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Cell and Tumor Biology

Identification of Src-Specific Phosphorylation Site on Focal Adhesion Kinase: Dissection of the Role of Src SH2 and Catalytic Functions and Their Consequences for Tumor Cell Behavior

Valerie G. Brunton, Egle Avizienyte, Valerie J. Fincham, Bryan Serrels, Chester A. Metcalf III, Tomi K. Sawyer and Margaret C. Frame
DOI: 10.1158/0008-5472.CAN-04-1949 Published February 2005

Abstract

Src tyrosine kinase expression and activity are elevated during colon cancer progression. How this contributes to the malignant phenotype is not fully understood. We show that in KM12C colon carcinoma cells, expression of kinase-deficient Src proteins (SrcMF and Src251) does not alter cell growth. Src kinase activity is required for turnover of cell-matrix adhesions and, in particular, the Src-dependent phosphorylation of focal adhesion kinase (FAK) is required for their disassembly. Surprisingly, we found that expression of SrcMF or Src251 resulted in increased tyrosine phosphorylation of FAK on Tyr407, Tyr576, Tyr577, and Tyr861, which are considered to be Src kinase substrates. This Src kinase–independent phosphorylation of FAK required an intact Src SH2 domain that mediates association of Src and FAK at peripheral adhesions. Use of a novel highly potent and selective Src kinase inhibitor AP23464 combined with experiments in Src/Fyn/Yes–deficient fibroblasts showed that increased phosphorylation of FAK in cells expressing SrcMF did not require Src-like kinases. However, specific phosphorylation on Tyr925 of FAK was not evident in SrcMF- or Src251-expressing cells, and lack of Src kinase–dependent phosphorylation on this site was associated with impaired adhesion turnover. Our data show that Src kinase activity is required for adhesion turnover associated with cell migration in cancer cells and that, in addition to the catalytic activity, Src also acts as an adaptor to recruit other kinases that can phosphorylate key substrates including FAK. These studies have implications for tumor progression with respect to the use of Src kinase inhibitors.

  • Src kinase
  • focal adhesion kinase
  • colon cancer
  • cell migration

Introduction

The Src family of tyrosine kinases (SFK) have multiple cellular functions including control of proliferation, survival, and adhesion dynamics (1–4) . In colon cancer, there is frequent elevation of Src kinase activity over that found in adjacent normal mucosa (5, 6) , with activation being linked to malignant potential (7). Further increases in Src expression and activity occur in metastatic tissues, suggesting an additional role for Src in metastatic progression of colon tumors (8, 9) . Although activating mutations of Src have been reported in a small subset of metastatic tumors (10), this does not seem to be the predominant mechanism of Src activation in tumors (11, 12) . The processes by which Src signaling may contribute to a more invasive and metastatic phenotype are not fully understood. Our data suggest that the predominant effect of deregulated Src signaling in KM12C colon epithelial cancer cells is on cell-matrix and cell-cell adhesions, whereas cell growth both in vitro and in vivo is unaltered (13, 14) .

Src is located at peripheral cell-matrix adhesions (15–17) , the assembly and turnover of which regulates cell motility. Numerous reports have implicated a role for Src in cell motility with fibroblasts derived from mice in which all three ubiquitously expressed SFKs (Src, Fyn, and Yes) have been knocked out having impaired motility (18), providing genetic conformation of the requirement of SFKs for fibroblast motility. This has been linked to a requirement for Src kinase activity in the turnover of focal adhesions during directed cell motility (19, 20) .

Another tyrosine kinase found in focal adhesions is focal adhesion kinase (FAK). Upon integrin engagement, FAK is autophosphorylated on Tyr397, creating a high-affinity binding site for Src (21, 22) . It has been proposed that Src can then phosphorylate FAK on five further tyrosine residues: Tyr407 in the amino terminal portion, Tyr576 and Tyr577 within the catalytic domain (and whose phosphorylation is required for maximal FAK enzymatic activity), and Tyr861 and Tyr925 at the carboxyl terminal of FAK (23–25) . Like Src, FAK-deficient fibroblasts have motility defects linked to an inability of the cells to turnover focal adhesions during cell migration (26). Restoration of the Src/FAK signaling complex in these cells is able to reverse the migration defect (27). In other cell types, signaling downstream of the Src/FAK complex is also required for cell migration (28) and invasion (29).

Here, we show that in a colon cancer cell line (KM12C), Src activity is not required for cell growth but is necessary for the turnover of integrin-dependent cellular adhesions. We further show that Src-dependent phosphorylation of FAK specifically promotes detachment of adhesions at the trailing edge of migrating cells. Surprisingly, phosphorylation of FAK on Tyr407, Tyr566, Tyr567, and Tyr861, previously attributed to Src kinase activity, was not mediated by Src kinase but was dependent on a functional Src SH2 domain. In contrast, phosphorylation of FAK on Tyr925 was identified as Src kinase dependent and lack of phosphorylation at this site was linked with an inability of cells to extend and retract their cellular protrusions associated with cell-matrix adhesions. These findings show for the first time that the migratory phenotype of colon cancer cells is controlled by the linked activities of Src and FAK, and that recruitment of FAK to adhesion sites results in its phosphorylation by Src and other peripheral tyrosine kinases. These studies have implications for the use of Src kinase inhibitors as antimetastatic therapeutic agents.

