Abstract
BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.
Highlights
BIM (BCL2-interacting mediator of cell death), a pro-apoptotic, BH3-only protein belonging to the BCL-2 protein family plays an important role in promoting cell death in response to several stimuli (Bouillet et al, 1999)
Use of immobilized ubiquitin-binding domains (UBDs) to demonstrate that BIM-extra long (BIMEL) is modified by K48-linked poly-ubiquitin chains K48-linked poly-Ub is recognized as a signal for degradation by the 26S proteasome, the nature of the Ub chain
HEK293 cells were transfected with empty vector or BIMEL, and cell extracts were incubated with GST–Dsk2 UBA immobilized on GSH-agarose beads
Summary
BIM (BCL2-interacting mediator of cell death), a pro-apoptotic, BH3-only protein belonging to the BCL-2 protein family plays an important role in promoting cell death in response to several stimuli (Bouillet et al, 1999). BIM-encoding mRNA and BIM protein levels increase upon growth factor withdrawal, and BIM promotes cell death under these conditions (Bouillet et al, 1999; Whitfield et al, 2001; Ewings et al, 2007). Among the common BIM splice variants (BIM-short, BIM-long and BIM-extra long) BIM-extra long (BIMEL) is the most abundant and exhibits the most dynamic increases in expression following cytokine withdrawal (Whitfield et al, 2001; Weston et al, 2003; Ewings et al, 2007). BIM is a tumour suppressor gene (Egle et al, 2004) and stabilization of BIMEL resulting from ERK1/2 inhibition is important in tumour cell death (Wickenden et al, 2008; Gillings et al, 2009). Understanding the mechanism of BIMEL degradation is of fundamental interest and will inform the use of new oncogene-targeted therapeutics
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
