This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kurenova, E.
Right arrow Articles by Cance, W. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurenova, E.
Right arrow Articles by Cance, W. G.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, May 2004, p. 4361-4371, Vol. 24, No. 10
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.10.4361-4371.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Focal Adhesion Kinase Suppresses Apoptosis by Binding to the Death Domain of Receptor-Interacting Protein

Elena Kurenova,1 Li-Hui Xu,2 Xihui Yang,2 Albert S. Baldwin Jr.,2 Rolf J. Craven,3 Steven K. Hanks,4 Zheng-gang Liu,5 and William G. Cance1*

Departments of Surgery, Biochemistry, and Molecular Biology, University of Florida, Gainesville, Florida 32610,1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,2 Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, Kentucky 40536,3 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,4 Department of Cell and Cancer Biology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208925

Received 8 September 2003/ Returned for modification 21 October 2003/ Accepted 11 February 2004


arrow
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.


arrow
INTRODUCTION
 
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays a key role in maintaining focal adhesion function and cell survival and is implicated in cell migration, adhesion, and cell cycle control (9, 13, 18, 20, 33, 44). Overexpression of FAK is a common event in numerous tumor systems, including breast, colon, and thyroid carcinomas (2, 24, 32, 41), and occurs at early stages of tumorigenesis, before a tumor has developed the capacity for invasion and metastasis (2). Importantly, FAK has been shown to be one of the critical factors protecting cells from apoptosis, but the exact mechanism is unknown (8, 9, 12, 19, 37, 43). Attenuation of FAK expression by antisense oligonucleotides led to apoptosis in tumor cells (42), and the treatment of cells with anti-FAK antibody (18, 26) or overexpression of the focal adhesion targeting (FAT) domain of FAK led to cell rounding, detachment, and apoptosis (19, 21, 40). We have created a model system for the attenuation of FAK function by adenoviral gene transduction of the carboxy-terminal domain of FAK (FAK-CD) and have demonstrated a loss of adhesion and apoptosis in breast cancer cells with this treatment (43). Both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain protein (FADD) and caspase 8, suggesting an important role for FAK in inhibiting death receptor-related apoptosis (43). This finding provided additional evidence that a death receptor-mediated apoptotic pathway or death receptor-related death domain proteins are involved in the apoptotic process triggered by the expression of FAK-CD.

The loss of adhesion and induction of apoptosis upon attenuation of FAK function by the expression of FAK-CD is similar to the phenomenon of anoikis (7-9). Intriguingly, there is evidence for the involvement of death receptor-related, death domain-containing proteins in anoikis (7, 35), whereby the silencer of death domain (SODD) and dominant-negative FADD efficiently inhibited anoikis in Madin-Darby canine kidney (MDCK) cells and in a number of untransformed epithelial cell lines. In these studies, it was also shown that cell matrix detachment activated caspase 8. However, the linkage of the signaling pathways to the death receptors remains unknown.

RIP is a serine/threonine kinase that contains a death domain (17, 38) and is named for its association with the death receptor complex. RIP interacts with the death domains of cell surface receptors of the tumor necrosis factor (TNF) superfamily and death domain adaptor proteins (3, 5, 17) and plays an indispensable role in NF-{kappa}B activation (23, 39). Recently, it was shown that TNF alpha-mediated activation of NF-{kappa}B depends on the association of RIP and FAK (11). TNF-induced NF-{kappa}B DNA binding activity and activation of I{kappa}B kinases were markedly impaired in FAK–/– cells (11). However, it has been well established that RIP has a dual function and is capable of either inducing apoptosis or activating cellular survival signals (14, 17, 23, 27, 38, 39). Similarly, it has been proposed that RIP is one of the switches between cell survival and apoptosis (28). However, the mechanisms by which such diverse functions are selected remain unclear.

To elucidate the possible protein(s) involved in apoptosis caused by the disruption of FAK signaling by expression of the C-terminal domain of FAK, we tested several death domain proteins for the ability to bind to FAK. We found that RIP bound to FAK in vitro and in vivo in different mammalian cell lines (normal and transformed), and the level of apoptosis caused by the attenuation of FAK correlated with the level of RIP expression in the particular cell line. In this report, we show a possible mechanism by which the death receptor machinery is involved in anoikis through the interaction of FAK and RIP.


arrow
MATERIALS AND METHODS
 
Cell culture. BT-474 and BT-20 human breast carcinoma cell lines were cultured as described previously (43). Tet-FAK cells, whereby the tetracycline repression system was implemented in FAK-null mouse embryonic fibroblasts to modulate FAK expression, were cultured as described previously (31). RIP–/– TNFR1–/– and RIP+/+ TNFR1–/– mouse embryonic fibroblasts (4) were kindly provided by P. Leder and M. Kelliher (Harvard Medical School, Boston, Mass.). HEK293 human embryonic kidney cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum. All cell lines were incubated at 37°C in 5% CO2.

