βTrCP- and Rsk1/2-Mediated Degradation of BimEL Inhibits Apoptosis

Abstract

Summary The BimEL tumor suppressor is a potent pro-apoptotic BH3-only protein. We found that in response to survival signals BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F-box protein βTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild type BimEL, a BimEL phosphorylation mutant unable to bind βTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either βTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that βTrCP promotes cell survival in cooperation with the ERK-RSK pathway, by targeting BimEL for degradation.

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

“…This mechanism of BimEL inactivation is similar to that observed after growth factor stimulation with the exception that the bTrCP phosphodegron is activated by Rsk1/2 downstream of growth factors rather than Aurora A. 20 We observed that the phosphorylation of S93/94/98 is dynamic and reversed by the activity of PP2A leading to the stabilization of BimEL proteins levels after Whole-cell extracts were analyzed by immunoblot to detect endogenous BimEL. P-T210 Plk1 and total Plk1 were used as controls to confirm the downstream effects of the aurora A inhibitor MLN8054.…”

“…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.…”

“…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. 293T cells were synchronized as shown in Figure 1a with the exception that cells were then left in Noc.…”

“…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.…”

“…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. Early reports suggested that this phosphorylation is catalyzed by ERK1/2 21 and ERK5 22 most likely as a pro-survival signal.…”