Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.
Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.
Upreti
Meenakshi
M
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Galitovskaya
Elena N
EN
Chu
Rong
R
Tackett
Alan J
AJ
Terrano
David T
DT
Granell
Susana
S
Chambers
Timothy C
TC
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology; D050257:Tubulin Modulators / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D050257:Tubulin Modulators; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D014750:Vincristine