1 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
2 Departments of Pediatrics, Cell Biology and Anatomy, and Anesthesiology, University of North Carolina at Chapel Hill, NC 27599-7220, USA
* Present address: Departments of Anesthesiology and Cell Biology, Johns Hopkins University, Blalock 904, Baltimore, MD 21215-4904, USA
Author for correspondence (e-mail: benny.geiger{at}weizmann.ac.il)
Accepted March 20, 2001
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SUMMARY |
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Key words: pp60c-src, Tensin, Focal contacts, Fibrillar adhesions, Cell-matrix adhesions, Tyrosine phosphorylation
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INTRODUCTION |
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Several lines of evidence implicate pp60c-src and its homologs pp59fyn and pp62c-yes, in focal contact formation and modulation. The oncogenic mutant pp60v-src has been shown to interact physically with focal contacts, extensively phosphorylate target molecules in them and affect their structure (Rohrschneider, 1980; Nigg et al., 1982; Maher et al., 1985; Hirst et al., 1986; Pasquale et al., 1986; Glenney and Zokas, 1989; Tapley et al., 1989; Volberg et al., 1991). Knockout of the Src gene was reported to suppress tyrosine phosphorylation in focal contacts (Kaplan et al., 1994; Bockholt and Burridge, 1995) and affect the adhesive properties of the cells (Kaplan et al., 1995). Conversely, increased pp60c-src or pp59fyn expression increased paxillin tyrosine phosphorylation in a FAK-dependent manner (Schaller et al., 1999). While these data suggest a role for pp60c-src in the establishment of matrix adhesions, the molecular mechanism of its effect remains unclear. Particularly intriguing is the apparent inconsistency between the role of pp60c-src in focal contact assembly and the destructive effect of the deregulated pp60v-src on cell-matrix adhesion.
Previous studies have indicated that focal contacts are relatively unaffected by the absence of Src family kinases (Bockholt and Burridge, 1995) except that Src-deficient cells exhibit decreased membrane ruffling (Boyce et al., 1993), smaller focal contacts with lower overall levels of tyrosine phosphorylation (Kaplan et al., 1994) and lower levels of pp130cas phosphorylation (Bockholt and Burridge, 1995). However, the specific effects of Src family kinases on the development, morphology and composition of matrix adhesions have not been quantitatively approached.
To gain insight into the mechanism of pp60c-src effects on the formation and reorganization of matrix adhesions, we have analyzed the composition of these sites in cells derived from mice in which the Src gene, or the Src, Fyn and Yes genes were deleted. In this study we have used quantitative microscopic analysis of matrix adhesions. We have recently applied this approach, and defined two distinct types of cell-matrix adhesions namely focal contacts and fibrillar adhesions (Zamir et al., 1999). The former, contain high levels of paxillin and vinculin, are highly tyrosine-phosphorylated and are primarily associated with vß3 integrin. Fibrillar adhesions, however, contain
5ß1 integrin, low levels of paxillin and vinculin, and essentially no phosphotyrosine. The most prominent cytoskeletal component of fibrillar adhesions is tensin (Zamir et al., 1999). These fibrillar adhesions emerge from focal contacts and translocate in an actomyosin-dependent manner towards the cell center, forming tensin-rich linear or dot-like arrays (Zamir et al., 2000). Their formation can be blocked by immobilization of fibronectin on the tissue culture substrate (Katz et al., 2000) or by inhibition of actomyosin contractility (Zamir et al., 2000).
The data presented here demonstrate that both the development and segregation of focal contacts and fibrillar adhesions are altered in mouse fibroblasts deficient for Src, or for Src, Fyn and Yes. This is manifested by an earlier onset of focal contact formation, lower levels of matrix adhesion-associated phosphotyrosine, and a marked increase in the size and intensity of tensin-containing mature adhesions. Apparent accumulation of tensin in focal adhesions was also noted in cells treated with a tyrphostin which inhibits the activity of FAK and pp60c-src, indicating that tyrosine phosphorylation is involved in the molecular reorganization of matrix adhesions. These findings suggest a central role for Src family kinases in the regulation of the initial assembly of cell-ECM adhesions and in their subsequent molecular maturation.
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MATERIALS AND METHODS |
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Immunochemical reagents
Primary antibodies that were used in this study include polyclonal anti-phosphotyrosine antibodies (PT40, kindly provided by Israel Pecht and Arie Licht, The Weizmann Institute) or PT-66 (purchased from Sigma Immunochemicals Ltd., Rehovot, Israel). Monoclonal antibodies against FAK, tensin and paxillin were purchased from Transduction Laboratories (Lexington, KY) and anti-vinculin (hVin1) was from Sigma. Cy3-conjugated goat anti-mouse IgG H+L (secondary antibodies for tensin staining), and Cy3-conjugated goat anti-mouse F(ab')2 fragment were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Alexa 488-conjugated goat anti-rabbit IgG (H+L) was purchased from Molecular Probes (Eugene, OR).
