The protein tyrosine kinase, focal adhesion kinase (FAK), is a central regulator of integrin-mediated signaling (Mitra et al., 2005; Parsons, 2003; Schlaepfer and Mitra, 2004). FAK is present in focal adhesions, adhesive structures that mediate attachment of cells to the extracellular matrix (ECM) (Hynes, 1992; Lauffenburger and Horwitz, 1996). In addition, FAK is present in other adhesion structures within the cell, most notably the dynamic adhesion complexes found at the periphery of lamellipodia of migrating cells (Webb et al., 2004). FAK is essential for the turnover (e.g. breakdown) of this latter class of cell adhesions and has also been implicated in the release of adhesion at the rear of the cell (Carragher et al., 2001; Webb et al., 2004). Inhibition of FAK activity or targeted deletion of FAK expression results in cell migration defects (Ilic et al., 1995; Richardson et al., 1997a; Schlaepfer and Mitra, 2004). Increased FAK expression is a hallmark of many cancers and may contribute to the metastatic phenotype of highly malignant cells (Gabarra-Niecko et al., 2003).

Clustering of cell surface integrins, as a consequence of cell attachment to ECM proteins or treatment with certain integrin-specific antibodies, leads to the activation of FAK catalytic activity and an increase in FAK tyrosine phosphorylation (Mitra et al., 2005; Parsons, 2003). Activation of FAK results in autophosphorylation of Y397, the recruitment of Src and Src-family kinases, and the increased phosphorylation of other proteins present in the adhesion complexes, notably paxillin and p130Cas (Mitra et al., 2005). The present evidence suggests that the initiation of this tyrosine phosphorylation cascade is essential for the downstream signaling mediated by cellular adhesion complexes. In addition to being a protein tyrosine kinase, FAK appears to perform a scaffolding function, binding to a variety of membrane-associated proteins including growth factor receptors and ERM proteins. In adhesions, FAK colocalizes with and binds to the focal adhesion proteins paxillin, p130Cas and talin, as well as SH3-domain-containing GTPase-activating proteins for the Rho and Arf family of small GTPases. In addition, the phosphorylation of specific tyrosine residues appears to be important for the recruitment of SH2-domain-containing signaling proteins, including Src and Src family kinases, PI 3-kinase and Grb2 (Mitra et al., 2005; Parsons, 2003; Parsons et al., 2000).

Little is known about the spatial and temporal organization of FAK in newly formed or more stable cellular adhesions. Further, little information is available about the factors that regulate the enzymatic activation of FAK or the interaction of FAK with its binding proteins. To understand the possible role of post-translational modifications on the regulation of FAK function, we have mapped the phosphorylation sites within FAK using immunoaffinity purification of FAK and mass spectrometry (MS). In this report, we identify 25 sites of phosphorylation (15 serine, 5 threonine and 5 tyrosine residues), including a number of the serine and tyrosine phosphorylation sites previously reported. In addition, we note the juxtaposition of phosphoserine-, phosphothreonine- and phosphotyrosine-containing residues, which suggests that coordinated phosphorylation of FAK by serine/threonine and tyrosine-specific kinases may be an important aspect of regulation of FAK function.

To identify the sites of phosphorylation in FAK, MS analysis was performed on FAK immune complexes prepared from HEK293 cells expressing chicken FAK. In some cases the cells were pretreated with phosphatase inhibitors to enhance the recovery of phosphorylated residues. Through this analysis, peptides representing 90% of the FAK protein were identified. Fig. 1 shows the sequence of FAK with all of the observed phosphorylation sites marked in red. The amino acid numbering in this report is based on the chicken sequence, which differs slightly from the numbering for human or mouse FAK. The regions of the protein that were not identified by this MS analysis are shown in lower case. The phosphorylation of residues within these regions is therefore unknown. In addition, there were two instances in which the specific phosphorylated amino acids within a peptide could not be assigned by the MS/MS spectra (shown in brackets). A total of 38 phosphopeptides were identified, and their sequences were confirmed by manual inspection of the spectra (Table 1). From these peptides, we have identified the phosphorylation of 15 serine, 5 threonine and 5 tyrosine residues.

