Involvement of Asp-Glu-Val-Asp-directed, caspase-mediated mitogen-activated protein kinase kinase 1 Cleavage, c-Jun N-terminal kinase activation, and subsequent Bcl-2 phosphorylation for paclitaxel-induced apoptosis in HL-60 cells.
Paclitaxel is a novel anticancer drug that has demonstrated efficacy toward treating several malignant tumor types. Here, we demonstrate that c-Jun NH(2)-terminal kinase (JNK), but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2, was persistently activated by paclitaxel or other microtubule-damaging agents within human leukemia HL-60 cells. Overexpression of a dominant-negative mutant, mitogen-activated protein kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotide prevented paclitaxel-induced JNK activation, Bcl-2 phosphorylation and apoptosis. Furthermore, we found that the full-length MEKK1 was cleaved to a 91-kDa carboxyl-terminal fragment at the earlier time of apoptosis induced by microtubule-damaging agents. This cleavage, however, occurred consistently with JNK activation and Bcl-2 phosphorylation, but preceded DNA fragmentation in cells in response to paclitaxel activity. The caspase inhibitor Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO), but not Ac-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO), effectively blocked MEKK1 cleavage, JNK activation, Bcl-2 phosphorylation, and subsequent apoptosis. Subcellular fractionation revealed that the 91-kDa C-terminal MEKK1 fragment was translocated to cytosol. Notably, the MEKK1 fragment could be coimmunoprecipitated with anti-JNK antibodies, suggesting that a signaling complex of C-terminal MEKK1/stress-activated protein kinase/extracellular-signal regulated kinase 1/JNK formed during apoptosis induced by microtubule-damaging agents. Taken together, our results suggest that disruption of cytoarchitecture by paclitaxel triggers a novel apoptosis-signaling pathway, wherein an active DEVD-directed caspase (DEVDase) initially cleaves MEKK1to generate a proapoptotic kinase fragment that is able to activate JNK and subsequent Bcl-2 phosphorylation, finally eliciting cell death.
Shiah
S G
SG
Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chuang
S E
SE
Kuo
M L
ML
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D017239:Paclitaxel / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; C108637:aspartyl-glutamyl-valyl-aspartal; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D010447:Peptide Hydrolases; D020169:Caspases; C108711:DEVDase; D017239:Paclitaxel