Immunofluorescence staining
Cells were plated on glass coverslips precoated with 25 µg/ml bovine plasma fibronectin (Sigma). The cells were simultaneously permeabilized and fixed for 2 minutes with 0.5% Triton X-100 (Sigma), 3% paraformaldehyde (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS), and then post-fixed with 3% paraformaldehyde for additional 20 minutes. The cells were washed with PBS, incubated with primary antibodies for 40 minutes, washed again and then incubated for 40 minutes with Cy3-conjugated goat anti-mouse and Alexa 488-conjugated goat anti-rabbit antibodies. The samples were washed again with PBS, and mounted on slides using Elvanol (Mowiol 4-88, Serafon, Ashdod, Israel).
Digital immunofluorescence microscopy
Quantitative fluorescence microscopy was carried out using the DeltaVision system (Applied Precision, Issaqua, WA) attached to an inverted Zeiss Axiovert microscope using a 100X/1.3 PlanNeofluoar objective (Zeiss, Oberkochen, Germany). Images were processed using the priism software of the DeltaVision system as previously described (Zamir et al., 1999). The processing employed here included the following routines:
(1) Image filtration: original images of immunostained cells were subjected to high-pass filtration subtracting the local average intensity surrounding each matrix adhesion site.
(2) Spectral presentation of fluorescence intensity: in order to visually compare fluorescence intensities, filtered images were presented using a blue-to-red linear spectrum scale.
(3) Fluorescence ratio imaging (FRI): cells were double-labeled for pairs of matrix adhesion proteins and the intensity ratio was computed per pixel as previously described (Zamir et al., 1999). The ratio images are presented in a logarithmic, spectrum scale.
(4) Segmentation and quantitation of matrix adhesions: adhesion sites in immunofluorescently labeled cells were identified and segmented using the water algorithm (Zamir et al., 1999) in order to generate quantitative data on the area and average fluorescence intensity of individual adhesion sites.
In typical experiments all the adhesion sites in 10 cells (typically, 50-150 adhesion sites/cell) were examined for each time point, fluorescence label and cell type. The significance of differences between the average intensity values obtained in different cells was determined by the Wilcoxon rank-sum test with alpha=0.001 using the Matlab software (MathWorks, MA, USA).
Immunoprecipitation and immunoblotting
Wild-type and Src-/- cells cultured for 24 hours, were lysed in lysis buffer (20 mM Tris-HCl buffer, containing 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 25 µg/ml leupeptin and 1% deoxycholic acid, pH 8.0). The lysates were clarified by centrifugation at 14,000 g for 10 minutes at 4°C and aliquots containing equal amounts of protein (determined by Bradford assay) were incubated with antibodies to tensin for 60 minutes at 4°C. Rabbit anti mouse IgG (Jackson ImmunoResearch) bound to protein A-Sepharose was then added to the samples and incubated for additional 60 minutes at 4°C. The beads were sedimented by brief centrifugation and washed extensively with washing buffer (20 mM Tris buffer, 150 mM NaCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM sodium orthovanadate, 25 µg/ml leupeptin, 0.1% Triton X-100 and 0.1% deoxycholic acid, pH 8.0). The samples were boiled in Laemmli sample buffer with 1 mM sodium orthovanadate and subjected to 7.5% SDS-PAGE. The proteins were then transferred to Hybond-C nitrocellulose membrane (Amersham Life Science, Buckinghamshire, UK) (Towbin et al., 1992). The nitrocellulose membrane was blocked with 2% BSA in buffer containing 10 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20, pH 7.6 (Buffer A), incubated with anti phosphotyrosine antibodies (PT-66, Sigma) at 4°C for 16 hours, washed extensively with Buffer A and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Life Science) for 60 minutes. The immunoreactive bands were detected by enhanced chemiluminescence (ECL, Amersham Life Science), and exposed to X-ray film. The antibodies were stripped from the Hybond-C nitrocellulose membrane by washing with 0.1 M glycine, pH 2.9 for 20 minutes and immunoblotted this time with tensin antibodies. The immunoreactive bands were detected again by ECL procedure. The bands were quantified by densitometry using an imaging densitometer, model GS-700 (BioRad laboratories, Hercules, CA) and analyzed by NIH Image software.
