Activation of the ERK1/2 Signaling Pathway Promotes Phosphorylation and Proteasome-dependent Degradation of the BH3-only Protein, Bim

Abstract

Both the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways can protect cells from apoptosis following withdrawal of survival factors. We have previously shown that the ERK1/2 pathway acts independently of PI3K to block expression of the BH3-only protein, Bim EL , and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced Bim EL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of Bim EL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum Bim EL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote Bim EL phosphorylation and that this leads to a substantial increase in turnover of the Bim EL protein. ERK1/2-dependent degradation of Bim EL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperaturesensitive mutation in the E1 component of the ubiquitinconjugating system. Finally, co-transfection of Bim EL and FLAG-ubiquitin causes the accumulation of polyubiquitinated forms of Bim, and this requires the ERK1/2 pathway. Our findings provide new insights into the regulation of Bim and the role of the ERK pathway in cell survival.

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

“…As BimEL degradation occurred concomitantly with phosphorylation, we examined two phosphorylation sites on BimEL previously shown to regulate proteolysis. Erk 1 and 2 were shown to phosphorylate BimEL on S69 10,12,18 and promote degradation and phosphorylation on S93/94/98 has been shown to induce degradation through the Skp1-Cullin1-F-Box (SCF) complex. 20 In order to assess the importance of these sites for BimEL degradation in mitosis, phosphospecific antibodies were used to monitor these modifications during mitosis.…”

“…4,7,8 Post-transcriptional mechanisms that have been shown to control Bim expression levels include both mRNA stability 9 and protein stability. [10][11][12] Signaling downstream of receptor tyrosine kinases (RTKs) including epidermal growth factor receptor induce the proteasomal degradation of Bim. [13][14][15][16] There are three splice variants of Bim: BimEL, BimL and BimS.…”

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

“…21 Subsequently, it was shown that activation of the ERK1/2 pathway was necessary and sufficient to accelerate the turnover of Bim EL and ERK1/2-dependent Bim EL degradation was shown to proceed via the proteasome. 34 Proteasome-dependent turnover has subsequently been confirmed in other cell types. 20,30,35,36 Phosphorylation at Ser69 seems to be an important signal for turnover of Bim EL since Ser69Ala or Ser69Gly mutants are defective for turnover, accumulate to higher levels in cells and so exhibit enhanced toxicity.…”

The BCL-2 protein family, BH3-mimetics and cancer therapy

“…Phosphorylation of BIM by ERK was reported to be critical for the antiapoptotic activity of this kinase. [56][57][58][59][60] However, a recent study has shown that ERK-mediated direct phosphorylation of BIM does not have a major role in the control of the proapoptotic activity of this BH3-only protein within the whole animal. 61 Both BIM and BMF were shown to be sequestered by binding to elements of the cytoskeleton, thereby restraining their pro-apoptotic activity.…”