Vinblastine-induced phosphorylation of Bcl-2 and Bcl-X-L is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and B cl-X-L. Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X-L phosphorylation in KB-3 cell s treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) an d p54(JNK2), were present in KB-3 cells, and both isoforms were activated b y vinblastine as determined by Mono Q chromatography. We used antisense oli gonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylatio n of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhi bited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X -L by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinb lastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl- 2/Bcl-X-L phosphorylation, confirming a bifurcation in JNK signaling involv ing both nuclear and non-nuclear substrates. Vinblastine-induced phosphoryl ation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/ Bcl-X-L phosphorylation.