Table 1.

Relative abundance of phosphopeptides identified by mass spectrometry

Relative abundance
Residue FAK peptide sequence (chicken) (+) Inhibitors (–) Inhibitors
T13   DPNLNHTPSSSAK   ++   
S29   THLGTGMERSPGAMERVLK   +++   ++++  
Y155   NDYMLEIADQVDQEIALK   ++++   
S386/T388   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
S386/T388   GVRSHTVSVSETDDYAEIIDEE   IMAC   
S386/S390   KQGVRSHTVSVSE   IMAC   
T388/S390   KQGVRSHTVSVSE   IMAC   
T388/S392   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
T388/Y397   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
S390   QGVRSHTVSVSETD   IMAC   
S390   QGVRSHTVSVSETDDYAEIIDEE   ++   IMAC  
S390   GVRSHTVSVSET   IMAC   
S390/[T394-Y397]   QGVRSHTVSVSE [TDDY] AEIIDEE    IMAC  
[T394-Y397]   QGVRSHTVSVSE [TDDY] AEIIDEE    IMAC  
Y397   QGVRSHTVSVSETDDYAEIIDEE    ++  
T406/Y407   DTYTMPSTRDYE   IMAC   
Y570   DFGLSRYME   IMAC   +  
T700/S708   RMRMESRRQVTVSWDSGGSD   ++++   
S722/[S725-S726]   DEAPPKPSRPGYPSPR [SS] EGF   IMAC   
S732   GFYPSPQHMVQPNHYQVSGYSGSHGIPAMAGSIYPGQASLLDQTDSWNHRPQE   IMAC   
S766   SHGIPAMAGSIYPGQASLL   IMAC   
T793   DSGTLDVRGMGQVLPTHLM   IMAC   
S845   RFLVMKPDVRLSRGSIE   IMAC   
S888   KPPRPGAPHLGSLASLNSPV   ++++   
S888   KPPRPGAPHLGSLASLNSPVDSYNEGVK   +++   
S888/S891   KPPRPGAPHLGSLASLN   IMAC   
S888/S891   KPPRPGAPHLGSLASLNSPV   IMAC   
S888/S891/S894   KPPRPGAPHLGSLASLNSPV   IMAC   
S888/S894   KPPRPGAPHLGSLASLNSPV   +++   
S888/S894   KPPRPGAPHLGSLASLNSPVDSYNEGVK   +++   
S894   KPPRPGAPHLGSLASLNSPV   ++++   
S894   KPPRPGAPHLGSLASLNSPVDSYNE   IMAC   
S894   KPPRPGAPHLGSLASLNSPVDSYNEGVK   ++++   
S894/S898   KPPRPGAPHLGSLASLNSPVDSYNEGVK   ++   
S894/S898/Y899   KPPRPGAPHLGSLASLNSPVDSYNEGVK   IMAC   
S894/Y899   SLNSPVDSYNEGVK   IMAC   
S911   IKPQEISPPPTANL   ++++   ++++  
S911   IKPQEISPPPTANLDRSNDK   ++++   
Relative abundance
Residue FAK peptide sequence (chicken) (+) Inhibitors (–) Inhibitors
T13   DPNLNHTPSSSAK   ++   
S29   THLGTGMERSPGAMERVLK   +++   ++++  
Y155   NDYMLEIADQVDQEIALK   ++++   
S386/T388   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
S386/T388   GVRSHTVSVSETDDYAEIIDEE   IMAC   
S386/S390   KQGVRSHTVSVSE   IMAC   
T388/S390   KQGVRSHTVSVSE   IMAC   
T388/S392   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
T388/Y397   QGVRSHTVSVSETDDYAEIIDEE   IMAC   
S390   QGVRSHTVSVSETD   IMAC   
S390   QGVRSHTVSVSETDDYAEIIDEE   ++   IMAC  
S390   GVRSHTVSVSET   IMAC   
S390/[T394-Y397]   QGVRSHTVSVSE [TDDY] AEIIDEE    IMAC  
[T394-Y397]   QGVRSHTVSVSE [TDDY] AEIIDEE    IMAC  
Y397   QGVRSHTVSVSETDDYAEIIDEE    ++  
T406/Y407   DTYTMPSTRDYE   IMAC   
Y570   DFGLSRYME   IMAC   +  
T700/S708   RMRMESRRQVTVSWDSGGSD   ++++   
S722/[S725-S726]   DEAPPKPSRPGYPSPR [SS] EGF   IMAC   
S732   GFYPSPQHMVQPNHYQVSGYSGSHGIPAMAGSIYPGQASLLDQTDSWNHRPQE   IMAC   
S766   SHGIPAMAGSIYPGQASLL   IMAC   
T793   DSGTLDVRGMGQVLPTHLM   IMAC   
S845   RFLVMKPDVRLSRGSIE   IMAC   
S888   KPPRPGAPHLGSLASLNSPV   ++++   
S888   KPPRPGAPHLGSLASLNSPVDSYNEGVK   +++   
S888/S891   KPPRPGAPHLGSLASLN   IMAC   
S888/S891   KPPRPGAPHLGSLASLNSPV   IMAC   
S888/S891/S894   KPPRPGAPHLGSLASLNSPV   IMAC   
S888/S894   KPPRPGAPHLGSLASLNSPV   +++   
S888/S894   KPPRPGAPHLGSLASLNSPVDSYNEGVK   +++   
S894   KPPRPGAPHLGSLASLNSPV   ++++   
S894   KPPRPGAPHLGSLASLNSPVDSYNE   IMAC   
S894   KPPRPGAPHLGSLASLNSPVDSYNEGVK   ++++   
S894/S898   KPPRPGAPHLGSLASLNSPVDSYNEGVK   ++   
S894/S898/Y899   KPPRPGAPHLGSLASLNSPVDSYNEGVK   IMAC   
S894/Y899   SLNSPVDSYNEGVK   IMAC   
S911   IKPQEISPPPTANL   ++++   ++++  
S911   IKPQEISPPPTANLDRSNDK   ++++   

