Filamin B is a target for ISGylation. (A) A549 cells were incubated for 48 h with or without 1,000 U/ml of IFNβ. Cell lysates were immunoprecipitated (IP) with filamin B or filamin A antibodies, followed by immunoblot with ISG15 antibodies. They were also probed directly with ISG15 antibodies. (B) HisMax (HM)-c-filamin B were expressed with Flag–ISG15gg or Flag–ISG15aa. E1 and E2 were also expressed by co-transfection of cells with pcDNA-UBE1L and pcDNA-Myc–UBCH8, respectively. Cell lysates were immunoprecipitated with Flag antibodies, followed by immunoblot with Xpress antibodies. Cell lysates were also subjected to NTA pull-down (PD: NTA) under denaturing conditions followed by immunoblot with Xpress or Flag antibodies. (C) HM–c-filamin B, Flag–ISG15 and UBP43 were expressed with E1 and E2 as indicated. Cell lysates were then subjected to NTA pull-down as in (B). c-Filamin B, the carboxy-terminal H1–R24 region of filamin B; IFN, interferon; ISG, interferon-stimulated gene; NTA, nitrilotriacetic acid; UBC, ubiquitin-conjugating enzyme; UBE1L, ubiquitin-activating enzyme E1-like; UBP43, ubiquitin-specific processing protease 43.

Lys 2467 of filamin B is the ISGylation site. (A) All deletion constructs were tagged with HisMax (HM) to their amino termini. Each was expressed in HeLa cells with Flag–ISG15, E1 and E2. Cell lysates were subjected to NTA pull-down followed by immunoblot (IB) with Xpress antibodies. Whether each deletion was modified by ISG15 or not are marked as + or –. ABD indicates the actin-binding domain, and H1 and H2 are the hinge regions. The numerals show the numbers of IgG-like repeats. (B) The Lys-to-Arg mutants of HM–R22–24 were expressed in HeLa cells with Flag–ISG15, E1 and E2. Cell lysates were subjected to NTA pull-down (PD: NTA) followed by immunoblot with Flag or Xpress antibodies. The numerals indicate the positions of lysine in filamin B. The dot indicates a 55-kDa protein that can be stained with Xpress antibodies but not with Flag antibodies. The asterisks indicate nonspecific bands. (C) HM–filamin B (wt) or its K2467R mutant (mt) were expressed in HeLa cells with Flag–ISG15, E1 and E2. Cell lysates were subjected to SDS–PAGE in 6% gels followed by immunoprecipitation (IP) or NTA pull-down. Precipitates were immunoblotted with Xpress antibodies. ISG, interferon-stimulated gene; mt, mutant; NTA, nitrilotriacetic acid; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; wt, wild type.

ISGylation of filamin B blocks its interaction with RAC1 and JNK cascade members. (A) HM–c-filamin B (wt) or HM–c-K2467R mutant (mt), Myc–RAC1, Flag–ISG15, E1 and E2 were expressed in HeLa cells as indicated. Cell lysates were immunoprecipitated (IP) with Myc antibodies, followed by immunoblot with Xpress and Myc antibodies; they were also subjected to NTA pull-down (PD: NTA) followed by immunoblot with Xpress or Flag antibodies. (B) HM–c-Filamin B and Myc–RAC1 were expressed in HeLa cells with Flag–ISG15gg or Flag–ISG15aa, E1 and E2. Cell lysates were analysed as above. (C–E) HM–c-filamin B (wt), HM–c-K2467R (mt), Flag–ISG15, E1 and E2 were expressed in HeLa cells with (C) Myc–JNK1, (D) HA–MEKK1 or (E) Myc–MKK4 as indicated. Cell lysates were immunoprecipitated with HA or Myc antibodies, followed by immunoblot with Xpress, HA or Myc antibodies. The asterisks in (C,E) indicate IgG heavy chain. (F) M2 cells complemented with HM–filamin B (left panel) or HM–K2467R (right panel) were treated with IFNα. Cell lysates were subjected to NTA pull-down followed by immunoblot analysis. They were also probed directly with ISG15 antibodies. (G) M2 cells transfected with shControl or shUBE1L were complemented with HM–filamin B. After incubation with IFNα, cell lysates were subjected to NTA pull-down followed by immunoblot analysis. They were also probed directly with ISG15 or UBE1L antibodies. c-Filamin B, the carboxy-terminal H1–R24 region of filamin B; HA, haemagglutinin; HM, HisMax; IFN, interferon; IgG, immunoglobulin G; ISG, interferon-stimulated gene; JNK, Jun N-terminal kinase; NTA, nitrilotriacetic acid; Sh, Short hairpin; UBE1L, ubiquitin-activating enzyme E1-like; wt, wild type.