Cultured cells were washed with cold PBS, scraped off the culture dish, and extracted with Laemmli sample buffer containing 1 mM sodium orthovanadate. The extracts were subjected to SDS-electrophoresis on 5-15% polyacrylamide gradient gels under reducing conditions and immunoblotted with the relevant antibodies. The detection of the bands and their analysis were conducted as described above.
Effect of tyrphostin AG1007 on Src kinase and FAK activity
For Src kinase assay, microtiter plates (96-well Maxisorp, Nunc) were coated with Poly Glu-Tyr (4:1, Sigma, 0.1 mg/ml in PBS; 100 µl/well). The plates were covered with Parafilm and incubated at 37°C for 16 hours. Excess poly Glu-Tyr was discarded and the plates were washed with TBS containing 0.2% Tween 20 (TBST) and allowed to dry at 37°C for 1-2 hours. GST-Src (kindly provided by Rothem Karni and Alex Levitzki, the Hebrew University, Jerusalem, Israel; 50 ng/well) was added to the wells in the presence or absence of 50 µM tyrphostin AG1007 (kindly provided by Aviv Gazit and Alex Levitzki, the Hebrew University, Jerusalem, Israel) (Ohmichi et al., 1993) in kinase assay buffer (20 mM Tris, pH 7.5 and 10 mM MgCl2). The kinase reaction was started by adding 20 µM ATP. The plates were incubated at 30°C for 20 minutes, on a shaker. The reaction was stopped by adding 200 mM EDTA and the plate was washed with TBST and blocked with TBST containing 5% low-fat milk. Rabbit anti-phosphotyrosine antibodies were added (100 µl /well) for 1 hour at room temperature, then washed four to six times with TBST. HRP-conjugated anti rabbit IgG (Amersham Life Science) was added and incubated for another 45 minutes at room temperature, washed 5 times with TBST and once with PBS. Then 0.5 mg/ml ABTS (2,2'-Azino-bis (3-Ethylbenzen-thiazoline-6-sulfonic acid, Sigma) and 0.004% H2O2, in citrate-phosphate buffer (100 mM citric acid and 200 mM Na2HPO4, pH 4.0) were added and incubated for 10 minutes and the optical density (405 nm) was read, using an Eliza Reader (ELX 800, Bio-Tek Industry).
For FAK autophosphorylation assay, pp125FAK was immunoprecipitated from Swiss 3T3 cells extracts using antibodies to FAK (Transduction Laboratories) and protein A-sepharose. The beads were washed with lysis buffer (20 mM Tris-HCl buffer, containing 1% Triton X-100, 150 mM NaCl, 1mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 25 µg/ml leupeptin and 1% deoxycholic acid, pH 8.0) followed by TBS and then transferred to TBS containing 3 mM MnCl2 and 10 mM piperazine-N,N'-bis (2-ethanesulfonic acid) pH 7.4. The beads were incubated with 100 µM AG1007 for 15-20 minutes at room temperature and then incubated for additional 20 minutes with 6 µCi per sample of 32P-ATP (Amersham Life Science). Samples were then boiled for 3 minutes in Laemmli sample buffer containing 1 mM sodium orthovanadate and subjected to 10% SDS-PAGE. The gel was dried and the radioactive bands were detected by using a phosphoimager (BAS 1000, Fujix).
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RESULTS |
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Immunofluorescence labeling of the cells following short (15 minutes) or long (24 hours) incubation indicated that the general morphology of tyrosine-phosphorylated focal contacts (size and subcellular location) was comparable in the various cell types, whereas the phosphorylation level of the adhesion sites in the Src-/- and SYF cells was considerably lower than that found in wild-type or wt6 cells (Fig. 1). This can be appreciated both from the fluorescence intensity patterns (Fig. 1A) and the quantitative analysis (intensity versus area) presented in the scattergrams (Fig. 1B). Plotting immunofluorescence intensity against the area of labeled adhesion sites revealed dramatic reduction in the number of intense and/or large phosphotyrosine-containing adhesion sites per cell (two- to fivefold reduction in Src-/- and SYF cells, compared with wild-type or wt6 controls). This effect was observed following either short or long incubation.
