Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407*

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr407 was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr407 increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr397 phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr397 and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr407 phosphorylation negatively regulates the enzymatic and biological activities of FAK.

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr 407 was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr 407 increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr 397 phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr 397 and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr 407 phosphorylation negatively regulates the enzymatic and biological activities of FAK.
Focal adhesion kinase (FAK) 3 is a nonreceptor cytoplasmic tyrosine kinase that modulates various cell functions, including survival, proliferation, and migration (1,2). Occupying an important receptor-proximal position in the signaling cascade, FAK mediates signal transduction in response to adhesion and/or binding of growth factors. The ability of FAK to transduce downstream signal(s) is dependent on its phosphorylation at tyrosine residues (3). Activation and phosphorylation of FAK leads to recruitment of a number of SH2 and SH3 domaincontaining proteins that mediate signaling via multiple down-stream pathways. These include the Src family kinases (4), phosphatidylinositol 3-kinase (PI3K), p130CAS (5,6), and Grb2 (7). Six tyrosine phosphoacceptor sites have been identified in FAK, Tyr 397 , Tyr 407 , Tyr 576 , Tyr-577, Tyr 861 , and Tyr 925 , most of which appear to play positive regulatory roles. As an example of FAK signaling, when integrin is activated, FAK is recruited to focal contacts and autophosphorylated at Tyr 397 (8). This creates a high affinity binding site for the SH2 domain of Src family tyrosine kinases and the p85 subunit of phosphoinositide 3-kinase, allowing their recruitment and activation. The recruitment of Src leads to additional phosphorylation on Tyr 576 , Tyr 577 , and Tyr 925 . Phosphorylation at Tyr 576 and Tyr 577 further activates FAK kinase activity (9), whereas that at Tyr 925 creates a binding site for the protein complex containing the adaptor Grb2 and the ras guanosine 5Ј-triphosphate exchange factor mSOS, which in turn activates the mitogen-activated protein kinase pathway. Phosphorylation at Tyr 861 is less well understood but has been detected in several cell lines, including prostate carcinoma cells (10), metastatic breast cancer cells (11), vesicular endothelial cells (12), and Ras-transformed fibroblasts (13). Leu and Maa (14) suggested that phosphorylation at Tyr 861 might enhance Tyr 397 phosphorylation of Tyr 397 , and our group recently reported that Tyr 861 phosphorylation was crucial for H-ras-induced transformation (15), implying that Tyr 861 phosphorylation is likely to potentiate the functions of FAK.
In contrast to the other phosphotyrosine residues, phosphorylation at Tyr 407 has not been extensively studied. Src has been proposed to regulate phosphorylation of FAK Tyr 407 , because phosphorylation of Tyr 407 was significantly and adhesion-dependently elevated in the presence of c-Src (16). However, Src kinase-independent phosphorylation at Tyr 407 has been also proposed, based on increased tyrosine phosphorylation of FAK Tyr 407 in a colon cancer cell line (KM12C) expressing kinasedeficient Src proteins (17).
Furthermore, although it is well known that other tyrosine phosphorylation events are linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr 407 remains unknown. A recent study showed that exposure of endothelial cells to vascular endothelial growth factor induced Tyr 407 phosphorylation, suggesting that Tyr 407 phosphorylation may play a role in transducing the vascular endothelial growth factor signals, which trigger assembly of focal adhesions and endothelial cell migration (18). Therefore, it is highly possible that phosphorylation of FAK at Tyr 407 plays one or more roles in FAK functions. Here we show for the first time that FAK Tyr 407 phosphorylation appears to function in the negative regulation of FAK activity and function.

Construction of Mutant FAK Mammalian Expression Vectors-
The full-length cDNA encoding FAK was subjected to site-directed mutagenesis using the Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA). The synthetic oligonucleotides GGA AGA CAC ATT TAC CAT GCC CTC G (Tyr 407 3 Phe) and GGA AGA CAC AGA AAC CAT GCC CTC G (Tyr 407 3 Glu) were used to change Tyr 407 to phenylalanine or glutamic acid. The cDNAs encoding wild-type FAK and mutant Y407F and Y407E were inserted into pRC/cytomegalovirus at the NotI/XbaI cloning sites to generate inframe fusions with a 3Ј sequence encoding three HA epitopes (YPYDVPDYA).