Each of the phosphopeptides identified from FAK is listed, with the phosphorylated residue(s) shown in red. The phosphorylated amino acids in brackets could not be distinguished. In some experiments, the cells were treated with phosphatase inhibitors before lysis. All of the cells were lysed in the presence of inhibitors. Relative phosphopeptide abundance is expressed in terms of ion counts (peak heights) observed for the most abundant charge state. Ion counts for the most abundant phosphopeptides are displayed as ++++; those that exhibit ion currents decreased by a factor of 10, 100 and 1000 are shown as +++, ++ and +, respectively. The relative abundance of peptides can only be compared within an individual experiment and, thus, peptides that were identified under different conditions may have been present at different levels in different experiments. We have indicated the highest relative amount that was observed. Peptide abundance with `+' inhibitors cannot be compared to abundance without `–' inhibitors. In some cases, peptides were only observed after enrichment with immobilized metal-affinity chromatography (IMAC), indicating that these peptides were present in limited amounts.

Fig. 2 shows the superposition of the detected phosphorylation sites on the domain structure of FAK. A number of features of the phospho-landscape are notable. The N-terminal FERM domain serves an autoinhibitory function (Cooper et al., 2003; Dunty et al., 2004) and as a bridge to growth factor receptors and membrane adapter proteins such as ezrin (Poullet et al., 2001; Sieg et al., 2000). Two sites of phosphorylation, T13 and S29, were observed N-terminal to the beginning of the FERM domain. Both residues are conserved in FAK from man, mouse and frog, indicating conservation of function in this region (Table 2). This region is unique to FAK and does not share sequence similarity with the related FAK kinase, PYK2/CAKβ. In the middle of the FERM domain, Y155 is a site of phosphorylation. Y155, which is conserved in FAK from man, mouse and frog, as well as in PYK2, is proximal to the previously identified site of FAK sumoylation, and phosphorylation of this residue may play a role in regulating nuclear trafficking of FAK (Kadare et al., 2003).