Materials and Methods

Cell Culture. Generation of KM12C cells expressing constitutively active Src (Src527F), Src251GFP, wild-type FAK, and FAK 407-925F were as described previously (13). Chicken SrcMF (K295M, Y527F, a gift from K. Kaplan, Massachusetts Institute of Technology, Boston, MA) was inserted into the BamH1 and Sal1 sites of the retroviral expression vector pBabe. The QuikChange Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used to generate W118A (SrcMFW) and R175L (SrcMFR) mutations in SrcMF and Y925F in FAK, and the sequences were verified by sequencing. Constructs were introduced into KM12C cells, Src, Yes, and Fyn–deficient fibroblasts (SYF−/− cells, American Type Culture Collection, LGC Promochem, Teddington, United Kingdom) and FAK−/− fibroblasts (a kind gift from D. Ilic, University of California, San Francisco, San Francisco, CA) by retroviral transfer as described previously (13).

Time Lapse Video Microscopy. Cells were plated at 2.5 × 105 per well of a six-well collagen-coated (10 μg/mL, BD Biosciences, Oxford, United Kingdom) plate. After 24 hours, the medium was replaced with low calcium (0.03 mM) KGM medium (Clonetics, San Diego, CA; ref. 13) supplemented with 2.5% fetal bovine serum and individual cells monitored using a Axiovert 200 M Zeiss microscope with a 20× objective. Images were taken every 30 minutes for 18 hours from five different fields in each well using AQM Advance software (Kinetic Imaging, Nottingham, United Kingdom). Tracking Analysis software (Kinetic Imaging) was used to further analyze the motility of the protrusions. At least 100 cells were monitored for each cell line within an experiment and the experiments were repeated at least thrice.

Confocal Immunofluorescence Microscopy. Confocal imaging was carried out as described previously (13) using 1:250 antiphosphotyrosine (PY100), 1:200 anti-FAK, 1:500 antipaxillin (all from BD PharMingen, Oxford, United Kingdom), 1:500 anti-Src (EC10, Upstate Biologicals, Milton Keynes, United Kingdom), 1:100 anti-phospho-FAK Tyr407, 1:100 anti-phospho-FAK Tyr576, 1:500 anti-phospho-FAK Tyr861, or 1:500 anti-phospho-FAK Tyr925 (all from Biosource International, Nivelles, Belgium) antibodies.

Immunoprecipitation and Immunoblotting. Cell lysates were prepared in 10 mmol/L Tris (pH 7.6), 150 mmol/L sodium chloride, 10 mmol/L sodium PPi, 1 mmol/L EDTA, 10 μg/mL aprotinin, 125 mmol/L phenylmethylsulfonyl fluoride, 100 μmol/L sodium orthovanadate, and 0.5 mmol/L sodium fluoride. Following sonication (2 × 5 seconds, 10 microns; only the KM12C lysates were sonicated), cleared lysates were immunoprecipitated with either 2 μL anti-myc (9E10, Sigma, Poole, United Kingdom), 15 μL anti-Src agarose conjugate (Merck Biosciences, Nottingham, United Kingdom), 5 μL antipaxillin, 5 μL anti-p130CAS (both from BD PharMingen), 10 μL anti-FAK (30), or phosphospecific FAK antibodies (13). Immune complexes were analyzed by 7% SDS-PAGE using either 1:1,000 antiphosphotyrosine (PY100), 1:1,000 antipaxillin, 1:1,000 anti-p130CAS, 1:500 anti-FAK (all from BD PharMingen), or 1:1,000 anti-Src (clone 327, Merck Biosciences) antibodies as indicated. Total cell lysates were probed with either of the following: 1:2,000 anti-myc (clone 9B11), 1:1,000 anti-phospho-Src Tyr416 (both from New England Biolabs, Hitchin, United Kingdom), or 1:1,000 anti-Src (clone 327, Merck Biosciences) antibodies. Bound antibody was detected by incubation with anti-mouse or anti-rabbit horseradish peroxidase–conjugated secondary antibody and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, United Kingdom). Src inhibitor AP23464 (ARIAD Pharmaceuticals, Cambridge, MA) was dissolved in DMSO and added to the cells for 16 hours before preparation of cell lysates. DMSO controls had no effect on FAK phosphorylation or Src activity (results not shown).