Plasmids, reagents, and antibodies. The mammalian expression vector encoding hemagglutinin (HA)-FAK has been described previously (43). pXpress-RIP and pMyc-RIP death domain were kindly provided by Zheng-gang Liu (National Cancer Institute, National Institutes of Health, Bethesda, Md.). The glutathione transferase (GST)-RIP construct was prepared by subcloning RIP into the SmaI site of the pGEX-3X vector. The FAT sequence (nucleotides 2987 to 3391 of FAK) was subcloned by PCR into the pGEX-4T-1 vector. GST-FAK-N terminus (NT), green fluorescent protein (GFP)-FAK-NT, and GFP-FAK-CD were previously described (1). Staurosporine (Calbiochem) was used in 200 and 500 nM concentrations. We used anti-FAK 4.47 (Upstate Biotechnology, Inc.) monoclonal antibody and C20 (Santa Cruz Biotechnology) polyclonal antibody; anti-HA antibody (12CA5, Roche Molecular Biochemicals) and anti-RIP monoclonal antibody (Transduction Laboratory); anti-FAK phosphotyrosine Y397 antibody (Biosourse International, Inc); and monoclonal anti-ß-actin and anti-GST antibody (Sigma), anti-Xpress antibody and anti-Myc antibody (Invitrogen), and anti-GFP antibody (Clontech).

Transfection. Cells were plated at a density of 2 x 106 cells per 100-mm culture plate or 2 x 105 cells per well in six-well plates and allowed to attach for 24 h and then were transfected by using Lipofectamine (Invitrogen) for BT-474 and BT-20 cells and Effectene (QIAGEN) for HEK293 cells according to the manufacturer's instructions.

Immunoprecipitation and Western blotting. For cell lysate preparation, 1% NP-40 lysis buffer was used (20 mM Tris-HCl [pH 8.0], 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin/ml, 20 µg of leupeptin/ml). The protein concentration of lysates was determined by using the bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.). For immunoprecipitation, lysates (500 µg of total protein) were precleared with protein A/G-agarose beads (Oncogene Research Products Inc.) at 4°C for 1 h and then incubated with 5 µl of antibody for 2 h followed by overnight incubation with protein A/G-agarose beads at 4°C. Precipitates were washed three times in lysis buffer, and beads were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, boiled for 5 min, and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane, probed with appropriate antibody, and detected by chemiluminescence (KPL, Gaithersburg, Md.).

In vitro binding. GST fusion proteins were produced and purified as previously described (43). For in vitro binding assay, cell lysates were precleared with GST protein (5 µg), and the cleared supernatants (500 µg) were incubated for 1 h at 4°C with 1, 5, or 10 µg of GST fusion protein immobilized on glutathione-agarose beads. Cell lysates were prepared from different cell lines or from 293 cells transfected with expression vectors encoding RIP, FAK, or its fragments. The beads were washed twice with lysis buffer and twice with phosphate-buffered saline (PBS). The bound proteins were analyzed by Western blotting.

Adenoviral infections. Adenovirus (Ad) FAK-CD (amino acids 693 to 1052 of FAK fused to the HA epitope tag) was described previously (43). The Ad carrying the lacZ gene was provided by J. Samulski (University of North Carolina, Chapel Hill, N.C.). Cells were plated at a density of 2 x 106 cells per 100-mm culture plate or 2 x 105 cells per well in six-well plates, allowed to attach for 24 h, and then infected with Ad FAK-CD or Ad LacZ for various times in complete medium. We typically infected cells with 200 focus-forming units (FFU)/cell, and expression was verified by Western blotting with an anti-HA antibody that allowed us to estimate that infectivity and expression of Ad were equal in all types of cells used.

Immunofluorescence staining. Cells were incubated in the presence or absence of staurosporine or Ad and stained with anti-FAK monoclonal antibody 4.47 as previously described (43).

siRNA synthesis and transfection. Small interfering RNAs (siRNAs) were prepared according to the Silencer siRNA construction kit protocol (Ambion). RIP siRNA H87 corresponded to the coding region 1129-1149 relative to the first nucleotide of the start codon, and the RIP mRNA target sequence is 5'-ACTCCAAGACGAAGCCAAC-3'. siRNA H121 corresponded to the coding region 1693-1713, and the RIP mRNA target sequence is 5'-AGAAGAGCCAGCTGCTAAG-3'. siRNA H121Scr is a scrambled sequence of H121 siRNA. siRNA GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was synthesized according to the Silencer kit protocol (Ambion). The transfection of siRNAs to target the endogenous genes was carried out by using Oligofectamine (Invitrogen) according to the manufacturer's protocol.

PolyHEMA-coated tissue culture dishes. Tissue culture dishes were coated with a film of poly(2-hydroxyethylmethacrylate) (polyHEMA; Sigma) as described by Folkman and Moscona (6). Cells were trypsinized, resuspended, and added to the plates coated with polyHEMA at a density of 2 x 106 cells per dish in a serum-containing medium. Cells were maintained in suspension culture for various times and assayed for cell survival by methods such as terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and methyl thiazole tetrazolium (MTT) assays.