Table 2.

Conservation of phosphorylation sites between species

Species conservation
Residue Human Mouse Xenopus
T13   +   +   -  
S29   +   +   +  
Y155   +   +   +  
S386   -   -   -  
T388   -   -   -  
S390   +   +   +  
S392   +   +   +  
[T394]   +   +   +  
Y397   +   +   +  
T406   +   +   +  
Y407   +   +   +  
Y570   +   +   +  
T700   +   +   +  
S708   +   +   +  
S722   +   +   +  
[S725]   +   +   +  
[S726]   +   +   +  
S732   +   +   +  
S766   +   +   +  
T793   -   -   -  
S845   +   +   +  
S888   +   -   -  
S891   +   +   -  
S894   +   +   +  
S898   +   +   -  
Y899   +   +   +  
S911   +   +   +  
Species conservation
Residue Human Mouse Xenopus
T13   +   +   -  
S29   +   +   +  
Y155   +   +   +  
S386   -   -   -  
T388   -   -   -  
S390   +   +   +  
S392   +   +   +  
[T394]   +   +   +  
Y397   +   +   +  
T406   +   +   +  
Y407   +   +   +  
Y570   +   +   +  
T700   +   +   +  
S708   +   +   +  
S722   +   +   +  
[S725]   +   +   +  
[S726]   +   +   +  
S732   +   +   +  
S766   +   +   +  
T793   -   -   -  
S845   +   +   +  
S888   +   -   -  
S891   +   +   -  
S894   +   +   +  
S898   +   +   -  
Y899   +   +   +  
S911   +   +   +  

The sequence of chicken FAK was aligned with the sequences from human (GenBank accession no. L13616), mouse (GenBank accession no. M95408) and the frog, Xenopus laevis (GenBank accession no. L33920) by the Clustal method using the DNAstar MegAlign program. The phosphorylated amino acids that are identical are shown as `+'. The numbering of residues was based on the sequence of chicken FAK and may vary slightly for other species.

Clustered sites of phosphorylation were observed proximal to the major site of autophosphorylation, Y397 (Schaller et al., 1994), and included five newly identified sites of phosphorylation (S386, T388, S390, S392 and T406), as well as Y407, which was previously identified as a site of Src phosphorylation (Calalb et al., 1995). S390, S392 and T406 are conserved amongst FAK proteins from mouse, man and frog, whereas S386 and T388 are chicken specific. The proximity of this cluster of phosphorylation sites to Y397 suggests that phosphorylation of these residues plays a role in the regulation of FAK enzymatic activity. Current evidence indicates that FAK autophosphorylation takes place via the dimerization of two FAK molecules (Toutant et al., 2002). The efficiency of dimerization and/or Y397 phosphorylation may be influenced by the proximal serine/threonine phosphorylations. In addition, phospho-Y397 constitutes a docking site for the SH2 domain of Src and Src family kinases (Schaller et al., 1994). It is possible that phosphorylation influences the association of FAK with Src or other binding partners that use phospho-Y397 as a docking site.

Fig. 1.

Identified sites of serine, threonine and tyrosine phosphorylation in FAK. The sequence of chicken FAK (GenBank accession no. M86656) is shown with the amino acids that were identified by mass spectrometry (MS) shown in capital letters. The phosphorylated residues identified by the MS analysis are shown in red. There were two peptides in which the phosphorylated residue could not be distinguished (indicated by brackets). Because T394 was only observed within a peptide containing Y397, a known site of phosphorylation, the phosphorylation status of T394 is questionable.

