Phosphorylation and Inactivation of Myeloid Cell Leukemia 1 by JNK in Response to Oxidative Stress

Keio University, Edo, Tōkyō, Japan
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2002; 277(46):43730-4. DOI: 10.1074/jbc.M207951200
Source: PubMed


Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H(2)O(2) and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H(2)O(2). JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage.

Download full-text


Available from: Akihiro Umezawa, Jun 19, 2014
25 Reads
  • Source
    • "The critical role of ASK1 in apoptosis induction has been reported [36-38]. We have found that BSO triggers activation of ASK1 in ATO-treated cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death. HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression. HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO. The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.
    BMC Cancer 01/2014; 14(1):27. DOI:10.1186/1471-2407-14-27 · 3.36 Impact Factor
  • Source
    • "Its interactions with other Bcl-2 family members can favour its stabilization or its degradation, depending of the partner [17]. Mcl-1 degradation can involve the proteasome pathway following JNK and GSK3 associated Mcl-1 phosphorylations [18], but can also be a consequence of caspases activity [19]. This last possibility has been excluded in our model since Mcl-1 was decreased even in condition in which apoptosis is not detected (24 h). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Our work has been carried out in the context of the therapeutic failure in ovarian carcinoma, which remains the leading cause of death by gynecologic malignancy. In these tumours, recurrence and subsequent acquired chemoresistance constitute major hurdles to successful therapy. Here we studied the interest of a member of the tripentone chemical family, MR22388, for the treatment of chemoresistant ovarian cancer cells. Findings MR22388 activity has been assessed in vitro on cisplatin-resistant (SKOV3 and IGROV1-R10) ovarian cancer cell lines by conventional analysis, alone or combined to a BH3-mimetic molecule, ABT-737. MR22388 exerts its activity on cisplatin resistant cells, and we showed that it induces a decrease of the Mcl-1 anti-apoptotic protein expression. Considering our previous work demonstrating that the efficiency of Bcl-xL targeting strategies is conditioned to the concomitant inhibition of Mcl-1 we studied the interest of the association of this MR22388 with ABT-737, and showed that this combination was highly cytotoxic in chemoresistant cells. Conclusions This work thus opens new perspectives for the use of this promising molecule for the treatment of highly chemoresistant ovarian cancer cells and for sensitization of emerging Bcl-xL targeting strategies such as the use of BH3-mimetic molecules.
    Journal of Ovarian Research 06/2013; 6(1):38. DOI:10.1186/1757-2215-6-38 · 2.43 Impact Factor
  • Source
    • "The subcellular localization of Ser64A (Fig. 2i, ii) and the phospho-mimetic, Ser64E (Fig.2iii, iv), were very similar to those of the wild type protein, being localized to mitochondria, but with some additional cytoplasmic and nuclear localization. Serine 121 is reported to be phosphorylated by JNK in response to oxidative stress [31], [32], while Ser159 is phosphorylated by Glycogen Synthase Kinase (GSK) 3 [33]–[35]. Mutation of these residues to the phospho-null residue Ala, had no effect on the subcellular localization of Mcl-1 (Fig. 2 v–viii). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mcl-1 is an anti-apoptotic member of the Bcl-2 family that plays a key role in normal development, but also in pathologies such as cancer. It has some unusual properties compared to other anti-apoptotic members of the Bcl-2 family, and its expression and function are dynamically regulated by a variety of post-transcriptional and post-translational processes. Of note, Mcl-1 protein has a very short half life, and its stability and function may be regulated by reversible phosphorylation. There is also evidence to suggest that it may be localized to different subcellular compartments. The aim of this work was to determine whether residues within the PEST region of Mcl-1 that may undergo reversible phosphorylation, also regulate its subcellular distribution. We show that EGFP:Mcl-1 localizes mainly to the mitochondria of HeLa cells, with some additional cytoplasmic and nuclear localization. The mutations, S64A, S64E, S121A, S159A, T163A and T163E did not significantly affect the localization of Mcl-1. However, mutation of Ser162 to the phospho-null residue, Alanine resulted in an essentially nuclear localization, with some cytoplasmic but no mitochondrial localization. This mutant Mcl-1 protein, S162A, showed significantly decreased stability and it decreased the ability to protect against Bak-induced apoptosis. These data identify a new molecular determinant of Mcl-1 function, localization and stability that may be important for understanding the role of this protein in disease.
    PLoS ONE 09/2012; 7(9):e45088. DOI:10.1371/journal.pone.0045088 · 3.23 Impact Factor
Show more