Assays of cell viability. Detection of apoptosis was performed by TUNEL assay and Hoechst 33342 staining. Cells were collected by centrifugation, fixed in 3.7% formaldehyde in 1x PBS for 10 min, washed twice with 1x PBS, and stained with Hoechst 33342 or spread on the slide for TUNEL staining. TUNEL assay was done with the ApopTag kit (Intergen, Purchase, N.Y.) according to the manufacturer’s protocol. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to the total number of cells counted with a fluorescence microscope in several fields of three independent experiments. Simultaneous staining and quantification of apoptotic cells with the Hoechst 33342 and TUNEL methods produced very similar results. Cell survival was assayed by measuring mitochondrial dehydrogenase activity, the conversion of soluble MTT into insoluble formasan product, as described by Mosmant (29).


arrow
RESULTS AND DISCUSSION
 
Association between FAK and RIP in vivo and in vitro. To determine whether there is a direct interaction between FAK and RIP, we tested their binding in breast cancer cell lines. Lysates were prepared from the BT-474 and BT-20 cell lines, and coimmunoprecipitation experiments were performed with antibody against FAK and RIP. We found a strong association of FAK and RIP in both the BT-474 (Fig. 1A) and the BT-20 (data not shown) cell lines. To confirm the specificity of this binding, we used an inducible system of FAK expression (Tet-FAK cells) in FAK-null mouse embryonic fibroblasts, where FAK expression is under the control of a tetracycline repressor (31). Between 12 and 24 h after tetracycline withdrawal, cells expressed high levels of FAK (Fig. 1B). Immunoprecipitation of FAK from Tet-FAK cells demonstrated the association of FAK and RIP, with the level of coimmunoprecipitation proportional to the amount of FAK protein expression upon the withdrawal of tetracycline from the medium. Taken together, these results show that FAK and RIP bind together in both breast cancer cell lines and mouse embryonic fibroblasts.



View larger version (27K):
[in this window]
[in a new window]
  		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.

To define the RIP-binding domain in FAK as well as the FAK-binding domain in RIP, we coexpressed different RIP and FAK plasmids in HEK293 cells. Full-length RIP bound to full-length FAK in HEK293 cells, and this binding appeared to occur at the amino terminus of FAK, since antibody against the C terminus of FAK (C20) did not reveal the FAK-CD protein in immunoprecipitates (Fig. 1C). When we expressed the death domain of RIP with different FAK constructs, RIP again bound both to full-length FAK and to the amino terminus of FAK (GFP-FAK-NT; Fig. 1D). To confirm this binding in vitro, we used GST-FAK fusion proteins to test their ability to bind to endogenous RIP in HEK293 cells. As shown in Fig. 1E, RIP bound only to the amino terminus of FAK (GST-FAK-NT), and did not bind to the focal adhesion targeting sequence in the C terminus of FAK (GST-FAT) or to control GST. From these experiments, we conclude that RIP binds to the amino terminus of FAK through the death domain both in vivo and in vitro.

Staurosporine-induced apoptosis causes RIP-dependent dephosphorylation and degradation of FAK. Next, we sought to determine the biological significance of the FAK-RIP interaction. First, we tested staurosporine, a bacterial alkaloid with broad inhibitory activities against a variety of serine/threonine kinases (34) and several tyrosine kinases (10, 30, 36). It also induces apoptosis in a variety of transformed cell lines and has been associated with FAK-mediated apoptosis in endothelial cells (22). In breast cancer cells, we showed that staurosporine caused a displacement of FAK from focal adhesions (Fig. 2A) and led to rapid dephosphorylation and slower degradation of FAK (Fig. 2B). We also found a decrease in the amount of RIP protein associated with FAK in BT-474 cells 2 to 3 h after treatment with staurosporine (data not shown), consistent with disruption of the interaction of FAK and RIP.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 2. Staurosporine causes dephosphorylation and degradation of FAK in breast carcinoma cell lines. A total of 2 x 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.

In parallel with the degradation of FAK, we observed an attenuation of RIP expression, resembling the proteosomal degradation of RIP, that has been described during the disruption of the RIP-Hsp90 complex with geldanamycin (25). Staurosporine induced different rates of apoptosis in these two cell lines (Fig. 2C), and intriguingly, the apoptotic rate was higher for the BT-474 cells that expressed significantly more RIP than that for the BT-20 cells (Fig. 2B). Based on densitometry analysis (data not shown), we estimated that BT-474 cells express 60% more RIP, suggesting that RIP may be providing proapoptotic signals in these breast cancer cells.

To specifically address this question, we tested staurosporine on embryonic fibroblasts derived from normal and RIP–/– mice (4). Staurosporine induced a higher rate of apoptosis in the RIP+/+ cells than in the RIP–/– cells (Fig. 3A). Furthermore, staurosporine caused a dose-dependent dephosphorylation and degradation of FAK in the RIP+/+ cells that was only minimal in the RIP–/– cells (Fig. 3B). The time course analysis of staurosporine treated RIP+/+ and RIP–/– mouse fibroblasts by immunostaining and Western blot analysis showed that after 6 h of treatment, there were no focal adhesions in the rare, attached RIP+/+ cells, while there were many attached RIP–/– cells with visible focal adhesion structures (data not shown). In addition, RIP–/– cells were resistant to FAK dephosphorylation and degradation, while FAK in RIP+/+ cells was rapidly dephosphorylated and degraded (data not shown). These results show that the presence of RIP enhanced the rate of staurosporine-induced apoptosis and the level of FAK dephosphorylation and degradation, suggesting that RIP provides proapoptotic signals in these cells as well.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Staurosporine causes dephosphorylation and degradation of FAK, which depends on the presence of RIP. A total of 2 x 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.

