Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis

Abstract

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.

Fig. 1.

Loss of MLKL protects against cell death in necrotic conditions but not apoptosis. (A) MLKL shRNA clones target full-length MLKL variant (shRNA-A) or the short isoform (shRNA-B). (B) HT-29 clones infected with either control shRNA (FFshRNA) or MLKLshRNA-A (clone shMLKL 1–3-2) or MLKLshRNA-B (clone shMLKL 1–3-1), were treated with TNF (30 ng/mL), Smac mimetic (SM-164 10 nM), and z-VAD-fmk (20 μM) (TSZ) for 24 or 48 h. Cell survival was determined by MTT. (C, Left) MLKLshRNA-A cells cotransfected with pCMV-LacZ and either control FLAG-vector or FLAG-MLKL-rWT(MLKL shRNA-resistant mutation). After 24 h, cells were either untreated (control) or treated with TSZ for 24 h. Cell survival was determined by counting LacZ⁺ cells and normalized to untreated cells. (Right) Immunoblot analysis of MLKLshRNA-A cells transfected with either control FLAG-vector or FLAG-MLKL using anti-FLAG or antiactin antibodies. (D) HT-29 clone MLKLshRNA-A cells were treated with apoptotic conditions [TNF (30 ng/mL) and Smac mimetic (SM-164 10 nmol) (TS) or TNF (30 ng/mL) and CHX (10 μg/mL) (TC)] or necrotic conditions (TSZ or TCZ) for 48 h. Cell survival was determined by MTT. (Right) Immunoblot analysis of HT-29 clones infected with FFshRNA or MLKLshRNA-A with anti-MLKL or anti-Actin antibodies.

Fig. 2.

MLKL interacts with RIP3. (A) HT-29 clones FFshRNA and MLKLshRNA-A were treated with TSZ for the indicated times; cell lysates were immunoprecipitated with anti-RIP1 antibody (IP:RIP1) and analyzed by immunoblot with anti-RIP3, anti-RIP1, or anti-Actin antibodies. Input, 1% of extract before immunoprecipitation (control). * indicates phosphorylated RIP3. (B) HEK293 cells were transfected with Myc-RIP1, RIP3-GFP, or FLAG-MLKL as indicated. After 24 h, cell lysates were immunoprecipitated with anti-FLAG antibody (IP:FLAG) and analyzed by immunoblot with anti-Myc, anti-GFP, or anti-FLAG antibodies. Input, 3% of extract before immunoprecipitation (control). (C) HEK293 cells were transfected with Myc-RIP1, FLAG-MLKL, RIP3-GFP, or RIP3D160N-GFP as indicated. After 24 h, cell lysates were immunoprecipitated with anti-GFP antibody (IP:GFP) and analyzed by immunoblot with anti-FLAG, anti-Myc, or anti-GFP antibodies. Input, 3% of extract before immunoprecipitation (control). (D) HT-29 cell lysates were immunoprecipitated with anti-MLKL antibody (IP:MLKL) and analyzed by immunoblot with anti-RIP3, anti-RIP1, and anti-MLKL antibodies. Input, 1.5% of extract before immunoprecipitation (control). * indicates phosphorylated RIP3.

Fig. 3.

MLKL kinase activity is required for necrosis. (A) HEK293 cells were transfected with either FLAG-vector, FLAG-MLKL, FLAG-RIP3, or FLAG-RIP1. After 24 h, cell lysates were immunoprecipitated with anti-FLAG antibody and used for in vitro kinase assay using MBP as substrate. Lysates were analyzed by Western blot with anti-FLAG antibody. (B) HEK293 cells were transfected with either FLAG-vector, or FLAG-MLKL, or FLAG-MLKL-K230A/K331A mutant. After 24 h, cell lysates were immunoprecipitated with anti-FLAG antibody and used for in vitro kinase assay using MBP as substrate. Lysates were analyzed by Western blot with anti-FLAG antibody. (C) HEK293 cells were transfected with FLAG-MLKL, RIP3-GFP, or FLAG-MLKL-K230A/K331A as indicated. After 24 h, cell lysates were immunoprecipitated with anti-FLAG antibody (IP:FLAG) and analyzed by immunoblot with anti-FLAG or anti-GFP antibodies. Input, 5% of extract before immunoprecipitation (control). (D, Left) MLKLshRNA-A cells were cotransfected with pCMV-LacZ plasmid and either control FLAG-vector or MLKL shRNA-resistant plasmids, FLAG-MLKL-rWT, or FLAG-MLKL-rK230A/K331A for 24 h followed by treatment with TSZ for 24 h. Survival was determined by counting LacZ⁺ cells and normalized to untreated cells. (Right) Western blot analysis of MLKLshRNA-A cells cotransfected with pCMV-LacZ plasmid and either control FLAG-vector, or FLAG-MLKL-rWT, or FLAG-MLKL-rK230A/K331A. After 24 h, cell lysates were immunoblotted with anti-FLAG and antiactin.

Fig. 4.

Loss of MLKL affects JNK phosphorylation. (A) HT-29 clones FFshRNA and MLKLshRNA-A were treated with TSZ for the indicated times; cell lysates were analyzed by immunoblot with anti-pJNK or anti-JNK1 antibodies. (B) HT-29 clones FFshRNA and MLKLshRNA-A were treated with TCZ for the indicated times; cell lysates were analyzed by immunoblot with anti-pJNK or anti-JNK1 antibodies. (C) HEK293 cells were transfected with either FLAG-vector, or FLAG-MLKL, and/or HA-JNK1 as indicated. After 24 h, cell lysates were immunoprecipitated with anti-HA antibody and used for in vitro kinase assay using GST-cJun (1–79) as substrate. Lysates were analyzed by immunoblot with anti-FLAG and anti-HA antibodies. * indicates nonspecific band. (D) HT-29 clones FFshRNA and MLKLshRNA-A were treated with TSZ for the indicated times, stained with DCFDA, and analyzed by flow cytometry. Gray line (NT) represents background ROS in untreated samples. (E) L929 cells were infected with lentivirus, nontargeting (NT) or MLKL shRNA for 24 h followed by selection with puromycin for 48 h. Cells were then untreated or treated with TNFα (30 ng/mL) for 8 h. Cell survival was determined by MTT assay. (Right) RT-PCR of L929 cells infected with NT or MLKL shRNA with MLKL or GAPDH primers. (F) Diagram of the role of MLKL in RIP1/RIP3-dependent necrosis.