Vinblastine-induced Apoptosis Is Mediated by Discrete Alterations in Subcellular Location, Oligomeric Structure, and Activation Status of Specific Bcl-2 Family Members

Abstract

To gain a broader insight into the role of Bcl-2 proteins in apoptosis induced after mitotic arrest, we investigated the subcellular location, oligomeric structure, and protein interactions of Bax, Bcl-2, and Bcl-xL in vinblastine-treated KB-3 cells. Vinblastine induced the translocation of Bax from the cytosol to the mitochondria, which was accompanied by conformational activation and oligomerization of Bax. Bcl-2 was located in the mitochondria, underwent multisite phosphorylation after vinblastine treatment, and was strictly monomeric under all conditions. In contrast, in control cells, Bcl-xL existed in both monomeric (30 kDa) and oligomeric (150

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

“…Thus, the ability of wild-type Bcl-xL to bind Bax was diminished after vinblastine treatment, whereas phospho-defective Bcl-xL retained the ability to bind Bax after vinblastine treatment. In untreated KB-3 cells, Bax is cytosolic, and Bcl-xL is present both in the cytosol and mitochondrial compartments (17), with Bax translocating to the mitochondria upon vinblastine treatment (18). Thus, it would appear that a key role for cytosolic Bcl-xL in this system is to sequester Bax, and upon Bcl-xL phosphorylation, Bax is freed to translocate to the mitochondria.…”

“…Although the findings suggest that phosphorylation normally antagonizes Bcl-xL anti-apoptotic function, they do not address the underlying mechanisms. Previously we showed by co-immunoprecipitation that Bcl-xL and Bax interact in untreated KB-3 cells but not after vinblastine treatment (18). In the present report we have taken advantage of the availability of the phospho-defective mutant to formally demonstrate that phosphorylation negatively modulates Bcl-xL/Bax interaction.…”

“…After mixing for 2 h, anti-rabbit goat IgG-agarose beads were added and mixed overnight. After centrifugation, the supernatant was recovered, and the immunoprecipitate was washed as described previously (18) and resuspended in SDS sample buffer. Equivalent portions of supernatant and precipitate were subjected to 12.5% acrylamide SDS-PAGE, and immunoblotting which was performed with monoclonal mouse antibodies.…”

“…Cells stably overexpressing HA-Bcl-xL or phospho-mutants were prepared by transfecting KB-3 cells with human Bcl-xL tagged with HA epitope in pCDNA3.1 vector and maintained in 0.5 mg/ml G418 as described (18).…”

“…Samples were resolved by 13.5% acrylamide SDS-PAGE using separation gels 15 cm in length and stained in Colloidal Coomassie Blue (Pierce). Examination of the interaction of Bcl-xL and Bax by co-immunoprecipitation was performed as described previously (18).…”