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. 2007 Jan 1;176(1):43-9.
doi: 10.1083/jcb.200608015. Epub 2006 Dec 26.

The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1

Yi-Chun Liao  1 , Lizhen Si, Ralph W deVere White, Su Hao Lo
Affiliations

The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1

Yi-Chun Liao et al. J Cell Biol. .

Abstract

The tensin family member cten (C-terminal tensin like) is an Src homology 2 (SH2) and phosphotyrosine binding domain-containing focal adhesion molecule that may function as a tumor suppressor. However, the mechanism has not been well established. We report that cten binds to another tumor suppressor, deleted in liver cancer 1 (DLC-1), and the SH2 domain of cten is responsible for the interaction. Unexpectedly, the interaction between DLC-1 and the cten SH2 domain is independent of tyrosine phosphorylation of DLC-1. By site-directed mutagenesis, we have identified several amino acid residues on cten and DLC-1 that are essential for this interaction. Mutations on DLC-1 perturb the interaction with cten and disrupt the focal adhesion localization of DLC-1. Furthermore, these DLC-1 mutants have lost their tumor suppression activities. When these DLC-1 mutants were fused to a focal adhesion targeting sequence, their tumor suppression activities were significantly restored. These results provide a novel mechanism whereby the SH2 domain of cten-mediated focal adhesion localization of DLC-1 plays an essential role in its tumor suppression activity.