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Anti-phosphotyrosine immunoblot analysis of lysates obtained from cells following different periods of incubation after plating revealed differences in the levels of protein tyrosine phosphorylation of specific bands, between the Src-/- cells and the wild-type or wt6 controls (Fig. 2A). Specifically, phosphorylation of a 125 kDa band, corresponding to FAK, increased progressively in the wild-type cells, while in the Src-/- cells its levels were low and did not increase significantly upon incubation. At 24 hours FAK phosphorylation in the wild-type cells and wt6 cells were 1.8- and 1.4-fold higher, respectively, than those of the Src-null cells (determined by densitometry). Phosphorylation of the 68 kDa band, corresponding to paxillin, was also much lower in the Src-/- cells, compared with wild type, and increased only after 24 hours of incubation. The wt6 cells exhibited intermediate levels of phosphorylation of both proteins and the levels of their phosphorylation increased upon incubation. It is noteworthy that the total levels of FAK and paxillin in wild-type and Src-/- cells were essentially the same (Fig. 2C). To determine the effect of pp60c-src knockout on tyrosine phosphorylation of tensin, extracts of wild-type and Src-/- cells were subjected to immunoprecipitation by tensin antibodies, followed by immunoblotting for phosphotyrosine. As shown in Fig. 2B, the levels of tyrosine-phosphorylated tensin (as analyzed by densitometry and normalized for changes in total tensin levels) were markedly reduced (by 62%) in the mutant cells.
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DISCUSSION |
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In this study we have used quantitative microscopy to determine the levels of the different molecular components and physical properties of single adhesion sites. These quantitative microscopic assays were based on well calibrated immunofluorescence labeling, ratio analysis and computerized morphometry, essentially as previously described (Zamir et al., 1999; Katz et al., 2000; Zamir et al., 2000). In addition, we have classified the immunolabeled adhesion sites into two groups according to their size and intensity. The processing thus included two stages of image analysis:
(1) A general segmentation step, based on the application of the Water algorithm (Zamir et al., 1999), revealing structures with a broad spectrum of sizes and intensities. These adhesion sites are usually dominated by small and faint structures, and thus, average values of size and intensity do not reflect changes in the stress-fiber associated focal contacts or large fibrillar adhesions.
(2) Specific counting of the number of large or intense adhesions per cell. Uniform cut-off values were set so that direct comparative information can be obtained on the formation and molecular composition of large and/or intensely labeled phosphotyrosine- and tensin-containing adhesion sites in the Src-containing cells and the two mutant cell lines. As may be appreciated from Figs 1 and 3, this segmentation appears to be rather robust and small changes in the threshold levels have nearly no effect on the final conclusions.
Comparing wild-type fibroblasts to Src-/- and triple knockout cells (SYF), we detected major changes in the molecular organization of matrix adhesions. The most striking differences between Src- (or Src/Fyn/Yes-null cells) and the wild-type (or wt6) cells is the reduction in tyrosine labeling in focal contacts and the massive accumulation of tensin in these sites in the mutants. These effects appear to be primarily attributable to the absence of pp60c-src, as the single and triple knockout cells display a similar phenotype, and the re-introduction of pp60c-src into Src-/- cells (yielding the wt6 clone) fully restores the normal phenotype (e.g. focal contact phosphorylation and tensin segregation). These effects reflect changes in the molecular organization of the adhesion sites, and not merely overall change in their formation, since other components of focal contacts, like vinculin (Fig. 4) and paxillin (data not shown) did not change significantly.
An additional manifestation of Src knockout is the more exuberant recruitment of FAK into nascent focal contacts, suggesting that the initial binding of FAK is Src-independent, or even suppressed by pp60c-src. After long incubation, FAK distribution in wild-type and mutant cells was quite similar, though its enzymatic activity was probably reduced, based on the lower levels of phosphotyrosine labeling in these sites, and the notion that phosphorylation of focal contacts is primarily attributable to FAK.
Taken together, these observations raise the possibility that pp60c-src-induced phosphorylation (either direct phosphorylation by pp60c-src or phosphorylation by FAK which is activated by pp60c-src) is responsible for maturation of focal contacts, manifested here by the exit of tensin. This hypothesis should be evaluated in light of recent information on the diversity of matrix adhesions. It has been previously shown, by photobleaching recovery assays, that focal contacts are highly dynamic structures and that their constituents (like vinculin, -actinin and actin) continuously exchange with a diffusible cytoplasmic pool (Kreis et al., 1985). Recently, we have discovered a novel mechanism for the dynamic reorganization of adhesion sites. It was shown (Zamir et al., 1999) that matrix adhesions are molecularly heterogeneous and consist of classical focal contacts, which are usually large, exhibit peripheral distribution and are enriched with phosphotyrosine, paxillin, vinculin and
vß3 integrin, and fibrillar adhesions, which are centrally located and are enriched with tensin and
5ß1 integrin. It was further demonstrated that fibrillar adhesions emerge from focal contacts and translocate, in an actomyosin-dependent manner, towards the cell center (Zamir et al., 2000). The dynamic properties of focal contacts may be different in motile and stationary cells (Smilenov et al., 1999) and may be involved not just in regulating adhesion and motility but also in matrix reorganization (e.g. fibrillogenesis; Pankov et al., 2000).
