Fig 2: CRAF is the key target for USP15 regulation of ERK signaling. A, HeLa cells were treated with control reagents or CRAF siRNA for 60 h before starvation for 12 h. EGF (1 ng/ml) was added for various times before lysis. IB, immunoblot. B, direct comparison of the effect of USP15 and CRAF depletion in HeLa cells stimulated for 5 min with EGF (n = 3, error bars; S.E., paired two-tailed t test compared with 5 min; control, CRAF (5 min), p < 0.01; USP15 (5 min), p < 0.025; CRAF (10 min), p < 0.005; USP15 (10 min), p < 0.001). C, WM266-4 BRAF (V600D) expressing melanoma cells were treated with control reagents or target specific siRNA, and Nonidet P-40 cell lysates were probed with antibodies indicated. Depletion of USP15 by two individual oligos (#1 and #17) leads to reduced CRAF levels without affecting pMEK and pERK levels. * indicates a nonspecific band detected by the CRAF antibody. D, a working model is shown. i, USP15 positively controls signal output by regulating CRAF levels. ii, USP15 may also negatively regulate MAPK signaling by directly stabilizing BRAP.

Fig 3: USP15 depletion in U2OS cells decreases CRAF expression levels and inhibits PDGF dependent pMEK activation. A, U2OS cells were treated >72 h with 20 nm of siRNA oligos targeting BRAP, USP4, and USP15 (two individual oligos). Cells were serum-starved for 16 h and then stimulated with 10 ng/ml PDGF for 5 min. RIPA lysates were probed with the indicated antibodies to assess knockdown efficiencies and expression levels or activation status of the key kinases of the MAPK cascade (CRAF, BRAF, MEK, and ERK). B, the ratio of pMEK/MEK, CRAF, and BRAP levels at 5 min post stimulation with PDGF, averaged from three independent experiments, are shown relative to control samples (error bars, S.D.; two-tailed paired t test compared with control; pMEK-USP15-1, p < 0.05; USP15-2, p < 0.05; CRAF-USP15-1, p < 0.01; USP15-2: p < 0.05).

Fig 4: 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).

Fig 5: 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).