Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.

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

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.