Transfection-Transient transfections were carried out using Lipofectamine reagent (Invitrogen) as described by the manufacturer. In brief, NIH3T3 cells were plated in 60-mm dishes and grown to ϳ80% confluence for 24 h. The cells were then transfected with a mixture of 15 l of Lipofectamine and 4 g of plasmid DNA. After 5 h, the transfection mixture was removed and replaced with medium containing 10% FBS. For stable transfections, transfected cell populations were selected by the continuous presence of 250 g/ml of G418.
Synthesis and Transfection of siRNA Constructs-To design oligonucleotides targeting the mouse FAK mRNA for degradation, siRNA Design of Ambion was used. The chosen targeted FAK siRNA sequence (5Ј-AATGCCCTAGAGAAGAAGTCC-3Ј) was chemically synthesized by Ambion (Austin, TX), and the negative control siRNAs were purchased from Ambion. In vitro cotransfections were performed with 500 nM of mouse siRNAs and 4 g of constructs encoding chicken FAK with and without introduced mutations (407Y, 407F, and 407E), using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocols.
Cell Proliferation Assay-Cell proliferation was measured by a colorimetric assay using MTT. In brief, NIH3T3 cells transfected with each of the generated plasmids were harvested with 0.05% trypsin/EDTA and seeded into 35-mm dishes at 1 ϫ 10 5 cells/dish. After the cells were allowed to attach, the medium containing 0.5 mg/ml MTT was added to each plate in a volume of 100 l, and the cells were incubated for 1 h. The medium was then removed, 200 l of dimethyl sulfoxide was added, and the plates were incubated for half an hour at room temperature. The mean concentration of absorbance at 570 nm in each set of all samples was measured using a 96-well microtiter plate reader (Dynatech, Chantilly, VA).
BrdUrd Incorporation Assay-The cells were pulsed with 10 M BrdUrd for 2 h, harvested, fixed in 70% ethanol at 4°C for 30 min, and resuspended in 2 N HCl and 0.5% Triton X-100 for 30 min at room temperature. After the reactions were neutralized with 0.1 M sodium tetraborate for 2 min, the cells were stained with fluorescein isothiocyanate-conjugated anti-BrdUrd antibody in 0.5 ml of 1% bovine serum albumin and 0.5% Tween 20 in phosphate-buffered saline (PBS) for 1 h at room temperature in the dark. The cells were then washed once and incubated in 0.5 ml of PBS containing 5 g/ml propidium iodide for 30 min prior to analysis with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and the CellQuest TM software.
Cell Adhesion Assays-Fibronectin was diluted to 10 g/ml in serum-free medium (SFM) and dispensed to 12-well plates (10 g/ml) that were incubated at room temperature for at least 1 h to allow adsorption. The plates were then washed with PBS, blocked with 0.2% heat-inactivated bovine serum albumin for 1 h, and then washed with SFM (2 ϫ 10 min). Cultured cells were detached from culture plates with 0.05% trypsin and 0.53 mM EDTA, suspended in SFM containing 0.25 mg/ml of soybean trypsin inhibitor, and centrifuged. The cells were then resuspended in SFM, plated onto fibronectin-coated plates, and incubated for the indicated durations at 37°C. For analysis of cell morphology, the cells were visualized with an inverted microscope (Zeiss) at 20ϫ magnification.
Migration Assay-Fibronectin (10 g/ml) was added to each well of a 24-well Transwell plate (8-m pore size; Costar), and the membranes were allowed to dry for 1 h at 25°C. Cells transfected with the various constructs (5 ϫ 10 4 cells) were added to the upper compartment of each well, and the plate was incubated for 6 h at 37°C in a 5% CO 2 atmosphere. Nonmigrated cells on the upper membrane were removed with a cotton swab. Migrated cells (located on the lower surface of the filters) were fixed for 5 min in methanol, stained with 0.6% hematoxylin and 0.5% eosin, and then counted.
