Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein - PubMed (original) (raw)

Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein

Elena Kurenova et al. Mol Cell Biol. 2004 May.

Abstract

Tumor cells resist the apoptotic stimuli associated with invasion and metastasis by activating survival signals that suppress apoptosis. Focal adhesion kinase (FAK), a tyrosine kinase that is overexpressed in a variety of human tumors, mediates one of these survival signals. Attenuation of FAK expression in tumor cells results in apoptosis that is mediated by caspase 8- and FADD-dependent pathways, suggesting that death receptor pathways are involved in the process. Here, we report a functional link between FAK and death receptors. We have demonstrated that FAK binds to the death domain kinase receptor-interacting protein (RIP). RIP is a major component of the death receptor complex and has been shown to interact with Fas and tumor necrosis factor receptor 1 through its binding to adapter proteins. We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK. We thus propose that FAK overexpression in human tumors provides a survival signal function by binding to RIP and inhibiting its interaction with the death receptor complex.

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Figures

FIG. 1.

FIG. 1.

Association between FAK and RIP in vivo and in vitro. (A) Human breast carcinoma cell line BT-474 was grown in monolayer. Binding was analyzed by immunoprecipitation with anti-RIP antibody and then followed by immunoblotting with anti-FAK antibody or by immunoprecipitation with anti-FAK antibody against N terminus 4.47 or C terminus C20 followed by IB with anti-RIP antibody. Probe without antibody was added as a control for background binding. (B) Tet-FAK cell lysates were immunoprecipitated with anti-FAK antibody followed by immunoblotting with anti-RIP antibody. A Western blot of Tet-FAK cell lysates with RIP antibody was performed to evaluate RIP synthesis in the cells during the time course of tetracycline withdrawal. Probe without antibody was added as a control for background binding. (C) HEK293 cells were transiently cotransfected with pXpress-RIP along with pcDNA3, pcDNA3-FAK, pGFP C1 vector, pGFP-FAK-CD, or pGFP-FAK-NT. pCMV-CrmA was also included in the transfections to protect the cells from apoptosis induced by the overexpression of RIP. After 24 h, cell lysates were subjected to immunoprecipitation with anti-Xpress antibody. The immunopellets were analyzed by immunoblotting and probing with anti-FAK4.47 and C20 antibody and anti-RIP antibody. The expression of all constructs was confirmed with the corresponding antibody (data not shown). (D) HEK293 cells were transiently cotransfected with pcDNA-Myc-RIP death domain along with pcDNA3-FAK, pGFP C1 vector, pGFP-FAK-NT, or pGFP-FAK-CD. pCMV-CrmA was also included in the transfections. After 24 h, cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The immunopellets were analyzed by immunoblotting and probing with anti-FAK 4.47 and C20 antibody (data not shown) and anti-GFP antibody. The expression of all constructs was confirmed by Western blot with the corresponding antibody (anti-Myc and anti-GFP antibody). (E) Association of endogenous RIP with GST-FAK-NT fusion protein. GST protein and GST-fused FAK fragments (NT or FAT domain) were bound to glutathione-Sepharose beads and used to pull RIP down from extracts of HEK293 cells, followed by SDS-10% PAGE. The immunodetection first used anti-RIP antibody to detect pulled-down RIP, and then the blot was reprobed with anti-GST antibody. IP, immunoprecipitation; IB, immunoblotting; −Ab, probe without antibody; WB, Western blot.

FIG. 2.

FIG. 2.

Staurosporine causes dephosphorylation and degradation of FAK in breast carcinoma cell lines. A total of 2 × 105 cells from BT-20 or BT-474 cell culture were plated on six-well plates and were treated with staurosporine (200 nM) after 24 h. After 6 and 18 h of treatment, cells were immunostained with anti-FAK 4.47 antibody, detached cells were counted, and cells were collected for TUNEL assay and Western blot analysis. (A) Immunostaining of untreated cells and cells treated with staurosporine for 6 h (white arrowheads mark some focal adhesions). (B) Western blot (WB) analysis of BT-20 and BT-474 lysates treated with staurosporine. Control cells were treated with dimethyl sulfoxide for 18 h. (C) TUNEL assay data from one of three representative experiments.

FIG. 3.

FIG. 3.

Staurosporine causes dephosphorylation and degradation of FAK, which depends on the presence of RIP. A total of 2 × 105 cells from RIP+/+ or RIP−/− cell culture were plated on six-well plates and were treated with staurosporine after 24 h. After 18 h of treatment, detached cells were counted, and cells were collected for TUNEL assay and Western blot analysis. (A) TUNEL assay data from one of three representative experiments. (B) Western blot (WB) analysis of RIP+/+ and RIP−/− lysates treated with staurosporine.

FIG.4.

FIG.4.

RIP is involved in transducing the proapoptotic signals initiated by the disruption of FAK signaling. A total of 2 × 105 cells from RIP+/+ or RIP−/− cell culture were plated on six-well plates and were infected with Ad LacZ or Ad FAK-CD (200 FFU/cell) after 24 h. After 6 and 18 h of treatment, cells were immunostained with anti-FAK 4.47 antibody (A) (white arrowheads mark some focal adhesions), detached cells were counted, and cells were collected for TUNEL assay and Western blot analysis. (B) Detachment and apoptosis data from one of four representative experiments. (C) Western blot (WB) analysis of RIP+/+ and RIP−/− lysates treated with Ad LacZ or Ad FAK-CD.

FIG. 5.

FIG. 5.

RIP is involved in transducing the detachment-induced proapoptotic signals. (A) A total of 2 × 106 RIP+/+ and RIP−/− cells were plated on tissue culture dishes coated with polyHEMA and were grown in complete medium for 24 to 72 h. TUNEL assay and Hoechst staining were performed at the indicated time points. TUNEL assay data from one of three representative experiments are shown. (B) A total of 2 × 105 RIP+/+ and RIP−/− cells were plated on tissue culture dishes coated with polyHEMA and were grown in complete medium for 24 to 72 h and then transduced with either Ad LacZ control or Ad FAK-CD for 24 h. TUNEL assay and Hoechst staining were performed at the indicated time points. TUNEL assay data from one of three representative experiments are shown.

FIG. 6.

FIG. 6.

Downregulation of RIP with siRNA leads to higher resistance to apoptosis caused by the attenuation of FAK. A total of 2 × 105 BT474 cells were plated on six-well plates and were transfected with siRNA (25 nM) after 24 h. (A) Forty-eight hours after transfection, cells were collected and analyzed by Western blotting (WB). Controls: scrambled RIP H121 siRNA (H121Scr), GAPDH siRNA, and cells treated with transfection reagent alone (lanes 3, 4, and 5, respectively). (B and C) After 48 h of transfection, cells were treated with 200 nM staurosporine and collected 1 h later to analyze tyrosine phosphorylation of FAK (B) or collected 18 h later for TUNEL assay (C). (D and E) After 48 h of transfection, adenoviral infection was performed and Ad LacZ and Ad FAK-CD were added to the cell cultures (200 FFU/cell). After 18 h of infection, detached cells were counted, and cells were collected for TUNEL assay and Western blot analysis. (D) Western blot analysis with anti-FAK phosphotyrosine Y397 antibody. (E) TUNEL assay data from one of three representative experiments.

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