The results presented here (mainly the early recruitment of FAK) are consistent with the hypothesis that pp60c-src does not promote, but rather suppresses, the establishment of initial adhesions and that it plays an important role in the dynamic segregation of mature focal contacts. This is in line with experimental data showing that excessive Src-mediated tyrosine phosphorylation, caused by the constitutively active oncogenic form, pp60v-src, might be detrimental to focal contacts, either by interfering with their early assembly or by unleashing their reorganization into fibrillar adhesions, leading to a dramatic loss of focal contacts and stress fibers (Rohrschneider, 1980; Volberg et al., 1991). It is interesting to note that excessive activity of pp60v-src is attributable to at least two factors: the insensitivity of the kinase activity to Csk-mediated downregulation (Kmiecik and Shalloway, 1987; Nada et al., 1991) and to the constitutive association of the molecule with adhesion sites (Kaplan et al., 1995; Felsenfeld et al., 1999).
Further support for the notion that focal contact reorganization (e.g. segregation of focal contacts and fibrillar adhesions) depends on tyrosine phosphorylation is obtained from the accumulation of tensin in focal contacts of the triple knockout (SYF) cells and from additional experiments in which tyrosine phosphorylation in wild-type cells was suppressed by specific tyrphostins. Thus, cells treated with AG1007, which was shown to inhibit pp60c-src and FAK in vitro, developed a Src-/- phenotype. These studies were performed in order to determine whether the effects of pp60v-src on focal contact reorganization are attributable to tyrosine phosphorylation per se, or to its capacity to act as an adapter protein, irrespective of its enzymatic activity (Kaplan et al., 1995). The data presented here favor the former possibility. It remains, however, to be determined whether pp60v-src is directly responsible for the phosphorylation events which drive focal contact reorganization or whether it exerts its effect indirectly (by activating FAK, for example).
The molecular mechanism underlying the effect of pp60c-src on focal contact reorganization is not known. Furthermore, even the full repertoire of specific molecular targets of pp60c-src, the phosphorylation of which might be responsible for the phenotype described here is not clear. The difficulty in defining the precise molecular targets of the pp60c-src effects on matrix adhesions is primarily attributable to the fact that many structural components of focal contacts are potential pp60c-src substrates. These include FAK, paxillin, tensin, talin, pp130cas, ß-integrin and P-I3K (Hirst et al., 1986; Pasquale et al., 1986; DeClue and Martin, 1987; Glenney and Zokas, 1989; Reynolds et al., 1989; Findik et al., 1990; Davis et al., 1991; Fukui and Hanafusa, 1991; Wu et al., 1991; Schaller et al., 1992, Liu et al., 1993; Sakai et al., 1994; Turner and Miller, 1994; Calalb et al., 1995; Haefner et al., 1995; Fincham and Frame, 1998; Schlaepfer and Hunter, 1998). Moreover, a variety of signaling molecules that might regulate focal contact assembly and integrin-mediated signaling were shown to be tyrosine-phosphorylated by the Src kinase (i.e. Ellis et al., 1990; Nakanishi et al., 1993). For example, the Rho family of small GTPases may be one means by which Src regulates matrix adhesion assembly. Recent data (Arthur et al., 2000) indicate that integrin-mediated cell-matrix adhesion inactivates RhoA in a Src-dependent fashion via the tyrosine phosphorylation of p190RhoGAP. This mechanism is deficient in SYF-null cells (Arthur et al., 2000).
How could tyrosine phosphorylation promote focal contact reorganization? One attractive speculation that we offer is inspired by the conformational changes that occur in pp60c-src itself after phosphorylation by Csk at position Y527 (Nada et al., 1991). It has been shown that after such phosphorylation pp60c-src folds into an enzymatically inactive closed conformation, where the SH2 domain binds to the phospho-Y527. Folding of cytoskeleton-associated molecules such as ezrin or vinculin, owing to intramolecular interactions (induced by phosphorylation or other modifications) could also modulate their interactions with the cytoskeleton and affect the cytoskeletal networks with which they interact (Bretscher et al., 1997, Johnson and Craig, 1994; Johnson and Craig, 1995). An attractive candidate for a pp60c-src target protein whose phosphorylation might affect its cytoskeletal interactions is tensin. As previously reported (Davis et al., 1991; Bockholt and Burridge, 1993) and confirmed in this study (Fig. 2). tensin is tyrosine phosphorylated in a Src-dependent manner. Tensin also contains one SH2 domain and several additional binding sites for actin (Davis et al., 1991; Lo et al., 1994), vinculin, ß-integrin (Lin and Lin, 1996) and PI3K (Auger et al., 1996), and was also found to associate with p130cas (Salgia et al., 1996).
