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Am J Physiol Gastrointest Liver Physiol 291: G491-G499, 2006; doi:10.1152/ajpgi.00292.2005
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MUCOSAL BIOLOGY

Focal adhesion kinase protein levels in gut epithelial motility

Marc D. Basson, Matthew A. Sanders, Ruben Gomez, James Hatfield, Richard VanderHeide, Vijayalakshmi Thamilselvan, Jianhu Zhang, and Mary F. Walsh

Departments of Surgery and Pathology, John D. Dingell Veterans Affairs Medical Center and Wayne State University, Detroit, Michigan

Submitted 29 June 2005 ; accepted in final form 12 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK397) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by ≥30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK397 phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.

healing; migration; restitution; ulcer

ALTHOUGH MOST PREVALENT in the stomach and duodenum, ulcers are found throughout the gastrointestinal tract. Regardless of site, mucosal ulcer healing is a complex process that involves restitution by migration of epithelial cells from the wound margins and eventual filling of the defect by proliferating epithelial and connective tissue cells (31, 43). Various factors have been shown to stimulate epithelial restitution and proliferation, but the intracellular signal proteins that control epithelial cellular migration are less well understood. Among these signals, activation of focal adhesion kinase (FAK) has been linked to gastric wound healing in vivo (41, 44). FAK is a tyrosine kinase that associates with the cytoplasmic tail of clustered integrins in focal adhesion complexes. On activation, autophosphorylation of FAK on tyrosine 397 is followed by further phosphorylation of FAK itself and other proteins (36, 56). Although FAK was originally described as being rapidly activated on integrin-mediated cell-matrix adhesion, this kinase has now been reported to be activated during epithelial cell motility and phosphorylated at both serine and tyrosine residues by many factors including occupancy of trans-activating G protein-coupled receptors (GPCRs) (10, 23, 35, 51). Modulating FAK activity and/or abundance affects epithelial motility (5, 28, 55) and tumor cell invasion differently in different cell types and on different matrix substrates (3, 8, 38, 47, 52), and FAK phosphorylation has recently been implicated in an inside-out signal pathway by which integrin-mediated adhesion is itself regulated (45).

Most studies of FAK function have focused on rapid phosphorylation events (34). Less is known about the regulation and relevance of FAK protein levels in cell biology in general or in the gut mucosa in particular. FAK is abnormally expressed in some cancers (1, 17, 25, 49) and increased during Caco-2 intestinal epithelial cell differentiation (18), suggesting that changes in FAK protein levels per se may play a role in both normal and pathological states. We previously reported that immunoreactivity for FAK protein is decreased in motile compared with static Caco-2 cells over 4 days in a differential density seeding model that maintains otherwise equivalent levels of some differentiation markers (55). In particular, FAK expression was lower at the lamellipodial edge of the migrating cells but easily visualized at cell-cell contacts in the static cells (54). Therefore, we hypothesized that FAK protein levels might be altered at sites of chronic gut epithelial injury and that these changes in FAK protein expression might influence intestinal epithelial cell motility and subsequent wound repair.

To test this hypothesis, we first used a linear scrape model of epithelial sheet migration for immunohistochemical analysis of FAK and FAK397 in Caco-2 colonic and IEC-6 small intestinal epithelial cells and less-differentiated HT-29 colon cancer cells. Caco-2 cells were originally derived from a human colon cancer, but they express many highly differentiated features and are a common model for normal gut mucosal biology (30). IEC-6 cells are a nonmalignant rat intestinal epithelial cell line. Our observations of sharply lower total and activated FAK (FAK397) protein immunoreactivity in migrating gut epithelial cells in culture were confirmed by Western blot analysis in two different models of cell migration. Having demonstrated reductions in FAK protein in migrating gut epithelial cells, we next sought to determine whether such reductions in FAK protein may alter gut epithelial cell motility. Therefore, small interfering RNAs (siRNAs) targeted toward human and rodent FAK were used to investigate the effects of decreased FAK protein expression on Caco-2 and IEC-6 cell motility. Finally, cycloheximide decay curves, in situ hybridization, and quantitative RT-PCR were used to begin to characterize potential mechanisms by which motility-induced changes in FAK protein might occur.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and cell culture. Caco-2BBE cells were originally obtained from M. Peterson (29) and maintained as previously described (2). IEC-6 and HT-29 cells were purchased from the American Tissue Culture Collection (ATCC; Rockville, MD) and maintained as per ATCC recommendations. All cells were propagated in tissue culture plastic flasks but studied in dishes precoated with type I collagen (Sigma, St. Louis, MO) as previously described (2).

Migration assays. Two models of epithelial sheet migration and the differential-density-seeding and multiple scrape models of static and migrating cells were used to study FAK biology of migrating cells. The first two models allow quantitation of migration and immunostain visualization of the migrating cells. 1) In linear or "scrape" wounding assays, confluent cell monolayers were wounded with a razor blade in a straight line, and the cells were allowed to migrate into the denuded area for 12, 24, 36, or 48 h before immunostaining. Two models were used for assessment of motility in cells with lowered FAK expression: the linear scrape model in Caco-2, IEC-6, and HT-29 cells where monolayer wounds were made and fixed loci were photographed after 24 h to quantitate the area of movement beyond the scrape; and 2) a circular wound model in which a small circular area in confluent Caco-2 or IEC-6 cell monolayers was denuded with a pipette tip. The extent of wound closure was measured by quantitation of photomicrographs of the original wound and of the same wound after 6 or 24 h. Images obtained from both models were transferred to a Kodak 440cf ImageStation for computer analysis. FAK protein abundance was assessed in the multiple scrape model. To create small areas of confluent cells from which cells might migrate, a broad-tooth comb was scraped in two directions (at a 90° angle) across the culture dish. Finally, homogeneous populations of static and motile cells were generated for biochemical studies, e.g., FAK protein expression and stability, by varying seeding density so that at 4 days after plating, one population of cells was static and confluent (13,000/cm2 initial density in 35 x 10-mm dishes) and the other consisted of small islands of radially migrating cells (750/ cm2 in 100 x 20-mm dishes). Previous studies (20, 21, 54, 55) have demonstrated that this model yields highly reproducible results and that the expression of some conventional differentiation markers is equivalent between these two cell populations.

