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      Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

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          Abstract

          Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.

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          Most cited references 28

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          Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.

          Apoptosis induced by TNF-receptor I (TNFR1) is thought to proceed via recruitment of the adaptor FADD and caspase-8 to the receptor complex. TNFR1 signaling is also known to activate the transcription factor NF-kappa B and promote survival. The mechanism by which this decision between cell death and survival is arbitrated is not clear. We report that TNFR1-induced apoptosis involves two sequential signaling complexes. The initial plasma membrane bound complex (complex I) consists of TNFR1, the adaptor TRADD, the kinase RIP1, and TRAF2 and rapidly signals activation of NF-kappa B. In a second step, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II). When NF-kappa B is activated by complex I, complex II harbors the caspase-8 inhibitor FLIP(L) and the cell survives. Thus, TNFR1-mediated-signal transduction includes a checkpoint, resulting in cell death (via complex II) in instances where the initial signal (via complex I, NF-kappa B) fails to be activated.
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            Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

            The ability to direct functional domains to specific DNA sequences is a long sought-after goal for studying and engineering biological processes. Transcription activator like effectors (TALEs) from Xanthomonas sp. present a promising platform for designing sequence-specific DNA binding proteins. Here we describe a robust and rapid method for overcoming the difficulty of constructing TALE repeat domains. We synthesized 17 designer TALEs (dTALEs) that are customized to recognize specific DNA binding sites, and demonstrate that dTALEs can specifically modulate transcription of endogenous genes (Sox2 and Klf4) from the native genome in human cells. dTALEs provide a designable DNA targeting platform for the interrogation and engineering of biological systems.
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              Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis.

              Tumor necrosis factor (TNF) is an important inflammatory cytokine and induces many cellular responses, including inflammation, cell proliferation, apoptosis, and necrosis. It is known that receptor interacting protein (RIP) kinases, RIP1 and RIP3, are key effectors of TNF-induced necrosis, but little is known about how these two RIP kinases mediate this process, although reactive oxygen species (ROS) generation and JNK activation have been suggested to be two downstream events of RIP kinases. Here we report the identification of mixed lineage kinase domain-like, MLKL, as a key RIP3 downstream component of TNF-induced necrosis. Through screening a kinase/phosphatase shRNA library in human colon adenocarcinoma HT-29 cells, we found that knockdown of MLKL blocked TNF-induced necrosis. Our data suggest that MLKL functions downstream of RIP1 and RIP3 and is recruited to the necrosome through its interaction with RIP3. Finally, we found that MLKL is required for the generation of ROS and the late-phase activation of JNK during TNF-induced necrosis. However, because these two events are not involved in TNF-induced necrosis in HT-29 cells, the target of MLKL during TNF-induced necrosis remains elusive. Taken together, our study suggests that MLKL is a key RIP3 downstream component of TNF-induced necrotic cell death.
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                Author and article information

                Journal
                Cell Res
                Cell Res
                Cell Research
                Nature Publishing Group
                1001-0602
                1748-7838
                January 2014
                24 December 2013
                1 January 2014
                : 24
                : 1
                : 105-121
                Affiliations
                [1 ]State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University , Xiamen, Fujian 361005, China
                Author notes
                [*]

                These three authors contributed equally to this work.

                Article
                cr2013171
                10.1038/cr.2013.171
                3879712
                24366341
                Copyright © 2014 Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

                This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0

                Categories
                Original Article

                Cell biology

                plasma membrane translocation, mlkl, necroptosis, sodium influx, rip3

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