RIP is involved in transducing the proapoptotic signals initiated by the disruption of FAK signaling. To further define the relationship between RIP and FAK, we studied apoptosis that was induced by specific attenuation of FAK. We used our model system in which the transduction of an adenoviral FAK-CD construct into cells causes tyrosine dephosphorylation of FAK and displacement of FAK from the focal adhesions. In breast cancer cell lines, this subsequently led to detachment and apoptotic cell death, while in normal mammary epithelial cells, the displacement of FAK appeared to have no deleterious effect on the cells (43). Then, we applied this model to test the effect of FAK attenuation on the RIP–/– and RIP+/+ fibroblasts. As shown in Fig. 4A, treatment of RIP–/– cells with Ad FAK-CD did not lead to the displacement of FAK from focal adhesions, while RIP+/+ cells showed displacement of FAK beginning 6 h after treatment. In addition, RIP–/– cells did not detach from their substratum and had a very low level of apoptosis (Fig. 4B). Furthermore, RIP–/– cells had no significant changes in their level of FAK tyrosine phosphorylation and did not have significant degradation of FAK (Fig. 4C). In contrast, the RIP+/+ cells detached from their substratum and underwent apoptosis (Fig. 4B) with corresponding changes in tyrosine phosphorylation and a resultant degradation of FAK (Fig. 4C). From these data, we conclude that the presence of FAK in the focal adhesions provides antiapoptotic signals to the cells, and once FAK has been displaced from focal adhesions, proapoptotic signals from RIP promote apoptosis and degradation of FAK.



View larger version (36K):
[in this window]
[in a new window]
  		FIG.4. RIP is involved in transducing the proapoptotic signals initiated by the disruption of FAK signaling. A total of 2 x 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.

RIP is involved in transducing the detachment-induced apoptotic signals. Different groups have shown that FAK controls adhesion-dependent cell survival in normal epithelial and endothelial cells (9, 12, 16, 19). Results from previous studies suggest that in breast tumor cells, FAK has two separate functions, one promoting the adhesive interactions between tumor cells and their extracellular matrix and the other acting as a survival signal that is independent of cellular adhesion (43). Thus, we compared the viability of RIP+/+ and RIP–/– cells in the absence of matrix attachment. Cells were maintained on polyHEMA-coated tissue culture plates that create anchorage-independent conditions. Both RIP+/+ and RIP–/– cells remained viable for at least 72 h, as determined by mitochondrial dehydrogenase activity using the MTT assay (data not shown). However, the viability of the suspended RIP+/+ cells was lower than the viability of attached cells, with an average of 17% of the cells undergoing apoptosis after 72 h in anchorage-independent conditions (Fig. 5A) compared to 2% for the RIP–/– cells (Fig. 5A). This result suggests that RIP may be involved in transducing proapoptotic signals in anoikis. To examine whether protection from anoikis required FAK, we attenuated FAK with Ad FAK-CD. When RIP–/– cells grown in suspension were transduced with Ad FAK-CD, their rate of apoptosis was not different from RIP–/– cells transduced with Ad LacZ (Fig. 5B). In contrast, RIP+/+ cells had a much higher level of apoptosis and became apoptotic much faster than RIP–/– cells when FAK signaling was disrupted (Fig. 5B). Thus, RIP+/+ cells were still sensitive to FAK attenuation under anchorage-independent conditions. Taken together, these results demonstrate that RIP provides proapoptotic signals in fibroblasts grown in the absence of adhesion and that FAK plays an additional role as an adhesion-independent survival signal not only in transformed cells but also in normal fibroblasts.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5. RIP is involved in transducing the detachment-induced proapoptotic signals. (A) A total of 2 x 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 x 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.

Downregulation of RIP with siRNA in breast cancer cells leads to higher resistance to apoptosis caused by the disruption of FAK signaling. While these results have provided evidence of a proapoptotic role for RIP, we returned to our breast cancer cell model to determine whether the downregulation of RIP expression in BT-474 breast cancer cells could rescue these cells from apoptosis induced by FAK attenuation. To downregulate RIP expression, we synthesized two pairs of silencing siRNAs corresponding to human RIP (H87 and H121), which caused a moderate decrease in RIP expression in the BT-474 cells (Fig. 6A). After 48 h of siRNA transfection, cells were treated with either staurosporine (Fig. 6B and C) or FAK-CD (Fig. 6D and E). In the cells treated with staurosporine, a decrease in RIP expression by both RIP siRNAs protected FAK from rapid dephosphorylation during the first hour of treatment, while control siRNAs (H121Scr and GAPDH) had no effect. Longer treatment with staurosporine caused apoptosis, the level of which was lower in the cells treated with RIP siRNA (Fig. 6C). In the cells where FAK was specifically downregulated by FAK-CD, the reduction of RIP expression by siRNA also preserved a higher level of FAK phosphorylation even after a long exposure to FAK-CD. In contrast, FAK was dephosphorylated in untreated control cells (Fig. 6D). Furthermore, the rate of apoptosis (Fig. 6E) decreased from 42% in control cells to 22 and 15% in the H87 and H121 siRNA-treated cells, respectively. These results suggest that increased levels of FAK expression, accompanied by decreased levels of RIP expression, enhance the survival signals in breast cancer cells and make them more resistant to apoptotic stimuli.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6. Downregulation of RIP with siRNA leads to higher resistance to apoptosis caused by the attenuation of FAK. A total of 2 x 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.

