Interaction of KLHL20 with Cul3 and DAPK. (A) Identification of KLHL20 as a DAPK-binding protein. Yeast strain L40 contransformed with Gal- and LexA-based constructs as indicated was assayed for His3 phenotype (−His) or β-galactosidase activity (β-Gal). (B) Endogenous KLHL20 interacts with endogenous DAPK. Lysates of HeLa cells were used for immunoprecipitation with anti-DAPK antibody or control IgG and the immunoprecipitates and cell lysate were subjected to western blot with antibodies as indicated. (C) The DD of DAPK is involved in binding KLHL20. Lysates of 293T cells transfected with DAPK-Flag or DAPKΔDD-Flag were used for immunoprecipitation and western blot analyses with antibodies as indicated. (D, E) Mapping the KLHL20 domain responsible for binding DAPK (D) or Cul3 (E). 293T cells were cotransfected with various constructs as indicated. Cells were lysed for immunoprecipitation and western blot analyses with antibodies as indicated. The position of immunoglobulin heavy chain is marked with an asterisk. (F) Cul3 coprecipitates with DAPK. Lysates of 293T cells cotransfected with various constructs were analysed by immunoprecipitation and/or western blot with indicated antibodies. (G) KLHL20 mediates the interaction between DAPK and Cul3. HeLa cells were infected with lentivirus carrying indicated siRNA and then selected with puromycin. The resulting stable knockdown cells were analysed by immunoprecipitation followed by western blot with antibodies as indicated. KLHL20, Cul3, and DAPK expression levels were assayed by western blot (bottom panel).

KLHL20–Cul3–ROC1 complex is a ubiquitin ligase for DAPK. (A, B) KLHL20–Cul3–ROC1 complex promotes DAPK ubiquitination in vivo. 293T cells were transfected with constructs expressing Myc-ubiquitin (Myc-Ub), ROC1 (not indicated), Cul3 (or mutant), KLHL20 (or mutant), and/or DAPK-Flag and treated with MG132 for 16 h. Cell lysates were used to detect DAPK ubiquitination by immunoprecipitation with anti-Flag antibody, followed by western blot with anti-Myc antibody. The expression levels of Cul3 and KLHL20 were examined by western blot (bottom panel). (C) The DD of DAPK is important for DAPK ubiquitination by KLHL20–Cul3–ROC1 complex. 293T cells transfected with indicated constructs and treated with MG132 were assayed for DAPK ubiquitination as in (A). (D) Endogenous KLHL20 promotes DAPK ubiquitination. HeLa cell derivatives as in Figure 1G were transfected with Myc-Ub and/or DAPK-Flag and assayed for DAPK ubiquitination as in (A). (E–F) In vitro ubiquitination of DAPK by the KLHL20–Cul3–ROC1 E3 ligase. Cul3 complex was purified by Glutathione Sepharose from lysates of 293T cells transfected with GST-Cul3 (or its mutant), Myc-ROC1, and KLHL20-Myc (or its mutant). The copurification of ROC1 and/or KLHL20 with GST-Cul3 was shown in Supplementary Figure S5. The Cul3 complex was subjected to in vitro ubiquitination assay in the presence of E1, E2, ubiquitin, and/or Flag-DAPK purified from baculovirus (see Materials and methods). The reaction was resolved on SDS–PAGE and analysed by western blot with anti-Flag antibody.

KLHL20 enhances DAPK proteasomal degradation and attenuates DAPK proapoptotic activity. (A) KLHL20 promotes proteasomal degradation of DAPK. 293T cells transfected with indicated constructs were treated with 1 μM MG132 or DMSO (−) for 12 h, and then lysed for western blot analyses. (B) KLHL20 promotes DAPK turnover. HeLa cells transfected with indicated constructs were treated with 100 μg/ml cycloheximide for various time points before lysis. Cell lysates were analysed by western blot. The amounts of Flag-DAPK relative to that of untreated cells are indicated. (C) Knockdown of KLHL20 increases DAPK steady-state level. HeLa cell derivatives as in Figure 1G were analysed for DAPK level by western blot. (D) KLHL20 inhibits the proapoptotic function of DAPK. NIH3T3 cells were transiently transfected with DAPK (or DAPKΔCaM) together with or without KLHL20 (or KLHL20ΔK). Apoptosis was assayed by the Cell Death Detection ELISA kit. Data are represented as mean±s.e.m. (^*P<0.05; ^**P<0.005; n=3). The expression levels of various proteins are shown on the bottom panel.

IFN blocks KLHL20-dependent DAPK ubiquitination and degradation. (A) IFN upregulates DAPK. 293T cells transfected with DAPK-Flag were treated with 1000 U/ml IFN-γ for indicated time points and then lysed for western blot analysis. (B) IFN inhibits proteasomal degradation of DAPK. HeLa cells were treated with 1500 U/ml IFN-α or 1000 U/ml IFN-γ for 18 h and then with 30 μM MG132 (or DMSO) for 3 h. The level of endogenous DAPK was analysed by western blot. (C, D) IFN-α or IFN-γ blocks KLHL20-dependent DAPK ubiquitination. 293T cells were transfected with Myc-Ub, Cul3, ROC1, KLHL20, and/or DAPK-Flag. Two days after transfection, cells were treated with IFN-γ for indicated time points (C) or IFN-α for 18 h (D). DAPK ubiquitination was assayed as in Figure 2A.