Fig. 1.

Identified sites of serine, threonine and tyrosine phosphorylation in FAK. The sequence of chicken FAK (GenBank accession no. M86656) is shown with the amino acids that were identified by mass spectrometry (MS) shown in capital letters. The phosphorylated residues identified by the MS analysis are shown in red. There were two peptides in which the phosphorylated residue could not be distinguished (indicated by brackets). Because T394 was only observed within a peptide containing Y397, a known site of phosphorylation, the phosphorylation status of T394 is questionable.

Fig. 2.

Distribution of phosphorylation sites in FAK. The domain structure of FAK is shown with the mapped sites of serine (blue stars), threonine (green stars) and tyrosine (red stars) phosphorylation shown below. The sites of sumoylation (♦) and caspase cleavage (*) are indicated.

Fig. 2.

Distribution of phosphorylation sites in FAK. The domain structure of FAK is shown with the mapped sites of serine (blue stars), threonine (green stars) and tyrosine (red stars) phosphorylation shown below. The sites of sumoylation (♦) and caspase cleavage (*) are indicated.

One new site of phosphorylation was identified in the kinase domain. Y570 is conserved in FAK from man, mouse and frog, as well as in PYK2. It is located proximal to Y576 and Y577, both of which reside within the catalytic lip of FAK and have been reported to be important for the adhesion-dependent activation of FAK (Calalb et al., 1995).

Two additional clusters of phosphorylation sites were observed within the C-terminal region of FAK, where 14 sites of phosphorylation were mapped (11 serines, 2 threonines and 1 tyrosine). Two of these newly identified sites, T700 and S708, surround a potential site of caspase cleavage (Gervais et al., 1998) and reside just downstream of the acidic amino acid linker region that separates the kinase domain from the C-terminal domain. Both sites are conserved in human, mouse and frog. In addition to these two residues, phosphorylation of S722, S725/726 and S732 were mapped proximal to site I, the proline-rich site of binding to p130Cas (Harte et al., 1996; Polte and Hanks, 1995). S722 is phosphorylated by GSK3 (Bianchi et al., 2005), whereas S732 is a reported site for CDK5 (Xie and Tsai, 2004). Serines 725/726 are newly identified sites that are conserved between species; however, this analysis was unable to determine which of the two residues was phosphorylated.

Isolated phosphorylation sites were observed at S766, which is conserved between species and lies adjacent to another caspase cleavage site (Gervais et al., 1998), T793 (not conserved) and S845, which is phosphorylated by PKA (Richardson et al., 1997b).

Finally, a cluster of phosphorylation sites were observed proximal to site II and the beginning of the focal adhesion targeting (FAT) domain. Site II is the proline-rich domain that interacts with SH3 domains from ASAP1 and Graf (Hildebrand et al., 1996; Liu et al., 2002), whereas the FAT domain interacts with a number of focal adhesion proteins including paxillin, talin and Grb2 (Chen et al., 1995; Hildebrand et al., 1995; Schlaepfer et al., 1994). This cluster of phosphorylated residues includes S888, S891, S894, S898, Y899 and S911. S888 is present only in chicken and human FAK; S891 and S898 are conserved in human and mouse; and S894, Y899 and S911 are conserved from human to frog. Ser911 (S910 in human and mouse) is the only site from this group that has been characterized and is reported to be phosphorylated in an ERK-dependent manner (Hunger-Glaser et al., 2003) Because of the large number of phosphorylation sites in this region, it is likely that differential phosphorylation by several different kinases modulate the binding of proteins to this region.