ISGylation of filamin B inhibits IFNα-induced JNK signalling. (A) HM–c-filamin B (wt) or HM–c-K2467R (mt) was expressed in M2 cells with or without Flag–ISG15, E1 and E2. After incubation with or without IFNα for 1 h, cell lysates were subjected to immunoblot (IB) analysis. They were also subjected to NTA pull-down (PD: NTA) followed by Flag antibodies. (B) HM–c-filamin B (wt) or HM–c-K2467R (mt) was expressed in M2 cells with or without ISG15, E1 and E2. After incubation with or without IFNα for 30 min, cell lysates were assayed for the activation of RAC1. (C) Myc–MKK4 and either HM–c-filamin B or HM–c-K2467R were expressed in HeLa cells with HA–MEKK1, Flag–ISG15, E1 and E2 as indicated. Cell lysates were immunoblotted with p-MKK4 antibodies. (D) JNK1–Myc–His and either HM–c-filamin B or HM–c-K2467R were expressed in HeLa cells with Myc–MKK4, Flag–ISG15, E1 and E2. Cell lysates were subjected to in vitro kinase assay for JNK using recombinant cJun as a substrate followed by immunoblot with p-cJun antibodies. (E) JNK1–Myc–His and either HM–c-filamin B or HM–c-K2467R were expressed in HeLa cells with HA–MEKK1, Flag–ISG15, E1 and E2. Cell lysates were subjected to in vitro kinase assay for JNK. (F) HM–c-filamin B (wt) or HM–c-K2467R (mt) was expressed in M2 cells with or without Flag–ISG15, E1 and E2. After incubation with or without IFNα for 12 h, cell lysates were immunoblotted. (G) M2 cells prepared as in (F) were incubated with or without IFNα for 12 h followed by TUNEL assay. Error bars indicate the mean±s.d. c-Filamin B, the carboxy-terminal H1–R24 region of filamin B; HM, HisMax; IFN, interferon; ISG, interferon-stimulated gene; JNK, Jun N-terminal kinase; mt, mutant; NTA, nitrilotriacetic acid; TUNEL, TdT-mediated dUTP nick-end labelling; wt, wild type.

Colocalization of UBCH8 and filamin B in membrane ruffles, and a model for the control of type I IFN-induced JNK signalling by ISGylation of filamin B. (A) Myc–UBCH8 was expressed in HeLa cells with or without HM–filamin B. Cells were stained with Xpress or Myc antibodies or phalloidin. The arrows indicate membrane ruffles; scale bar, 10 μm. (B) Early IFN response leads to the promotion of filamin B scaffold function and thus to the activation of cJun; late IFN response leads to the ISGylation of filamin B, dissociation of RAC1, MEKK1 and MKK4 from filamin B, and termination of the early response. GEF indicates a putative guanine nucleotide exchange factor that links type I IFN signal to RAC1. HM, HisMax; IFN, interferon; IFNAR, type I IFN receptor; ISG, interferon-stimulated gene; JNK, Jun N-terminal kinase; UBC, ubiquitin-conjugating enzyme.