Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 1. A, direct association of p53 with the N-terminal domain of FAK in vitro. Upper panel, the scheme of the GST-FAK constructs used for the pull-down assay. Cloning of FAK-GST constructs was performed by PCR amplification of the N-terminal, kinase, and C-terminal domains of FAK and then subcloning into the pGEX-4T1 GST vector. All sequences were analyzed by automatic sequencing (University of Florida Sequencing Facility). The FAK-GST constructs were full-length GST-N terminus (aa 1–423), GST-kinase domain (aa 416–676), and GST-C-terminal domain (aa 677–1052). All of the constructs covered full-length FAK. Recombinant human p53 protein (0.1 g) expressed with the baculovirus system (BD Pharmingen) was precleared in radioimmune precipitation-lysis buffer with GST on beads (2 g) by rotating at 4 °C for 1 h. Precleared p53 (input 10%), loaded on the first lane, was incubated with GST alone, GST-FAK-NT, GST-kinase, or GST-FAK-CD proteins. The p53 protein associated with these GST-FAK proteins was analyzed by Western blot (WB) with p53 antibody. Western blot analysis detected binding of p53 with the GST-FAK-NT protein. The same pull-down assay (above) was performed with purified human p73- protein (American Proteomics). A Western blot with anti-p73- antibody did not detect binding with GST-FAK proteins. Lower panel, the GST proteins used for the pull-down assay were analyzed by SDS-PAGE and Coomassie Blue staining. The GST-FAK proteins are marked by asterisks. The GST-FAK proteins were loaded on parallel SDS-polyacrylamide gel, and Western blotting was performed with antibodies specific to different FAK domains. The GST-FAK-NT protein reacted with anti-FAK antibody specific to the N terminus of FAK (monoclonal antibody) (clone 77; BD Transduction Laboratories). The kinase domain and the N-terminal FAK-NT domain GST proteins were recognized by polyclonal BC2 antibody (see “Experimental Procedures”). The GST-FAK-CD proteins were analyzed by Western blotting with anti-FAK antibody, specific to the C-terminal domain of FAK, C-20 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Molecular weight marker sizes are shown in the left lanes. B–F, specific and direct binding of full-length FAK with p53 protein. B, we used the baculovirus system (Invitrogen) to isolate and purify a full-length FAK protein and a FAK family member, PYK-2 protein. As a control, we used purified recombinant GFP protein. Coomassie-stained proteins are shown. C–F, pull-down assays of baculovirus proteins with GST and GST-p53 proteins immobilized on glutathione-agarose beads. Proteins expressed with the baculovirus system and used for the pull-down assay were full-length FAK (C), PYK-2 (D), and GFP (E). The GST and GST-p53 proteins were analyzed by Western blotting with GST antibody (F). Full-length FAK directly physically and specifically interacted with p53, whereas PYK-2 and GFP did not bind to p53 protein.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: In Vitro, Construct, Pull Down Assay, Cloning, Amplification, Subcloning, Plasmid Preparation, Sequencing, Recombinant, Lysis, Incubation, Western Blot, Binding Assay, Purification, SDS Page, Staining, Transduction, Molecular Weight, Marker, Control

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 2. A, association of FAK and p53 proteins in 293 cells in vivo. The lysate of 293 cells was precleared with the A/G-agarose beads and used for immunoprecipitation (IP) with p53 antibody (left panel). The samples were analyzed by Western blot (WB) and probed with FAK antibody. FAK was co-immunoprecipitated with p53. The blot was stripped and probed with p53 antibody to detect immunoprecipitated p53 protein. The reverse experiment was performed with immunoprecipitation using FAK antibody and Western blot using p53 antibody (right panel). The blot was stripped and probed with FAK antibody to detect immunoprecipitated FAK protein. Ten g of precleared cell lysate is shown in the left lanes. Immunoprecipitation without antibody was used as a control (middle lanes). B, silencing of p53 expression with p53 siRNA decreases the complex of FAK and p53 in 293 cells in vivo. Upper panel, 293 cells were transfected on 100-mm dishes with 50 nM of small interfering p53 siRNA or control siRNA (Dharmacon). Western blot with p53 shows significant inhibition of p53 expression by p53 siRNA compared with control siRNA. Western blot with -actin antibody was used for control of equal protein loading. Lower panel, immunoprecipitation of lysates from untreated and p53 siRNA 293 cells was performed with FAK antibody. Immunoprecipitation of 293 cells without antibody was used as a control, and Western blotting with anti-p53 antibody was performed. The blot was stripped and probed with FAK antibody. p53 siRNA treatment significantly inhibited the

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Control, Expressing, Transfection, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 3. A, co-localization of FAK-NT and FAK proteins with p53 protein in breast cancer BT474 cells by confocal microscopy. BT474 cells were transfected with GFP, GFP-FAK-NT, GFP-FAK-CD, and GFP-FAK plasmids. Co-localization of endogenous FAK and p53 was performed in nontransfected cells. Cells were fixed and processed for immunofluorescence analysis by confocal microscopy (see “Experimental Procedures”). Localization of endogenous FAK was detected in nontransfected BT474 cells with FAK polyclonal C-20 antibody (Santa Cruz Biotechnology) and probed with secondary fluorescein isothiocyanate-conjugated anti-rabbit antibody. For detection of p53, immunostaining with p53 mouse mono- clonal antibody and with secondary rhodamine-conjugated anti-mouse antibody was performed. The nuclei were stained by Hoechst 33342. GFP staining was cytoplasmic. GFP-FAK-NT was mainly co-localized with p53 in the nuclei in BT474 cells (marked by the white arrows). GFP-FAK-CD was localized in the cytoplasm and in the focal adhesions (marked by the white arrowheads). GFP-FAK mainly localized in the focal adhesions (white arrowheads) and cytoplasm. GFP-FAK co-localized with p53 in the cytoplasmic and nuclear speckles (marked by the white arrows). The same co-localization with p53 was observed with endogenous FAK (lower panel, white arrows). B–E, FAK and p53 associate in cytoplasmic and nuclear fractions. Upper panels, BT474 (B) and 293 (C) cells were fractionated into cytoplasmic (labeled C) and nuclear (labeled N) fractions (see “Experimental Procedures”). 15 g of each fraction lysate was run on SDS-polyacrylamide gels. Western blots (WB) were performed with anti-FAK antibody to detect FAK protein and with anti-p53 antibody to detect p53 protein. Western blot with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was performed for detection of the cytoplasmic fraction, and Western blot with anti-histone H3 was performed for detection of the nuclear fraction. D and E, immunoprecipitation (IP) with FAK antibody was performed using cytoplasmic and nuclear fraction of BT-474 (D) and 293 cells (E). No antibody immunoprecipitation was used as a control. Western blot with p53 antibody was performed to detect a complex of p53 with FAK protein. Then blots were stripped and probed with anti-FAK antibody.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Confocal Microscopy, Transfection, Immunofluorescence, Immunostaining, Staining, Labeling, Western Blot, Immunoprecipitation, Control

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 4. A, scheme of the GST-p53 constructs used for mapping of FAK and p53 binding. Upper panel, all p53 construct; transactivation, TAD (aa 1–92) (called N-p53); DNA binding (aa 102–292) (C-p53); tetramerization (T-p53) (aa 325–393); and NC (aa 1–292) domains (called NC-p53) were constructed by PCR and cloned into pGEX-4T1 GST vector. B, the N-terminal transactivation domain of p53 binds FAK-NT protein. Upper panel, human FAK-NT protein eluted by thrombin digestion from recombinant GST-FAK-NT protein (10% input, first left lane) was used for pull-down analysis with GST (control) and GST-p53 proteins: GST-p53, GST-N-p53, GST-C-p53, GST-T-p53, and GST-NC-p53. The pull-down assays demonstrate that FAK-NT protein binds to the N-terminal domain of p53. Isolated GST-p53 proteins were analyzed by SDS-PAGE and Coomassie Blue staining (lower panel). Recombinant p53-GST and GST proteins are marked by the arrows.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Construct, Binding Assay, Clone Assay, Plasmid Preparation, Recombinant, Control, Isolation, SDS Page, Staining

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 5. A and B, the FAK-NT (aa 206–422) protein binds p53. A, left upper panel, scheme of GST-FAK-NT proteins used for pull-down assay. We used full-length GST-FAK-NT (aa 1–423), first half of FAK-NT (aa 1–205), and second half of FAK-NT protein (aa 206–422). Middle panel, the GST-FAK-NT proteins were isolated and analyzed by Coomassie staining. The GST and GST-FAK-NT proteins are marked by asterisks. GST-FAK-proteins were analyzed by Western blot with FAK antibody, specific to the second half of FAK-NT (clone 77; BD Transduction Laboratories). The full-length of GST-FAK-NT and terminal GST-FAK-NT (aa 206–422) domain reacted with FAK antibody (clone 77). The proteins were also analyzed with FAK 4.47 antibody (Upstate Biotechnology) specific to the first half of FAK-NT (lower panel). Full-length GST-FAK-NT and GST-FAK-NT (aa 1–205) were effectively recognized with 4.47 antibody. B, pull-down assay with GST-FAK-NT proteins. Baculovirus-expressed p53 protein was used for pull-down analysis with GST-FAK-NT proteins, shown in A. p53 protein binds to the second half of FAK-NT and not to the first half of FAK-NT. All experiments were repeated for three times, and the representative blot is shown. WB, Western blot.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Pull Down Assay, Isolation, Staining, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 6. A, FAK expression blocks p53 apoptosis in SAOS-2 cells. SAOS-2 p53-null cells were transfected with p53-pCMV4 plasmid alone (1 g), FAK-pcDNA3 plasmid alone (at doses of 0.5 and 1 g), -FAK-NT plasmid alone (at doses of 0.5 and 1 g), or FAK-pcDNA3 and -FAK-NT plasmids (with deleted N-terminal domain of FAK) at doses (0.5 and 1 g) together with p53 (1 g) plasmid. In all transfec- tions, GFP was added to control for transfection efficiency. An equal amount of DNA was used for each transfection. 48 h after transfections,