FAK immunocytochemistry in expanding cell monolayers. FAK protein abundance in migrating Caco-2, IEC-6, and HT-29 cells established by linear wounding was assessed in periodate, lysine, formaldehyde-fixed cells 12, 24, 36, and 48 h after wounding. Cells were permeabilized with 0.5% Triton X-100 on ice and washed with PBS. To quench nonspecific reactive sites, the cells were incubated with 2.5% horse serum followed by incubation for 1 h at 37°C with anti-FAK antibody (mouse monoclonal 1:100, BD Biosciences, San Diego, CA), antibody directed at tyrosine-phosphorylated activated FAK397 (rabbit polyclonal 1:500, Biosource International, Camarillo, CA), or anti-ERK antibody as control (rabbit polyclonal 1:100, Cell Signaling, Beverly, MA). After exposure to a biotynilated universal second antibody (Vector Laboratories, Burlingame, CA), staining was accomplished with streptavidin-horseradish peroxidase and diaminobenzidene (DAB) chromogen (R&D Systems, Minneapolis, MN). Some samples were also immunostained with the Upstate anti-FAK monoclonal antibody routinely used for Western immunoblots in our laboratory. This antibody is directed at amino acids 1–423 (NH2 terminal) of the human FAK sequence, whereas the BD monoclonal was generated against amino acids 354–533 in the kinase domain of chicken FAK. Although both antibodies yielded similar results, staining was more prominent with the BD antibody, and we chose this particular antibody for our immunocytochemistry. Cells were then counterstained with Mayer's hematoxylin before mounting (Gel/Mount, Biomedia, Foster City, CA).

FAK protein inhibition with siRNA. The human TTT GGC GGT TGC AAT TGT A dt/dTdT, rat AGA AAT AGC TGA TCA AGT A dt/dTdT siRNAs, and nontargeting duplexes were synthesized by Dharmacon (Dallas, TX). The appropriate duplex and its nontargeting control were introduced into Caco-2 (120 nM) or IEC-6 (200 nM) cells with a mix of Oligofectamine and Plus Reagents in OptiMEM using a slight modification of the manufacturer's protocol (Invitrogen, Carlsbad, CA). After 6 h, complete DMEM with 2x FBS was added to the OptiMEM and the cells used for migration studies 72 h after transfection. At that time, efficiency of inhibition was ascertained by Western immunoblot. Briefly, cells exposed to siRNA, nontargeting RNA, or mock transfection (OptiMEM, Plus Reagent, and Oligofectamine) conditions were collected at the time of wounding in ice-cold lysis buffer (20 mM sodium phosphate, 50 mM NaCl, 5 mM EDTA, 0.5% SDS, pH 7.4), supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 10 µg/ml leupeptin, and 1 µg/ml aprotinin. Nuclei and other organelles were not removed before sonication on ice for 15 s. The lysates were subsequently centrifuged at 1,000 rpm for 10 min to remove cellular debris. Equal protein samples were resolved by SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were then incubated with antibody to total FAK (BD Biosciences or Upstate, Lake Placid, NY); results did not differ between the two antibodies, but those shown were obtained with the Upstate monoclonal. Protein bands were visualized with enhanced chemiluminescence (ECL; Amersham, Little Chalfont, Buckinghamshire, England). The blots were stripped and reprobed for FAK397 (Biosource). Some blots were first probed for FAK397, then stripped and reprobed for total FAK. In some studies, both tFAK and FAK397 were compared directly to the tubulin control. Each method yielded the same results. Imaging and analysis of band density were performed on a Kodak 440cf Image Station.

Cycloheximide decay curves in static and motile cells and after FAK activity inhibition by transient transfection with HA-tagged FAK Y397F. Lower FAK expression at the migrating edge of the in vitro samples could be the result of FAK protein degradation, perhaps related to the proportionally greater activation of the remaining FAK. Therefore, we transfected the cells with a dominant-negative construct that cannot be activated by autophosphorylation at tyrosine 397 before assessment of protein stability by cycloheximide decay. Briefly, HA-tagged FAK Y397F and wild-type FAK (wtFAK) were transfected into 30–50% confluent Caco-2 cells via Lipofectamine Plus and OptiMEM for 6 h (Invitrogen), and the cells were incubated in normal medium for a further 24 h before a study of FAK stability. Either static and motile Caco-2 cells or Caco-2 cells transiently transfected with HA-tagged wtFAK or FAK Y397F were pretreated with 10 µg/ml cycloheximide to block new protein synthesis. Cells were harvested at 0, 2, 4, and 6 h and every 6 h thereafter for 42 h. To compare rates of FAK degradation, cells were lysed for Western immunoblot for total FAK, and wtFAK and FAK397 decay was quantitated with an antibody to the HA tag (Covance, Berkeley, CA).

RT-PCR for FAK expression. The FAK primer pairs were designed using the MIT Prime3 online primer design protocol from the FAK gene sequence of Whitney et al. (48). The FAK primers 5'-ATT GCT GCC TCG GAA TGT TCT-3' and 5'-GCT GAG GTA AAA CGT CGA AAA-3' yielded a 167-base product. RNA was isolated from static and motile cells using the Qiagen Total RNA isolation kit (Qiagen, Valencia, CA) with digestion of DNA with DNAseI. Complete digestion was tested for by standard PCR with primers for the 18S gene as control. RT-PCR was performed on the cDNA template transcribed from total RNA extracted from Caco-2 cells using the Taq DNA polymerase kit and 10 mM DNTP mix, and the product DNA was sequenced and confirmed to be FAK. RT-PCR was accomplished using SYBRGreen, and the ABI 7700 sequence detection system (Invitrogen). Relative mRNA levels were determined by the comparative CT method (Applied Biosystems User Manual) after ascertaining that the efficiencies of amplification of the control 18S and FAK primers were similar. The resulting number of mRNA molecules was calculated by negative log transformation of the differences between CT for FAK and 18S.

In situ hybridization for FAK. Bsu36 I and SacI were used to cut a 654-bp fragment (2476–3130) from wild-type pcDNA3WTFAK. The fragment was cloned into the pBluescript vector and transcribed in vitro, digested out with RNAse-free DNAse, and purified on an acrylamide gel. The probe was then labeled with digoxigenin. The cells were fixed with 4% formaldehyde, washed with PBS, and treated with proteinase K (10 µg/ml). Prehybridization with 100 µg/ml salmon sperm was followed by hybridization with the digoxigenin labeled FAK sense or antisense. The cells were exposed to anti-digoxigenin antibody coupled to alkaline phosphatase overnight and developed with NBT/X-phos for 1 h. Hybridization with a scrambled probe of equal length in parallel migrating monolayers did not yield any staining (not shown).

