The LD-like motif in DLC1 is responsible for the interaction with the central region of talin. (A) Mapping the region in DLC1 required for binding the talin central region. (A Upper) Schematic representation of full-length GFP-DLC1 and its derivatives; the LD-like motif is shown to highlight its location in DLC1 and its presence or absence in the derivatives. (A Lower) After transient transfection of 293T cells, the GFP-DLC1 constructs were pulled down by GST or GST-talin (1,288–1,646) fusion protein followed by immunoblotting (IB) with anti-GFP. The whole-cell extracts (WCE) containing transfected proteins are shown as loading controls. (B) Pull-down assays using DLC1 internal deletion mutants in transfected 293T cells. The WT DLC1 and DLC1 with small internal deletions within or outside the LD motif as well as GFPDLC3α were pulled down by GST or GST-talin (1,288–1,646). (C) Mapping the minimal talin fragment in the central region (1,288–1,646) required for DLC1 binding in pull-down assays. Transfected GST-talin fragments with N- or C-terminal truncations of 1,288–1,646 were used to pull down GFPDLC1. Expression of the transfected proteins is shown as loading controls.

DLC1 binds FAK and interferes with paxillin binding to the FAT domain of FAK: dependence on the LD-like motif. (A) Endogenous DLC1 and FAK form a complex in human cell lines. A skin fibroblast line (Skin) and NSCLC line H1703 were analyzed by reciprocal coimmunoprecipitation. (B) Colocalization of DLC1 with FAK in NSCLC lines. Endogenous DLC1 in H1703 cells or transfected DLC1 in H358 cells were stained with anti-DLC1 antibody (red) and endogenous FAK with anti-FAK antibodies (green). The confocal images are representative of the majority of cells observed. (Scale bar: 10 μm.) (C) Association of DLC1 with talin or FAK depends on the LD-like motif. Pull down of endogenous talin and FAK by GST-fusion DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. (D) Interaction between DLC1 and FAK is talin-independent. Pull-down of 293T cells, with or without talin siRNA treatment, of endogenous FAK and talin by DLC1 GST (448–500) WT or delLD. (E) DLC1-FAK interaction is through the FAT-domain of FAK and requires the LD-like motif of DLC1. Pull down of GFP-FAT, derived from FAK, by GST-tagged DLC1 fragment (448–500) with WT LD-like motif or delLD in 293T cells. (F) DLC1 can compete with paxillin but not with talin for binding the FAT-domain. GST-FAT was cotransfected with increasing amounts of DLC1 at the indicated ratios in 293T cells. The pull down of DLC1, endogenous paxillin, and endogenous talin are shown, and the transfected proteins are also shown. (G) Schematic representation of the similar region in the FAT domain of FAK binds paxillin and the LD-like motif, whereas talin binds a distinct region of the FAT domain as well as the LD-like domain.

The DLC1 targets talin and FAK contribute to cell migration and anchorage-independent growth. (A) Verification of siRNA knockdown in NSCLC line H1299. The specific siRNA knockdown of DLC1, talin, and FAK was confirmed by immunoprecipitation/IB of anti-DLC1, anti-talin, and anti-FAK blots. The Rho-GTP level was also analyzed, and the totalt Rho-immunoblo is shown as loading control. (B) Effect of indicated siRNA on cell migration. H1299 cells were analyzed by wound healing and transwell assays 48 h after siRNA transfection. The quantitation of migrated cells in the transwell assay is shown. (C) Effect of indicated siRNA on anchorage-independent cell growth. Equal numbers of siRNA-transfected H1299 cells were seeded in soft agar for growth and quantitation.

Immune complex formation between endogenous DLC1 and talin in human cell lines. (A) Schematic representation of talin and DLC1 proteins and identified yeast two-hybrid (Y2H) interaction between a central region (residues 1,288–1,646) in the talin rod and DLC1 (residues 260–630). (B) Immune complex formation between endogenous talin and DLC1 in human cells was analyzed by reciprocal immunoprecipitation in 1634 fibroblasts and H1703 NSCLC cells (B Left) and human skin fibroblast line and H1703 (B Right). (C) Colocalization of endogenous DLC1 with talin; 1634 cells were stained with anti-talin (red) and anti-DLC1 (green) antibodies. The confocal images are representative of the majority of cells. (Scale bar: 5 μm.)

The LD mutation but not the GAP dead mutation in full-length DLC1 reduces complex formation and colocalization with endogenous talin. (A) Pull-down assay in transfected 293T cells. The WT DLC1 and the GAP dead DLC1 R718A with or without deletion of LD motif (delLD) were pulled down by GST-talin (1,288–1,646) followed by immunoblotting. The expression of transfected proteins from WCE is shown as loading controls. (B) Complex formation of DLC1 with endogenous talin in stably transfected H358 cells, which do not express detectable endogenous DLC1. Characterization of H358 stable clones expressing R718A with or without delLD: expression, Rho-GTP, and coimmunoprecipitation with endogenous talin. (C) Colocalization of DLC1 with endogenous talin in H358 stable cell lines. Coimmunostaining of endogenous talin (red) and DLC1 (green) in H358 parental cells and stable clones expressing DLC1-R718A with or without delLD mutation. The confocal images are representative of the majority of cells observed. (Scale bar: 10 μm.) (D Upper) Pull down of endogenous talin in 293T cells cotransfected with GST or GST-fusion and DLC1 fragment (448–500) containing WT or mutant LD motif (mtLD) followed by immunoblotting. (D Lower) The conserved WT LD-like motif in DLC1, the three substitution mutations (underlined) in mtLD, and the deleted LD amino acids in delLD are shown.

Mutant DLC1 is less active than WT in inhibiting the bioactivity of H358 cells in vitro and in vivo. (A) Rho-GAP activity in H358 cells. H358 parental cells and derived DLC1 stable clones were analyzed by Rhotekin pull-down assay. The Rho-GTP level, total Rho-A protein, and expression level of DLC1 in each stable clone are shown. (B and D) H358 parental cells and derived DLC1-expressing cells were assayed by transwell migration (B) and soft agar colony growth (D). The migrated cells and the number of colonies are shown, respectively. (C) Xenographic tumors of H358 cells expressing WT or mutant DLC1. H358 stable clones were injected s.c. into NOD/SCID mice. Tumors were weighted 5 wk later. The results are expressed as means ± SD (tests shown in A and E had four animals, and tests shown in B–D had six animals). Statistical significance was analyzed by the nonparametric Mann–Whitney t test.