Vinblastine-induced phosphorylation of Bcl-2 and Bcl-XL 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 Bcl-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 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited 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 vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation 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.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
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Goodwin
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D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D020935:MAP Kinase Signaling System; D048055:Mitogen-Activated Protein Kinase 8; D048056:Mitogen-Activated Protein Kinase 9; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000494:pharmacology*; D051020:bcl-X Protein
D048868:Adaptor Proteins, Signal Transducing; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D007527:Isoenzymes; C490666:MAPK8IP1 protein, human; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D014747:Vinblastine; D048056:Mitogen-Activated Protein Kinase 9; D019908:Proto-Oncogene Proteins c-raf; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases