The microtubule inhibitor vinblastine promotes subcellular redistribution, posttranslational modifications, and protein-protein interactions of Bcl-2 family proteins

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The function of Bcl-2 family proteins is regulated primarily by subcellular location, post-translational modification, and specific protein-protein interactions. We examined the subcellular distribution of the pro-survival proteins Bcl-2 and Bcl-xL and the pro-apoptotic protein Bax in KB-3 cells treated with the microtubule inhibitor vinblastine. Cells were treated with 30 nM vinblastine for varying times, and cytosolic and mitochondrial fractions were subjected to immunoblotting. Bcl-2 and Bcl-xL were phosphorylated in response to vinblastine, and both were primarily mitochondrial in control and drug-treated cells. However, Bax was cytosolic in control cells and translocated to the mitochondria after vinblastine treatment. These results were confirmed by immunostaining followed by confocal microscopy. Chemical crosslinking using dithiobis(succinimidyl propionate) (DSP) was performed to examine protein:protein interactions. DSP treatment of cell fractions after permeabilization revealed that Bax was exclusively monomeric (∼20 kDa) in control cells but a substantial proportion migrated with a higher molecular mass of ∼40 kDa in extracts from vinblastine-treated cells. These results suggest that the lethal actions of vinblastine may involve not only phosphorylation of Bcl-2 and Bcl-xL, but also specific changes in Bax location and functional interaction. (Supported by NIH CA75577 and CA10982).