Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M413165200 on April 6, 2005

J. Biol. Chem., Vol. 280, Issue 25, 23516-23522, June 24, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/25/23516    most recent
M413165200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanders, M. A.
Right arrow Articles by Basson, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanders, M. A.
Right arrow Articles by Basson, M. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

p130cas but Not Paxillin Is Essential for Caco-2 Intestinal Epithelial Cell Spreading and Migration on Collagen IV*

Matthew A. Sanders{ddagger}§ and Marc D. Basson{ddagger}||

From the {ddagger}Department of Surgery, Wayne State University, §Research Service and ||Surgical Service, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201-1932

Received for publication, November 22, 2004 , and in revised form, March 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intestinal epithelial cells exist in vivo on a basement membrane that is rich in type IV collagen and laminins and also contains fibronectin. The functional unit of the intestinal epithelium, the crypt-villus axis, includes two main distinct cell populations, the proliferating, poorly differentiated crypt cells and the mature enterocytes of the villus. Intestinal epithelial cells become progressively more differentiated as they move from the crypt up the villus and are eventually released into the lumen, being renewed over a 3–5 day period in the human intestinal epithelium (reviewed in Refs. 1–3). Although intestinal epithelial cell adhesion and migration on matrix proteins have been described by several investigators, including our own group (4–10), the signaling mechanisms by which matrix regulates intestinal epithelial cells are poorly understood. Our previous work in human Caco-2 intestinal epithelial cells, a cell line that progressively differentiates as it grows past confluence and is thus a widely used human intestinal epithelial cell in vitro model system, suggests that Src kinase is an important regulator of intestinal epithelial cell spreading and sheet migration on collagen IV and that this regulation proceeds through extracellular signal-regulated kinase (ERK)1-independent pathways (11).

Potential mediators of Src regulation of Caco-2 spreading and migration on collagen IV include the adaptor proteins p130cas and paxillin, each of which are rapidly tyrosine-phosphorylated after Caco-2 adhesion to collagen IV (12). Most studies have suggested that paxillin (13–15) and p130cas (16–18) positively regulate cell migration. Other studies have suggested, however, that paxillin inhibits cell migration (18, 19) and that p130cas is not required for migration (19). Although studies of fibroblasts obtained from knock-out mice have yielded valuable insights into the role of paxillin (20) and p130cas (16) in cell migration in this cell type, studies of this kind have not been performed in intestinal epithelial cells because of difficulties in obtaining and culturing these cells from knock-out mice. In the present work we utilized small interfering RNAs (siRNAs) specific for p130cas and paxillin to inhibit expression of these proteins and examine their role in Caco-2 intestinal epithelial cell spreading and migration on collagen IV.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium, Oligofectamine, Lipofectamine, and Plus Reagent were obtained from Invitrogen. Western blot stripping reagent was obtained from Chemicon International (Temecula, CA). Human transferrin was obtained from Roche Applied Science. Trypsin, soybean trypsin inhibitor, collagen IV, poly-L-lysine (Mr = 70,000–150,000), and horseradish peroxidase-conjugated rabbit anti-mouse IgG were obtained from Sigma. Paxillin, Crk, and p130cas monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). Phosphospecific polyclonal antibodies to focal adhesion kinase (FAK) Tyr(P)397 or FAK Tyr(P)925 were obtained from, respectively, BIOSOURCE International (Camarillo, CA) or Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific polyclonal antibodies to paxillin Tyr(P)31 or paxillin Tyr(P)118 were obtained from BIOSOURCE International. CrkL monoclonal antibody and phosphospecific polyclonal antibody to p130cas Tyr(P)165 was obtained from Cell Signaling Technology (Beverly, MA). {alpha}-Tubulin monoclonal antibody was obtained from Calbiochem. Horseradish peroxidase-conjugated donkey anti-rabbit IgG was obtained from Amersham Biosciences. Double-stranded siRNAs targeting human forms of p130cas, Crk, CrkL, and paxillin and control nontargeting siRNA 1 (NT1 siRNA) were purchased from Dharmacon (Lafayette, CO). Sequences targeted by siRNA were selected using Dharmacon Smartdesign®: p130cas, GGTCGACAGTGGTGTGTAT; Crk, ACACTATTTGGACACTACA; CrkL, GTCACAAGGATGAATATAA; and paxillin, GTGTGGAGCCTTCTTTGGT. Two additional siRNAs that targeted different paxillin sequences (CCACACATACCAGGAGATT and GAACGACAAGCCTTACTGT) were used to confirm results of the paxillin siRNA cell spreading studies. Expression vectors with hemagglutinin-tagged rat p130cas, hemagglutinin-tagged rat p130cas with a deletion of the tyrosine-phosphorylated substrate domain, and the control vector pSSR{alpha} were a generous gift from Dr. Tetsuya Nakamoto and Dr. Hisamaru Hirai (University of Tokyo).