IFN triggers the disruption of KLHL20–DAPK complex by sequestrating KLHL20 in PML-NBs. (A) IFN blocks the interaction of KLHL20 with endogenous DAPK. HeLa cells transfected with KLHL20-Flag were treated with IFN-γ for 18 h and lysed for immunoprecipitation with anti-Flag antibody. The immunoprecipitates and cell lysates were analysed by western blot with indicated antibodies. (B) IFN triggers the enrichment of KLHL20 in PML-NBs. HeLa cells were treated with or without IFN-γ for 18 h. Cells were fixed, triple stained by DAPI, anti-KLHL20 antibody and anti-PML antibody (upper panel) or by DAPI, anti-DAPK antibody and anti-PML antibody (bottom panel), and examined by confocal microscopy. The box area was amplified to show the colocalization of KLHL20 and PML. Bar, 20 μm. (C) Interaction of KLHL20 with PML. HeLa cells transfected and treated as in (A) were lysed for immunoprecipitation with anti-Flag or a control antibody (IgG). The immunoprecipitates and cell lysates were analysed by western blot with antibodies as indicated. (D) DAPK and PML compete for binding KLHL20. 293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Flag, followed by western blot with indicated antibodies.

PML depletion reverses the inhibitory effects of IFN on DAPK ubiquitination and degradation. (A) Generation of PML knockdown cells. HeLa cells were infected with lentivirus carrying indicated siRNA and selected with puromycin. Cells were treated with or without IFN-γ for 18 h and then lysed for western blot analysis. (B) PML siRNA rescues the interaction between DAPK and KLHL20 in IFN-γ-treated cells. Cells as in (A) were transfected with KLHL20-Flag and treated with IFN-γ for 18 h. Cells were lysed for immunoprecipitation with anti-Flag antibody. The immunoprecipitates and cell lysates were analysed by western blot with antibodies as indicated. (C) PML siRNA rescues KLHL20-mediated DAPK ubiquitination in IFN-γ-treated cells. Cells as in (A) were transfected with indicated constructs and treated with IFN-γ for 18 h. DAPK ubiquitination was analysed as in Figure 2A. The expression levels of various proteins are shown on the bottom. (D) IFN-γ fails to upregulate DAPK in PML null cells. PML^+/+ or PML^−/− MEFs were treated with IFN-γ for 18 h and then lysed for western blot analysis with indicated antibodies. (E) PML is required for IFN-γ-induced DAPK stabilization. Cells as in (D) were treated with IFN-γ for 18 h and then with 50 μg/ml cycloheximide for indicated time points. Cells were lysed for western blot analysis and the level of DAPK was normalized to that of tubulin and plotted on the right.

Blockage of KLHL20-mediated DAPK degradation contributes to MM cell responsiveness to IFN. (A, B) IFN-α induces PML-NBs and KLHL20 relocation in H929 cells but not in XG1 cells. Cells treated with IFN-α (1000 U/ml) for 16 h were fixed, stained by DAPI (blue), anti-KLHL20 antibody and anti-PML antibody, and examined by confocal microscopy. Bar, 10 μm. (C) IFN-α induces the disruption of KLHL20–DAPK complex in H929 cells but not in XG1 cells. Cells as indicated were treated as in (A) and then subjected to immunoprecipitation with anti-KLHL20 antibody or control antibody (IgG), followed by western blot with anti-KLHL20 or anti-DAPK antibody. (D) IFN-α induces the upregulation of DAPK in H929 cells but not in XG1 cells. Cells treated as in (A) were analysed by western blot with antibodies as indicated. (E) H929 cells were infected with lentivirus-expressing KLHL20, KLHL20m6 or DAPK siRNA and then selected with blasticidin (for KLHL20 construct) or puromycin (for siDAPK construct). To generate cells stably expressing both KLHL20 and DAPKΔDD, H929 cells were first infected with retrovirus carrying DAPKΔDD and selected with puromycin. The resulting stable line was then infected with lentivirus-expressing KLHL20 and selected with blasticidin. Cell lysates were analysed by western blot with antibodies as indicated (bottom panel). Alternatively, cells were treated with IFN-α and apoptosis was assayed by Annexin V staining followed by flow cytometry analysis (upper panel). (F) XG1 cells were infected with lentivirus carrying indicated siRNAs and then selected with hygromycin (for DAPK siRNA construct) and/or puromycin (for KLHL20 siRNA construct). Cells were lysed for western blot analysis (bottom panel). Alternatively, IFN-α-induced apoptosis was assayed as in (C). Data are represented as mean±s.e.m. (^*P<0.05; ^**P<0.005; ^***P<0.0005; n=3).

Blockage of KLHL20-mediated DAPK degradation contributes to IFN-induced autophagy. (A) MCF7-LC3 cells were transfected with various constructs together with mCherry-expressing construct and then treated with or without IFN-γ. The GFP-LC3 signal was examined by confocal microscopy (for image presentation) and epifluorescent microscopy (for quantification). Bar, 20 μm. The number of GFP-LC3 dots per cell was quantified from 50 mCherry-positive cells and plotted (bottom panel). Data are represented as mean±s.e.m. (^***P<0.0005; n=3). (B) MCF7 parental cells were transfected and treated as in (A) and then analysed by western blot with indicated antibodies. The positions of LC3-I and LC3-II are indicated. (C) MCF7 cells transfected and treated as in (B) except for the addition of E64d and pepstatin A at 16 h before harvest. Cell lysates were analysed by western blot with antibodies as indicated. (D) Models for the DAPK degradation pathway mediated by KLHL20–Cul3–ROC1 E3 ligase complex and for the inhibition of this pathway in IFN-treated cells.