Src Kinase Inhibitor AP23464. Using structure-based design and focused synthetic libraries of 2,6,9-trisubstituted purine analogues, a highly potent and selective ATP-binding site inhibitor, AP23464, was identified. 3 The chemical structure of AP23464 and its Src family kinase inhibition properties are described below ( Fig. 4A). The compound is highly potent (IC50 < 1 nmol/L) against Src, Fyn, and Yes kinase. Furthermore, AP23464 shows similar potent inhibition of Abl kinase, including mutant forms (31), as well as activity against a small set of other oncogenic protein kinases relative to its remarkable selectivity (≥1,000-fold) to >40 other protein kinases and ∼60 additional receptor/enzyme targets tested to date. 3

In vitro Kinase Assay. FAK immunoprecipitates were resuspended in kinase buffer [20 mmol/L Tris (pH 7.2), 10 mmol/L magnesium chloride, and 100 μmol/L sodium orthovanadate] containing 20 μmol/L ATP and incubated with a peptide encompassing amino acids 361 to 463 of FAK. The reaction was started by addition of 5 μCi [γ-32P]ATP and after 10 minutes at 30°C stopped by addition of 2× Laemmli sample buffer. The phosphorylated peptide was then detected by autoradiography following separation by 15% SDS-PAGE.

Tumorigenicity Assay. Cells (1 × 107) were injected s.c. into the right flank of 4- to 6-week-old female nude mice (Charles Rivers, Harlan, United Kingdom). Bidimensional tumor measurements were made every 3 days and the average of these measurements was used to calculate tumor volume. Ten animals were injected per cell line and mean time to sacrifice (when the tumor had reached 1,000 mm3) following injection calculated. Tumors excised at the termination of the experiment were analyzed by immunoblotting using anti-Src antibody EC10 (1:1,000, Upstate Biologicals) as described above or immunohistochemistry using standard methods. In brief, formalin-fixed, paraffin-embedded tumors were deparaffinized, rehydrated, and labeled with 1:100 anti-Src antibody (clone 327, Merck Biosciences). Detection was carried out using a biotin/streptavidin system (DAKO, Ely, United Kingdom). Slides were counterstained with hematoxylin before mounting.

Results

Expression of Kinase-Defective Src Does Not Alter Cell Growth. As Src has been implicated in the growth of tumor cells, we looked at whether expression of kinase-defective Src proteins could alter the growth properties of KM12C cells. Neither expression of SrcMF nor Src251 (20) altered the growth of the cells in culture ( Fig. 1A ). As growth of tumor cells in vitro does not always correspond to their growth in vivo, we also looked at the ability of the cells to grow as s.c. tumors in mice. There was no difference in the growth rate of tumors expressing SrcMF and Src251 compared with vector controls ( Fig. 1B). Immunohistochemical analysis of the tumors revealed that the mutant Src proteins were expressed at significantly higher levels than endogenous Src ( Fig. 1C). Western blotting with an antibody that only recognizes the exogenously expressed chicken Src proteins ( Fig. 1D) showed that the kinase-defective Src proteins were still expressed in the tumors excised at the end of the experiment. Because expression of both kinase-defective Src mutant proteins did not alter the growth of the KM12C cells, we analyzed the integrin-dependent adhesions in these cells.

Figure 1.
Figure 1.

Expression of kinase-defective Src does not alter cell growth. A, cells were plated on 24-well plates; after 24 hours, cell number was determined and thereafter for every 24 hours up to 120 hours. Points, mean of four replicate wells from a representative experiment in a series of three. B, the mean time to sacrifice of mice following s.c. injection of cells was determined as the time the tumors had taken to reach a volume of 1,000 mm3. Columns, mean; bars, SD (n = 10 animals). C, immunohistochemical images of formalin-fixed, paraffin-embedded sections of tumors following staining with an anti-Src antibody (clone 327). Scale bars, 2 μm. D, lysates from tumor xenografts analyzed by immunoblotting with an anti-Src antibody (EC10).

Src Kinase Activity Is Required for Protrusion Dynamics. We have previously shown that expression of constitutively active Src (Src527F) in KM12C cells results in the formation of integrin-dependent adhesions associated with cellular protrusions. This is a kinase-independent function of Src as expression of Src251 was sufficient to induce the formation of these peripheral adhesion complexes (13). Here, we used time lapse video microscopy to examine the movement of the peripheral protrusions present in Src-expressing cells. Movies of the images shown here are available as Supplementary Data. Cells expressing Src527F have dynamic protrusions that are constantly being extended and retracted. A representative cell is shown in Fig. 2A (top), where the cell is seen to extend a number of protrusions that can then be retracted as the cell moves. The black arrowheads show the formation of new protrusions at the leading edge of the cell, whereas the white arrowheads indicate sites of detachment at the rear. In contrast to the Src527F cells, cells expressing SrcMF ( Fig. 2A, bottom) were static with no net movement over the period of the experiment. Analysis of the movie revealed that although there was some fluctuations of the cell body, the cell remained tethered to the substratum at the same points of contact for the duration of the experiment (12 hours). Quantitation of protrusion motility (the ability of cells to extend and retract protrusions) showed that in Src527F-expressing cells, around 85% of cells had motile protrusions, which was reduced to 35% in SrcMF-expressing cells and 21% in Src251-expressing cells ( Fig. 2B). Thus, Src kinase activity is required for optimal dynamic protrusion and retraction of these Src-induced peripheral structures.