Statistical analysis. Results are expressed as means ± SE, and differences between groups were evaluated using Student's t-test or linear regression for cycloheximide decay curves. RT-PCR data were analyzed by the Wilcoxon's signed-rank test (SigmaStat, SYSTAT Software, Point Richmond, CA). Statistical significance was set a priori at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FAK expression and activation are decreased at the migrating edge of expanding epithelial cell monolayers. Motility itself has been reported to stimulate the phosphorylation of FAK in intestinal epithelial and other cells. However, we reported previously that subconfluent Caco-2 cells characterized by lamellipodial extension exhibit less phosphorylated FAK than confluent smaller and nonmotile Caco-2 cells, perhaps as a result of lower FAK protein levels (54, 55). We sought to determine whether a migrating intestinal epithelial sheet more representative of intestinal restitution would also display decreased FAK protein expression. Therefore, the linear scrape technique was used to examine FAK abundance in well-differentiated human Caco-2 colonocytes, in normal rat intestinal epithelial cells (IEC-6) and less-differentiated HT-29 cells. Representative total (left) and activated (right) FAK immunocytochemistry in Caco-2 cells at 24, 36, and 48 h are shown in the top of Fig. 1. At every time point, total FAK and FAK397 immunostaining intensity were diminished in the cells migrating across the scrape compared with the cells behind the scrape. ERK immunostaining was not substantially different in the motile cells (data not shown). At the earlier time point of 24 h, only cells migrating actively across the scrape line exhibited lowered FAK immunoreactivity. After 36 and 48 h, cells at the edge of the migrating front stained more lightly, whereas those behind the front, although across the scrape line, showed higher levels of immunostaining, suggesting that motility itself was the stimulus for the reduced FAK expression. Nuclear FAK staining was also prominent, but this was masked by the hematoxylin counterstain. As shown in Fig. 1, bottom, in cells stained only with DAB, FAK immunostaining is already reduced at 12 h after wounding but nuclear staining appears to be similar in the motile and static Caco-2 cells (left). After 48 h, the intensity of nuclear staining is increased in the motile cells. However, the perinuclear region also appears to be highly reactive in these cells (right). This was true as early as 24 h after wounding (data not shown).


Figure 1
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  		Fig. 1. Total and active focal adhesion kinase (FAK) FAK397 immunostaining is lower at the migrating edge of expanding Caco-2 cell monolayers. Top: time course of Caco-2 cells migrating over a linear wound. Cells were fixed, immunostained for total (left) and activated FAK (right) and counterstained with hematoxylin. After 24 h, both total FAK and FAK397 immunoreactivity are substantially diminished in cells migrating over the wound. These decreases are maintained in motile cells 36 and 48 h after wounding. Arrows indicate direction of migration; all images are x100 magnification. Bottom: motile Caco-2 cells show lower FAK immunostaining as early as 12 h after a linear scrape (left). Nuclear FAK immunostaining in both static and motile cells appears similar in these cells not counterstained with hematoxylin. However, intensity of both nuclear and perinuclear FAK immunostaining appears elevated 48 h after wounding (right).

 
Although IEC-6 cells migrate more quickly, changes in FAK levels in the motile cells were similar to those in the slower Caco-2 cells (Fig. 2). After 12 h, immunostaining of the extensive migration zone shows lower total (top) and activated (middle) FAK, with no change in ERK expression (bottom). These results were replicated in the HT-29 colon cancer cells (data not shown).


Figure 2
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  		Fig. 2. Total and activated FAK immunoreactivity is also lower in migrating rat intestinal epithelial cell (IEC-6). After 12 h, parallel decreases in total (top) and phosphorylated immunoreactive FAK (middle) are also evident at the migrating edge of IEC-6 enterocyte monolayers with no change in the ERK control (bottom). Arrows indicate direction of migration. Each image (x100) is representative of at least 10 similar.

 
Caco-2 and IEC-6 cell motility on collagen I is increased when FAK protein levels are reduced. We then evaluated the effect of a specific exogenous reduction in FAK protein (i.e., not related to motility itself) on Caco-2 intestinal epithelial sheet migration using siRNA. In Fig. 3, left, the bars represent densitometric analysis of three separate experiments expressed as the ratio of FAK band density to that of the tubulin control; representative Western blots are shown above the bars. In initial experiments with siRNA, FAK protein levels were reduced to 32 ± 9% (P = 0.001) of mock transfected cells or cells exposed to the nontargeting scrambled control by transient transfection with siRNA targeted to FAK in Caco-2 (Fig. 3A). In the same blots, stripped and reprobed for activated FAK, FAK397 levels were also reduced (72 ± 5% of scrambled control, P = 0.007; Fig. 3B), although the proportion of phosphorylated FAK (expressed as the ratio of FAK397 to total FAK) was actually increased to 235 ± 37%, P = 0.04 (Fig. 3C), suggesting that reductions in FAK protein levels may stimulate compensatory increases in the phosphorylation of the FAK that remains.


Figure 3
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  		Fig. 3. FAK protein reduction with a specific small interfering (si) RNA (siRNA) induces a compensatory increase in FAK phosphorylation in Caco-2 cells. A: FAK protein levels, expressed as the ratio of total FAK to the tubulin control, are significantly lower in the siFAK-treated cells [70% compared with the nontargeting scrambled control (sc) (NT1), *P = 0.001]. B: the same Western blots, stripped and reprobed for the active autophosphorylated form FAK397 (pFAK), also show a significant decrease (30%, *P = 0.007) in the amount of phosphorylated FAK following siRNA treatment compared with the tubulin control. However, when pFAK is expressed as a ratio of total FAK, analysis demonstrates an increase in the proportion of the remaining FAK that is phosphorylated on Y-397 (C). Bars represent means ± SE of densitometric analysis of at least 3 experiments each; representative Western blots are shown above the bars. In A-C, protein ratios of the NT1 siRNA and the FAK siRNA-transfected cells are expressed relative to the protein ratio of the mock control.

