betaTrCP- and Rsk1/2-mediated degradation of BimEL inhibits apoptosis

Abstract

The BimEL tumor suppressor is a potent proapoptotic 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 betaTrCP. 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 betaTrCP 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 betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation.

Figure 1. βTrCP controls the degradation of BimEL

(A) BimEL specifically interacts with βTrCP. HEK293 cells were transfected with the indicated FLAG-tagged F-box proteins (FBPs) or an empty vector (EV). Twenty-four hours post-transfection, cells were treated for three hours with PMA and MG132 before harvesting. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-FLAG resin (α-FLAG) and immunobloting for the indicated proteins. (B) The interaction of βTrCP1 with BimEL is promoted by PMA and MG132 and is inhibited by a MEK inhibitor. HEK293 cells were transfected with FLAG-tagged βTrCP or an empty vector. Twenty-four hours post-transfection, cells were treated with the indicated drugs for three hours before harvesting. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-FLAG resin (α-FLAG) and immunobloting for the indicated proteins. (C) βTrCP silencing stabilizes BimEL. HEK293 cells were treated with a control siRNA (Ctrl) or an siRNA targeting βTrCP. Forty-eight hours post-transfection, cells were treated with either PMA alone or PMA and MG132 for the indicated times. Extracts were then immunoblotted for the indicated proteins. The accumulation of Cdc25A (a known βTrCP substrate) demonstrates efficient βTrCP knockdown [also confirmed by RT-PCR (Fig. S10)]. (D) HEK293T cells were transfected with HA-tagged BimEL, Skp1, Cul1, and Roc1 in the presence of an empty vector (EV), FLAG-tagged βTrCP or FLAG-tagged βTrCP(ΔF-box). After immunopurification with anti-FLAG resin, in vitro ubiquitylation of BimEL was performed. Samples were analyzed by immunoblotting with an anti-HA antibody. The asterisk denotes a non-specific band.

Figure 2. Identification of the BimEL degron

(A) Ser93, Ser94, and Ser98 are required for the interaction of BimEL with βTrCP1. HEK293 cells were transfected with an empty vector (EV), HA-tagged βcatenin (positive control), HA-tagged wild type BimEL, or the indicated HA-tagged BimEL mutants. Twenty-four hours post-transfection, cells were treated for three hours with PMA and MG132. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-HA resin (α-HA) and immunobloting for the indicated proteins. (B) The BimEL degron requires phosphorylation to bind βTrCP1. ³⁵S-labeled, in vitro-translated βTrCP1, Fbxw2, and Fbxw4 were used in binding reactions with beads coupled to the BimEL peptide ⁸⁸CLSRSSSGYFSFD¹⁰⁰ (lane 2) or the phosphopeptide ⁸⁸CLSRSpSpSGYFpSFD¹⁰⁰ (lane 3). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS-PAGE and autoradiography. The first lane shows 10% of the in vitro-translated protein inputs. (C) In vivo phoshorylation of BimEL on Ser93/94/98 is induced by mitogens. HEK293 cells were serum deprived (SD) for 24 hours (lane 1), and then either serum (S) (lanes 2-3) or PMA (lane 4-5) was added for 20 minutes in the absence or presence of UO126, as indicated. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with an anti-Bim antibody and immunobloting for the indicated proteins. (D) Ser69 promotes phoshorylation of BimEL on Ser93/94/98. HEK293 cells were transfected with HA-tagged wild type (WT) BimEL (lane 1-3), HA-tagged BimEL(S69A) (lane 4), or HA-tagged BimEL(S94/98A) (lane 5). Cells were serum deprived (SD) for 24 hours (lane 1), and then either serum (S) (lane 2) or PMA (lane 3-5) was added for 20 minutes. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) with anti-HA resin (α-HA) and immunobloting for the indicated proteins. (E) Cytokines induce phosphorylation of BimEL on Ser93/94/98. Activated primary mouse T cells were deprived of IL2 for 5 hours and Ba/F3 and FL5.12 cells were deprived of IL3 for 5 hours. Then cells were stimulated for 15 minutes with IL2 or IL3, in the absence or presence of UO126, as indicated. Whole cell extracts (WCE) were subjected to immunoprecipitation with an anti-Bim antibody and immunobloting for the indicated proteins.

