FIGURE 10.

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.