In Vitro Kinase Assays-FAK immunoprecipitates were washed twice with 1ϫ radioimmune precipitation assay buffer and once with 10 mM Tris buffer. The pellets were dissolved in 20 l of kinase buffer (10 mM Tris, pH 7.4, 10 mM MnCl 2 , 2 mM MgCl 2 , 0.02% Triton X-100), and the reactions were started by adding 10 Ci of [␥-32 P]ATP, 1 M cold ATP, and purified either GST-FAK on glutathione-Sepharose 4B columns or with 1 mg/ml poly(Glu-Tyr) as a substrate, and incubated at 25°C for 5 min. The reactions were stopped by boiling in Laemmli buffer and then analyzed in SDS-PAGE gel.

RESULTS
FAK Tyr 407 Phosphorylation Appears to Be Inversely Correlated with Cell Proliferation-To investigate the role of phosphorylation at FAK residue Tyr 407 , we examined this site-specific phosphorylation under various cell conditions (Fig. 1). FAK Tyr 407 phosphorylation was found to be higher in NIH3T3 cells treated with the cell cycle arresting agents, CQ and deferoxamine (Fig. 1A), as well as in serum-starved cells (Fig.  1B) and cells grown to achieve higher density saturation at confluence versus exponentially growing cells (Fig. 1C). Because both cell proliferation and FAK activity are decreased under conditions of serum starvation, contact inhibition, and cell cycle arrest, these results seem to suggest that FAK Tyr 407 phosphorylation might decrease the ability of FAK to regulate cell proliferation. Consistent with this notion, we found that FAK Tyr 407 phosphorylation was inversely related with activation-associated phosphorylation at FAK Tyr 397 (Fig. 1).
To further investigate the potential negative role of FAK Tyr 407 phosphorylation in cell proliferation, we used site-directed mutagenesis to replace Tyr 407 with a nonphosphorylatable phenylalanine residue (407F) and transfected vectors encoding HA-tagged 407F into NIH3T3 cells (Fig. 2). Com-pared with vector-transfected cells, those expressing wild type (FAK) and FRNK showed similar cell numbers. In contrast, for 72 h mutant FAK 407F-transfected cells showed slight but sig-  nificant increased in cell numbers versus those expressing wildtype FAK (Fig. 2A). Consistent with this increased proliferation, ERK phosphorylation, which is involved in cell cycle progression (19,20) was increased (Fig. 2B), and BrdUrd incorporation into newly synthesized DNA in response to serum stimulation was 1.4-fold higher (Fig. 2C) in FAK 407F-transfected cells versus wild-type FAK-transfected cells. In NIH3T3 cells, the G 1 population peaked between 6 and 10 h following activation of serum, and the population of cells in S phase began to increase 12 h after serum starvation (data not shown). We then examined the effect of expression of 407F mutant FAK. NIH3T3 cells stably expressing HA-tagged FAK 407F showed significantly more cell proliferation. Western blot analysis of HA-FAK 407F expression levels in individual clones (Fig. 3A, top left panel) reveal that NIH3T3 cells expressing higher levels of FAK 407F showed correspondingly higher proliferation rate (Fig. 3A, bottom left panel) and higher overall tyrosine phosphorylation levels in total cell lysates (Fig. 3A, right panel). Similarly, cell counting and BrdUrd incorporation revealed that NIH3T3 cells stably overexpressing FAK 407F showed delayed cell cycle arrest in response to serum starvation (Fig. 3B) and CQ treatment (Fig. 3C). Furthermore, the duration of delay paralleled the expression level of FAK 407F. Taken together, these data strongly suggest that FAK Tyr 407 phosphorylation might be involved in negative regulation of cell proliferation.