Figure 2.
Figure 2.

Src-dependent phosphorylation sites in FAK are required for protrusion dynamics. A, KM12C cells expressing Src527F (top) or SrcMF (bottom) were plated and images taken every 30 minutes over an 18-hour period. Individual stills taken at 2-hour intervals are presented from these time lapse movies and show representative cells from Src527F and SrcMF cells. The arrowheads show the relative position of an individual protrusion in each cell. Scale bars, 5 μm. B, the ability of KM12C cells expressing activated Src (Src527F), SrcMF, Src251, Src527F-FAK-wt, or Src527F-FAK-407-925F to extend or retract the protrusions was quantitated by tracking the protrusions in each individual cell in a field over the time course of the movie. Columns, mean of five individual fields, representing at least 100 cells, from a representative experiment in a series of three; bars, SD. C, individual stills depicting Src527F-FAK-407-925F-expressing cells. Scale bars, 5 μm.

The Src-Dependent Phosphorylation Sites in FAK Are Required for Protrusion Motility. To address whether Src-induced phosphorylation of FAK was involved in protrusion dynamics in KM12C cells, we expressed a FAK mutant in which the Src-dependent tyrosine phosphorylation sites (Tyr407, Tyr576, Tyr577, Tyr861, and Tyr925) were mutated to phenylalanine (FAK-407-925F). A nonmutated wild-type FAK protein (FAK-wt) was also expressed. Both proteins were expressed at equivalent levels in Src527F cells and around 10 times higher than the endogenous FAK levels (results not shown). Expression of FAK-407-925F reduced the number of cells with motile protrusions (from 85% in parental Src527F cells to 38% in FAK-407-925F-expressing cells), whereas the number of cells with motile protrusions in empty vector and wild-type FAK-expressing cells was similar to that seen in parental Src527F cells ( Fig. 2B). In many cases, the cells were able to assemble protrusions, whereas there was an inability to break the point of contact with the substratum as the cell body moves forward. An example of this is shown in Fig. 2C (movie available in Supplementary Data). This suggests that Src-dependent phosphorylation of FAK is required for release of adhesions at the rear of migrating cells.

Increased FAK Phosphorylation in Cells Expressing Kinase-Defective Src Mutants. Immunofluorescence analysis revealed an increased phosphotyrosine content associated with peripheral adhesions in cells expressing SrcMF and Src251 compared with empty vector ( Fig. 3A ). Immunoprecipitation of paxillin or the adaptor protein p130CAS, which are known substrates of Src associated with integrin adhesion complexes, showed that there was decreased phosphorylation of both in cells expressing kinase-defective Src ( Fig. 3B and C). However, the phosphotyrosine content of FAK was increased in cells expressing SrcMF or Src251 compared with vector cells ( Fig. 3D).

Figure 3.
Figure 3.

Increased FAK phosphorylation is associated with overexpression of kinase-defective Src. A, immunofluorescence images of cells following antiphosphotyrosine staining. Scale bars, 100 μm. B, cell lysates were immunoprecipitated with an antipaxillin antibody and the associated proteins analyzed by immunoblotting with antiphosphotyrosine antibody (top). The antiphosphotyrosine antibody was removed and the blot reprobed with an antipaxillin antibody (bottom). The same experiments were carried out as in B and lysates were immunoprecipitated with anti-p130CAS (C) or anti-FAK antibodies (D).

FAK Phosphorylation Occurs Independently of Src Kinase Activity. As Src is proposed to be the major kinase responsible for phosphorylation of FAK, we addressed whether inhibition of endogenous SFKs in SrcMF cells prevented the increased phosphorylation of FAK in these cells. Treatment with a highly potent and selective Src inhibitor AP23464 ( Fig. 4A ) reduced the levels of FAK phosphorylation in cells expressing Src527F to a level comparable with that seen in SrcMF cells, whereas treatment of SrcMF cells had no effect on the levels of FAK phosphorylation ( Fig. 4B, top). The concentration of AP23464 used in these experiments was able to completely block Src activity in the cells using phosphorylation of the autophosphorylation site of Src (Tyr416) as a measure of Src activity in the cells ( Fig. 4B, middle), whereas Src protein levels were unaltered ( Fig. 4B, bottom). This compound also inhibits the activity of Yes and Fyn at similar concentrations but has no effect on FAK kinase activity ( Fig. 4A). This suggests that a fraction of the FAK phosphorylation observed in cells expressing constitutively active Src cannot be attributed to endogenous SFK activity, and that in SrcMF-expressing cells the increased phosphorylation of FAK is independent of Src family kinase activity.