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Expressing, Transfection, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 7. Overexpression of FAK in- hibits p21-luciferase promoter activ- ity in HCT 116 p53/ cells. A, co-trans- fection of HA-tagged FAK-pcDNA3 plasmid with wild type p21-pGL3 lucifer- ase, containing two p53 binding sites in- side the p21 promoter sequence or p21- mut-pGL3 luciferase without p53 binding site constructs, described in Ref. 41, and PRL-Renilla plasmid was performed in HCT p53/ and HCT p53/ cells. An equal amount of DNA was used in each transfection. After 24 h, cells were lysed, and dual luciferase assays were per- formed, as described (see “Experimental Procedures”). The bars show mean data S.E. values of four independent experi- ments. The -FAK-NT-pcDNA3.1/Xpress plasmid, containing a deletion of the N- terminal FAK-NT fragment (aa 1–416) was co-transfected into p53/ cells to- gether with the p21-wild type and p21- mutant-PGL3 luciferase constructs. Overexpression of wild type FAK signifi- cantly inhibits p53-mediated p21 tran- scriptional activity in HCT116 p53/

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Over Expression, Luciferase, Plasmid Preparation, Binding Assay, Sequencing, Construct, Transfection, Mutagenesis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 8. A and B, overexpression of FAK inhibits MDM-2 luciferase promoter activity in HCT 116 p53/ cells. A, co-transfection of HA- tagged FAK-pcDNA3 plasmid was performed with MDM-2-pGL2 lucif- erase construct (see “Experimental Procedures”) and PRL-Renilla plas- mid in HCT p53/ and HCT p53/ cells, as described in the legend to Fig. 7A. The bars show mean data S.E. values of three independent experiments. The -FAK-NT-pcDNA3.1/Xpress plasmid was co-trans- fected into p53/ cells together with the MDM-2 luciferase construct. Overexpression of wild type FAK significantly inhibits p53-mediated MDM-2 transcriptional activity in HCT116 p53/ cells, whereas the -FAK-NT did not significantly affect luciferase activity. *, p 0.05 compared with vector control p53/ HCT116 cells. B, FAK inhibits MDM-2-luciferase promoter activity in HCT 116 p53/ cells in a dose- dependent manner. Co-transfection of different amounts of HA-tagged FAK-pcDNA3 plasmid (0.25, 0.5, and 1 g) with MDM-2-pGL2 lucifer- ase construct (1 g) and PRL-Renilla plasmid (0.1 g) was performed in HCT p53/ cells. An equal amount of DNA was used in each transfec- tion. After 24 h, cells were lysed, and dual luciferase assays were performed, as described (see “Experimental Procedures”). The bars show mean data S.E. of three independent experiments. FAK inhib- ited MDM-2-luciferase activity in a dose-dependent manner in HCT 116 p53/ cells.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Over Expression, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Construct, Control, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

doi: 10.1074/jbc.m414172200

Figure Lengend Snippet: FIG. 9. A and B, overexpression of FAK inhibits p53-induced BAX luciferase promoter activity in HCT116 and SAOS-2 cells. HCT 116 p53/ cells (A) and SAOS-2 cells (B) were transfected with 1 g of p53-pCMV4 plasmid alone or together with HA-tagged FAK-pcDNA3 plasmid (1 g) or -FAK-NT-pcDNA3 (1 g) plasmids with the BAX- promoter luciferase construct (1 g). The total amount of DNA was kept constant with pcDNA3 (Vector) DNA. PRL-Renilla plasmid (0.1 g) was co-transfected together with the above plasmids and used as a normal- ization control. After 48 h, cells were lysed, and dual luciferase assays were performed, as described (see “Experimental Procedures”). The bars show mean data S.E. values of three independent experiments. Overexpression of wild type FAK significantly inhibits p53-mediated BAX-luciferase activity in both HCT116 and SAOS-2 cell lines, whereas -FAK-NT plasmid did not affect BAX-luciferase activity. *, p 0.05 versus vector control sample.

Article Snippet: FAK / p53 / and FAK / p53 / mouse embryo fibroblasts (MEFs) were obtained from ATCC and were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 g/ml penicillin/streptomycin.

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Control