FAK Tyr 407 Phosphorylation Negatively Regulates Autophosphorylation and Kinase Activity-Our finding that the phosphorylation levels of Tyr 407 and Tyr 397 were inversely correlated promptly us to investigate whether FAK Tyr 407 phosphorylation might negatively regulate FAK activity. NIH3T3 cells were transfected with the empty vector, wild-type FAK (407Y), nonphosphorylatable mutant FAK (407F), or phosphorylation-mimicking mutant FAK (407E). Interestingly, both total tyrosine phosphorylation of FAK and autophosphorylation at Tyr 397 was increased in cells transfected with FAK 407F and decreased in cells transfected with FAK 407E, compared with cells expressing wild-type FAK (Fig. 4A, top panel). Similarly, in vitro kinase assay showed that FAK kinase activity was increased in cells expressing FAK 407F and decreased in cells expressing FAK 407E, compared with cells expressing wild-type FAK, FAK 407Y (Fig. 4A, bottom panel). Phosphorylation of FAK Tyr 397 creates a high affinity binding site for PI3K and the Src family kinases. Consistent to the altered levels of Tyr 397 phosphorylation, interactions of FAK with PI3K and Src were decreased in cells transfected with FAK 407E mutant and increased in cells transfected with FAK 407F mutant versus those expressing wild-type FAK (Fig. 4B). These results collectively suggest that FAK Tyr 407 phosphorylation may inhibit FAK Tyr 397 phosphorylation.
Because Tyr 397 and Tyr 407 are located close to one another in the linker region of the FAK protein, we examined whether phosphorylation of one affects that of the other. We generated  a recombinant GST-FAK 407 polypeptide containing phosphorylatable Tyr 397 and wild-type Tyr 407 (407Y) or mutants containing either Glu (407E) or Phe (407F). FAK immunocomplexes containing these peptides were used for in vitro kinase assays. Interestingly, Tyr 397 phosphorylation was 2-fold higher in experiments containing GST-FAK 407F and decreased in those containing GST-FAK 407E, compared with assays involving GST-FAK 407Y (Fig. 4C). These findings seem to indicate that FAK Tyr 407 phosphorylation might inhibit the access of additional phosphates on Tyr 397 , perhaps because of increased negative charge repulsion. It has been known that FAK molecules phosphorylated at Tyr 397 reside close to the plasma membrane (21,22), and the absence of Tyr 397 phosphorylation decreases its residency at focal adhesions but not in cytosol (22). Consistent with this notion, higher levels of Tyr 407 phosphorylation were observed in cytosolic FAK versus membrane-bound FAK (Fig. 4D). Collectively, these data suggest that FAK Tyr 407 phosphorylation negatively regulates autophosphorylation at Tyr 397 and FAK kinase activity.
FAK Tyr 407 Phosphorylation Negatively Regulates FAK Functions-Because FAK plays a critical role in integrin-mediated signal transduction, where it acts as a cytosolic kinase to phosphorylating cytoskeletal proteins, and because integrin-mediated signal transduction requires Tyr 397 phosphorylation (23), we speculated that FAK Tyr 407 phosphorylation might negatively regulate FAK function. To examine this possibility, we designed a unique 21-bp small interfering RNA (siRNA) sequence targeted against the mouse FAK (mFAK) mRNA and used this siRNA to knock down mFAK expression in NIH3T3 cells (Fig. 5A). Expectedly, NIH3T3 cells transfected with mFAK siRNA showed decreased expression of FAK (Fig. 5A, compare first and second lanes) as well as decreased cell adhesion and migration on fibronectin (data not shown, but refer to Figs. 6A and 7). To further investigate the role of phosphorylation at FAK residue Tyr 407 , we then re-expressed recombinant HA-tagged chicken FAK (cFAK) in the knockdown NIH3T3 cells and compared the knockdown cells with those expressing recombinant wild-type or mutant FAK proteins. Consistent with the results reported above, FAK Tyr 397 phosphorylation was increased in knockdown cells transfected with cFAK 407F and decreased in those expressing cFAK 407E, as compared with the knockdown cells transfected with cFAK 407Y (data not shown but refer to Fig. 6B). Similarly, proliferation and BrdUrd incorporation were increased in knockdown cells expressing cFAK 407F and decreased in those expressing cFAK 407E, as compared with the knockdown cells transfected with cFAK 407Y (Fig. 5, B and C). We then examined integrin-mediated cell adhesion in FAK knockdown cells and those re-expressing mutant or wild-type cFAK. The cells were detached and replated on fibronectin and observed for spreading. By 60 min about 38% of the mFAK knockdown NIH3T3 cells showed cell spreading on fibronectin. In contrast, about 82% of cells re-expressing wild-type cFAK completed cell spreading.