Cell Culture—The Caco-2 intestinal epithelial cell line used for this work (Caco-2BBE) was a subclone of the original Caco-2 cell line that was selected for its ability to differentiate in culture as indicated by formation of an apical brush border and expression of brush border enzymes (21, 22). Passage 45–67 Caco-2 cells were maintained at 37 °C with 8% CO2 in Dulbecco's modified Eagle's medium with 4500 mg/liter D-glucose, 4 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml transferrin, 0.1 mM minimum Eagle's medium nonessential amino acids solution, 10 mM HEPES, pH 7.4, and 3.7 g/liter NaHCO3 supplemented with 10% fetal bovine serum.

Coating of Cell Culture Dishes—Cell culture dishes were coated with a saturating concentration (23) of collagen IV (12.5 µg/ml) in precoating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.4). Collagen IV-coated tissue culture dishes were overlaid with 1% heat-inactivated (80 °C, 30 min) bovine serum albumin in phosphate-buffered saline (2.8 mM NaH2PO4, 7.2 mM Na2HPO4, 150 mM NaCl) for 30 min at 37 °C prior to spreading studies. Migration studies were performed on collagen IV-coated Petri dishes.

Cell Lysis—Cells were lysed on ice in modified radioimmunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 1 mM ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N'N'-tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). Lysates were centrifuged at 15,000 x g for 10 min at 4 °C, and supernatants were stored at –80 °C. Protein concentrations were determined by the BCA method (Pierce). Samples were resolved on SDS-polyacrylamide gels. After transfer of gels to Immobilon P membranes (Millipore, Bedford, MA), immunoblots were detected by the ECL method (Amersham Biosciences).

Transfections—For transfection with siRNA, Caco-2 cells were plated on collagen I-coated dishes at 15–20% confluence 1 day before transfection. siRNAs were combined with Plus reagent in Opti-MEM as described previously for plasmid DNA transfection (12). Oligofectamine in Opti-MEM was used for transfection at 10 µg/ml according to the manufacturer's protocol. The final siRNA concentration was 100 nM unless indicated otherwise. After 5–8 h of transfection, 0.5 volume of Caco-2 medium containing 30% serum was added to the cells, and transfection was continued overnight.

For experiments in which cells were transfected with rat p130cas plasmid DNA, cells transfected with siRNA as described above were transfected with plasmid DNA (4 µg of vector control or rat p130cas plasmid DNA for each p100 dish) 1 day after siRNA transfection. Plasmid transfection was performed as described previously (12) except that Lipofectamine was used at 5 µg/ml. Because plasmid DNA transfection efficiency in Caco-2 cells is only 25% (10), cells were cotransfected at this time with a {beta}-galactosidase expression vector (1 µg for each p100 dish) to mark the successfully transfected cells.

Cell Spreading Assays—Transfected cells were switched to medium with 0.3% serum 18–24 h prior to the start of the experiment. Cells were then harvested using 0.02% trypsin and 0.27 mM EDTA in phosphate-buffered saline, and trypsinization was stopped using soybean trypsin inhibitor. Cells were then rinsed twice with serum-free medium containing 0.1% bovine serum albumin; after recovery for 45 min at 37 °C the cells were allowed to initially adhere for 15 min at 37 °C to collagen IV-coated dishes blocked with bovine serum albumin. Nonadherent cells were then rinsed off with serum-free medium, and cells were allowed to continue spreading in serum-free medium at 37 °C for 60 min. Cells were then fixed with 10% formalin solution and stained with Mayer's hematoxylin (Dako Corp., Carpinteria, CA). Measurements of cell area were based on at least 150 cells for each condition in each experiment. For experiments in which cells were cotransfected with rat p130cas and {beta}-galactosidase plasmid DNA, cells were stained for {beta}-galactosidase expression after spreading, and measurements of cell area were based on at least 150 lacZ-positive cells for each condition in each experiment.

Cell Migration Assays—siRNA-transfected cells were harvested by trypsinization 1 day after transfection and replated in 0.5% serum medium on collagen IV-coated Petri dishes. Cells were maintained in reduced serum medium for 2 days until reaching complete confluence, and migration assays were then performed in serum-free medium as described previously (11). Migration assays were stopped 20 h after the start of migration. Migration area was determined from the area of five or more randomly chosen fields for each condition in each experiment.

