Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads

Abstract

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.

Figure 1.

Whole-genome RNAi screening in S2R⁺ cells uncovered new components in the Dpp–Mad pathway. (A) Phosphorylation-dependent nuclear accumulation of Flag-Mad. S2R+ cells stably transfected with plasmids for Flag-Mad only, or Flag-Mad with punt and tkv were induced to express these proteins with CuSO₄ for the indicated time. Flag-Mad was detected by immunofluorescence staining with anti-Flag, and the nuclei were marked with DAPI. Bar, 10 μm. (B) Flag-Mad distribution patterns in wells containing indicated dsRNA in genome-wide RNAi screening. S2R+ cells expressing Flag-Mad, Punt, and Tkv were stained with anti-Flag after indicated RNAi, and the images were obtained with Discovery1 automated microscopy (Molecular Devices). Bar, 10 μm. (C) In S2R+ cells induced to express Flag-Mad and Punt/Tkv, RNAi targeting either punt plus tkv or msk (with dsRNA different from that in B) blocked nuclear concentration of Flag-Mad. The cells and experimental procedure were as in A. Bar, 10 μm. (D) Same RNAi experiment as in C, and proteins were extracted from the cells and analyzed by immunoblotting with antibodies against phospho-Mad (Mad-P) or Flag.

Figure 2.

msk, but not the importin β homologue ketel, is required for nuclear accumulation of endogenous Mad in Dpp-treated Drosophila S2 cells. (A) S2 cells were treated with indicated dsRNA and then subject to Dpp stimulation (1 nM for 1 h). Distribution of phospho-Mad (Mad-P) was detected by immunofluorescence staining using the PS1 antibody. The phospho-Mad signal per unit area in the nucleus and cytoplasm was measured using NIH ImageJ, and the nucleus/cytoplasm (N/C) ratios are shown (>50 cells were counted per sample). Bar, 10 μm. (B) S2 cells treated with indicated dsRNA were stimulated with Dpp as in A. Subcellular fractions were prepared and examined for phospho-Mad (Mad-P) and lamin levels (C: cytoplasm; N: nucleus). (C) S2R+ cells were subject to indicated RNAi. The cells were then stimulated with Dpp (1 nM) for 2 h and the mRNA level of dad was measured by real-time RT-PCR. The expression level of Rp49 was used as the internal standard for quantitation. The plotted data are derived from multiple experiments. Error bars indicate SD. (D) Co-immunoprecipitation of endogenous Msk with Flag-Mad. Whole-cell extract (WCE) was prepared from S2 cells transfected with Flag-Mad and Punt/Tkv as indicated and subject to immunoprecipitation using anti-Flag antibody conjugated to agarose beads. The bound proteins as well as input extract were analyzed by immunoblotting with indicated antibodies.

Figure 3.

msk null mutant cells in the developing eye imaginal disc did not have distinct nuclear staining of phospho-Mad. (A) Third instar eye imaginal discs (anterior to the left) were stained with PS1, which specifically recognizes phospho-Mad (Mad-P, red). The nuclei were marked with DAPI (blue). The boxed area was magnified and shown as the three panels on the right. Bars, 10 μm. (B) msk⁵ (null) or wild-type clones were generated using FLP recombinase driven by the eyeless promoter. The third instar eye imaginal discs were stained for phospho-Mad (red) and nuclei (blue). The clones were marked as negative for GFP signal (black) and are outlined. Bar, 10 μm. (C) msk⁵ or wild-type clonal cells falling within the posterior phospho-Mad–positive stripe (5–6 cells wide) were scored as having concentrated phospho-Mad staining in the nucleus or not. The numbers are obtained from more than eight discs in each case.

Figure 4.

Imp7 and 8 are required for nuclear accumulation of BMP-activated Smad1 in HeLa cells. (A) HeLa cells transfected with indicated siRNA duplexes (40 nM) were analyzed for mRNA levels of Imp7 or Imp8 by real-time RT-PCR. GAPDH was used as the internal standard for quantitation. Error bars indicate SD. (B) HeLa cells transfected with siRNAs were treated with BMP2 (100 ng/ml, 1 h) as indicated and analyzed by immunoblotting using antibodies specific for phospho-Smad1 or total Smad1. (C) HeLa cells transfected and treated as in B were immunostained with the PS1 antibody recognizing phospho-Smad1 and analyzed by fluorescence microscopy. Bar, 10 μm. (D) BMP2-induced transcriptional up-regulation of Smad6 was inhibited by siRNA targeting Imp7 or Imp8. HeLa cells transfected and treated as in B were analyzed for Smad6 mRNA level by real-time RT-PCR, with GAPDH serving as the standard. The plotted data are derived from three experiments and the error bars indicate SD.

Figure 5.

