Figure 4.

ERK1 and ERK2 can directly phosphorylate Bcl2 at Ser-70 in a stauro-resistant manner. (A) Wt or the phosphorylation-defective S70A mutant Bcl2 was immunoprecipitated from cell lysates and incubated with purified, activated ERK1 or ERK2 in an in vitro kinase assay as described in Materials and Methods. (B) As in A, immunoprecipitated wt Bcl2 was used as substrate for purified, active ERK1 or ERK2 or PKCα with or without 1 μM stauro as indicated. (C) Transfection of Cos7 cells using constitutively active MEK1, wt, activatable ERK1 or ERK2, and HA-Bcl2 expression plasmids was performed as described in Materials and Methods. At 48 h after transfection, the cells were pretreated with either 10 μM PD98059 or 1 μM stauro for 1 h as indicated and then metabolically labeled with ³²P-labeled orthophosphate and treated with the same inhibitor for an additional 4 h. HA-Bcl2 was immunoprecipitated with monoclonal HA-11 antibody and analyzed as in Fig. 1.