Figure 8.

ERK1/2-dependent phosphorylation of Bim_EL at Ser⁶⁵ is required for dissociation of Bim_EL from Mcl-1. (A) HR1 cells were transfected with plasmids encoding HA-Bim_EL (WT), or HA-Bim_EL with individual point mutations at Ser⁵⁵Ala, Ser⁶⁵Ala or Ser⁷³Ala. After 24 h, cells were serum starved for 6 h before treating for a further 15 min with 100 nM 4-HT to activate ERK1/2. Whole-cell extracts (input WCE) were used for immunoprecipitation of Mcl-1 and samples were then immunoblotted with antibodies to Mcl-1, HA(Bim), P-ERK1/2 and ERK1/2. (B) HR1 cells were transfected with HA-Bim_EL. Twenty-four hours later, cells were serum starved for 1 h in the presence of 20 μM U0126 to inactivate ERK1/2. Cells were then washed thoroughly and stimulated with 100 nM 4-HT±20 μM U0126 for 1 h. Whole-cell extracts (input WCE) were used to immunoprecipitate Mcl-1 or HA-Bim_EL. Samples were then immunoblotted with antibodies to Mcl-1, HA(Bim), Bim, Phospho-Ser⁶⁵-Bim, P-ERK1/2 and ERK1/2. The arrowheads indicate the position of the HA-Bim_EL, while the asterisk indicates the position of the endogenous Bim_EL. Note that ΔRaf-1:ER^* induces phosphorylation of Bim_EL at Ser⁶⁵, but phospho-Ser⁶⁵-Bim_EL is not detected in Mcl-1 IPs.