Statistical Analysis—Where indicated, results were compared using the Student's t test and considered statistically significant when p < 0.05. All experiments were done independently at least three times unless indicated otherwise.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 1.
Effect of Src inhibition on paxillin phosphorylation. Prior to adhesion cells were treated with either 10 µM PP2 or PP3 as described under "Experimental Procedures." Lysates of cells adherent to the control substrate poly-L-lysine (PLL) or collagen IV (Col) were immunoblotted with phosphospecific antibodies to paxillin Tyr(P)31 or paxillin Tyr(P)118. The paxillin Tyr(P)118 blot was stripped and reprobed for total paxillin protein. Results from one of three similar experiments are shown.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Paxillin Tyr31 and Tyr118 Phosphorylation Is Regulated by Src—We have previously observed that collagen IV-stimulated tyrosine phosphorylation of p130cas is strongly inhibited by the Src inhibitor PP2 (11). Immunoblotting with phosphospecific antibodies to paxillin Tyr(P)31 and paxillin Tyr(P)118 indicated that phosphorylation of both sites was stimulated in Caco-2 cells following adhesion to collagen IV and that this phosphorylation was also strongly inhibited by PP2 treatment. Treatment with the inactive structurally similar compound PP3 did not affect paxillin tyrosine phosphorylation (Fig. 1).

Transfection with p130cas but Not paxillin siRNA Inhibits Spreading of Caco-2 Cells on Collagen IV—Transfection with paxillin or p130cas siRNAs inhibited expression of each of these proteins by more than 90% in Caco-2 cells (Fig. 2A). p130cas siRNA transfection significantly inhibited cell spreading on collagen IV as indicated by both mean and median cell area (Fig. 2B). Transfection with paxillin siRNA, however, did not significantly inhibit either mean or median cell area (Fig. 2C). Two additional paxillin siRNAs that were equally effective in inhibiting paxillin expression also did not affect Caco-2 cell spreading (data not shown). In a subset of the experiments summarized in Fig. 2, B and C, we also examined the combined effect of p130cas and paxillin siRNA transfection on cell spreading. Combined transfection with both siRNAs resulted in significantly more inhibition of mean cell area compared with transfection with only p130cas siRNA (36.6 ± 2.7 versus 25.0 ± 2.5% inhibition, n = 4, p < 0.05). Combined treatment with these siRNAs also increased inhibition of median cell area compared with transfection with p130cas siRNA alone, but this increase did not achieve statistical significance (26.7 ± 1.1 versus 21.2 ± 1.7% inhibition, n = 4, p = 0.056). Taken together, these results suggest that p130cas has a more important role than paxillin in regulation of Caco-2 cell spreading on collagen IV.

siRNA Knock-out of p130cas Expression Does Not Affect Src or FAK Phosphorylation—In parallel with the spreading studies described above, cells transfected with p130cas siRNA were allowed to adhere to collagen IV and were lysed. p130cas can bind to Src via the Src SH2 and SH3 domains, and Src activity in C3H10T1/2–5H murine fibroblasts is modulated by p130cas protein levels (24). Because we have previously observed that Src regulates Caco-2 spreading and migration on collagen IV (11), we examined whether siRNA knock-out of p130cas may have inhibited cell spreading through an effect on Src activity. Additionally, because p130cas binds to FAK in several cell types (25, 26) and our previous work suggests a role for FAK in Caco-2 cell migration (10), we also examined whether FAK Tyr397 autophosphorylation and Src-dependent phosphorylation of FAK Tyr925 (11) were inhibited by siRNA knock-out of p130cas. Phosphorylation of Src (phosphorylated Tyr416 and nonphosphorylated Tyr527) and FAK following Caco-2 cell adhesion to collagen IV was not significantly affected by siRNA knock-out of p130cas expression (Fig. 3). This suggests that the effects of p130cas siRNA on cell spreading were not exerted through an effect on Src or FAK activity but through other proteins that interact with p130cas.



View larger version (70K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of p130cas or paxillin siRNA on cell spreading on collagen IV. A, lysates of cells transfected with NT1 control, p130cas, or paxillin (Pax) siRNAs as described under "Experimental Procedures" were immunoblotted for p130cas, paxillin, or {alpha}-tubulin (loading control). Results from one of three or more similar experiments are shown. B, p130cas siRNA-transfected cells were allowed to spread on collagen IV for 1 h after an initial 15-min adhesion as described under "Experimental Procedures." ***, p < 0.001 compared with NT1 siRNA-transfected cells. C, paxillin siRNA-transfected cells were allowed to spread on collagen IV for 1 h after an initial 15-min adhesion as described under "Experimental Procedures." For both p130cas and paxillin siRNAs, results are based on seven independent experiments. In each experiment the cell area was determined from measurements of at least 150 cells for each condition.

 
Src-phosphorylated Substrate Domain of p130cas Is Required for Cell Spreading—We examined whether the Src-phosphorylated substrate domain of p130cas is required for cell spreading by expressing rat p130cas in Caco-2 cells after siRNA knock-out of endogenous human p130cas. The region of human p130cas targeted by the p130cas siRNA used in these studies differs from rat p130cas at 6 of 19 nucleotides and thus would not be expected to inhibit rat p130cas expression. Expression of full-length rat p130cas rescued 71% of the inhibition of cell spreading caused by siRNA knock-out of human p130cas. Expression of substrate domain-deleted rat p130cas at similar levels, however, had no effect on cell spreading in p130cas siRNA-transfected cells (Fig. 4, A and B).