 
We next assessed whether an equal or greater level of FAK protein inhibition would alter Caco-2 cell motility in both linear scrape and circular wound assays. Cells were grown to confluence after exposure to the requisite siRNA or nontargeting control before wounding. The initial wound was photographed. The cells were allowed to migrate for 6 and 24 h, and the wounds were photographed again. Only 24-h data are shown because Caco-2 cells do not migrate quickly, with only 8–10% of the initial wound being covered after 6 h. Fig. 4A demonstrates that after 24 h, migrating siRNA treated (siFAK) cells covered a 20% greater area (P = 0.05) after a linear wound than cells exposed to the nontargeting siRNA (NT1; left); a representative Western blot documenting FAK protein expression levels is shown. The computed area of relative migration for four experiments is shown in the bars (right). Typical Caco-2 cell migration into a circular wound 24 h after nontargeting control (NT1) or siFAK treatment is depicted in Fig. 4B. In the panels on the left, the original wound area is delineated by the white line, and the remaining wound area after 24 h is delineated by the dashed black line. Wound closure area, calculated from 12 circular wounds in two separate experiments, is 16% greater (P = 0.007) in the siRNA-treated cells than in the control cells (NT1; right).


Figure 4
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  		Fig. 4. FAK reduction with siRNA enhances Caco-2 cell migration. A: in the linear scrape model, migrating siRNA-treated Caco-2 cells cover a 20% greater area on collagen I (P = 0.05) 24 h after wounding than NT1-treated cells. A representative migration is depicted on the left; computer analysis of relative migration for 4 experiments is shown in the bars on the right. B: in the circular wound model, wound closure area, expressed as % closure of the original wound, is 16% greater (*P = 0.007) in the siFAK-treated cells. In the panels on the left, original wound area is delineated by the white line and final wound area at 24 h by the dashed black line (the top right is rotated to match orientation). Analysis of 12 circular wounds in 2 separate experiments is shown on the right.

 
To confirm that the effects of FAK protein reduction on cell motility were not limited to a colon cancer cell line, the studies were repeated in the circular wound model in normal rat intestinal epithelial IEC-6 cells (Fig. 5). Treatment with siFAK significantly reduced FAK expression to 35 ± 7% (P = 0.008) that in the NT1 cells after 72 h (Fig. 5A). Data in the bars represent densitometric analysis of five separate experiments as a ratio of tFAK to the tubulin control; typical Western blots are shown above (left). However, as in Caco-2 cells, although total FAK397 is lower in the siFAK-treated IEC-6 cells (not shown), the FAK397/tFAK ratio is elevated, suggesting a compensatory FAK activation occurs in these cells also (right). Because IEC-6 cells migrate more quickly than the Caco-2, the extent of closure of circular wounds was assessed 6 h after wounding (Fig. 5B). To the left represents circular wounds in NT1- and siFAK-treated IEC-6 cells. The white line delineates the original wound, and the broken black line delineates the remaining wound area after 6 h. To the right, as in the Caco-2, wound closure was greater (19%; P = 0.009) in the siFAK-treated cells than in either the mock (not shown) or nontargeting (NT1) siRNA-treated cells. Bars represent computer analysis of 6–11 circular wounds each in two separate experiments. Although the wounds were nearly closed at 24 h, the extent of wound closure was still greater in siRNA-treated IEC-6 cells (89 ± 2% vs. 83 ± 2% in NT-1-treated cells; P = 0.01). Thus reductions in FAK protein expression enhance cell motility in two different cell types in two different wound models. The possibility exists, however, that lowered total FAK protein levels and concurrent compensatory increases in FAK397 are coupled with upregulation of another protein or proteins that may affect cell motility.


Figure 5
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  		Fig. 5. Decreasing FAK protein abundance also increases IEC-6 cell motility on collagen I. A: siFAK lowered FAK protein abundance to 35 ± 7% (*P = 0.008) of that in IEC-6 cells treated with the NT1. Densitometric analysis of 5 separate experiments is shown in the bars on the left; a representative Western blot is shown above the bars. Although pFAK levels are lower in the siFAK-treated cells, when expressed as the ratio of total FAK, the remaining FAK is more highly phosphorylated (bars and Western blot on the right; pFAK/tFAK ratio of the NT1 siRNA and the FAK siRNA-transfected cells is expressed relative to the pFAK/tFAK ratio of the Mock control, *P = 0.01, n = 5). B: IEC-6 cell motility in the circular wound model. Left: original wound area is outlined by the white line, and final wound area after 6 h by the dashed black line. Quantitation of cell migration as % closure of the original wound is shown in the bars at the right. Wound closure is 19% greater (*P = 0.009) in the siFAK-treated compared with the NT1-treated cells. Five to eleven wounds in two separate experiments were analyzed.

 
FAK protein reductions in motile cells are the result of transcriptional control and not changes in protein degradation. Because FAK protein levels appeared reduced during migration and because the siRNA studies suggested that such reductions could be biologically significant, we next asked whether this reduction might be regulated by alterations in protein stability and/or mRNA expression. Neither the linear scrape nor the circular wound models readily provide the uniform cell populations required for biochemical and/or molecular analysis. We therefore seeded Caco-2 cells at different densities as previously described (55) so that 4 days later, one population of cells was static and contact inhibited, whereas the other consisted of small islands of migrating cells. We have previously shown that these two populations of Caco-2 cells established in this manner are characterized by similar increases in cell number and similar cell differentiation as assessed by alkaline phosphatase and dipeptidyl dipeptidase specific activity (55). To confirm that our results in the differential density seeding model were not unique to the model, we also quantitated FAK expression in the multiple-scrape model of static/migrating cells. As assessed by Western immunoblot and shown in Fig. 6A, total FAK expression was significantly decreased (32%; P = 0.036, n = 5 separate experiments) in sparsely seeded motile cells compared with confluent static cells. These results were replicated in the multiple-scrape model (Fig. 6B). These levels do not mirror the fairly large reductions observed with immunocytochemistry, perhaps because the differential density seeding and multiple-scrape models are not homogeneous and contain islands of confluent cells that express higher FAK levels. Cycloheximide decay curves did not demonstrate substantial differences in FAK protein stability between static and motile cells in three separate experiments (Fig. 6C); the slope of decay in static (–0.8 ± 0.08) and in motile cells (–0.83 ± 0.18, P = 0.877) was similar by linear regression analysis (superimposed dashed lines). However, the y-intercepts were significantly different (79.7 ± 2.3 in static vs. 59.7 ± 5.3 in motile; P = 0.0130), reflecting the decreases in FAK expression in motile cells and corroborating previously published observations (55). In a parallel study, we transfected motile Caco-2 cells with hemaglutinnin-tagged wild-type FAK or a FAK Y397F mutant that does not autophosphorylate, and then we examined the decay of these constructs after cycloheximide blockade of new protein synthesis. The stability of the construct that could not be autophosphorylated did not differ from that of HA-tagged wild-type FAK (n = 3; Fig. 6D), suggesting that the higher ratio of phosphorylated FAK in motile cells does not contribute to the lower FAK protein levels seen in those cells.