These results provide a possible mechanistic explanation for the prosurvival function of FAK in human breast cancer. FAK binds to RIP in both normal and transformed cells, and the disruption of the balance between these two kinases in a cell may result in amplification of pro- or antiapoptotic stimuli. This result is consistent with other reports, where it has been shown that RIP may provide proapoptotic signals in some cases or antiapoptotic signals in others (14, 23, 28). Thus, our model suggests that the upregulation of FAK expression in cancer cells shifts the FAK-RIP balance towards resistance to apoptosis and promotes survival of the cells. Finally, these results may provide the link between FAK and the death receptor for the phenomenon of anoikis. Tumor cells have been postulated to be resistant to anoikis to allow them to survive during the processes of invasion and metastasis that require anchorage-independent survival (15). As tumor cells become detached and proceed with invasion and metastasis, our model predicts that FAK would bind to RIP and provide survival signals to these cells. Thus, this proposed mechanism suggests that targeting the FAK-RIP association might provide a novel approach for cancer therapeutics.


arrow
ACKNOWLEDGMENTS
 
We thank Philip Leder and Michelle Kelliher for providing the RIP-deficient cell line and Scott Kaufmann and Keith Burridge for critical discussions.

This work is supported by NIH grant CA65910 to W.G.C.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Health Science Center, P.O. Box 100286, 1600 SW Archer Rd., Gainesville FL 32610. Phone: (352) 265-0622. Fax: (352) 338-9809. E-mail: cance{at}surgery.ufl.edu. Back