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 3.
Effect of p130cas siRNA on FAK and Src phosphorylation. Lysates of NT1 control siRNA and p130cas siRNA-transfected cells after adhesion to collagen IV for 30 min were immunoblotted for p130cas Tyr(P)165, FAK Tyr(P)397, FAK Tyr(P)925, Src Tyr(P)416, and nonphosphorylated Src Tyr527. Results from one of three similar experiments are shown.

 
To further confirm a role for the p130cas substrate domain in regulation of Caco-2 cell spreading on collagen IV, we examined the effect of siRNAs targeting Crk and CrkL on cell spreading. These related adaptor proteins bind via their SH2 domains to the phosphorylated substrate domain of p130cas (27, 28) and are important for cell migration in many cell types (reviewed in Ref. 29). As observed with p130cas siRNA, transfection with Crk siRNA significantly inhibited Caco-2 cell spreading on collagen IV, whereas combined transfection with Crk and CrkL siRNAs more strongly inhibited cell spreading (Fig. 4C).

p130cas siRNA Inhibits Caco-2 Sheet Migration and Lamellipodial Extension on Collagen IV—We next examined Caco-2 sheet migration and lamellipodial extension on collagen IV in a model that more closely resembles the in vivo movement of intestinal epithelial cells. Although migration assays were started 3–4 days post-transfection, examination of paxillin and p130cas expression in lysates of cells that were scraped from a region of the dish away from the migrating front when the migration assay was finished confirmed a persistent 90% inhibition of protein expression by both paxillin and p130cas siRNAs (Fig. 5A). Both p130cas siRNA and combined Crk and CrkL siRNAs strongly inhibited sheet migration of Caco-2 cells on collagen IV (Fig. 5, B and C). Compared with cells transfected with NT1 control siRNA, both p130cas siRNA and combined Crk and CrkL siRNA-transfected cells at the migrating front had fewer lamellipodial extensions (Fig. 5B, arrows). Paxillin siRNA inhibited migration to a much lesser extent than p130cas and combined Crk and CrkL siRNAs (Fig. 5C), and paxillin siRNA-transfected cells at the migrating front did not appear noticeably different from NT1 control siRNA-transfected cells (not shown). In a subset of the experiments summarized in Fig. 5C we also examined the combined effect of p130cas and paxillin siRNA transfection on cell migration. Combined transfection with p130cas and paxillin siRNAs did not significantly increase inhibition of migration compared with transfection with p130cas siRNA alone (35.5 ± 5.2% inhibition for combined siRNAs compared with 32.0 ± 4.0% inhibition for p130cas siRNA alone, n = 3).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The differential expression of integrins in intestinal epithelial cells (reviewed in Ref. 2) and basement membrane extracellular matrix proteins (reviewed in Ref. 30) along the intestinal epithelial crypt-villus axis in vivo suggests a potential role for cell-matrix interactions in regulating intestinal epithelial cell differentiation and migration. Additionally, the extracellular matrix influences restitution of mucosal wounds not only as a physical substrate but also by modulating the function of the relevant intracellular proteins and the receptors for extracellular soluble factors, which influence cell migration (reviewed in Refs. 3, 31, and 32). The effects of physical forces such as repetitive deformation on intestinal epithelial cells also appear to be matrix- and integrin-mediated (33). Our previous work (11) indicated that Src is an important regulator of Caco-2 spreading and migration on collagen IV and that Src regulates collagen IV-initiated tyrosine phosphorylation of p130cas. In this previous work we observed a small but not statistically significant inhibition of Caco-2 cell spreading on collagen IV by substrate domain-deleted p130cas expression as indicated by the percent of cells that were spread (11) rather than measurements of cell area as were done in the current study. Overexpression of dominant negative forms of multifunctional adaptor proteins with mutations or deletions of Src-phosphorylated tyrosines, however, may up-regulate other functions of these proteins. Additionally, the low transfection efficiency of plasmid DNA in Caco-2 cells makes this method inappropriate for examining protein function in more physiologically relevant sheet migration assays. We therefore used siRNA to reexamine the role of p130cas in Caco-2 cell spreading and migration on collagen IV and also to characterize the role of the adaptor protein paxillin in these processes. p130cas—Although previous studies indicate that p130cas regulates cell spreading and haptotactic migration (reviewed in Refs. 34 and 35), the role of p130cas in epithelial cell sheet migration has not been examined in as much detail. Yano et al. (19) recently observed that siRNA knock-out of p130cas expression in HeLa cells did not affect sheet migration of HeLa cells on collagen I. In contrast, our data indicated an important role for p130cas in both spreading and sheet migration of Caco-2 cells on collagen IV (Figs. 2, 4, and 5). Our results from Fig. 4 also suggested that the tyrosine-phosphorylated substrate domain of p130cas plays an important role in Caco-2 spreading. The adaptor proteins Crk, CrkL, Nck, and the phosphatase SHIP2 have been reported to interact with the phosphotyrosines in the p130cas substrate domain via their SH2 domains (reviewed in Refs. 34 and 35). Of these proteins our data indicated an important role for Crk and CrkL in regulation of Caco-2 cell spreading and migration on collagen IV (Figs. 4C and 5, B and C). Determining the role of other p130cas substrate domain-binding proteins in regulating p130cas-dependent spreading and migration of Caco-2 cells will be an interesting topic for future studies.