Figure 6
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  		Fig. 6. FAK protein degradation is not a major contributor to lowered FAK expression in motile Caco-2 cells. A: in the differential-density-seeding model, motile (subconfluent) cells express less FAK protein than their static (confluent) counterparts (*P = 0.036). Bars represent densitometric analysis of 5 separate experiments; a representative Western blot is shown above the bars. B: similar results were obtained in the multiple scrape model. C: in the differential-density-seeding model, linear regression and correlation analysis demonstrates that, although initial FAK expression is lower in motile cells (y-intercept = 59.7 ± 5.3 vs. 79.7 ± 2.3 in static cells, *P = 0.013), timed FAK protein decay in the presence of 10 µg/ml cycloheximide is similar. The slope of decay in the motile cells, –0.83 ± 0.18, is not different from that in the static cells, –0.80 ± 0.08. (dashed line; calculated linear regression; SE are not shown). D: FAK phosphorylation does not affect the rate of protein degradation because motile Caco-2 cells transfected with the FAK-397F kinase inactive construct show the same rate of decay as cells transfected with wild-type FAK (wtFAK); n = 3 in each case.

 
In contrast, quantitative real time RT-PCR using 18S primers as control demonstrated a significant (P = 0.008 by Wilcoxon signed rank test) decrease in FAK mRNA levels in the motile cells (Fig. 7A). Relative expression in subconfluent and confluent cells was calculated from the difference in the relative number of cycles required to amplify the signal compared with that of the 18S control and is the aggregate of eight experiments. In situ hybridization in the linear scrape model also revealed lower FAK mRNA levels at the migrating front of Caco-2 monolayers, and some increase in nuclear staining in the motile cells (Fig. 7B). These mRNA changes parallel those in FAK protein that we had observed in migrating gut epithelial sheets in vitro (Fig. 1), as well as previously in the differential density seeding model (55).


Figure 7
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  		Fig. 7. Motile Caco-2 cells express less FAK mRNA. A: RT-PCR analysis indicates that FAK mRNA expression in motile (subconfluent) cells is significantly lower than that in static (confluent) cells (P < 0.05 by Wilcoxon's ranked sum test). Error bars are not shown because of log transformation of the data. 18S primers were used as control. B: similarly, in situ hybridization demonstrates less staining for FAK mRNA at the migrating edge of Caco-2 cells. Arrow denotes direction of migration.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although FAK function has generally been characterized as acutely regulated by phosphorylation, these results suggest that FAK protein levels may also be critically modulated in intestinal epithelial cells and gut mucosal wounds. Changes in the amount of FAK protein can then affect the total amount of phosphorylated (active) FAK, which appears to be more important in the regulation of motility than changes in the proportion of Y397 phosphorylated to total FAK. Finally, our studies in Caco-2 cells raise the possibility that FAK may actually be regulated at the mRNA level as well.

FAK may be phosphorylated at a number of tyrosine and/or serine sites by autoactivation on binding to integrins or in response to a number of growth factors and other stimuli (10, 19, 47). Because the ulcer environment may differ in terms of growth factors or other mediators of FAK phosphorylation, we chose to focus on auto-activated FAK397. Although changes in FAK phosphorylation can clearly affect diverse aspects of cell biology, it has been less clear whether the important factor was the amount of phosphorylated FAK or the proportion of FAK that is phosphorylated. The siRNA studies suggest that the amount of phosphorylated FAK may be more critical, at least for Caco-2 and IEC-6 cell motility, and that alterations in FAK protein levels can affect the amount of phosphorylated FAK in important ways.

Interestingly, the amount of 397-phosphorlylated FAK decreased less than the amount of total FAK in response to siRNA targeted to FAK. This apparent increase in the proportion of phosphorylated FAK immunoreactivity to total FAK immunoreactivity after siRNA transfection could reflect a compensatory pathway that triggers increased phosphorylation and activation of the remaining FAK in cells where total FAK is markedly reduced. Alternatively, the relative inaccessibility of nuclear mRNA to siRNA targeting might contribute to this observation. It is possible that the nuclear FAK mRNA does not get shuttled out of the nucleus in time to be bound by the siRNA. The resulting FAK product could include a preponderance of alternatively spliced forms that could be more likely to be 397-phosphorylated than the more common protein. Such mechanisms have been proposed for other proteins (24). A similar nuclear retention of FAK mRNA could be responsible for the enhanced nuclear staining seen in motile cells by in situ hybridization.

In any case, it is clear that decreasing FAK protein by siRNA decreases the total amount of phosphorylated FAK as well and that this, in turn, may increase Caco-2 and IEC-6 cell motility. This observation appears to be at odds with previous reports that inhibiting FAK phosphorylation reduces epithelial motility (5) and malignant invasion (37). Our results may reflect differences in cell type and matrix, method, and extent of FAK reduction or stimulus for migration. For example, a 70% reduction of FAK protein levels by siRNA decreased colony formation and migration in soft agar in response to serum or EGF in the H1299 lung cancer cell line (7). Although there are exceptions (11), most previous studies investigating the relationship of FAK activation and motility were carried out in fibroblasts plated on fibronectin (10, 27). Westhoff et al. (47) reported that expression of the 4–9F-FAK interfering mutant in FAK-null fibroblasts disrupts F-actin assembly, adhesion, and spreading, focusing on the relationship of FAK activation and motility. However, fibroblasts migrate as single cells, whereas epithelial cells exhibit sheet migration, an effect that is slowed or inhibited by fibronectin in some cell types (52). In confluent HeLa cells plated on collagen, a >90% reduction in FAK expression by siRNA resulted in increased motility across a linear wound, corroborating our results. The authors replicated their results in BJ human diploid fibroblasts on collagen. HeLa cells overexpressing the kinase-deficient FAK-related nonkinase also exhibited enhanced migration on collagen (52), suggesting that matrix may be more important than cell type in its effects on FAK regulation of motility (55). The fact that in our studies, small intestinal IEC-6 cell motility was also greater when FAK protein levels were lower indicates that this effect is not isolated to one cell line and may be important to diverse cells that undergo restitution by epithelial sheet migration. Furthermore, although reductions in FAK expression may influence proliferation, the early time point of the effect (6 h) in the IEC-6 cells strongly suggests that wound closure in our model is primarily related to changes in cell motility.