Figure 4.
Figure 4.

FAK phosphorylation is independent of Src kinase activity. A, chemical structure and protein kinase inhibition properties of AP23464. B, cells were treated for 16 hours with the Src inhibitor AP23464 (1 μmol/L), after which lysates were prepared and immunoprecipitated with an anti-FAK antibody (top). The same lysates were also analyzed by immunoblotting with an anti-phospho-Src Tyr416 antibody (middle). After removal of the anti-phospho-Src Tyr416 antibody, the blot was reprobed with an anti-Src antibody (bottom). C, lysates from SYF−/− cells expressing vector or SrcMF were immunoprecipitated with an anti-FAK antibody and the associated proteins analyzed by immunoblotting with an antiphosphotyrosine antibody (top). After removal of the antiphosphotyrosine antibody, the blot was reprobed with an anti-FAK antibody (bottom). D, in vitro kinase assay of FAK immunoprecipitates from SYF−/− cells expressing vector or SrcMF. [γ-32P]ATP incorporation into a fragment of FAK encompassing the FAK autophosphorylation site (Tyr397; top). Levels of FAK peptide in each assay as determined by Western blotting with an anti-FAK antibody (middle) and FAK protein in immunoprecipitates (bottom). E, FAK−/− fibroblasts reexpressing wild-type FAK or kinase-dead FAK were infected with vector or SrcMF. Phosphorylation of immunoprecipitated FAK was analyzed using an anti-phospho-Tyr397 antibody (top). Lysates were probed with an anti-myc antibody (middle) and an anti-Src antibody (bottom). F, in vitro kinase assay of FAK immunoprecipitates from FAK−/− cells expressing kinase-dead FAK as in D.

Further support for a Src kinase–independent mechanism of FAK phosphorylation came from studies in fibroblasts that lack Src, Yes, and Fyn (SYF−/− mouse embryo fibroblasts). Immunoprecipitation of FAK revealed an increased FAK phosphorylation in cells expressing SrcMF when compared with vector cells ( Fig. 4C). No other Src family members are believed to be present in the fibroblasts although Lyn expression has been found in early embryos. We found no Lyn protein in the SYF−/− cells in accordance with previous reports (ref. 18 and data not shown). Thus, the globally increased phosphorylation of FAK seen in both fibroblasts and epithelial cells by overexpression of kinase-defective Src proteins is independent of Src tyrosine kinase activity.

To address whether the increase in FAK phosphorylation was due to increased catalytic activity of FAK itself, we carried out a FAK kinase assay. We made FAK immunoprecipitates from SYF−/− cells, which alleviates the problem of Src-associated kinase activity in FAK immunoprecipitates (32). The ability of FAK immunoprecipitates to phosphorylate a small peptide encompassing the autophosphorylation site of FAK (Tyr397) showed no increase in FAK kinase activity in SrcMF cells ( Fig. 4D, top). Equivalent levels of both the exogenously added FAK peptide and the immunoprecipitated FAK ( Fig. 4D, middle and bottom) were present. Furthermore, co-expression of SrcMF and kinase-dead FAK (K454R) in FAK−/− cells revealed an increase in both total FAK phosphorylation and Tyr397 phosphorylation ( Fig. 4E). Thus, induction of FAK phosphorylation upon expression of kinase-defective Src is not due to FAK autophosphorylation in these cells.

Phosphorylation of kinase-deficient FAK on Tyr397 in FAK−/− cells has previously been reported and may be due to phosphorylation by growth factor receptors, such as epidermal growth factor and platelet-derived growth factor (32). Treatment of FAK−/− cells expressing SrcMF with the Src inhibitor AP23464 resulted in only a slight decrease in tyrosine phosphorylation of kinase-deficient FAK, suggesting that residual phosphorylation is modulated via non-Src-like kinases (results not shown). Furthermore, in vitro phosphorylation of immunoprecipitates of kinase-deficient FAK from these cells resulted in phosphorylation of FAK, which was not inhibited by AP23464, indicating association of a non-Src-like kinase with kinase-deficient FAK ( Fig. 4F).