Several phosphatases, kinases, and adaptor proteins also interact with p130cas via its SH3 domain, its proline-rich region, or its C-terminal region (reviewed in Refs. 34 and 35; see also Refs. 36 and 37). Among those proteins that associate with p130cas under various conditions we have previously observed a role for Src in the regulation of Caco-2 spreading and migration on collagen IV (11) and for FAK in the regulation of Caco-2 migration on collagen I (10). A role for p130cas in regulating Src activity has been reported in other cell types. For example, p130cas overexpression enhances Src phosphorylation of cortactin, paxillin, and FAK in murine C3H10T1/2 fibroblasts that overexpress Src. This was not dependent on p130cas tyrosine phosphorylation, because a C-terminal fragment of p130cas lacking the tyrosine-phosphorylated substrate domain was sufficient to stimulate Src activity (24). In addition, p130cas overexpression enhances Src Tyr416 phosphorylation in HEK 293 cells (38). Our results in Fig. 3, however, suggested that siRNA knock-out of p130cas did not inhibit cell spreading on collagen IV through an inhibition of Src activation, because Src Tyr416 and Tyr527 phosphorylation and Src-dependent phosphorylation of FAK Tyr925 were not affected by p130cas siRNA. Furthermore, FAK Tyr397 autophosphorylation, which is not regulated by Src activity in this experimental system (11), was also unaffected by knock-out of p130cas, suggesting that p130cas did not affect cell spreading through an effect on FAK autophosphorylation.

Paxillin—Paxillin phosphorylation at Tyr31 and Tyr118 creates potential binding sites for the Crk and CrkL adaptor proteins (39, 40), which we observed to play an important role in regulation of Caco-2 cell spreading and migration on collagen IV (Figs. 4C and 5, B and C). In addition, C-terminal Src tyrosine kinase, which negatively regulates Src activity through phosphorylation of Src Tyr527 (41), also associates with tyrosine-phosphorylated paxillin (42). Tyr31 phosphorylation of paxillin promotes random migration of NBII-T bladder epithelial cells on collagen I, although Tyr31 phosphorylation does not affect cell spreading on this matrix protein (14). In another study, however, overexpression of paxillin but not a tyrosine phosphorylation site mutant of paxillin inhibits haptotactic migration of mouse mammary epithelial NMuMG cells, COS7 cells, and MM1 cells toward type I collagen (18). siRNA knockout of paxillin also enhances spreading and movement of HeLa cells on type I collagen, interferes with the formation of N-cadherin-mediated cell-cell contacts, and enhances activation of Rac (19). Both paxillin Tyr31 and Tyr118 underwent Src-dependent phosphorylation in collagen IV-adherent Caco-2 cells (Fig. 1). Inhibition of paxillin expression by three different siRNAs, however, did not significantly affect cell spreading on collagen IV (Fig. 2) and only slightly inhibited cell migration (Fig. 5C). Although this observation would suggest that Src phosphorylation of paxillin is not important in cell spreading and migration under these conditions, it is possible that knocking out potential binding sites for both the C-terminal Src kinase and Crk could exert opposing effects on cell spreading and migration. The strong inhibition of cell migration by p130cas siRNA, however, suggests that Crk binding to paxillin cannot compensate for the loss of Crk binding to p130cas. Although we obtained greater than 90% reduction of paxillin expression by each of the paxillin siRNAs used in the current study, it is also possible that the remaining paxillin suffices to perform its normal function in regulating Caco-2 cell spreading and migration on collagen IV.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 4.
Full-length rat p130cas but not substrate domain-deleted rat p130cas rescues cell spreading on collagen IV. Cells transfected with either NT1 or p130cas siRNA were transfected with either SSR-{alpha} control plasmid or rat p130cas expression plasmids as described under "Experimental Procedures." Cells were cotransfected with a lacZ expression plasmid to indicate transfected cells. A, remaining cells not used for spreading studies were lysed and immunoblotted for p130cas. Note that because the p130cas monoclonal antibody was raised against rat p130cas, it may bind more strongly to the transfected rat p130cas than endogenous human p130cas. Therefore, the difference in intensity of bands of human p130cas in NT1 control siRNA-transfected cells and of rat p130cas in cells transfected with rat p130cas expression vectors may not be indicative of relative expression. {Delta}SD p130Cas, rat p130cas with deletion of tyrosine-phosphorylated substrate domain. B, measurements of cell area were based on three independent experiments. In each experiment the size of at least 150 lacZ-positive cells for each condition was measured. hCas, human p130cas; rCas, rat p130cas. *, p < 0.05 compared with cells transfected with p130cas siRNA and pSSR{alpha} plasmid DNA. C, Caco-2 cells transfected with NT1 (200 nM), NT1 (100 nM) + Crk (100 nM), NT1 (100 nM) + CrkL (100 nM), or Crk (100 nM) + CrkL (100 nM) siRNAs were allowed to spread on collagen IV for 1 h after an initial 15-min adhesion as described under "Experimental Procedures." Results are based on three independent experiments. The Crk siRNA targeted a sequence common to both CrkI and CrkII. The CrkII band is shown in the immunoblot. In each experiment the cell area was determined from measurements of at least 150 cells for each condition. **, p < 0.01 compared with NT1 control siRNA; ##, p < 0.01 compared with all other siRNA treatments.