Cycloheximide decay curves suggest that FAK protein levels in cells in which protein synthesis has been blocked are decreased by ~50% at 24 h regardless of whether the cells are static or motile in the 4-day differential density seeding model. FAK immunoreactivity in motile cells at the edge of a confluent monolayer in which protein synthesis has not been blocked appears even less intense than that which is observed in static cells in which protein synthesis has not been blocked. This apparent difference could reflect differences in the linearity of the assays used to visualize immunoreactivity, Western blot and immunocytochemistry, because the latter is not designed to be truly quantitative as well as differences in the models used. In addition, the FAK redistribution in motile cells described by us previously (55) may give the overall appearance of much lighter staining in these low-power micrographs. Finally, it remains possible that increased cell areas in the motile cells may spread cellular proteins over a broader area, decreasing the intensity of immunoreactivity at any given point somewhat. However, our comparative immunocytochemical stains for ERK suggest that the decrease in FAK immunoreactivity in cells at the migrating fronts is much more substantial than the decrease in immunoreactivity for this other intracellular signal protein. In addition, although the cycloheximide studies presented here do not indicate a contribution of FAK protein degradation to these differences, it remains possible that there is some difference in FAK protein degradation in motile cells too small to be detected in our experiment.

Immunostaining data in Caco-2 cells also demonstrated nuclear immunoreactivity for FAK, suggesting that FAK, or some of its degradation products, may be retained in the nucleus or shuttled back to the nucleus as a consequence of motility. Although the nuclear presence of full-length FAK protein has been documented, NH2-terminal fragments of FAK are normally more abundant in the nucleus and may accumulate there in response to membrane receptor activation, pathology, or apoptosis (6, 13, 14, 22, 53). Some of these NH2-terminal fragments may include the kinase domain, but all such fragments that have been described lack the COOH-terminal focal adhesion targeting domain, and they are usually not phosphorylated on Y397 (14, 22, 40, 53). The monoclonal antibody used for immunocytochemical staining for total FAK was targeted at the FAK kinase domain and may have recognized some FAK fragments as well as the total molecule. Thus cytosolic FAK immunocytochemical staining may be lower in motile cells, in part, as a result of nuclear aggregation of FAK or FAK degradation products. However, the antibody used for Western analysis of FAK abundance in motile cells was directed at the NH2 terminus and would have recognized both FAK and all of the relevant FAK fragments. These Western blots also clearly demonstrate a significant decrease in FAK protein levels and would not have been artifactually affected by distinctions between FAK itself and FAK fragments. Whereas the precise magnitude of the decrease in FAK protein in motile cells indubitably depends on many factors, taken together, all of our results are clearly consistent with an overall substantial and statistically significant decrease in FAK protein in motile gut epithelial cells in vitro.

The mechanisms by which FAK may affect motility are likely to be complex and beyond the scope of the current study. FAK activation can affect integrin binding affinity for matrix proteins via inside-out signaling (45). Activated FAK also participates in a complex intracellular signal cascade that might regulate processes as diverse as cytoskeletal function (41), activation of the myosin motor (42), integrin organization (12), and responses to growth factors (26, 33). Indeed, it has been suggested that the increased cell spreading seen with FAK inhibition may be related to upregulation of some component involved in the regulation of cytoskeletal dynamics (52). Furthermore, it seems likely that the consequences of FAK activation may also depend on the location of FAK within the cell (e.g., lamellipodium vs. trailing edge) (9).

The cell culture studies suggest that the decrease in FAK we observed at the migrating edge of gut epithelial wounds may reflect a decrease in FAK protein as a consequence of motility itself. Indeed, at the later time points, cells that had migrated over the scrape line and were no longer at the migrating front exhibited FAK immunostaining levels similar to those in the cells behind the scrape. This is consistent with previous reports that both FAK protein and mRNA levels are higher in proliferating prostatic epithelial cells (46). It is possible that cell motility and its associated FAK phosphorylation invoke some sort of negative feedback loop that, over time, inhibits FAK synthesis in a compensatory manner. Alternatively, lesser FAK protein reductions or modulation of FAK within specific parts of the cell (such as the leading or trailing edge) might yield different effects. Taken together with our in vitro findings that decreasing FAK protein levels enhances motility, the observation that motile cells express less FAK raises the possibility that this decrease in FAK may be teleologically appropriate because it could be presumed to potentiate mucosal healing.

These observations do not contradict the numerous reports that have elucidated control of FAK by influencing its phosphorylation in response to an increasingly wide variety of factors, including matrix proteins and integrins (50), motility itself (54, 55), GPCRs (32, 39), and other growth factor receptors (15, 16). Rather, the present results suggest that alterations in the amount of FAK available to be phosphorylated after exposure to such stimuli may have important consequences for the amplitude of subsequent biological responses. Because the cycloheximide decay curves suggest that FAK protein is relatively stable, it is possible that the importance of FAK protein regulation might not be appreciated in cell culture models that evaluate more rapid responses to stimuli. Indeed, although our results do not preclude a role for FAK regulation by changes in protein stability (4), they suggest that at least one fundamental control mechanism for FAK may reside at the mRNA level.