Imp7 and Imp8 are required for TGF-β–activated Smad2/3 to translocate into the nucleus. (A) HeLa or HaCaT cells transfected with indicated siRNAs (40 nM) were analyzed by immunostaining using anti-Smad2/3 antibody, with or without prior TGF-β stimulation as indicated (100 pM, 30 min). The nuclei were marked by DAPI. Bars, 10 μm. (B) HaCaT cells transfected and treated as in A were examined by immunoblotting using antibodies recognizing phospho-Smad2 or total Smad2 and 3. (C) Total RNA was isolated from the same HaCaT cells as in B and the mRNA level of Smad7 was measured by quantitative real-time PCR. The plotted data are derived from three experiments and the error bars indicate SD. (D) HeLa cells were transfected with siRNA against Imp7 or Imp8 individually or in combination (20 nM each) and were stimulated with TGF-β before immunostaining as in A. Non-targeting control siRNA was used to balance the final concentration of total siRNA in each transfection (40 nM final). Bar, 10 μm. (E) Cells in D were categorized as nuclear (Smad2/3 predominantly in the nucleus) or cytoplasmic (Smad2/3 evenly distributed in the cytoplasm and nucleus) based on anti-Smad2/3 immunostaining pattern. Over 400 cells were counted in each case.

Figure 6.

Effects of Imp7 and 8 overexpression on Smad localization. (A) Overexpression of siRNA-insensitive Imp7 and 8 rescued the defect in Smad2/3 nuclear accumulation in response to TGF-β. HeLa cells were transfected with siRNAs targeting endogenous Imp8 (top) or Imp7 and 8 combined (bottom) first, followed with expression vectors encoding HA-tagged mutant Imp7 and 8 that are no longer recognized by the siRNAs. After TGF-β stimulation (100 pM, 30 min), cells were co-stained with anti-Smad2/3 (red) and anti-HA (green) antibodies, and DAPI for the nuclei (blue). Cells expressing HA-tagged mutant Imp7 and 8 are marked with arrows. Bar, 10 μm. (B) HA-tagged wild-type Imp7 or 8 were transfected into Hela cells. After indicated treatments, cells were stained with antibodies and DAPI as in A. Bar, 10 μm.

Figure 7.

Interaction of Smads with Imp7 and Imp8, and the regulation by Ran-GTP. (A) Co-immunoprecipitation of Smad1 with Imp7 and Imp8. 293T cells were transfected with indicated expression plasmids and the whole-cell extract (WCE) was immunoprecipitated with anti-Flag antibody. Protein A/G bead was used as the control (c). The bound proteins and the input extract (WCE) were analyzed by immunoblotting as indicated. (B) Co-immunoprecipitation of Smad2 with Imp7 and Imp8. Same experimental design as in A, but with different expression plasmids transfected as indicated. (C) Mapping of Smad3 domains involved in interaction with Imp7/8. Recombinant GST fusions of indicated Smad3 or Smad2 fragments were used to pull down endogenous Imp7/8 in HeLa cells. The bound proteins were analyzed by an antibody that recognized both Imp7 and 8. Comparable amount of GST proteins was used in the pull down as judged by the Coomassie stain intensity. The arrowheads mark GST fusion proteins on the SDS-PAGE gel. S3MH1: aa 1–155; S2MH1: aa 1–185; S3MH2: aa 231–425; S3(L+MH2): aa 145–425; S3FL: full-length. Schematic drawing of Smad3 is also shown. (D) Purified GST-Imp8 on glutathione beads was used to pull down purified recombinant Smad1 and Smad3. The bound proteins were examined by immunoblotting using indicated antibodies. GST was used as the control. (E) Ran-GTP interrupts association between Smad3 and Imp8. GST-fusion of full-length Smad3 (GST-S3FL) was used in a pull-down experiment as in C. The bound proteins were further incubated with RanQ69L-GTP or BSA, and proteins released into the supernatant were collected and analyzed at indicated time points (15 min and 45 min elution). At the 45-min time point, the beads were washed again and proteins remaining bound to GST-S3FL (bound) were also examined by anti-HA immunoblotting.

Figure 8.

Roles of Msk, Imp7, and Imp8 in nuclear import of Smads at basal state. (A) S2R+ cells transfected with Flag-Mad expression vector were subject to RNAi as indicated. After inducing the expression of Flag-Mad, the cells were analyzed by anti-Flag immunofluorescence staining (green). Bar, 10 μm. (B) HeLa cells were transfected with indicated siRNAs. 3 d later, cells were stained with anti-Smad2/3 antibody (red) without prior TGF-β treatment. Bar, 10 μm. (C) 2 d after HeLa cells were transfected with indicated siRNAs, the cells were further transfected with a Myc-Fox H1 expression vector. Double- immunofluorescence staining with anti-Myc (green) and anti-Smad2/3 (red) was performed 1 d after Fox H1 transfection with no TGF-β stimulation. Bar, 10 μm.