 



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 5.
Effect of p130cas, combined Crk and CrkL, or paxillin siRNA transfection on Caco-2 sheet migration on collagen IV. A, representative immunoblot demonstrating persistent siRNA inhibition of p130cas or paxillin expression at the end of migration assays 4 days after transfection. B, representative sheet migration of control NT1, p130cas, and combined Crk and CrkL siRNA-transfected Caco-2 cells on collagen IV. Arrows indicate lamellipodial extensions present in NT1 siRNA-transfected cells but greatly reduced in both p130cas and combined Crk and CrkL siRNA-transfected cells. Migration studies were performed as described under "Experimental Procedures." C, measurement of collagen IV sheet migration of Caco-2 cells transfected with NT1 control, p130cas, combined Crk and CrkL, or paxillin (Pax) siRNAs. Measurements of migration area are based on at least five randomly chosen fields for each condition in each experiment. **, p < 0.01 compared with NT1 siRNA-transfected cells; *, p < 0.05 compared with NT1 siRNA-transfected cells.

 
Studies also indicate roles for serine phosphorylation of paxillin by the p38 mitogen-activated protein kinase (43) and c-jun N-terminal kinase (44) as positively regulating, respectively, PC-12 neurite extension and rat NBII-T bladder epithelial cell migration. Other studies describe ERK1/2-mediated phosphorylation of paxillin (45, 46). Although we have observed Src-dependent ERK1/2 activation following Caco-2 adhesion to collagen IV, the ERK1/2 inhibitor PD98059 only inhibits Caco-2 migration on collagen IV by 10% (11). In addition, we have not detected activation of the p38 and c-jun N-terminal kinases following Caco-2 adhesion to collagen IV (data not shown). Thus, serine/threonine phosphorylation of paxillin by mitogen-activated protein kinases does not appear to play a major role in regulating Caco-2 migration under the experimental conditions studied here.

In conclusion, our data suggest that p130cas is an important regulator of Caco-2 intestinal epithelial cell spreading and migration on collagen IV, a major component of the intestinal epithelial basement membrane. Contrary to observations in some other cell types, however, our data suggest that paxillin is not essential for these processes. Our results also suggest that the Src-phosphorylated substrate domain of p130cas and the Crk and CrkL adaptor proteins that bind to this region are important for Caco-2 cell spreading and migration on collagen IV. Taken together with our previous observations, the results described in this manuscript suggest that Src-dependent phosphorylation of p130cas initiated by intestinal epithelial basement membrane matrix proteins such as type IV collagen may regulate intestinal epithelial cell migration.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R01DK60771 and a Veterans Affairs Merit Award (to M. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Research Service, John D. Dingell Veterans Affairs Medical Center, 4646 John R. St., Detroit, MI 48201-1932. Tel.: 313-576-1000 (ext. 4455); E-mail: aj2198{at}wayne.edu.

1 The abbreviations used are: ERK, extracellular-signal regulated kinase; FAK, focal adhesion kinase; NT1 siRNA, nontargeting control siRNA 1; siRNA, small interfering RNA; SH, Src homology. Back


