Survival factor-induced extracellular signal-regulated kinase phosphorylates BIM, inhibiting its association with BAX and proapoptotic activity

Abstract

The “BH3-only” proapoptotic BCL-2 family members initiate the intrinsic apoptotic pathway. A small interfering RNA knockdown of BIM confirms this BH3-only member is important for the cytokine-mediated homeostasis of hematopoietic cells. We show here that the phosphorylation status of BIM controls its proapoptotic activity. IL-3, a hematopoietic survival factor, induces extracellular signal-regulated kinase/mitogen-activated protein kinase-mediated phosphorylation of BIM on three serine sites (S55, S65, and S100). After IL-3 withdrawal, only nonphosphorylated BIM interacts with the multidomain proapoptotic effector BAX. Phosphorylation of BIM on exposure of cells to IL-3 dramatically reduces the BIM/BAX interaction. A nonphosphorylatable BIM molecule (S55A, S65A, and S100A) demonstrates enhanced interaction with BAX and enhanced proapoptotic activity. Thus, ERK/mitogen-activated protein kinase-dependent phosphorylation of BIM in response to survival factor regulates BIM/BAX interaction and the pro-death activity of BIM

Regulatory phosphorylation of Bim: sorting out the ERK from the JNK

“…Stimulation of FL5.12 pro-B cells with IL-3 promotes the ERK1/2-dependent phosphorylation of Bim EL . 32 In common with other studies, 27 this has no effect on the interaction of Bim EL with Bcl-2 or Mcl-1, but rather prevented Bim EL from interacting with Bax; 32 this would presumably have the effect of preventing the oligomerization of Bax and cell death. Mutation of three phosphorylation sites in Bim EL (Ser59Ala, Ser69Ala and Ser104Ala) abolished phosphorylation in vivo, enhanced Bim EL -Bax interactions and enhanced cell death following withdrawal of IL-3.…”

“…There is some precedent for this in the ability of c-Jun to provide a docking site for JNK, which then phosphorylates the c-Jun dimer partner, JunD, which otherwise fails to bind JNK directly. 40 Despite these reports, 26,32 other studies suggest that Bim L is not phosphorylated following activation of ERK1/2. [29][30][31]33 Aside from caveats regarding different cell types or stimulation conditions, an alternative explanation for this confusion is that another Bim splice variant is actually being studied.…”

“…These additional sites may be phosphorylated by ERK1/2 or by other ERK-dependent kinases; the identity of these sites is not known, but the proline-directed sites at Ser59 and Ser104 (Ser55 and Ser100 in rat and mouse) are good candidates. 31,32 Efficient phosphorylation by ERK1/2 in vivo requires an appropriate phospho-acceptor site in the substrate (such as PPASP), but also requires a distinct docking domain for the kinase. Using GST-Bim fusion proteins as bait, it has been shown that Bim EL , but not Bim L or Bim S , can bind to activated ERK1/2 in cell lysates.…”

“…NGF stimulation of PC12 cells was reported to promote the ERK1/2-dependent phosphorylation of Bim L as well as Bim EL , 26 while mutation of Ser44 (Ser104 in Bim EL ) abolished the ERK1/2-dependent phosphorylation of Bim L in FL5.12 cells. 32 Since Ser44 is a prolinedirected site, this suggested that ERK1/2 might directly phosphorylate Bim L . At first this seems difficult to reconcile with the fact that Bim L lacks an ERK1/2 docking domain and is not phosphorylated by ERK1/2 in vitro.…”

BH3-only proteins: key regulators of neuronal apoptosis

“…47 In addition, it has been suggested that phosphorylation of Bim by ERK1/2 inhibits its interaction with Bax. 48 Whether ERKs phosphorylate Bim EL at serine 65 in sympathetic neurons maintained in the presence of NGF is unknown.…”