Several features of the MS analysis allow an approximation of the the different phosphorylated peptides. Within an individual experiment, the relative phosphopeptide abundance can be expressed in terms of ion counts (peak heights) observed for the most abundant charge state. Assigning ion counts for the most abundant phosphopeptides as ++++, peptides that exhibited ion currents decreased by a factor of 10, 100 and 1000 can be indicated by +++, ++, and +, respectively (Table 1). Using this method of pseudo-quantitation, the abundance of peptides identified in FAK prepared from cells treated with or without phosphatase inhibitors was ranked from + to ++++. Peptides were often identified at different relative quantities depending on the conditions of each experiment. We have indicated the highest relative abundance that was observed by C18 analysis. In addition, some peptides were only identified following IMAC enrichment, suggesting that such phosphopeptide species were present at lower levels.

Using this method of quantitation, we observed that peptides containing Y155, T700/S708, S888, S894 and S911 were represented in the highest amounts in cells pretreated with phosphatase inhibitors. These residues may represent phosphorylation sites that are tightly regulated by phosphatases. Of these sites, it is noteworthy that only one, S911, was observed in high yield in the absence of phosphatase inhibitors. The other major species observed in the absence of inhibitors was S29. The pretreatment of cells with phosphatase inhibitors increased the yield of phosphopeptides (only seven peptides were recovered without inhibitors). Interestingly, phosphopeptides containing either Y397 or Y570 were present at higher amounts in cells that were not treated with phosphatase inhibitors. Additional studies will be required to understand the dynamics of phosphorylation of these and the other sites reported herein.

This MS analysis has greatly increased our knowledge about the phosphorylation sites in FAK. Previous studies had identified serines 722, 732, 842, 845 and 911, and tyrosines 397, 407, 576, 577, 863 and 926 as sites of phosphorylation (Calalb et al., 1995; Calalb et al., 1996; Ma et al., 2001; Richardson et al., 1997b; Schaller et al., 1994; Schlaepfer and Hunter, 1996; Xie and Tsai, 2004). Under the conditions of these experiments, we observed all but five of the known phosphorylation sites (Y576/577, S842, Y863 and Y926). Furthermore, these experiments have identified 19 new sites of phosphorylation (Table 1). Including the previously reported sites, there are now a total of 30 sites of phosphorylation in FAK. The task now is to determine the differential regulation and biological consequences of this large number of phosphorylation sites.

The data discussed herein were obtained as a part of a study supported by the Cell Migration Consortium. A summary of the data and methods used, as well as analyses of other migration related proteins, can be accessed at the Cell Migration Gateway (http://www.cellmigration.org).

HEK293 cells expressing low levels of FLAG-epitope tagged FAK (one- to twofold greater than endogenous levels) were grown on 135 mm cell culture dishes (Nunclon). The cells were treated for 30 minutes with a serine/threonine phosphatase inhibitor calyculin (0.1 μM) and/or a tyrosine phosphatase inhibitor sodium orthovanadate (100 μM), or they were processed without any inhibitor treatment. FLAG-FAK was captured from clarified cell extracts made in CSK-NP buffer [150 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.4, 10% glycerol, 10 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1% NP-40, 1× EDTA-free protease inhibitors (Roche)] using M2-agarose affinity resin (anti-FLAG antibody conjugated to agarose, Sigma). The M2 agarose beads were sequentially washed with CSK-buffer, MS buffer (150 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.4) and 1 M buffer (1 M NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.4) in order to remove non-specific binding proteins. The FAK protein was eluted with 0.3 mg/ml FLAG peptide in MS buffer. Typically 30-40 pmol of purified FAK was recovered from 107 cells, and 2-10 pmol of protein was used to initiate C18 reverse phase (RP)-HPLC analysis and mass spectrometry. Aliquots of purified FAK protein were digested with either GluC, LysC or AspN or a combination of LysC and AspN to maximize the coverage of peptides analyzed in the mass spectrometer. The resulting peptides were separated by RP-chromatography coupled to online detection by nanoflow LC and microcapillary ESI tandem mass spectrometry (LTQ-FTMS, Thermo Electron, San Jose, CA). In some cases enrichment of phosphopeptides was performed with immobilized metal affinity chromatography (IMAC) (Ficarro et al., 2002), prior to RP chromatography. In those cases, 10-30 pmol of protein were loaded onto IMAC columns.

