Phosphorylation of Bcl-x_L by polo-like kinase 1 results in the inactivation of the anti-apoptotic activity during tubulin binder-induced apoptosis Free

A109

Plk1 (polo-like kinase 1), a serine/threonine protein kinase, is involved in multiple events during the mitosis, such as centrosome maturation, spindle assembly, and chromosome segregation. In humans, there are 4 Plk family members, which contain an N-terminal catalytic domain and C-terminal polo-box domain that contributes to the phospho-dependent substrate recognition. It has been reported that Plk1 is also overexpressed in many human tumors; therefore, Plk1 has attracted attention as a molecular target for anti-tumor drugs. Recently, our group has shown that Plk1 is stabilized by Hsp90, and phosphorylates somatic type Wee1, leading to the degradation of Wee1 via ubiquitin-proteasome pathway. Although many substrates of Plk1, including Wee1, tubulins, and Myt1, are already known, the identification of additional substrates will enhance our general understanding of the important roles played by Plk1.
 Bcl-x_L is a 29-kDa mitochondrial membrane protein that inhibits apoptosis induced by various stimuli, such as UV irradiation, anti tumor drug treatment, and growth factor withdrawal. It has been reported that Bcl-x_L is overexpressed in many human tumors, and contributes to their treatment resistance during chemotherapy. Bcl-x_L contains 4 regions of BH and has a flexible loop domain between BH4 and BH3. This loop region is required for the post-translational modifications of Bcl-x_L. Also, the BH1 and BH2 regions of Bcl-x_L are required for heterodimerization with Bax to prevent apoptosis. Bcl-2 family proteins are modified post-translationally, and their function, localization, and protein-protein interaction are controlled by the modifications. Although Bcl-x_L is found to be phosphorylated by Hsp90 inhibitor-sensitive kinase(s), there are inconsistent results as to whether the proposed kinase(s) can actually phosphorylate Bcl-x_L. Upon the treatment with tubulin binder, Bcl-x_L is phosphorylated, but the role of the phosphorylation and its responsible kinase(s) remains poorly understood.
 In this study, we found that Plk1 phosphorylates Bcl-x_L at 13 Ser/Thr residues in vitro. When all 13 phosphorylation sites were mutated to alanine residues, cells expressing the mutant became more resistant to tubulin binder-induced apoptosis than those expressing wild-type Bcl-x_L. Thus, Plk1 controls the anti-apoptotic activity of Bcl-x_L through the phosphorylation in tubulin binder-induced apoptosis. These observations suggest that Plk1-mediated phosphorylation is an important key on the decrease of the anti-apoptotic activity of Bcl-x_L, and the drugs, that could induce Bcl-x_L phosphorylation, such as tubulin binders may provide effective therapeutic drugs and/or a novel chemosensitizers for those human cancers that are expressing Bcl-x_L.