arrow
REFERENCES
 
    1
  1. Beviglia, L., V. Golubovskaya, L. Xu, X. Yang, R. J. Craven, and W. G. Cance. 2003. Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis. Biochem. J. 373:201-210.[CrossRef][Medline]
    2. 2
  2. Cance, W. G., J. E. Harris, M. V. Iacocca, E. Roche, X. Yang, J. Chang, S. Simkins, and L. Xu. 2000. Immunohistochemical analyses of focal adhesion kinase expression in benign and malignant human breast and colon tissues: correlation with preinvasive and invasive phenotypes. Clin. Cancer Res. 6:2417-2423.[Abstract/Free Full Text]
    3. 3
  3. Chinnaiyan, A. M., C. G. Tepper, M. F. Seldin, K. O'Rourke, F. C. Kischkel, S. Hellbardt, P. H. Krammer, M. E. Peter, and V. M. Dixit. 1996. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J. Biol. Chem. 271:4961-4965.[Abstract/Free Full Text]
    4. 4
  4. Cusson, N., S. Oikemus, E. D. Kilpatrick, L. Cunningham, and M. Kelliher. 2002. The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor type 2-induced cell death. J. Exp. Med. 196:15-26.[Abstract/Free Full Text]
    5. 5
  5. Duan, H., and V. M. Dixit. 1997. RAIDD is a new 'death' adaptor molecule. Nature 385:86-89.[CrossRef][Medline]
    6. 6
  6. Folkman, J., and A. Moscona. 1978. Role of cell shape in growth control. Nature 273:345-349.[CrossRef][Medline]
    7. 7
  7. Frisch, S. M. 1999. Evidence for a function of death-receptor-related, death-domain-containing proteins in anoikis. Curr. Biol. 9:1047-1049.[CrossRef][Medline]
    8. 8
  8. Frisch, S. M., and H. Francis. 1994. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124:619-626.[Abstract/Free Full Text]
    9. 9
  9. Frisch, S. M., K. Vuori, E. Ruoslahti, and P. Y. Chan-Hui. 1996. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 134:793-799.[Abstract/Free Full Text]
    10. 10
  10. Fujita-Yamaguchi, Y., and S. Kathuria. 1988. Characterization of receptor tyrosine-specific protein kinases by the use of inhibitors. Staurosporine is a 100-times more potent inhibitor of insulin receptor than IGF-I receptor. Biochem. Biophys. Res. Commun. 157:955-962.[CrossRef][Medline]
    11. 11
  11. Funakoshi-Tago, M., Y. Sonoda, S. Tanaka, K. Hashimoto, K. Tago, S. Tominaga, and T. Kasahara. 2003. Tumor necrosis factor-induced nuclear factor kappaB activation is impaired in focal adhesion kinase-deficient fibroblasts. J. Biol. Chem. 278:29359-29365.[Abstract/Free Full Text]
    12. 12
  12. Gilmore, A. P., A. D. Metcalfe, L. H. Romer, and C. H. Streuli. 2000. Integrin-mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization. J. Cell Biol. 149:431-446.[Abstract/Free Full Text]
    13. 13
  13. Gilmore, A. P., and L. H. Romer. 1996. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol. Biol. Cell 7:1209-1224.[Abstract]
    14. 14
  14. Grimm, S., B. Z. Stanger, and P. Leder. 1996. RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways. Proc. Natl. Acad. Sci. USA 93:10923-10927.[Abstract/Free Full Text]
    15. 15
  15. Grossmann, J. 2002. Molecular mechanisms of "detachment-induced apoptosis-anoikis." Apoptosis 7:247-260.[CrossRef][Medline]
    16. 16
  16. Hisano, C., R. Tanaka, H. Fujishima, H. Ariyama, T. Tsuchiya, T. Tatsumoto, K. Mitsugi, M. Nakamura, and S. Nakano. 2003. Suppression of anoikis by v-Src but not by activated c-H-Ras in human gallbladder epithelial cells. Cell Biol. Int. 27:415-421.[CrossRef][Medline]
    17. 17
  17. Hsu, H., J. Huang, H. B. Shu, V. Baichwal, and D. V. Goeddel. 1996. TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4:387-396.[CrossRef][Medline]
    18. 18
  18. Hungerford, J. E., M. T. Compton, M. L. Matter, B. G. Hoffstrom, and C. A. Otey. 1996. Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J. Cell Biol. 135:1383-1390.[Abstract/Free Full Text]
    19. 19
  19. Ilic, D., E. A. Almeida, D. D. Schlaepfer, P. Dazin, S. Aizawa, and C. H. Damsky. 1998. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol. 143:547-560.[Abstract/Free Full Text]
    20. 20
  20. Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, T. Yamamoto, et al. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377:539-544.[CrossRef][Medline]
    21. 21
  21. Jones, G., J. Machado, Jr., and A. Merlo. 2001. Loss of focal adhesion kinase (FAK) inhibits epidermal growth factor receptor-dependent migration and induces aggregation of NH2-terminal FAK in the nuclei of apoptotic glioblastoma cells. Cancer Res. 61:4978-4981.[Abstract/Free Full Text]
    22. 22
  22. Kabir, J., M. Lobo, and I. Zachary. 2002. Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis. Biochem. J. 367:145-155.[CrossRef][Medline]