If tyrosine phosphorylation of tensin induces intramolecular interaction between the phosphorylated site and the SH2 domain of the molecule (analogous to the effect of Y527 phosphorylation of pp60c-src) some of these cytoskeletal interactions may be perturbed and the molecule may be more readily mobilized away from the focal contact by the associated actomyosin system (Zamir et al., 2000). Direct attempts to test this hypothesis are currently underway.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Arthur, W. T., Petch, L. A. and Burridge, K. (2000). Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism. Curr. Biol. 10, 719-722.[Medline]
Auger, K. R., Songyang, Z., Lo, S. H., Roberts, T. M. and Chen, L. B. (1996). Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes. J. Biol. Chem. 271, 23452-23457.
Bockholt, S. M. and Burridge, K. (1993). Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin. J. Biol. Chem. 268, 14565-14567.
Bockholt, S. M. and Burridge, K. (1995). An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. Cell Adhes Commun 3, 91-100.[Medline]
Boyce, B. F., Chen, H., Soriano, P. and Mundy, G. R. (1993). Histomorphometric and immunocytochemical studies of src-related osteopetrosis. Bone 14, 335-340.[Medline]
Bretscher, A., Reczek, D. and Berryman, M. (1997). Ezrin: a protein requiring conformational activation to link microfilament to the plasma membrane in the assembly of cell surface structures. J. Cell Sci. 110, 3011-3018.
Burridge, K., Turner, C. E. and Romer, L. H. (1992). Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119, 893-903.[Abstract]
Burridge, K. and Chrzanowska-Wodnicka, M. (1996). Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12, 463-518.[Medline]
Calalb, M. B., Polte, T. R. and Hanks, S. K. (1995). Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol. Cell. Biol. 15, 954-963.[Abstract]
Cary, L. A., Chang, J. F. and Guan, J. L. (1996). Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109, 1787-1794.
Cary, L. A. and Guan, J. L. (1999). Focal adhesion kinase in integrin-mediated signaling. Front. Biosci. 4, D102-D113.[Medline]
Courtneidge, S. A., Fumagalli, S., Koegl, M., Superti-Furga, G. and Twamley-Stein, G. M. (1993). The Src family of protein tyrosine kinases: regulation and functions. Development 117, Suppl., 57-64.
Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q. and Chen, L. B. (1991). Presence of an SH2 domain in the actin-binding protein tensin. Science 252, 712-715.[Medline]
DeClue, J. E. and Martin, G. S. (1987). Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells. Mol. Cell. Biol. 7, 371-378.[Medline]
Ellis, C., Moran, M., McCormick, F. and Pawson, T. (1990). Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature 343, 377-381.[Medline]
Felsenfeld, D. P., Schwartzberg, P. L., Venegas, A., Tse, R. and Sheetz, M. P. (1999). Selective regulation of integrincytoskeleton interactions by the tyrosine kinase Src. Nat. Cell Biol. 1, 200-206.[Medline]
Fincham, V. J. and Frame, M. C. (1998). The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility. EMBO J. 17, 81-92.
Findik, D., Reuter, C. and Presek, P. (1990). Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase. FEBS Lett. 262, 1-4.[Medline]
Frisch, S. M., Vuori, K., Ruoslahti, E. and Chan-Hui, P. Y. (1996). Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 134, 793-799.[Abstract]
Fukui, Y. and Hanafusa, H. (1991). Requirement of phosphatidylinositol-3 kinase modification for its association with p60src. Mol. Cell Biol. 11, 1972-1979.[Medline]
Garratt, A. N. and Humphries, M. J. (1995). Recent insights into ligand binding, activation and signalling by integrin adhesion receptors. Acta Anat. 154, 34-45.[Medline]
Geiger, B., Yehuda-Levenberg, S. and Bershadsky, A. D. (1995). Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions. Acta Anat. 154, 46-62.[Medline]
Giancotti, F. G. and Ruoslahti, E. (1999). Integrin signaling. Science 285, 1028-1032.
Gilmore, A. P. and Romer, L. H. (1996). Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol. Biol. Cell 7, 1209-1224.[Abstract]
Glenney, J. R., Jr and Zokas, L. (1989). Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J. Cell Biol. 108, 2401-2408.[Abstract]
Guan, J. L. (1997). Role of focal adhesion kinase in integrin signaling. Int. J. Biochem. Cell Biol. 29, 1085-1096.[Medline]
Haefner, B., Baxter, R., Fincham, V. J., Downes, C. P. and Frame, M. C. (1995). Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain. J. Biol. Chem. 270, 7937-7943.