Src SH2 Domain is Required for the Increased Phosphorylation of FAK. As the observed phosphorylation of FAK was apparently independent of Src tyrosine kinase activity and as both SrcMF and Src251 have functional SH2 and SH3 domains, we examined their involvement in the phosphorylation of FAK. Mutant versions of SrcMF, which lack either a functional SH2 or SH3 domain, were utilized. SrcMFW (SrcMF-W118A) has a point mutation in the SH3 domain (33, 34) and SrcMFR (SrcMF-R175L) has a point mutation in the SH2 domain, which disrupts binding to phosphotyrosine-containing substrates (15). Cells expressing Src527F were used for comparison. The mutants were expressed to equivalent levels in cells ( Fig. 5A, bottom ) and the phosphotyrosine content of FAK determined in FAK immunoprecipitates. As shown previously, expression of SrcMF resulted in increased phosphorylation of FAK above that seen in vector cells (3.6-fold; Figs. 3 and 5), although it was less than that observed in cells expressing Src527F (4.8-fold; Fig. 5A). Mutation of the SH3 domain of SrcMF had no effect on the phosphotyrosine content of FAK, whereas in cells expressing the SrcMF SH2 mutant there was no increase in FAK phosphorylation with levels being equivalent to those in the vector cells ( Fig. 5A, top). This could not be attributed to differences in FAK protein levels in the immunoprecipitates ( Fig. 5A, middle).

Figure 5.
Figure 5.

Src SH2 domain is required for phosphorylation of FAK. A, cell lysates were immunoprecipitated with an anti-FAK antibody; then, the associated proteins were analyzed by immunoblotting with an antiphosphotyrosine antibody (top). The antiphosphotyrosine antibody was removed and the blot reprobed with an anti-FAK antibody (middle). Cell lysates were analyzed by immunoblotting with an anti-Src antibody (bottom). B, cell lysates were immunoprecipitated with anti-Src antibodies and the associated proteins analyzed by immunoblotting with an anti-FAK antibody (top). The anti-FAK antibody was removed and the blot reprobed with an anti-Src antibody (bottom). C, immunofluorescence images of KM12C cells expressing vector, SrcMF, SrcMFW, or SrcMFR following anti-FAK staining (a-d) or anti-Src staining (e-h) are shown. Scale bars, 100 μm.

As FAK is known to associate with Src at focal adhesions, we looked at the ability of SrcMF mutants to associate with FAK. FAK co-immunoprecipitated with constitutively active Src and to a lesser extent with SrcMF (2.5-fold less association with SrcMF; Fig. 5B). Mutation of the SH3 domain in SrcMF had no effect on the ability of SrcMF to associate with FAK, whereas mutation of the SH2 domain abolished the association ( Fig. 5B). We then examined the localization of FAK within the cells by immunofluorescence. In vector-expressing cells, a very small amount of FAK localizes to the periphery of cell clusters where it is found in discrete punctate structures ( Fig. 5C, a), which co-localize with paxillin (data not shown). In cells expressing SrcMF, there is an increase in intensity of FAK staining at the cell periphery ( Fig. 5C, b). Expression of SrcMFW also resulted in increased association of FAK at the cell periphery ( Fig. 5C, c). In contrast, in cells expressing the Src SH2 mutant, there were barely detectable levels of FAK at the periphery similar to the pattern seen in vector cells ( Fig. 5C, d). An antibody that recognizes only the exogenously expressed SrcMF mutants was used to determine the localization of the SrcMF proteins within the cell. Both SrcMF and SrcMFW were found at peripheral adhesion sites located at the edge of cell clusters ( Fig. 5C, f and g), although the number of Src containing adhesions were reduced in cells with the Src SH3 mutant ( Fig. 5C, g). In contrast, mutation of the Src SH2 domain resulted in a Src protein that could not localize to peripheral adhesion sites on the outer edge of cell clusters ( Fig. 5C, h). Thus, mutation of the Src SH2 domain prevented both the formation of a Src-FAK complex and recruitment of FAK and Src to peripheral adhesion sites and indicates that the Src SH2 domain is required for Src-independent phosphorylation of FAK. SrcMF therefore recruits FAK to a signaling complex at peripheral adhesion sites where it can be phosphorylated.

Phosphorylation of FAK Tyr925 Is Required for Protrusion Motility. Phosphospecific antibodies raised against the different FAK tyrosine phosphorylation sites were used to look at peripheral FAK phosphorylation. Expression of SrcMF or Src251 resulted in increased phosphorylation of FAK at peripheral adhesions on Tyr407, Tyr576/577, and Tyr861 compared with vector cells ( Fig. 6A ). Undetectable levels of phosphorylation on FAK Tyr925 were seen in SrcMF and Src251 cells at the cell periphery ( Fig. 6A). In contrast, expression of Src527F resulted in increased phosphorylation of FAK on all tyrosine residues examined, including Tyr925 ( Fig. 6A). Increased phosphorylation of FAK on Tyr407, Tyr576/577, and Tyr861, but not Tyr925, in cells expressing kinase-defective Src proteins was confirmed by Western blotting ( Fig. 6B). The phosphorylation on Tyr407 was very low and could only be observed after longer exposures in some experiments (results not shown).

Figure 6.
Figure 6.

