Fig. 4.

ERK/RSK signalling promotes mTORC1-mediated eIF4F assembly and translation in melanoma. (A) Serum-starved melanoma cells were treated with PD184352, BI-D1870, KU-0063794 or rapamycin (50 nM) for 60 minutes. Time course analysis of [³H]leucine incorporation was performed as in Fig. 1, except that cells were collected at 0, 2, 4 and 6 h after addition of [³H]leucine. (B) Serum-starved Colo829 cells were treated with indicated inhibitors as in (A). Cell extracts were size-fractionated by centrifugation through sucrose gradients (20-50%). The absorbance of polysomes (P) and subpolysomal (S) particles was continuously monitored at 260 nm. Representative A₂₆₀ nm traces are shown (n = 3). The area under the curves was calculated and the P/S ratio refers to the percentage of ribosomes engaged in translation. The data are normalized to P/S ratio of control condition (DMSO) and presented as a mean ± S.E. (n = 3). (C) Association of 4E-BP1 and eIF4G to the 7-methylguanosine cap complex was monitored by immunoblotting 7-methylguanosine precipitates from serum-starved Colo829 cells, pre-treated with the indicated inhibitors for 60 minutes. Equal levels between precipitates were monitored through analysis of eIF4E. Phosphorylation and total protein level of endogenous 4E-BP1 and ERK1/2 were monitored by immunoblotting.