Hanks, S. K., Calalb, M. B., Harper, M. C. and Patel, S. K. (1992). Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc. Natl. Acad. Sci. USA 89, 8487-8491.[Abstract]
Hanks, S. K. and Polte, T. R. (1997). Signaling through focal adhesion kinase. BioEssays 19, 137-145.[Medline]
Hirst, R., Horwitz, A., Buck, C. and Rohrschneider, L. (1986). Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc. Natl. Acad. Sci. USA 83, 6470-6474.[Abstract]
Hungerford, J. E., Compton, M. T., Matter, M. L., Hoffstrom, B. G. and Otey, C. A. (1996). Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J. Cell Biol. 135, 1383-1390.[Abstract]
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]
Ilic, D., Damsky, C. H. and Yamamoto, T. (1997). Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci 110, 401-407.
Jockusch, B. M., Bubeck, P., Giehl, K., Kroemker, M., Moschner, J., Rothkegel, M., Rudiger, M., Schluter, K., Stanke, G. and Winkler, J. (1995). The molecular architecture of focal adhesions. Annu. Rev. Cell Dev. Biol. 11, 379-416.[Medline]
Johnson, R. P. and Craig, S. W. (1994). An intramolecular association between the head and tail domains of vinculin modulates talin binding. J. Biol. Chem. 269, 12611-12619.
Johnson, R. P. and Craig, S. W. (1995). F-actin binding site masked by the intramolecular association of vinculin head and tail domains. Nature 373, 261-264.[Medline]
Kaplan, K. B., Bibbins, K. B., Swedlow, J. R., Arnaud, M., Morgan, D. O. and Varmus, H. E. (1994). Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. EMBO J 13, 4745-4756.[Abstract]
Kaplan, K. B., Swedlow, J. R., Morgan, D. O. and Varmus, H. E. (1995). c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev. 9, 1505-1517.[Abstract]
Katz, B. Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K. M. and Geiger, B. (2000). Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions. Mol. Biol. Cell 11, 1047-1060.
Kmiecik, T. E. and Shalloway, D. (1987). Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 49, 65-73.[Medline]
Kries, T.E., Avnur, Z., Schlessinger, J. and Geiger, B. (1985). In: Molecular Biology of the Cytoskeleton (ed. G. Borisy, D. Cleveland and D. Murphy), pp. 45-57. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Lin, S. and Lin, D.C. (1996).Mapping of actin, vinculin, and integrin binding domains suggests a direct role of tensin in actin-membrane association Mol. Biol. Cell. 7, 389a
Liu, X., Marengere, L. E., Koch, C. A. and Pawson, T. (1993). The v-Src SH3 domain binds phosphatidylinositol 3'-kinase. Mol. Cell. Biol. 13, 5225-5232.[Abstract]
Lo, S. H., Janmey, P. A., Hartwig, J. H. and Chen, L. B. (1994). Interaction of tensin with actin and identification of its three distinct actin-binding domains. J. Cell Biol. 125, 1067-1075.[Abstract]
Maher, P. A., Pasquale, E. B., Wang, J. Y. and Singer, S. J. (1985). Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc. Natl. Acad. Sci. USA 82, 6576-6580.[Abstract]
Matsumoto, K., Nakamura, T. and Kramer, R. H. (1994). Hepatocyte growth factor/scatter factor induces tyrosine phosphorylation of focal adhesion kinase (p125FAK) and promotes migration and invasion by oral squamous cell carcinoma cells. J. Biol. Chem. 269, 31807-31813.
Nada, S., Okada, M., MacAuley, A., Cooper, J. A. and Nakagawa, H. (1991). Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature 351, 69-72.[Medline]
Nakanishi, O., Shibasaki, F., Hidaka, M., Homma, Y. and Takenawa, T. (1993). Phospholipase C-gamma 1 associates with viral and cellular src kinases. J. Biol. Chem. 268, 10754-10759.
Nigg, E. A., Sefton, B. M., Hunter, T., Walter, G. and Singer, S. J. (1982). Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide. Proc. Natl. Acad. Sci. USA 79, 5322-5326.[Abstract]
Ohmichi, M., Pang, L., Ribon, V., Gazit, A., Levitzki, A., Saltiel, A. R. (1993). The tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor. Biochemistry 32, 4650-4658[Medline]
Pankov, R. Cukierman, E., Katz, B. Z., Matsumoto, K., Lin, S., Hahn, C. and Yamada, K. M. (2000). Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 148, 1075-1090
Pasquale, E. B., Maher, P. A. and Singer, S. J. (1986). Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 83, 5507-5511.[Abstract]
Petch, L. A., Bockholt, S. M., Bouton, A., Parsons, J. T. and Burridge, K. (1995). Adhesion-induced tyrosine phosphorylation of the p130 src substrate. J. Cell Sci. 108, 1371-1379.