   ACKNOWLEDGMENTS
 
We thank the following for technical assistance and generous gifts of expression vectors: the laboratory of Dr. Paul Skoff (Department of Anatomy, Wayne State University Medical School) for assistance and use of microscopy equipment and Drs. Tetsuya Nakamoto and Hisamaru Hirai (University of Tokyo) for providing pSSR{alpha}, hemagglutinin-p130cas, and hemagglutinin-{Delta}S.D.-p130cas expression vectors.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Basson, M. D. (1998) Clin Exp. Pharmacol. Physiol. 25, 280–285[Medline] [Order article via Infotrieve]
  2. Lussier, C., Basora, N., Bouatrouss, Y., and Beaulieu, J. F. (2000) Microsc. Res. Tech. 51, 169–178[CrossRef][Medline] [Order article via Infotrieve]
  3. Podolsky, D. K. (1999) Am. J. Physiol. 277, G495–G499[Medline] [Order article via Infotrieve]
  4. Basson, M. D., Modlin, I. M., and Madri, J. A. (1992) J. Clin. Investig. 90, 15–23[Medline] [Order article via Infotrieve]
  5. De Arcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger, M., and Simon-Assmann, P. (1996) J. Cell Biol. 133, 417–430[Abstract/Free Full Text]
  6. Lotz, M. M., Nusrat, A., Madara, J. L., Ezzell, R., Wewer, U. M., and Mercurio, A. M. (1997) Am. J. Pathol. 150, 747–760[Abstract]
  7. Lotz, M. M., Rabinovitz, I., Mercurio, A. M., Nusrat, A., Madara, J. L., Ezzell, R., and Wewer, U. M. (2000) Am. J. Pathol. 156, 985–996[Abstract/Free Full Text]
  8. Vachon, P. H., and Beaulieu, J. F. (1995) Am. J. Physiol. 268, G857–G867[Medline] [Order article via Infotrieve]
  9. Wolpert, S., Wong, M. L., and Bass, B. L. (1996) Am. J. Surg. 171, 109–112[CrossRef][Medline] [Order article via Infotrieve]
  10. Yu, C. F., Sanders, M. A., and Basson, M. D. (2000) Am. J. Physiol. 278, G952–G966
  11. Sanders, M. A., and Basson, M. D. (2004) Am. J. Physiol. 286, G547–G557
  12. Sanders, M. A., and Basson, M. D. (2000) J. Biol. Chem. 275, 38040–38047[Abstract/Free Full Text]
  13. Lamorte, L., Rodrigues, S., Sangwan, V., Turner, C. E., and Park, M. (2003) Mol. Biol. Cell 14, 2818–2831[Abstract/Free Full Text]
  14. Petit, V., Boyer, B., Lentz, D., Turner, C. E., Thiery, J. P., and Valles, A. M. (2000) J. Cell Biol. 148, 957–970[Abstract/Free Full Text]
  15. Tsubouchi, A., Sakakura, J., Yagi, R., Mazaki, Y., Schaefer, E., Yano, H., and Sabe, H. (2002) J. Cell Biol. 159, 673–683[Abstract/Free Full Text]
  16. Huang, J., Hamasaki, H., Nakamoto, T., Honda, H., Hirai, H., Saito, M., Takato, T., and Sakai, R. (2002) J. Biol. Chem. 277, 27265–27272[Abstract/Free Full Text]
  17. Goldberg, G. S., Alexander, D. B., Pellicena, P., Zhang, Z. Y., Tsuda, H., and Miller, W. T. (2003) J. Biol. Chem. 278, 46533–46540[Abstract/Free Full Text]
  18. Yano, H., Uchida, H., Iwasaki, T., Mukai, M., Akedo, H., Nakamura, K., Hashimoto, S., and Sabe, H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9076–9081[Abstract/Free Full Text]
  19. Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S. K., Matsuda, M., and Sabe, H. (2004) J. Cell Biol. 166, 283–295[Abstract/Free Full Text]
  20. Hagel, M., George, E. L., Kim, A., Tamimi, R., Opitz, S. L., Turner, C. E., Imamoto, A., and Thomas, S. M. (2002) Mol. Cell. Biol. 22, 901–915[Abstract/Free Full Text]
  21. Peterson, M. D., Bement, W. M., and Mooseker, M. S. (1993) J. Cell Sci. 105, 461–472[Abstract]
  22. Peterson, M. D., and Mooseker, M. S. (1993) J. Cell Sci. 105, 445–460[Abstract]
  23. Madri, J. A., Pratt, B. M., and Yannariello-Brown, J. (1988) Am. J. Pathol. 132, 18–27[Abstract]
  24. Burnham, M. R., Bruce-Staskal, P. J., Harte, M. T., Weidow, C. L., Ma, A., Weed, S. A., and Bouton, A. H. (2000) Mol. Cell. Biol. 20, 5865–5878[Abstract/Free Full Text]
  25. Polte, T. R., and Hanks, S. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10678–10682[Abstract/Free Full Text]
  26. Harte, M. T., Hildebrand, J. D., Burnham, M. R., Bouton, A. H., and Parsons, J. T. (1996) J. Biol. Chem. 271, 13649–13655[Abstract/Free Full Text]
  27. Salgia, R., Pisick, E., Sattler, M., Li, J. L., Uemura, N., Wong, W. K., Burky, S. A., Hirai, H., Chen, L. B., and Griffin, J. D. (1996) J. Biol. Chem. 271, 25198–25203[Abstract/Free Full Text]
