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 SHINE-GWO SHIAH, SHUANG-EN CHUANG, and MIN-LIANG KUO Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (S.- G.S., M.-L.K.); and Division of Cancer Research, National Health Research Institute, Taipei, Taiwan (S.-E.C.) Received March 10, 2000; accepted October 22, 2000 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT 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 pre- vented paclitaxel-induced JNK activation, Bcl-2 phosphoryla- tion 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 phosphoryla- tion, and subsequent apoptosis. Subcellular fractionation re- vealed that the 91-kDa C-terminal MEKK1 fragment was trans- located 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-sig- naling 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. c-Jun N-terminal kinase (JNKs), known as stress-acti- vated protein kinases (SAPKs), is a member of the family of mammalian mitogen-activated protein (MAP) kinase that mediates intracellular signals originated from diverse extra- cellular stimuli, including growth factors, cytokines, UV light, heat shock, and a variety of anti-cancer drugs (Chen et al., 1996; Osborn and Chambers, 1996; Verheij et al., 1996). The activation of JNK involves a protein kinase cascade wherein mitogen-activated protein kinase kinase-1 (MEKK1) (Lange-Carter et al., 1993) activates SAPK kinase-1 (SEK1) (Yan et al., 1994) and ultimately activates JNK. The activa- tion of the JNK cascade in cells leading to a wide range of cellular functions including inflammation, cell growth, and cell death. Recently, a number of studies have referred to extensive efforts attempting to decipher the role of JNK in apoptosis. Various well known chemotherapeutic drugs, such as doxorubicin, vinblastine, VP-16, camptothecin, and pacli- taxel have been demonstrated to be capable of activating JNK (Seimiya et al., 1997), these drugs being critical for the apoptosis-triggering program for different cell lines. The ex- pression of a dominant-negative mutant of MEKK1 or JNK prevented the induction of apoptotic cell death by UV-C, -irradiation, or anticancer drugs (Faris et al., 1996). These observations suggest an essential role for JNK in the regu- lation of apoptosis induced by diversified stimuli. This work was supported by National Science Council of Taiwan Grant NSC 89-2320-B-002-015. ABBREVIATIONS: JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAP, mitogen-activated protein; MEKK, mitogen- activated protein kinase kinase; SAPK, stress-activated protein kinase; SEK, stress-activated protein kinase kinase; DEVDase, Asp-Glu-Val-Asp- directed caspase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; DN, dominant negative; Dex, dexamethasone; VCR, vincristine; VBL, vinblastine; ERK, extracellular signal-regulated kinase. 0026-895X/01/5902-254 –262$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 2 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 143/881149 Mol Pharmacol 59:254–262, 2001 Printed in U.S.A. 254