Extracellular Signal-regulated Kinases 1/2 Are Serum-stimulated “BimEL Kinases” That Bind to the BH3-only Protein BimEL Causing Its Phosphorylation and Turnover

Abstract

Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim EL , targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim EL phosphorylation remained unclear. We now show that Bim EL is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim EL occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim EL , but not Bim S or Bim L , in vitro, and mutation of Ser 65 to alanine blocks the phosphorylation of Bim EL by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GSTBim EL , but not GST-Bim L or GST-Bim S , in vitro. ERK1/2 also binds to full-length Bim EL in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim EL protein. Our findings provide new insights into the post-translational regulation of Bim EL and the role of the ERK1/2 pathway in cell survival signaling.

BimEL is phosphorylated at mitosis by Aurora A and targeted for degradation by βTrCP1

“…BimEL has been shown to form complexes with kinases that phosphorylate it. 19,20,24 Similarly, in Figure 6g we show that that GST-BimEL was able to pull down Aurora A from mitotic extracts derived from HeLa or 293T cells.…”

“…Figure 2b (lanes [19][20][21][22][23][24][25][26][27] show that taxol treatment stabilized the APC/C substrates, cyclin B and securin, but did not affect stability of BimEL or Wee1, suggesting that degradation of BimEL was APC/C independent. Interestingly, treatment with RO3306 stabilized BimEL (compare lanes 1-9 with lanes 28-36 of Figure 2b), suggesting that cdk1 activity was required for degradation.…”

“…17 Phosphorylation by extracellular signal-regulated kinase 1/2 (Erk1/2) on Serine-69 (S69) of human BimEL has been shown to induce degradation of the protein. 12,18,19 More recently, phosphorylation at S69 was shown to promote phosphorylation at additional sites on Serines 93/94/98 (S93/94/98) by ribosomal S6 kinase (Rsk1/2) within a conserved phosphodegron motif recognized by the F-box protein beta-transducin repeat containing E3 ubiquitin protein ligase (bTrCP). 20 In addition to being phosphorylated in response to growth factor signaling, BimEL is also phosphorylated during mitosis.…”

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

“…Exon 3 includes an ERK1/2 docking domain (FSF) and ERK1/2 phosphorylation sites, including Ser69. [29][30][31]33 The presence of exon 3 in Bima1, Bimb1 and Bimb2 also correlates with their ERK1/2-dependent phosphorylation in vivo. 33 ERK1/2-dependent phosphorylation of Bim EL targets it for proteasomal degradation and may prevent binding to Bax.…”

BH3-only proteins: key regulators of neuronal apoptosis

“…Mutation of this residue to alanine blocks the phosphorylation of Bim EL by ERK1/2 and prevents the degradation of the protein following activation of this pathway. 46 It has also been shown for osteoclasts that trophic factors, such as M-CSF, promote the phosphorylation, ubiquitylation and degradation of Bim, and that overexpression of a lysine-free Bim mutant that cannot be ubiquitylated in bim À/À cells abrogates the antiapoptotic effect of M-CSF. 47 In addition, it has been suggested that phosphorylation of Bim by ERK1/2 inhibits its interaction with Bax.…”