Rankin, S. and Rozengurt, E. (1994). 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.
Reynolds, A. B., Kanner, S. B., Wang, H. C. and Parsons, J. T. (1989). Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins. Mol. Cell. Biol. 9, 3951-3958.[Medline]
Richardson, A. and Parsons, T. (1996). A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature 380, 538-540.[Medline]
Rohrschneider, L. R. (1980). Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc. Natl. Acad. Sci. USA 77, 3514-3518.[Abstract]
Rozengurt, E. (1995). Convergent signalling in the action of integrins, neuropeptides, growth factors and oncogenes. Cancer Surv. 24, 81-96.[Medline]
Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y. and Hirai, H. (1994). A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J. 13, 3748-3756.[Abstract]
Salgia, R., Pisick, E., Sattler, M., Li, J. L., Uemura, N., Wong, W. K. et al. (1996). p130CAS forms a signaling complex with the adapter protein CRKL in hematopoietic cells transformed by the BCR/ABL oncogene. J. Biol. Chem. 271, 25198-25203
Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B. and Parsons, J. T. (1992). pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc. Natl. Acad. Sci. USA 89, 5192-5196.[Abstract]
Schaller, M. D., Hildebrand, J. D. and Parsons, J. T. (1999). Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases. Mol. Biol. Cell 10, 3489-3505.
Schlaepfer, D. D., Broome, M. A. and Hunter, T. (1997). Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol. Cell. Biol. 17, 1702-1713.[Abstract]
Schlaepfer, D. D. and Hunter, T. (1998). Integrin signalling and tyrosine phosphorylation: just the FAKs? Trends Cell Biol. 8, 151-157.[Medline]
Schlaepfer, D. D., Jones, K. C. and Hunter, T. (1998). Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol. Cell. Biol. 18, 2571-2585.
Sieg, D. J., Hauck, C. R. and Schlaepfer, D. D. (1999). Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J. Cell Sci. 112, 2677-2691.
Smilenov, L. B,. Mikhailov, A., Pelham, R. J., Marcantonio, E. E and Gundersen, G. G. (1999) Focal adhesion motility revealed in stationary fibroblasts. Science 286, 1172-1174.
Tapley, P., Horwitz, A., Buck, C., Duggan, K. and Rohrschneider, L. (1989). Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts. Oncogene 4, 325-333.[Medline]
Tarone, G., Cirillo, D., Giancotti, F. G., Comoglio, P. M. and Marchisio, P. C. (1985). Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp. Cell Res. 159, 141-157.[Medline]
Thomas, J. W., Ellis, B., Boerner, R. J., Knight, W. B., White, G. C., 2nd and Schaller, M. D. (1998). SH2- and SH3-mediated interactions between focal adhesion kinase and Src. J. Biol. Chem. 273, 577-583.
Thomas, S. M. and Brugge, J. S. (1997). Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513-609.[Medline]
Towbin, H., Staehelin, T. and Gordon, J. (1992). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979. Biotechnology 24, 145-149.[Medline]
Turner, C. E. and Miller, J. T. (1994). Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J. Cell Sci. 107, 1583-1591.
Volberg, T., Geiger, B., Dror, R. and Zick, Y. (1991). Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells. Cell Regul. 2, 105-120.[Medline]
Volberg, T., Zick, Y., Dror, R., Sabanay, I., Gilon, C., Levitzki, A. and Geiger, B. (1992). The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions. EMBO J. 11, 1733-1742.[Abstract]
Wu, H., Reynolds, A. B., Kanner, S. B., Vines, R. R. and Parsons, J. T. (1991). Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol. Cell. Biol. 11, 5113-5124.[Medline]
Yamada, K. M. and Geiger, B. (1997). Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol. 9, 76-85.[Medline]
Zachary, I. (1997). Focal adhesion kinase. Int. J. Biochem. Cell Biol. 29, 929-934.[Medline]
Zamir, E., Katz, B. Z., Aota, S., Yamada, K. M., Geiger, B. and Kam, Z. (1999). Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112, 1655-1669.
Zamir, E., Katz, M., Posen, Y., Erez, N., Yamada, K. M., Katz, B. Z., Lin, S., Lin, D. C., Bershadsky, A., Kam, Z. et al. (2000). Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2, 191-196.[Medline]
Zhao, J. H., Reiske, H. and Guan, J. L. (1998). Regulation of the cell cycle by focal adhesion kinase. J. Cell Biol. 143, 1997-2008.