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Figures

Figure 1.
Figure 1.
Identification of DLC-1 as a cten binding partner. (A) A549 cells were transfected with pEGFP (lane 1) or pEGFP–DLC-1 (lane 2). Cell lysates were coimmunoprecipitated with anti-GFP and analyzed by immunoblotting (IB) with anti-cten (left) or anti-GFP (right). The arrow indicates cten, and arrowheads show GFP and GFP–DLC-1. (B) NIH 3T3 cells in 24-well dishes were cotransfected with pCMV-AD-cten, and pCMV-BD vector with no insert as a negative control (column 1), pCMV-BD-DLC-1 (column 2), pCMV-BD-DLC-1S440A (column 3), or pCMV-BD-DLC-1Y442F (column 4), and together with the reporter plasmid pFR-Luc. The luciferase activities were measured by luminometry and shown as relative light units (RLU) per milligram of cellular proteins. Data are from two independent triplicate experiments. Error bars indicate mean ± SD. (C) MLC SV-40 cell lysates were coimmunoprecipitated with normal rabbit serum (lane 1) or anti–DLC-1 (lane 2) and analyzed by immunoblotting with anti-cten (left) or anti–DLC-1 (right). The arrow indicates cten, and the arrowhead shows DLC-1.
Figure 2.
Figure 2.
Determination of binding regions on cten and DLC-1 and their binding specificities. (A) Schematic diagram of cten and DLC-1 and their segments that were used for mapping the binding sites. (B) AH109 yeast cells transformed with the indicated plasmids and grown on two-dropout plates were restreaked on four-dropout plates. (C) Bacterial lysates containing Xpress–DLC-1 (113–535) were incubated with immobilized GST (lane 1) or GST-cten SH2 (lane 2). After washing, the associated proteins were analyzed by immunoblotting (IB) with anti-Xpress antibody. (D) 0.5 μg of purified GST-cten SH2 (lane 1), GST-Src SH2 (lane 2), and GST-p85 SH2 (lane 3) recombinant proteins were incubated with DLC-1 peptide (CSRLSIY442DNVPG) immobilized on agarose beads. After washing, the associated proteins were analyzed by immunoblotting with anti-GST antibody. (E) 0.5 μg of purified GST-cten SH2 recombinant proteins were incubated with DLC-1 peptide (CSRLSIY442DNVPG; lane 1), tyrosine-phosphorylated DLC-1 peptide (CSRLSIpY442DNVPG; lane 2), EGFR peptide (CSVQNPVY1086HNQP; lane 3), or tyrosine-phosphorylated EGFR peptide (CSVQNPVpY1086HNQP; lane 4) immobilized on agarose beads. After washing, the associated proteins were analyzed by immunoblotting with anti-GST antibody.
Figure 3.
Figure 3.
Subcellular localization of DLC-1 and its recruitment by cten SH2. (A) A549 cells grown on coverslips were transfected with pEGFP–DLC-1 (1–535), pEGFP–DLC-1 (1–535)S440A, or pEGFP–DLC-1 (1–535)Y442F. After labeling with anti-cten antibodies followed by Alexa Fluor 594–conjugated secondary antibody, cells were visualized with a confocal microscope. Arrows indicate cten and GFP fusion protein colocalized at focal adhesions. Arrowheads show only cten at focal adhesions. About 100 GFP-positive cells were examined in each transfection. More than 90% of GFP-positive cells showed focal adhesion localization when transfected with GFP–DLC-1 (1–535), and no GFP-positive cells transfected with DLC-1 mutants showed focal adhesion localization. Cell lysates from A549 transiently expressing GFP–DLC-1 (1–535; lane 1), GFP–DLC-1(1–535)S440A (lane 2), or GFP–DLC-1 (1–535)Y442F (lane 3) were immunoprecipitated with anti-GFP and analyzed by immunoblotting with anti-GFP to show similar amounts, and correct sizes of recombinant proteins were expressed. (B) A549 cells grown on coverslips were cotransfected with pDsRed1/pEGFP–DLC-1 (1–535) or pDsRed1-cten SH2-SKL/pEGFP-DLC-1 (1–535). Cells were visualized with a confocal microscope. Note that the DsRed-cten SH2-SKL and GFP–DLC-1 (1–535) were colocalized at peroxisomes (arrows), although some GFP–DLC-1 (1–535) proteins were still detected at focal adhesions (at different focus plane; not depicted), as predicted, because of the presence of endogenous tensins in A549 cells. Arrowheads show only GFP–DLC-1 (1–535) at focal adhesions. Bars, 10 μm.
Figure 4.
Figure 4.
Colony formation and cell growth assays in MDA-MB-468 cells transfected with wild-type and mutant DLC-1. (A) Cells were transfected with the indicated constructs. After being cultured in media containing 0.8 mg/ml G418 for 2 wk, G418-resistant colonies were stained with crystal violet. (B) The histogram shows the colony formation assay of GFP (column 1), GFP–DLC-1 (column 2), GFP–DLC-1S440A (column 3), GFP–DLC-1Y442F (column 4), GFP–FAB–DLC-1 (column 5), GFP–FAB–DLC-1S440A (column 6), and GFP–FAB–DLC-1Y442F (column 7) from four independent experiments. (C) G418-resistant MDA-MB-468 cells (2 × 105) expressing the indicated proteins were seeded in triplicate in 60-mm dishes. Cells were harvested at 24-h intervals for 4 d, and the numbers of viable cells were counted by trypan blue exclusion assay with a hematocytometer. Error bars indicate mean ± SD. (D) Cell lysates from MDA-MB-468 transfected with GFP vector (lane 1), GFP–DLC-1 (lane 2), GFP–DLC-1S440A (lane 3), GFP–DLC-1Y442F (lane 4), GFP–FAB–DLC-1 (lane 5), GFP–FAB–DLC-1S440A (lane 6), or GFP–FAB–DLC-1Y442F (lane 7) were immunoprecipitated with anti-GFP and analyzed by immunoblotting with anti-GFP to show similar expression levels and correct sizes of recombinant proteins. The arrow indicates GFP, and arrowheads show GFP–DLC-1 and GFP–FAB–DLC-1 fusion proteins. (E) A549 cells grown on coverslips were transfected with pEGFP–FAB–DLC-1, pEGFP–FAB–DLC-1S440A, or pEGFP–FAB–DLC-1Y442F. After labeling with anti-cten antibodies followed by Alexa Fluor 594–conjugated secondary antibody, cells were visualized with a confocal microscope. Arrows indicate cten and GFP fusion protein colocalized at focal adhesions. Bars, 10 μm.
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