BCL-X(L) IS PHOSPHORYLATED IN MALIGNANT-CELLS FOLLOWING MICROTUBULE DISRUPTION

The oncogenic protein Bcl-2 functions as a potent inhibitor of program med cell death. This survival activity has been shown in some settings to be influenced by the Bcl-2 phosphorylation state. It has been demo nstrated that treatment with microtubule-targeted agents results in ph osphorylation of both Raf-1 kinase and Bcl-2, The Bcl-2-related family member Bcl-x(L) also exhibits a death suppressive activity, but its p otential for phosphorylation following exposure to drugs that interact with microtubules has not been evaluated. Several tumor cell lines wi th low or undetectable levels of Bcl-2 protein expression were found t o express Bcl-x(L), A more slowly migrating Bcl-x(L) band was observed on immunoblots after cells were treated with microtubule-targeted age nts. The appearance of this band was responsive to dose and was absent when the cell lysates were treated with lambda protein phosphatase. U sing a Bcl-x(L)-specific monoclonal antibody, the phosphorylated form of Bcl-x(L) was immunoprecipitated from cells treated with paclitaxel and metabolically labeled with P-32-labeled inorganic orthophosphate, Herein, we report that Bcl-x(L) is phosphorylated in malignant cells a fter incubation with agents that target tubulin, including paclitaxel, vincristine, vinblastine, colchicine, and nocodazole. Moreover, pacli taxel-resistant ovarian carcinoma cell lines that have mutations in tu bulin failed to exhibit phosphorylation of Bcl-x(L) after paclitaxel e xposure, but they did demonstrate Bcl-x(L) phosphorylation in the pres ence of other tubulin-targeting agents. As observed for Bcl-2, phospho rylation of Bcl-x(L) was accompanied by phosphorylation of Raf-1, Inte restingly, phosphorylation of these three proteins failed to occur or was much less pronounced when cells grown at high density were challen ged with drug. Also, reduced Raf-1 expression, observed after treatmen t of cells with geldanamycin prior to and during incubation with the m icrotubule-active drugs, correlated with diminished Bcl-x(L) phosphory lation, Taken together, these results suggest that Bcl-x(L), like Bcl- 2, is phosphorylated by agents that disrupt microtubule architecture, By analogy with Bcl-2, this phosphorylation may play a critical role i n modulating Bcl-x(L) function and may be an important determinant of microtubule-directed chemotherapeutic efficacy in human tumors.