Identification and characterization of MT-1X as a novel FHL3-binding partner

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Abstract

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3- mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation. © 2014 Cai et al.

Figures

  • Table 1. The corresponding plasmids and culture medium for confirmation of interaction between full length MT-1X and FHL3d4.
  • Fig. 1. The interaction between full length MT-1X and FHL3 by yeast two hybrid assay. (A) Identification of recombinant plasmid pAS2-1/ FHL3d4 by restriction analysis. M:DNA marker; Lane1: Digested pAS2-1/FHL3d4 plasmid with NdeI and EcoRI. (B) Identification of recombinant plasmid pACT2/MT-1X by restriction analysis. M:DNA marker; Lane1-4: Digested pACT2/MT-1X plasmid with BamHI and EcoRI. (C) Detection of MT-1X autoactivation by yeast two hybrid analysis. Line1:1,2: positive control; Line1: 3,4: negative control; Line2:1-5:transform pACT2/MT-1X. (D) Coloration analysis to verify the interaction between full length MT-1X and FHL3. Line1:1,2:positive control; Line1: 3,4: negative control. Line2:1–7: Co-transform pAS2-1/FHL3d4 and pACT2/MT-1X. doi:10.1371/journal.pone.0093723.g001
  • Fig. 2. The interaction between FHL3 and MT-1X in vivo. 293T cells were transfected by Myc-MT-1X with HA-FHL3. Lane 1: lysate of cells transfected with Myc-MT-1X and HA-FHL3. 2: IP of cells with antiIgG monoclonal antibody as negative control. 3: IP of cells with anti-Myc monoclonal antibody; After 30 h, total cell lysates were analyzed before co-immunoprecipitation to verify expression of Myc-MT-1X and HAFHL3 (lane 1). After co-immunoprecipitation with anti-Myc tag antibody, MT-1X was detected only in the presence of immune complexes of Myc-FHL3 (lane 3). doi:10.1371/journal.pone.0093723.g002
  • Fig. 3. GST pull-down assay demonstrating in vitro binding between MT-1X and FHL3. (A) Purification of GST-FHL3 protein. Purification of GST protein (Lane1) and GST-FHL3 protein (Lane2) by Western blot analysis using anti-GST monoclonal antibody. (B) GST pull-down assay. Lane1 demonstrated the signal from purified His-MT-1X. GST protein and GST-FHL3 fusion protein were mixed with purified His-MT-1X (Lane2 and 3) and precipitated with Glutathione Agarose followed by Western blot analysis. doi:10.1371/journal.pone.0093723.g003
  • Fig. 4. Knockdown of MT-1X promoted the FHL3-dependent inhibitory effect on HepG2 cell. (A) The expression of MT-1X protein of HepG2 cells transfected by small interference RNA against MT-1X or negative control siRNA determined by western blot. 1, Negative control siRNA; 2, siMT-1X. GAPDH was used as loading control. The transfection efficiency of HepG2 is ,30%. (B) Indicated amounts of rhFHL3 were added to 96-well plates containing 5,000 cells/well in transfected with siMT-1X or siNC cells. After incubation for 48 h, cell growth was measured by MTT assay. The data represent the means of three independent experiments. doi:10.1371/journal.pone.0093723.g004
  • Fig. 5. MT-1X involved in the FHL3-mediated TGF-b-like response. (A) HepG2 cells were transiently transfected with NC siRNA or MT-1X siRNA as indicated, and cell lysates were analyzed by Western blot analysis with the indicated antibodies. (B) HepG2 cells were transiently transfected with pCMV-Myc/MT-1X or pCMV-Myc expression vector as indicated, and cell lysates were analyzed by Western blot analysis with the indicated antibodies. doi:10.1371/journal.pone.0093723.g005
  • Fig. 6. MT-1X regulated expression of cell cycle-related proteins. (A) Extracts from HepG2 cells transfected with MT-1X siRNA were used for Western blot analysis with the indicated antibodies. (B) Extracts from HepG2 cells transfected with MT-1X expression vector were used for Western blot analysis with the indicated antibodies. doi:10.1371/journal.pone.0093723.g006

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Cai, X., Wang, J., Huang, X., Fu, W., Xia, W., Zou, M., … Xu, D. (2014). Identification and characterization of MT-1X as a novel FHL3-binding partner. PLoS ONE, 9(4). https://doi.org/10.1371/journal.pone.0093723

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