Identification of Novel Isoforms of the BH3 Domain Protein Bim Which Directly Activate Bax To Trigger Apoptosis

Abstract

Bim (Bcl-2-interacting mediator of cell death) is a member of the BH3 domain-only subgroup of Bcl-2 family members, for which three splice variants have been described. Bim is expressed in many healthy cell types, where it is maintained in an inactive conformation through binding to the microtubule-associated dynein motor complex. Upon certain apoptotic stimuli, Bim is released from microtubules and mediates caspasedependent apoptosis through a mechanism that is still unclear. Here, we have identified and characterized novel splice variants of human Bim mRNA. In particular, we show that a newly discovered, small protein isoform, BimAD, is also able to induce apoptosis strongly in several human cell lines. BimAD and the previously characterized isoform BimS are shown to be capable of heterodimerizing in vivo with both death antagonists (Bcl-2 and Bcl-X L ) and death agonists (Bax). Mutants of BimAD that bind to Bax but not to Bcl-2 still promote apoptosis, indicating that Bim can regulate apoptosis through direct activation of the Baxmediated cell death pathway without interaction with antiapoptotic Bcl-2 family members. Furthermore, we have shown that the interaction of the BimS and BimAD isoforms with Bax leads to a conformational change in this protein analogous to that triggered by the BH3-only protein Bid.

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

“…The Bim locus encodes three major isoforms: Bim short (Bim S ), Bim long (Bim L ) and Bim extra long (Bim EL ), generated by alternative splicing. 4,5 More recently, additional isoforms have been reported [6][7][8] so that recent estimates suggest that there may be up to 18 different Bim splice variants. 8 However, the physiological relevance of these unique splice variants has not yet been addressed and while the protein products of endogenous Bim S , Bim L and Bim EL are readily detected, few, if any, studies have identified the naturally occurring protein products of these novel splice variants.…”

“…In addition, while all forms of Bim can bind to prosurvival Bcl-2 proteins, some forms may bind directly to Bax. 7 However, this is an area of considerable uncertainty; for example, while it is clear that the Bim BH3 domain can bind to all prosurvival Bcl-2 proteins with a physiologically relevant nM affinity in Biacore real-time binding assays, 10 no such evidence is available for Bim binding to Bax or Bak. Consequently, many in the field still believe that Bim and most other BH3-only proteins trigger apoptosis largely by binding to their prosurvival relatives and this remains an area of considerable debate.…”

“…The only question regarding this model is that others have argued that Bim EL fails to interact directly with Bax and this whole issue remains uncertain at the present time. 7,8 Finally, ERK1/2-dependent phosphorylation of Bim EL has been shown to occur at mitosis, although the functional relevance of this remains unclear. 37 Taken together, these results suggest that the presence of exon 3 in Bim EL provides an ERK1/2 docking domain and at least two ERK1/2 phosphorylation site(s), including Ser69.…”

BH3-only proteins and their roles in programmed cell death

“…The BH3 domain seems to be entirely responsible for this interaction, and is absolutely required for the killing activity of the BH3-only proteins. In addition, Bim, Bid and Puma were also found to interact with Bax (Marani et al, 2002;Cartron et al, 2004;Willis et al, 2007). Bim, Bad and Bmf are unstructured in the absence of a binding partner, and only their BH3 domain becomes structured upon binding a pro-survival protein (Hinds et al, 2007).…”

“…While binding of Bid to Bax is relatively easy to observe by coimmunoprecipitation in the presence of non-ionic detergents (Wei et al, 2000;Walensky et al, 2006), binding of Bim to Bax could be shown only for two minor isoforms, BimS and BimAD (Marani et al, 2002), whose physiological significance is also a matter of debate. No direct binding of the major isoform, BimEL, could be observed under the same conditions (Willis et al, 2007), and no binding of any Bim isoform to Bak was ever reported.…”