  28. Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y., and Hirai, H. (1994) EMBO J. 13, 3748–3756[Medline] [Order article via Infotrieve]
  29. Stupack, D. G., Cho, S. Y., and Klemke, R. L. (2000) Immunol. Res. 21, 83–88[CrossRef][Medline] [Order article via Infotrieve]
  30. Beaulieu, J. F. (1997) Prog. Histochem. Cytochem. 31, 1–78[Medline] [Order article via Infotrieve]
  31. Dignass, A. U. (2001) Inflamm. Bowel Dis. 7, 68–77[Medline] [Order article via Infotrieve]
  32. Basson, M. D. (2001) Life Sci. 69, 3005–3018[CrossRef][Medline] [Order article via Infotrieve]
  33. Zhang, J., Li, W., Sanders, M. A., Sumpio, B. E., Panja, A., and Basson, M. D. (2003) FASEB J.
  34. O'Neill, G. M., Fashena, S. J., and Golemis, E. A. (2000) Trends Cell Biol. 10, 111–119[CrossRef][Medline] [Order article via Infotrieve]
  35. Bouton, A. H., Riggins, R. B., and Bruce-Staskal, P. J. (2001) Oncogene 20, 6448–6458[CrossRef][Medline] [Order article via Infotrieve]
  36. Abassi, Y. A., Rehn, M., Ekman, N., Alitalo, K., and Vuori, K. (2003) J. Biol. Chem. 278, 35636–35643[Abstract/Free Full Text]
  37. Di Stefano, P., Cabodi, S., Boeri Erba, E., Margaria, V., Bergatto, E., Giuffrida, M. G., Silengo, L., Tarone, G., Turco, E., and Defilippi, P. (2004) Mol. Biol. Cell 15, 787–800[Abstract/Free Full Text]
  38. Xing, L., Ge, C., Zeltser, R., Maskevitch, G., Mayer, B. J., and Alexandropoulos, K. (2000) Mol. Cell. Biol. 20, 7363–7377[Abstract/Free Full Text]
  39. Schaller, M. D., and Parsons, J. T. (1995) Mol. Cell. Biol. 15, 2635–2645[Abstract]
  40. Birge, R. B., Fajardo, J. E., Reichman, C., Shoelson, S. E., Songyang, Z., Cantley, L. C., and Hanafusa, H. (1993) Mol. Cell. Biol. 13, 4648–4656[Abstract/Free Full Text]
  41. Murphy, S. M., Bergman, M., and Morgan, D. O. (1993) Mol. Cell. Biol. 13, 5290–5300[Abstract/Free Full Text]
  42. Sabe, H., Hata, A., Okada, M., Nakagawa, H., and Hanafusa, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3984–3988[Abstract/Free Full Text]
  43. Huang, C., Borchers, C. H., Schaller, M. D., and Jacobson, K. (2004) J. Cell Biol. 164, 593–602[Abstract/Free Full Text]
  44. Huang, C., Rajfur, Z., Borchers, C., Schaller, M. D., and Jacobson, K. (2003) Nature 424, 219–223[CrossRef][Medline] [Order article via Infotrieve]
  45. Liu, Z. X., Yu, C. F., Nickel, C., Thomas, S., and Cantley, L. G. (2002) J. Biol. Chem. 277, 10452–10458[Abstract/Free Full Text]
  46. Ku, H., and Meier, K. E. (2000) J. Biol. Chem. 275, 11333–11340[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
H. Shiratsuchi, Y. Kouatli, G. X. Yu, H. M. Marsh, and M. D. Basson
Propofol inhibits pressure-stimulated macrophage phagocytosis via the GABAA receptor and dysregulation of p130cas phosphorylation
Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1400 - C1410.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. A. Sanders, D. Ampasala, and M. D. Basson
DOCK5 and DOCK1 Regulate Caco-2 Intestinal Epithelial Cell Spreading and Migration on Collagen IV
J. Biol. Chem., January 2, 2009; 284(1): 27 - 35.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. H. Craig, B. Haimovich, and M. D. Basson
{alpha}-Actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1862 - C1874.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
J. Shi and J. E. Casanova
Invasion of Host Cells by Salmonella typhimurium Requires Focal Adhesion Kinase and p130Cas
Mol. Biol. Cell, November 1, 2006; 17(11): 4698 - 4708.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Shen, Z. Jia, R. G. Nagele, H. Ichikawa, and G. S. Goldberg
Src Uses Cas to Suppress Fhl1 in Order to Promote Nonanchored Growth and Migration of Tumor Cells
Cancer Res., February 1, 2006; 66(3): 1543 - 1552.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/25/23516    most recent
M413165200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sanders, M. A.
Right arrow Articles by Basson, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sanders, M. A.
Right arrow Articles by Basson, M. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement