Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine.
Microtubule inhibitors such as vinblastine cause mitotic arrest and subsequent apoptosis through the intrinsic mitochondrial pathway. However, although Bcl-2 family proteins have been implicated as distal mediators, their precise role is largely unknown. In this study, we investigated the role of Bak in vinblastine-induced apoptosis. Bak was mainly monomeric in untreated KB-3 cells, and multimers corresponding to dimer, trimer, and higher oligomers were observed after vinblastine treatment. The oligomeric Bak species were strongly diminished in cells stably overexpressing Bcl-xL. Immunoprecipitation with a conformation-dependent Bak antibody revealed that vinblastine induced Bak activation. Reciprocal immunoprecipitations indicated that vinblastine induced the interaction of active Bak with active Bax. Furthermore, Bcl-xL overexpression prevented Bak and Bax interaction and strongly inhibited apoptosis, whereas Bcl-2 overexpression did not prevent Bak-Bax interaction and only weakly inhibited apoptosis. The relative contributions of Bak and Bax were investigated using fibroblasts deficient in one or both of these proteins; double knockouts were highly resistant compared with single knockouts, with vinblastine sensitivities in the order of Bak(+)/Bax(+) > Bak(+)/Bax(-) > Bak(-)/Bax(+) > Bak(-)/Bax(-). These results highlight Bak as a key mediator of vinblastine-induced apoptosis and show for the first time activation and oligomerization of Bak by an antimitotic agent. In addition, our results suggest that the interaction of the activated forms of Bak and Bax represents a key distal step in the apoptotic response to this important chemotherapeutic drug.
Upreti
Meenakshi
M
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 West Markham Street, Little Rock, AR 72205-7199, USA.
Chu
Rong
R
Galitovskaya
Elena
E
Smart
Sherri K
SK
Chambers
Timothy C
TC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects; D014747:Vinblastine / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism
D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D014747:Vinblastine