Interestingly, re-expression of cFAK 407F further enhanced adhesion and spreading, whereas re-expression of cFAK 407E decreased adhesion and spreading, as compared with those re-expressing wild-type cFAK (Fig. 6A). During spreading on fibronectin, FAK Tyr 397 phosphorylation was increased in cells re-expressing cFAK 407F and decreased in cells re-expressing cFAK 407E, as compared with those re-expressing wild-type cFAK (Fig. 6B), indicating the involvement of FAK activation in this process. Because FAK plays a key role in the regulation of cell migration (1,3,23), we further investigated adhesion-mediated cell migration in FAK knockdown cells and those re-expressing mutant or wild-type cFAK. The experiments in Transwell chambers revealed that re-expression of wild-type cFAK enhanced cell migration of mFAK knockdown NIH3T3 cells. Consistently, FAK-mediated cell migration was further increased in cells re-expressing cFAK 407F and decreased in cells re-expressing cFAK 407E, as compared with those re-expressing wild-type cFAK (Fig. 7), indicating that FAK-mediated cell migration was inhibited by re-expression of cFAK in a Tyr 407 phosphorylation-dependent fashion. Taken together, these data strongly suggest that FAK Tyr 407 phosphorylation negatively regulates FAK functions during adhesion-dependent signal transductions. FAK Tyr 407 Phosphorylation Is Not Mediated by Src Family Kinase during Cell Cycle Arrest-Because Src has been proposed to regulate phosphorylation of FAK Tyr 407 (16), we investigated whether Src family kinase plays a role in FAK Tyr 407 phosphorylation during cell cycle arrest. NIH3T3 cell lysates were incubated with [␥-32 P]ATP and GST-FAK407 as a substrate to allow phosphorylation, and phosphorylated proteins were analyzed by autoradiography (Fig. 8). Interestingly, GST-FAK407 phosphorylation was observed only in CQ-treated cell lysate (Fig. 8A, left panels), and it was also seen in the presence of PP1, the Src family kinase inhibitors (Fig.  8A, right panels). In addition, consistent to the previous report (13), trichostatin A caused an increase of FAK Tyr 407 phosphorylation in H-ras-transformed NIH3T3 cells even in the presence of PP1. Therefore, it seems that FAK Tyr 407 phosphorylation is not mediated by Src family kinase during cell cycle arrest.

DISCUSSION
Although phosphorylation of FAK at Tyr 407 was previously reported (16,17), the function of this event was virtually unknown until this point. Here, we provided the first evidence that FAK Tyr 407 phosphorylation has a negative regulatory function. Unlike the other five phosphorylatable tyrosine residues, which showed increased phosphorylation upon activation of FAK, higher levels of FAK Tyr 407 phosphorylation were observed under conditions of basal FAK activity and relatively low levels of FAK Tyr 397 phosphorylation. Both Tyr 397 autophosphorylation and FAK kinase activity were decreased in cells transfected with the phosphorylation-mimicking FAK 407E mutant and increased in cells transfected with the nonphosphorylatable FAK 407F mutant (Fig. 4). In addition, FAK functions such as cell adhesion, spreading, migration, and proliferation were decreased in cells transfected with FAK 407E and increased in cells transfected with FAK 407F, as compared with cells transfected with FAK 407Y (Figs. 6 and 7). Collectively, these findings suggest that FAK Tyr 407 phosphorylation negatively regulates the enzymatic and biological activities of FAK.
