The extent of MEK inhibition correlated with the extent of loss in induction and ERK1 phosphorylation of 1 Bcl 2 relative, the proapoptotic BH3 only protein Bim. The inactivation of induction and ERK1/2 of Bim were accompanied by a decrease natural compound library in the apparent molecular weight of Bim, that has been indicative of dephosphorylation, verified by phosphatase analysis. Since ERK1/2 can phosphorylate Bim, thereby priming it for ubiquitination and proteasomal degradation, shutdown of this signaling pathway is likely to account for an important part of the accumulation of Bim. In agreement with this concept, the ranges of ERK1/2 phosphorylation correlated inversely with the total amount of Bim inside our panel of 4 B RAF mutant tumors and also in an array of other cell lines. Furthermore, it was recently demonstrated that ERK1/2 mediated phosphorylation of BimEL can also encourage its speedy dissociation from prosurvival Bcl 2 household members. We expect that MEK chemical induced shutdown with this ERK1/2 mediated process promotes apoptosis Endosymbiotic theory in B RAF mutant cells by facilitating the binding of BimEL to prosurvival Bcl 2 household members. Studies applying RNAi demonstrated that Bim was essential for MEK inhibition induced killing and loss in clonogenic poten Figure 4 MEK inhibition triggers induction and dephosphorylation of Bim in a variety of W RAF mutant tumor cells. MM200 1, SkMel 28, Mel RMU, and MCF 7 cancer derived cell lines were not treated, were treated with 20 m UO126 for the indicated Ibrutinib solubility time details, or were treated with the indicated concentrations of UO126 for 18 h, and were examined for degrees of Bim, phosphorylated ERK1/2, and full ERK1/2 by Western blotting. D, DMSO control. SkMel 28 cells were not treated or were treated for 18 h with 20 m UO126, harvested, and lysed. Lysates weren’t treated or were treated with phosphatase and then assessed by Western blotting for the migration of Bim on SDS PAGE. Arrow shows the weak diffuse band of Bim within untreated healthy cells. MM200 1, untreated PC3, SkMel 28, Mel RMU, and Colo205 cells were examined by Western blotting for the indicated apoptosis related proteins, all on a single membranes to permit direct comparisons. 3656 The Journal of Clinical Investigation. jci. Net Volume 118 Number 11 November 2008 tial of T RAF mutant cancer cells. The degree of protection afforded by Bim KD was similar to that afforded by Bcl 2 overexpression at early time points, but it was considerably less efficient after more protracted MEK inhibition. This is probably the effect of incomplete Bim KD, but it can be possible that activation of other BH3 only proteins or inactivation of prosurvival Bcl 2 family members led to MEK induced tumefaction cell-killing, even though we found no evidence in support of this possibility.