1. Cells were starved overnight in serum-free medium and stimulated with EGF (2 ng/ml, 30 min). MEK and MAPK (ERK) activation was assessed with phospho-specific antibodies (pMEK, pERK). CTRL, control. B and C, three individual siRNA oligos (#1, #2, #17) were used to deplete USP15 in HeLa cells, which were processed as in A. Shown are the relative phospho-MEK to total MEK levels in cells stimulated for 30 min with EGF, averaged over two (USP4, USP15-17) or three (all others) independent experiments carried out in duplicate (error bars, S.E., two-tailed paired t test compared with control. B, USP15-1: p < 0.05, USP15-2 p < 0.05. C, USP15-1: p < 0.05, USP15-2: p < 0.01).