Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members.
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 kDa) forms. Treatment with agents that induced Bcl-xL phosphorylation (microtubule inhibitors) caused loss of the 150-kDa form, but this species was unaffected by apoptotic stimuli that did not stimulate phosphorylation. Vinblastine also promoted Bax activation and Bax oligomerization in HCT116 colon cancer cells. Both wild-type and Bax-deficient HCT116 cells expressed the 150-kDa form of Bcl-xL, which was depleted similarly in both cell lines upon vinblastine treatment. Co-immunoprecipitation studies revealed that in untreated KB-3 cells inactive cytosolic Bax interacted with Bcl-xL, whereas in vinblastine-treated cells, activated mitochondrial Bax did not interact with Bcl-xL. Interaction of Bcl-2 with Bax was not observed under any condition. Overexpression of Bcl-xL inhibited vinblastine-induced Bax activation and Bax dimerization and in parallel inhibited apoptosis. The results indicate that vinblastine-induced apoptosis requires translocation, activation, and oligomerization of Bax and is associated with specific changes in the oligomeric properties of Bcl-xL, which occur independently of Bax.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, USA.
Meenakshi
M
Upreti
Christopher S
CS
Lyle
Brian
B
Skaug
Lihua
L
Du
Timothy C
TC
Chambers
D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D047468:Immunoprecipitation; D010766:Phosphorylation; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology*
D019253:Proto-Oncogene Proteins c-bcl-2; D014747:Vinblastine