    23. 23
  23. Kelliher, M. A., S. Grimm, Y. Ishida, F. Kuo, B. Z. Stanger, and P. Leder. 1998. The death domain kinase RIP mediates the TNF-induced NF-kappaB signal. Immunity 8:297-303.[CrossRef][Medline]
    24. 24
  24. Lark, A. L., C. A. Livasy, B. Calvo, L. Caskey, D. T. Moore, X. Yang, and W. G. Cance. 2003. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses. Clin. Cancer Res. 9:215-222.[Abstract/Free Full Text]
    25. 25
  25. Lewis, J., A. Devin, A. Miller, Y. Lin, Y. Rodriguez, L. Neckers, and Z. G. Liu. 2000. Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation. J. Biol. Chem. 275:10519-10526.[Abstract/Free Full Text]
    26. 26
  26. Liu, X. J., L. Yang, H. B. Wu, O. Qiang, M. H. Huang, and Y. P. Wang. 2002. Apoptosis of rat hepatic stellate cells induced by anti-focal adhesion kinase antibody. World J. Gastroenterol. 8:734-738.[Medline]
    27. 27
  27. Liu, Z. G., H. Hsu, D. V. Goeddel, and M. Karin. 1996. Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87:565-576.[CrossRef][Medline]
    28. 28
  28. Martinon, F., N. Holler, C. Richard, and J. Tschopp. 2000. Activation of a pro-apoptotic amplification loop through inhibition of NF-kappaB-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett. 468:134-136.[CrossRef][Medline]
    29. 29
  29. Mosmant, T. 1983. Rapid colorimetric assay for cellular growth and survival. J. Immunol. Methods 65:55-63.[CrossRef][Medline]
    30. 30
  30. Nakano, H., E. Kobayashi, I. Takahashi, T. Tamaoki, Y. Kuzuu, and H. Iba. 1987. Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60. J. Antibiot. (Tokyo) 40:706-708.[Medline]
    31. 31
  31. Owen, J. D., P. J. Ruest, D. W. Fry, and S. K. Hanks. 1999. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol. Cell. Biol. 19:4806-4818.[Abstract/Free Full Text]
    32. 32
  32. Owens, L. V., L. Xu, R. J. Craven, G. A. Dent, T. M. Weiner, L. Kornberg, E. T. Liu, and W. G. Cance. 1995. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res. 55:2752-2755.[Abstract/Free Full Text]
    33. 33
  33. Richardson, A., and T. Parsons. 1996. A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 380:538-540.[CrossRef][Medline]
    34. 34
  34. Ruegg, U. T., and G. M. Burgess. 1989. Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol. Sci. 10:218-220.[CrossRef][Medline]
    35. 35
  35. Rytomaa, M., L. M. Martins, and J. Downward. 1999. Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr. Biol. 9:1043-1046.[CrossRef][Medline]
    36. 36
  36. Secrist, J. P., I. Sehgal, G. Powis, and R. T. Abraham. 1990. Preferential inhibition of the platelet-derived growth factor receptor tyrosine kinase by staurosporine. J. Biol. Chem. 265:20394-20400.[Abstract/Free Full Text]
    37. 37
  37. Sonoda, Y., Y. Matsumoto, M. Funakoshi, D. Yamamoto, S. K. Hanks, and T. Kasahara. 2000. Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60. J. Biol. Chem. 275:16309-16315.[Abstract/Free Full Text]
    38. 38
  38. Stanger, B. Z., P. Leder, T. H. Lee, E. Kim, and B. Seed. 1995. RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81:513-523.[CrossRef][Medline]
    39. 39
  39. Ting, A. T., F. X. Pimentel-Muinos, and B. Seed. 1996. RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis. EMBO J. 15:6189-6196.[Medline]
    40. 40
  40. van De Water, B., F. Houtepen, M. Huigsloot, and I. B. Tijdens. 2001. Suppression of chemically induced apoptosis but not necrosis of renal proximal tubular epithelial (LLC-PK1) cells by focal adhesion kinase (FAK). Role of FAK in maintaining focal adhesion organization after acute renal cell injury. J. Biol. Chem. 276:36183-36193.[Abstract/Free Full Text]
    41. 41
  41. Weiner, T. M., E. T. Liu, R. J. Craven, and W. G. Cance. 1993. Expression of focal adhesion kinase gene and invasive cancer. Lancet 342:1024-1025.[CrossRef][Medline]
    42. 42
  42. Xu, L. H., L. V. Owens, G. C. Sturge, X. Yang, E. T. Liu, R. J. Craven, and W. G. Cance. 1996. Attenuation of the expression of the focal adhesion kinase induces apoptosis in tumor cells. Cell Growth Differ. 7:413-418.[Abstract]
    43. 43
  43. Xu, L.-H., X.-H. Yang, C. A. Bradham, D. A. Brenner, A. S. Baldwin, R. J. Craven, and W. G. Cance. 2000. The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. J. Biol. Chem. 275:30597-30604.[Abstract/Free Full Text]
    44. 44
  44. Zhao, J. H., H. Reiske, and J. L. Guan. 1998. Regulation of the cell cycle by focal adhesion kinase. J. Cell Biol. 143:1997-2008.[Abstract/Free Full Text]

