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Dynein light chain binding determines complex formation and posttranslational stability of the Bcl-2 family members Bmf and Bim

Abstract

The BH3-only class of Bcl-2 family proteins triggers mitochondrial apoptosis. Several mechanisms are used to restrain the pro-apoptotic activity of these proteins. Dynein light chain (DYNLL) 1 and 2 has been proposed to negatively regulate the activity of Bim and Bmf, respectively, and the Bim–DYNLL1 interaction leads to the formation of large protein complexes on mitochondria. Here we found that Bim and Bmf interact with both isoforms of DYNLL (DYNLL1 and DYNLL2). DYNLL1/2 not only induced homo-dimerization of Bim and Bmf but also led to the formation of ternary complexes (Bim–DYNLL–Bmf), both in cell-free and in cellular systems. DYNLL-induced oligomerization stabilized Bmf in cultured cells and inhibited its degradation by the ubiquitin-independent 20S proteasome in a cell-free system. Surprisingly, overexpression of wild-type Bmf but not of a DYNLL-binding-deficient mutant induced degradation of endogenous Bim in different cell lines, but both variants sensitized to apoptosis. Mutant Bmf incapable of interacting with anti-apoptotic Bcl-2 proteins and of inducing apoptosis still caused Bim degradation. These results suggest that Bmf overexpression-induced Bim degradation is not due to the displacement of Bim from anti-apoptotic Bcl-2 proteins but a direct consequence of the modulation of Bim–DYNLL association. A peptide derived from the DYNLL-binding domain of Bim also led to the degradation of Bim as well as of its preferred binding partner Mcl-1. Thus DYNLL regulates the mitochondrial pathway of apoptosis by determining the stability of Bmf, Bim, and Mcl-1 proteins.

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Acknowledgements

This study was supported by the German Research Council (DFG, FOR2036 to GH and AJG-S) and the Austrian Science Fund (FWF, project # I3271 to AV).

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Correspondence to Georg Häcker.

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The authors declare that they have no conflict of interest.

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Supplementary information

Bmf interacts with both isoforms of DYNLL

DYNLL1 induces Bmf dimerization

Recombinant full length Bmf interacts with both the isoforms of DYNLL in solution

DYNLL-binding-dependent oligomerization of Bmf in MEFs

Commercial antibodies exhibit limited specificity between DYNLL1 and DYNLL2

DYNLL1/2 induces Bim-Bmf hetero-dimerization

DYNLL-binding dependent hetero-oligomerization of Bim and Bmf in MEFs

Mutations in BH3-domain of Bmf abrogate its interaction with anti-apoptotic Bcl-2 proteins but not with Bim

Bmf levels in Noxa-/- and Bim-/- Noxa-/- Hoxb8 NPCs expressing HA-Bmf or BmfAA

Bmf overexpression sensitizes Hoxb8 neutrophil progenitor cells to apoptosis induced by Mcl-1 inhibitor and differentiation

Mass spectrophotometry analysis of Bmf-IP from Ba/F3 cells

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