Figure 3. Rsk1/2 control BimEL stability

(A) Inhibition of Rsk1/2 reduces phosphorylation of BimEL on Ser93/94/98. HEK293 were serum deprived (SD) and then treated for 15 minutes with PMA in the absence or presence of FMK, as indicated. Whole cell extracts (WCE) were subjected to immunoprecipitation with an anti-Bim antibody and immunobloting for the indicated proteins. (B) Inhibition of Rsk1/2 reduces BimEL binding to βTrCP1. HEK293 cells were transfected with FLAG-tagged βTrCP1. Twenty-four hours post-transfection, cells were treated with PMA and, where indicated, either FMK or LY294002 (LY) was added. Whole cell extracts (WCE) were subjected to immunoprecipitation (IP) using FLAG resin (α-FLAG) and immunobloting for the indicated proteins. (C) HEK293 cells were treated with a control siRNA (Ctrl) or an siRNA targeting both Rsk1 and Rsk2 mRNAs. Cells were deprived of serum for 24 hours (-) and then activated with PMA (+) for 20 minutes. Cell extracts were subjected to immunoprecipitation with an anti-Bim antibody and immunobloting for the indicated proteins. (D) Silencing Rsk1/2 stabilizes BimEL. HEK293 cells were treated with a control siRNA (Ctrl) or an siRNA targeting both Rsk1 and Rsk2. Forty-eight hours after siRNA treatment, cells were treated with PMA for the indicated times. Extracts were immunoblotted for the indicated proteins. (E) Rsk1 phosphorylates BimEL in vitro. Recombinant, purified BimEL was incubated for 30 minutes with ATP and the indicated purified kinases. Reaction products were subjected to immunoblotting for the indicated proteins. (F) In vitro binding of BimEL to βTrCP2 is dependent on Rsk1. Recombinant, purified wild type BimEL and BimEL(S94/98A) were phosphorylated as in (E) and incubated with in vitro translated, FLAG-tagged βTrCP2 (except sample shown in the last lane). Proteins were immunoprecipitated (IP) using FLAG resin (α-FLAG), and immunoblotting for the indicated proteins was performed.

Figure 4. SCF^βTrCP- and Rsk-mediated degradation of BimEL controls the apoptotic response

(A) Mutation of Ser94/98 stabilizes BimEL despite ERK activation. Cells were transfected with either wild type BimEL or BimEL(S94/98A) mutant. Twenty-four hours post-transfection, cells were treated with PMA and cyclohexamide (CHX) for the indicated times before immunoblotting for the indicated proteins. (B) Mutation of Ser94/98 augments the apoptotic activity of BimEL. Bim-/- and Bax-/-;Bak-/- MEFs were infected with a retrovirus expressing either wild type BimEL or different BimEL mutants. Apoptosis was measured 48 hours following infection using propidium iodide and Annexin-V staining, with flowcytometric analysis (n = 3, ± SD). (C) Silencing Rsk1/2 or βTrCP promotes Bim-dependent apoptosis in primary human T cells. Human T cells were transfected twice with the indicated siRNAs and collected 24 hours thereafter. Apoptosis (left panel) was determined as in (B), and cell extracts were analyzed by immunoblotting for the indicated proteins (right panel). (D) Silencing Rsk1/2 or βTrCP promotes apoptosis in NSCLC cells independent of their sensitivity to gefitinib. HCC827 and H1650 cells were transfected with the indicated siRNAs and collected at the indicated times. Apoptosis was determined as in (B) (n = 3, ± SD). (E) Silencing Bim rescues apoptosis induced by downregualtion of Rsk1/2 or βTrCP. HCC827 and H1650 cells were treated with gefitinib for 24 hours or transfected with the indicated siRNAs and collected 48 hours thereafter. Apoptosis was determined as in (B) (n = 3, ± SD). (F) HCC827 and H1650 cells, treated as in (E), were collected, and cell extracts were analyzed by immunoblotting for to the indicated proteins.