FAK phosphorylation in kinase-defective Src-expressing cells is site specific. A, immunofluorescence images of cells following staining with an anti-phospho-FAK Tyr407 antibody (a-d), an anti-phospho-FAK Tyr576 antibody (e-h), ananti-phospho-FAK Tyr861 antibody (i-l), or an anti-phospho-FAK Tyr925 antibody (m-p). Scale bars, 100 μm. B, cell lysates were immunoprecipitated with anti-phospho-FAK antibodies and the associated proteins analyzed by immunoblotting with an anti-FAK antibody. C, the ability of KM12C cells expressing Src527F and FAK-925F to extend or retract the protrusions was quantitated by tracking the protrusions in each individual cell in a field over the time course of the movie. Columns, mean of five individual fields, representing at least 100 cells, from a representative experiment in a series of three; bars, SD. D, cell lysates from KM12C cells expressing activated Src and FAK-925F were analyzed by immunoblotting with an anti-myc antibody.

As there is increased phosphorylation of FAK at all residues apart from Tyr925 in cells expressing the kinase-defective Src mutants, we generated a single point mutant in which only FAK Tyr925 was mutated to phenylalanine (Y925F) to address whether lack of phosphorylation on this site was linked to the reduction in protrusion dynamics. Expression of FAK-Y925F in active Src-expressing cells reduced the number of cells with motile protrusions ( Fig. 6C) and this correlated with the level of expression of the putative dominant-negative FAK-Y925F ( Fig. 6D). Results for two independent clones are shown. Thus, only phosphorylation of FAK on Tyr925 is dependent on Src kinase activity. Moreover, phosphorylation of this site is associated with the ability of cells to dynamically regulate matrix adhesions, thereby allowing extension and retraction of membrane protrusions required for cell movement.

Discussion

Role of Src in Tumor Cell Growth

Previously, we have shown that overexpression of Src does not confer a growth advantage in KM12C cells (14), which reveals that the level of endogenous Src expressed is not limiting for growth of these cells. Here, in the converse experiment, we show that expression of two distinct kinase-defective Src mutants did not alter the growth of KM12C cells, indicating that these tumor cells have become Src independent for proliferation. This is in agreement with work in the rat NBT-II carcinoma model where expression of a dominant-negative Src did not alter tumor cell growth (35). However, the situation seems to be context dependent as reduction of Src expression using antisense or treatment with Src kinase inhibitors is able to inhibit the proliferation of tumor cells (36–39) . One consistent finding is that interference with Src activity alters the invasiveness and metastatic potential of tumor cells (35, 38–40) .

Role of FAK Phosphorylation in Adhesion Turnover and Cell Migration

For epithelial cancer cells to invade and metastasize, they are required to remodel their cell-matrix adhesions, detach, migrate, and adhere at distant sites within the body. A number of signaling pathways are involved in this process and here we addressed the role of Src and FAK in adhesion remodeling in colon cancer epithelial cells. Tyrosine phosphorylation of FAK is required for the ability of KM12C cells to turnover their adhesions, which is associated with cell motility. This is in accordance with work on viral Src where the Src-induced phosphorylation of FAK is linked with proteolytic cleavage of FAK by calpain required for both cell transformation and focal adhesion turnover and migration (41, 42) . Specifically, expression of FAK 407-925F prevented focal adhesion turnover and cell migration in fibroblasts (43) and a similar pathway may be initiated upon expression of this interfering mutant in KM12C cells where analysis of adhesion dynamics in real time revealed a defect in the ability of cells to turnover integrin adhesions at the tips of cellular protrusions.

The presence of static protrusions in SrcMF cells correlated with an inability of this mutant to induce phosphorylation on Tyr925, whereas significant levels of phosphorylation were observed on all other sites. This suggests that FAK Tyr925 plays a role in adhesion turnover and protrusion dynamics in these cells. Phosphorylation of FAK Tyr925 results in the recruitment of growth factor receptor binding protein 2 and subsequent activation of the extracellular signal-regulated kinase/myosin light chain kinase pathway. However, the ability of growth factor receptor binding protein 2 to bind to FAK in the KM12C cells was not altered by the phosphorylation status of FAK Tyr925, suggesting that this pathway does not regulate protrusive motility (results not shown). As Tyr925 lies within the FAT domain, it is possible that phosphorylation at this site may alter its conformation and modify the binding of paxillin or other binding proteins in this region, which may alter FAK function (44).

In fibroblasts, phosphorylation of Tyr397 is critical for the ability of FAK to induce cell motility. However, in KM12C cells, we see phosphorylation of Tyr397, which is increased 3.2-fold in cells expressing active Src and 2-fold in cells expressing kinase-inactive Src (data not shown), suggesting that an inability of Tyr397 to be phosphorylated or indeed protected from dephosphorylation in SrcMF cells cannot account for their motility defect.