Protein tyrosine kinases such as FAK play crucial roles in signal transduction, but their constitutive activation often leads to cancer formation and progression (24,25). Thus, it seems reasonable to surmise that normal cells have regulatory mechanism(s) responsible for maintaining FAK activity at basal levels. In addition, the cells might also have a second level of negative regulation responsible for abruptly terminating FAK activity when necessary. Negative regulation by tyrosine phosphorylation is seen in another cytosolic tyrosine kinase, Src, which is inactivated by specific C-terminal tyrosine phosphorylation by C-terminal Src kinase. Upon phosphorylation, the C-terminal Src phosphotyrosine residue engages in intramolecular interactions that lock the Src molecule in an inactive conformation. Thus, Src can undergo both an activating autophosphorylation event at Tyr 416 and an inhibiting phosphorylation event at Tyr 527 . However, unlike the fairly well elucidated negative regulation of Src, that of FAK has not been extensively studied.
A recent paper proposed that the FERM domain, located in the N-terminal region of FAK, might repress the catalytic activity by intramolecular inhibition (26,27). Indeed, FAK lacking a region of the N terminus corresponding to amino acids 1-384 showed a striking increase in phosphorylation while retaining intact integrin-mediated signaling (27). Based on these and other reports, it has been proposed that the cytoplasmic  domains of integrins activate FAK by relieving FERM-mediated autoinhibition. Here, we provide the first evidence supporting a Tyr 407 phosphorylation-mediated negative regulatory mechanism for FAK. Specifically, we show that FAK-407 phosphorylation negatively regulates important FAK functions including cell adhesion, proliferation, and migration in NIH3T3 cells (Figs. 6 and 7). This negative regulation could exist in addition to FERM-mediated inhibition, or it could be a downstream event initiated by FERM.
Our experiments further revealed that FAK Tyr 407 phosphorylation appears to inhibit the activating phosphorylation of Tyr 397 . This may be mediated by physical hindrance of the interaction between ATP and Tyr 397 , i.e. phosphorylation at Tyr 407 creates an electrostatic environment that restricts access to Tyr 397 , leading to reduced phosphorylation at Tyr 397 . Consistent with this notion, we showed that the degree of in vitro phosphorylation at Tyr 397 of GST-FAK 407Y was higher than that of nonphosphorylatable mutant GST-FAK 407E but lower than that of phosphorylation-mimicking mutant GST-FAK 407E (Fig. 4).
Another possibility is that FAK Tyr 407 phosphorylation may be involved in FERM-mediated autoinhibition of FAK. If the FERM domain represses the catalytic activity of the enzyme by intramolecular autoinhibition, the linker domain of FAK, which contains Tyr 407 , might participate by stabilizing interactions between FERM and kinase domains. This hypothesis is supported by the observation that FERM contains a phosphotyrosine binding-like domain. Future work will be required to determine the precise mechanism(s) by which inactivating phosphorylation at Tyr 407 inhibits activating phosphorylation at Tyr 397 . In addition, Src has been proposed to regulate FAK phosphorylation at Tyr 407 (16). We found, however, that the recombinant FAK polypeptide containing Tyr 407 was not phosphorylated by Src family kinase but phosphorylated by cell lysate from serum-starved NIH3T3 cells (Fig. 8), implying negative regulation through FAK Tyr 407 phosphorylation by another cytosolic tyrosine kinase.
In sum, we herein show for the first time that FAK Tyr 407 phosphorylation contributes to negative regulation of kinase activity and decreased autophosphorylation at Tyr 397 , leading to negative regulation of FAK-related functions in adhesionmediated signal transduction. We are currently attempting to identify tyrosine kinase and tyrosine phosphatase that respectively phosphorylate and dephosphorylate FAK Tyr 407 , in an effort to clarify the precise negative regulatory mechanism(s) of FAK Tyr 407 phosphorylation. However, the present work pro-vides the first evidence for negative regulation of FAK activity and function by phosphorylation at Tyr 407 .