Molecular and Cellular Biology, May 2004, p. 4361-4371, Vol. 24, No. 10
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.10.4361-4371.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kasorn, A., Alcaide, P., Jia, Y., Subramanian, K. K., Sarraj, B., Li, Y., Loison, F., Hattori, H., Silberstein, L. E., Luscinskas, W. F., Luo, H. R. (2009). Focal Adhesion Kinase Regulates Pathogen-Killing Capability and Life Span of Neutrophils via Mediating Both Adhesion-Dependent and -Independent Cellular Signals. J. Immunol. 183: 1032-1043 [Abstract] [Full Text]  
  • Park, S., Hatanpaa, K. J., Xie, Y., Mickey, B. E., Madden, C. J., Raisanen, J. M., Ramnarain, D. B., Xiao, G., Saha, D., Boothman, D. A., Zhao, D., Bachoo, R. M., Pieper, R. O., Habib, A. A. (2009). The Receptor Interacting Protein 1 Inhibits p53 Induction through NF-{kappa}B Activation and Confers a Worse Prognosis in Glioblastoma. Cancer Res. 69: 2809-2816 [Abstract] [Full Text]  
  • Liu, W., Bloom, D. A., Cance, W. G., Kurenova, E. V., Golubovskaya, V. M., Hochwald, S. N. (2008). FAK and IGF-IR interact to provide survival signals in human pancreatic adenocarcinoma cells. Carcinogenesis 29: 1096-1107 [Abstract] [Full Text]  
  • Cance, W. G., Golubovskaya, V. M. (2008). Focal Adhesion Kinase Versus p53: Apoptosis or Survival?. Sci Signal 1: pe22-pe22 [Abstract] [Full Text]  
  • Kelly, K. J., Wu, P., Patterson, C. E., Temm, C., Dominguez, J. H. (2008). LOX-1 and inflammation: a new mechanism for renal injury in obesity and diabetes. Am. J. Physiol. Renal Physiol. 294: F1136-F1145 [Abstract] [Full Text]  
  • Schlaepfer, D. D., Hou, S., Lim, S.-T., Tomar, A., Yu, H., Lim, Y., Hanson, D. A., Uryu, S. A., Molina, J., Mitra, S. K. (2007). Tumor Necrosis Factor-{alpha} Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression. J. Biol. Chem. 282: 17450-17459 [Abstract] [Full Text]  
  • Slack-Davis, J. K., Martin, K. H., Tilghman, R. W., Iwanicki, M., Ung, E. J., Autry, C., Luzzio, M. J., Cooper, B., Kath, J. C., Roberts, W. G., Parsons, J. T. (2007). Cellular Characterization of a Novel Focal Adhesion Kinase Inhibitor. J. Biol. Chem. 282: 14845-14852 [Abstract] [Full Text]  
  • Beierle, E. A., Trujillo, A., Nagaram, A., Kurenova, E. V., Finch, R., Ma, X., Vella, J., Cance, W. G., Golubovskaya, V. M. (2007). N-MYC Regulates Focal Adhesion Kinase Expression in Human Neuroblastoma. J. Biol. Chem. 282: 12503-12516 [Abstract] [Full Text]  
  • Lunn, J. A., Jacamo, R., Rozengurt, E. (2007). Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress. J. Biol. Chem. 282: 10370-10379 [Abstract] [Full Text]  
  • Huang, D., Khoe, M., Befekadu, M., Chung, S., Takata, Y., Ilic, D., Bryer-Ash, M. (2007). Focal adhesion kinase mediates cell survival via NF-{kappa}B and ERK signaling pathways. Am. J. Physiol. Cell Physiol. 292: C1339-C1352 [Abstract] [Full Text]  
  • Halle, M., Liu, Y.-C., Hardy, S., Theberge, J.-F., Blanchetot, C., Bourdeau, A., Meng, T.-C., Tremblay, M. L. (2007). Caspase-3 Regulates Catalytic Activity and Scaffolding Functions of the Protein Tyrosine Phosphatase PEST, a Novel Modulator of the Apoptotic Response. Mol. Cell. Biol. 27: 1172-1190 [Abstract] [Full Text]  
  • Eiras, S., Fernandez, P., Pineiro, R., Iglesias, M. J., Gonzalez-Juanatey, J. R., Lago, F. (2006). Doxazosin induces activation of GADD153 and cleavage of focal adhesion kinase in cardiomyocytes en route to apoptosis. Cardiovasc Res 71: 118-128 [Abstract] [Full Text]  
  • Siesser, P. M.F., Hanks, S. K. (2006). The Signaling and Biological Implications of FAK Overexpression in Cancer.. Clin. Cancer Res. 12: 3233-3237 [Full Text]  
  • Zhang, H. M., Keledjian, K. M., Rao, J. N., Zou, T., Liu, L., Marasa, B. S., Wang, S. R., Ru, L., Strauch, E. D., Wang, J.-Y. (2006). Induced focal adhesion kinase expression suppresses apoptosis by activating NF-{kappa}B signaling in intestinal epithelial cells. Am. J. Physiol. Cell Physiol. 290: C1310-C1320 [Abstract] [Full Text]  
  • Romer, L. H., Birukov, K. G., Garcia, J. G.N. (2006). Focal Adhesions: Paradigm for a Signaling Nexus. Circ. Res. 98: 606-616 [Abstract] [Full Text]  
  • Garces, C. A., Kurenova, E. V., Golubovskaya, V. M., Cance, W. G. (2006). Vascular Endothelial Growth Factor Receptor-3 and Focal Adhesion Kinase Bind and Suppress Apoptosis in Breast Cancer Cells. Cancer Res. 66: 1446-1454 [Abstract] [Full Text]  
  • Aixinjueluo, W., Furukawa, K., Zhang, Q., Hamamura, K., Tokuda, N., Yoshida, S., Ueda, R., Furukawa, K. (2005). Mechanisms for the Apoptosis of Small Cell Lung Cancer Cells Induced by Anti-GD2 Monoclonal Antibodies: ROLES OF ANOIKIS. J. Biol. Chem. 280: 29828-29836 [Abstract] [Full Text]  
  • Golubovskaya, V. M., Finch, R., Cance, W. G. (2005). Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53. J. Biol. Chem. 280: 25008-25021 [Abstract] [Full Text]  
  • Griffin, M. A., Feng, H., Tewari, M., Acosta, P., Kawana, M., Sweeney, H. L., Discher, D. E. (2005). {gamma}-Sarcoglycan deficiency increases cell contractility, apoptosis and MAPK pathway activation but does not affect adhesion. J. Cell Sci. 118: 1405-1416 [Abstract] [Full Text]  
  • Lieman, J. H., Worley, L. A., Harbour, J. W. (2005). Loss of Rb-E2F Repression Results in Caspase-8-mediated Apoptosis through Inactivation of Focal Adhesion Kinase. J. Biol. Chem. 280: 10484-10490 [Abstract] [Full Text]  
  • Zhang, Y., Lu, H., Dazin, P., Kapila, Y. (2004). Squamous Cell Carcinoma Cell Aggregates Escape Suspension-induced, p53-mediated Anoikis: FIBRONECTIN AND INTEGRIN {alpha}v MEDIATE SURVIVAL SIGNALS THROUGH FOCAL ADHESION KINASE. J. Biol. Chem. 279: 48342-48349 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kurenova, E.
Right arrow Articles by Cance, W. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kurenova, E.
Right arrow Articles by Cance, W. G.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»