Src Kinase-Independent Functions. Although the majority of Src-dependent functions require the kinase activity of Src, there are a number of reports of Src kinase–independent functions (15, 18, 45, 46) . One surprising observation from our studies was the increased phosphorylation of FAK in both KM12C and SYF−/−cells expressing kinase-defective mutants of Src. Thus, FAK phosphorylation can occur in the absence of SFKs. However, expression of a Src protein in which a point mutation in the kinase domain has rendered the protein catalytically inactive (K295R) was unable to alter FAK phosphorylation in SYF−/− cells (47). This mutant differs from the other kinase-defective proteins in that it does not have an open conformation such that the SH2 and SH3 domains are available for protein-protein interactions. As such, it does not associate constitutively with adhesion complexes, which may account for the different levels of FAK phosphorylation observed between studies using various Src mutants (15, 48) . The apparent scaffolding function of elevated expression of SrcK295R in SYF−/− cells (47) suggests that in tumors where Src expression and activity is often deregulated, strategies to block the adaptor function of Src should be considered alongside inhibition of kinase activity.

The decreased phosphorylation of both p130CAS and paxillin in KM12C cells suggests that the increased phosphorylation of FAK is not due to a general increase in phosphorylation of Src substrates at integrin adhesion sites. Such results also argue against a role for the recruitment of other Src family members to these sites, resulting in phosphorylation of FAK. This possibility was more directly addressed by use of a highly potent and selective Src family kinase inhibitor AP23464, which did not inhibit the phosphorylation of FAK in the SrcMF-expressing cells. Candidate effectors for the phosphorylation of FAK include growth factor receptors and Abl; however, preliminary data have not revealed a role for these kinases in the phosphorylation of FAK in KM12C cells. 4 Growth factor–induced migration of fibroblasts is independent of FAK kinase activity and it has been proposed that FAK may be a substrate for other tyrosine kinases in this system (32).

Although expression of SrcMF or Src251 in KM12C cells was able to induce phosphorylation of FAK on Tyr407, Tyr576, Tyr577, and Tyr861, there was an inability of these cells to remodel their integrin adhesions and retract their cellular protrusions. Src kinase activity may be required to activate other pathways that act in concert with FAK phosphorylation to regulate adhesion turnover and migration. For example, phosphorylation of p130CAS and paxillin are important for cell motility, but their phosphorylation is abolished in cells expressing SrcMF or Src251. The partial ability of a kinase-defective Src to restore the motility defect in SYF−/− cells supports a role for Src in the integration of a number of factors controlling cell motility (18). The requirement for the Src SH2 domain for both increased phosphorylation of FAK and recruitment of the Src-FAK complex to peripheral adhesions suggests that, in addition to its role as a tyrosine kinase, the ability of Src to stabilize signaling complexes at adhesion sites is important. It is also possible that the Src SH2 domain may stabilize the phosphorylation of FAK via interaction with the phosphorylated protein, thus protecting phosphorylation sites from the action of phosphatases within the cell.

We have shown that phosphorylation of FAK Tyr925 is a Src-dependent phosphorylation event in the KM12C cells and that phosphorylation of this site correlates with the ability of cells to dynamically regulate integrin adhesions. Taken together with the increased levels of FAK and Src proteins found in tumors and the importance of the FAK-Src signaling complex to tumor cell behavior suggests that phosphorylation of Tyr925 on FAK may provide a useful indicator of increased signaling through the Src-FAK signaling complex in tumors. The identification of kinase-independent functions of Src associated with overexpression of the Src SH2 and SH3 domains also suggests that increased expression of Src in tumors may lead to formation of protein complexes that could alter signaling in tumor cells. An increased understanding of the Src-dependent pathways regulating tumor cell behavior and the establishment of suitable biomarkers will ultimately be essential to the clinical application of Src kinase inhibitors for the treatment of cancer metastasis (49, 50) . As shown in this investigation, AP23464 is a promising Src kinase inhibitor in a colon cancer cell model as exemplified by its ability to effectively block a critical Src-FAK molecular relay switch underlying migration. These studies are encouraging toward the clinical development of AP23464 or analogues thereof as a novel small molecule therapeutic for the treatment of cancer metastasis. Such efforts are under way at ARIAD Pharmaceuticals and will be reported elsewhere.

Acknowledgments

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.

We thank John Wyke for helpful discussions and critical reading of the manuscript.

Footnotes

  • Received June 3, 2004.
  • Revision received November 10, 2004.
  • Accepted December 1, 2004.

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Cancer Research: 65 (4)
February 2005
Volume 65, Issue 4

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Identification of Src-Specific Phosphorylation Site on Focal Adhesion Kinase: Dissection of the Role of Src SH2 and Catalytic Functions and Their Consequences for Tumor Cell Behavior
Valerie G. Brunton, Egle Avizienyte, Valerie J. Fincham, Bryan Serrels, Chester A. Metcalf III, Tomi K. Sawyer and Margaret C. Frame
Cancer Res February 15 2005 (65) (4) 1335-1342; DOI: 10.1158/0008-5472.CAN-04-1949
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