In summary, these results suggest that FAK protein levels are regulated in migrating gut epithelial cells, most probably at the mRNA level. The regulation of FAK signaling is likely to be a complex summation of cellular processes that alter the levels of the FAK protein substrate as well as the molecules that induce or inhibit its phosphorylation. Changes in FAK protein levels in healing gut mucosa may therefore be pathophysiologically significant for mucosal healing or the lack thereof. The reduction in FAK seen in migrating gut epithelial cells suggests that this molecule may in the future prove an attractive target for interventions to promote mucosal healing.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Basson, Chief, Surgical Service, John D. Dingell VA Medical Center, 4646 John R. St., Detroit, MI 48201–1932 (email: marc.basson{at}va.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Agochiya M, Brunton VG, Owens DW, Parkinson EK, Paraskeva C, Keith WN, and Frame MC. Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells. Oncogene 18: 5646–5653, 1999.[CrossRef][ISI][Medline]
  2. Basson MD, Modlin IM, and Madri JA. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J Clin Invest 90: 15–23, 1992.[ISI][Medline]
  3. Cao Y, Baig MR, Hamm LL, Wu K, and Simon EE. Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase. Biochem Biophys Res Commun 328: 560–566, 2005.[CrossRef][ISI][Medline]
  4. Gatti A. Nerve growth factor (NGF) induces a rapid and sustained downregulation of the focal adhesion kinase (FAK). Cell Mol Neurobiol 24: 461–475, 2004.[CrossRef][ISI][Medline]
  5. Gilmore AP and Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell 7: 1209–1224, 1996.[Abstract]
  6. Golubovskaya V, Finch R, and Cance WG. 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, 2005.[Abstract/Free Full Text]
  7. Han EK, McGonigal T, Wang J, Giranda VL, and Luo Y. Functional analysis of focal adhesion kinase (FAK) reduction by small inhibitory RNAs. Anticancer Res 24: 3899–3905, 2004.[ISI][Medline]
  8. Hauck CR, Sieg DJ, Hsia DA, Loftus JC, Gaarde WA, Monia BP, and Schlaepfer DD. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Res 61: 7079–7090, 2001.[Abstract/Free Full Text]
  9. Hsia DA, Mitra SK, Hauck CR, Streblow DN, Nelson JA, Ilic D, Huang S, Li E, Nemerow GR, Leng J, Spencer KS, Cheresh DA, and Schlaepfer DD. Differential regulation of cell motility and invasion by FAK. J Cell Biol 160: 753–767, 2003.[Abstract/Free Full Text]
  10. Hunger-Glaser I, Fan RS, Perez-Salazar E, and Rozengurt E. PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation. J Cell Physiol 200: 213–222, 2004.[CrossRef][ISI][Medline]
  11. Iliâc D, Genbacev O, Jin F, Caceres E, Almeida EA, Bellingard-Dubouchaud V, Schaefer EM, Damsky CH, and Fisher SJ. Plasma membrane-associated pY397FAK is a marker of cytotrophoblast invasion in vivo and in vitro. Am J Pathol 159: 93–108, 2001.[Abstract/Free Full Text]
  12. Ilic D, Kovacic B, Johkura K, Schlaepfer DD, Tomasevic N, Han Q, Kim JB, Howerton K, Baumbusch C, Ogiwara N, Streblow DN, Nelson JA, Dazin P, Shino Y, Sasaki K, and Damsky CH. FAK promotes organization of fibronectin matrix and fibrillar adhesions. J Cell Sci 117: 177–187, 2004.[Abstract/Free Full Text]
  13. Jones G, Machado J, and Merlo A. 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, 2001.[Abstract/Free Full Text]
  14. Jones G and Stewart G. Nuclear import of N-terminal FAK by activation of the FcepsilonR1 receptor in RBL-2H3 cells. Biochem Biophys Res Commun 314: 39–45, 2004.[CrossRef][ISI][Medline]
  15. Jones MK, Tomikawa M, Mohajer B, and Tarnawski AS. Gastrointestinal mucosal regeneration: role of growth factors. Front Biosci 4: D303–309, 1999.[Medline]
  16. Konturek PC, Konturek SJ, Brzozowski T, and Ernst H. Epidermal growth factor and transforming growth factor-alpha: role in protection and healing of gastric mucosal lesions. Eur J Gastroenterol Hepatol 7: 933–937, 1995.[ISI][Medline]
  17. Lark AL, Livasy CA, Calvo B, Caskey L, Moore DT, Yang X, and Cance WG. 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, 2003.[Abstract/Free Full Text]
  18. Levy P, Robin H, Kornprobst M, Capeau J, and Cherqui G. Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling. J Cell Physiol 177: 618–627, 1998.[CrossRef][ISI][Medline]
  19. Lim Y, Han I, Jeon J, Park H, Bahk YY, and Oh ES. Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts. J Biol Chem 279: 29060–29065, 2004.[Abstract/Free Full Text]
  20. Liu YW, Sanders MA, and Basson MD. Human Caco-2 intestinal epithelial motility is associated with tyrosine kinase and cytoskeletal focal adhesion kinase signals. J Surg Res 77: 112–118, 1998.[CrossRef][ISI][Medline]
  21. Liu YW, Sanders MA, and Basson MD. Loss of matrix-dependent cytoskeletal tyrosine kinase signals may regulate intestinal epithelial differentiation during mucosal healing. J Gastrointest Surg 3: 82–94, 1999.[CrossRef][ISI][Medline]
  22. Lobo M and Zachary I. Nuclear localization and apoptotic regulation of an amino-terminal domain focal adhesion kinase fragment in endothelial cells. Biochem Biophys Res Commun 276: 1068–1073, 2000.[CrossRef][ISI][Medline]
  23. Ma A, Richardson A, Schaefer EM, and Parsons JT. Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130(Cas). Mol Biol Cell 12: 1–12, 2001.[Abstract/Free Full Text]
  24. Martin W and Koonin EV. Introns and the origin of the nucleus-cytosol compartmentalization. Nature 440: 41–45, 2006.[CrossRef][Medline]
  25. Matkowskyj KA, Keller K, Glover S, Kornberg L, Tran-Son-Tay R, and Benya RV Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407. J Histochem Cytochem 51: 1041–1048, 2003.[Abstract/Free Full Text]
  26. Milani S and Calabro A. Role of growth factors and their receptors in gastric ulcer healing. Microsc Res Tech 53: 360–371, 2001.[CrossRef][ISI][Medline]
  27. Moissoglu K and Gelman IH. v-Src rescues actin-based cytoskeletal architecture and cell motility and induces enhanced anchorage independence during oncogenic transformation of focal adhesion kinase-null fibroblasts. J Biol Chem 278: 47946–47959, 2003.[Abstract/Free Full Text]
  28. Owen JD, Ruest PJ, Fry DW, and Hanks SK. 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, 1999.[Abstract/Free Full Text]
  29. Peterson MD, Bement WM, and Mooseker MS. An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci 105: 461–472, 1993.[Abstract]
  30. Peterson MD and Mooseker MS. An in vitro model for the analysis of intestinal brush border assembly. I. Ultrastructural analysis of cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci 105: 445–460, 1993.[Abstract]
  31. Polk DB and Tong W. Epidermal and hepatocyte growth factors stimulate chemotaxis in an intestinal epithelial cell line. Am J Physiol Cell Physiol 277: C1149–C1159, 1999.[Abstract/Free Full Text]
  32. Rankin S and Rozengurt E. Platelet-derived growth factor modulation of focal adhesion kinase (p125FAK) and paxillin tyrosine phosphorylation in Swiss 3T3 cells. Bell-shaped dose response and cross-talk with bombesin. J Biol Chem 269: 704–710, 1994.[Abstract/Free Full Text]
  33. Renshaw MW, Price LS, and Schwartz MA. Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase. J Cell Biol 147: 611–618, 1999.[Abstract/Free Full Text]
  34. Rhoads JM, Chen W, Gookin J, Wu GY, Fu Q, Blikslager AT, Rippe RA, Argenzio RA, Cance WG, Weaver EM, and Romer LH. Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism. Gut 53: 514–522, 2004.[Abstract/Free Full Text]
  35. Salazar EP and Rozengurt E. Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells. J Biol Chem 274: 28371–28378, 1999.[Abstract/Free Full Text]
  36. Schlaepfer DD, Hauck CR, and Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71: 435–478, 1999.[CrossRef][ISI][Medline]
  37. Schlaepfer DD, Mitra SK, and Ilic D. Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692: 77–102, 2004.[Medline]
  38. Schneider GB, Kurago Z, Zaharias R, Gruman LM, Schaller MD, and Hendrix MJ. Elevated focal adhesion kinase expression facilitates oral tumor cell invasion. Cancer 95: 2508–2515, 2002.[CrossRef][ISI][Medline]
  39. Seufferlein T, Withers DJ, Mann D, and Rozengurt E. Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor. Mol Biol Cell 7: 1865–1875, 1996.[Abstract]
  40. Stewart A, Ham C, and Zachary I. The focal adhesion kinase amino-terminal domain localises to nuclei and intercellular junctions in HEK293 and MDCK cells independently of tyrosine 397 and the carboxy-terminal domain. Biochem Biophys Res Commun 299: 62–66, 2002.[CrossRef][ISI][Medline]
  41. Szabo IL, Pai R, Jones MK, Ehring GR, Kawanaka H, and Tarnawski AS. Indomethacin delays gastric restitution: association with the inhibition of focal adhesion kinase and tensin phosphorylation and reduced actin stress fibers. Exp Biol Med (Maywood) 227: 412–424, 2002.[Abstract/Free Full Text]
  42. Tangkijvanich P, Melton AC, Chitapanarux T, Han J, and Yee HF. Platelet-derived growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase. Exp Cell Res 281: 140–147, 2002.[CrossRef][ISI][Medline]
  43. Tarnawski A, Szabo IL, Husain SS, and Soreghan B. Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways. J Physiol 95: 337–344, 2001.
  44. Tarnawski AS and Jones MK. The role of epidermal growth factor (EGF) and its receptor in mucosal protection, adaptation to injury, and ulcer healing: involvement of EGF-R signal transduction pathways. J Clin Gastroenterol 27, Suppl 1: S12–S20, 1998.[CrossRef][ISI][Medline]
  45. Thamiselvan V and Basson MD. Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and cytoskeletal signaling. Gastroenterology 126: 8–18, 2004.[CrossRef][ISI][Medline]
  46. Tremblay L, Hauck W, Nguyen LT, Allard P, Landry F, Chapdelaine A, and Chevalier S. Regulation and activation of focal adhesion kinase and paxillin during the adhesion, proliferation, and differentiation of prostatic epithelial cells in vitro and in vivo. Mol Endocrinol 10: 1010–1020, 1996.[Abstract]
  47. Westhoff MA, Serrels B, Fincham VJ, Frame MC, and Carragher NO. SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling. Mol Cell Biol 24: 8113–8133, 2004.[Abstract/Free Full Text]
  48. Whitney GS, Chan PY, Blake J, Cosand WL, Neubauer MG, Aruffo A, and Kanner SB. Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK. DNA Cell Biol 12: 823–830, 1993.[ISI][Medline]
  49. Withers BE, Hanks SK, and Fry DW. Correlations between the expression, phosphotyrosine content and enzymatic activity of focal adhesion kinase, pp125FAK, in tumor and nontransformed cells. Cancer Biochem Biophys 15: 127–139, 1996.[ISI][Medline]
  50. Wozniak MA, Modzelewska K, Kwong L, and Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta 1692: 103–119, 2004.[Medline]
  51. Wu SS, Chiu T, and Rozengurt E. ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase. Am J Physiol Cell Physiol 282: C1432–C1444, 2002.[Abstract/Free Full Text]
  52. Yano H, Mazaki Y, Kurokawa K, Hanks SK, Matsuda M, and Sabe H. Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. J Cell Biol 166: 283–295, 2004.[Abstract/Free Full Text]
  53. Yi X, Wang X, Gerdes AM, and Li F. Subcellular distribution of focal adhesion kinase and its related nonkinase in hypertrophic myocardium. Hypertension 41: 1317–1323, 2003.[Abstract/Free Full Text]
  54. Yu CF and Basson MD. Matrix-specific FAK and MAPK reorganization during Caco-2 cell motility. Microsc Res Tech 51: 191–203, 2000.[CrossRef][ISI][Medline]
  55. Yu CF, Sanders MA, and Basson MD. Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner. Am J Physiol Gastrointest Liver Physiol 278: G952–G966, 2000.[Abstract/Free Full Text]
  56. Zachary I. Focal adhesion kinase. Int J Biochem Cell Biol 29: 929–934, 1997.[CrossRef][ISI][Medline]



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