These studies were supported by an award from NIH-NIGMS 1U54 GM64346-Cell Migration Consortium. Additional support came from a grant from NIH-NCI CA40042 (to J.T.P.).

Bianchi, M., De Luicchini, S., Marin, O., Turner, D. L., Hanks, S. K. and Villa-Moruzzi, E. (
2005
). Regulation of FAK Ser 722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration.
Biochem. J.
(epub).
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.
Calalb, M. B., Zhang, X., Polte, T. R. and Hanks, S. K. (
1996
). Focal adhesion kinase tyrosine-861 is a major site of phosphorylation by Src.
Biochem. Biophys. Res. Commun.
228
,
662
-668.
Carragher, N. O., Fincham, V. J., Riley, D. and Frame, M. C. (
2001
). Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases.
J. Biol. Chem.
276
,
4270
-4275.
Chen, H. C., Appeddu, P. A., Parsons, J. T., Hildebrand, J. D., Schaller, M. D. and Guan, J. L. (
1995
). Interaction of focal adhesion kinase with cytoskeletal protein talin.
J. Biol. Chem.
270
,
16995
-16999.
Cooper, L. A., Shen, T. L. and Guan, J. L. (
2003
). Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction.
Mol. Cell. Biol.
23
,
8030
-8041.
Dunty, J. M., Gabarra-Niecko, V., King, M. L., Ceccarelli, D. F., Eck, M. J. and Schaller, M. D. (
2004
). FERM domain interaction promotes FAK signaling.
Mol. Cell. Biol.
24
,
5353
-5368.
Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F. and White, F. M. (
2002
). Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.
Nat. Biotechnol.
20
,
301
-305.
Gabarra-Niecko, V., Schaller, M. D. and Dunty, J. M. (
2003
). FAK regulates biological processes important for the pathogenesis of cancer.
Cancer Metastasis Rev.
22
,
359
-374.
Gervais, F. G., Thornberry, N. A., Ruffolo, S. C., Nicholson, D. W. and Roy, S. (
1998
). Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide.
J. Biol. Chem.
273
,
17102
-17108.
Harte, M. T., Hildebrand, J. D., Burnham, M. R., Bouton, A. H. and Parsons, J. T. (
1996
). p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase.
J. Biol. Chem.
271
,
13649
-13655.
Hildebrand, J. D., Schaller, M. D. and Parsons, J. T. (
1995
). Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.
Mol. Biol. Cell
6
,
637
-647.
Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (
1996
). An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.
Mol. Cell. Biol.
16
,
3169
-3178.
Hunger-Glaser, I., Salazar, E. P., Sinnett-Smith, J. and Rozengurt, E. (
2003
). Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation.
J. Biol. Chem.
278
,
22631
-22643.
Hynes, R. O. (
1992
). Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69
,
11
-25.
Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M. and Yamamoto, T. (
1995
). Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
Nature
377
,
539
-544.
Kadare, G., Toutant, M., Formstecher, E., Corvol, J. C., Carnaud, M., Boutterin, M. C. and Girault, J. A. (
2003
). PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation.
J. Biol. Chem.
278
,
47434
-47440.
Lauffenburger, D. A. and Horwitz, A. F. (
1996
). Cell migration: a physically integrated molecular process.
Cell
84
,
359
-369.
Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V. and Parsons, J. T. (
2002
). The association of ASAP1, an ADP ribosylation factor-GTPase activating protein, with focal adhesion kinase contributes to the process of focal adhesion assembly.
Mol. Biol. Cell
13
,
2147
-2156.
Ma, A., Richardson, A., Schaefer, E. M. and Parsons, J. T. (
2001
). Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130(Cas).
Mol. Biol. Cell
12
,
1
-12.
Mitra, S. K., Hanson, D. A. and Schlaepfer, D. D. (
2005
). Focal adhesion kinase: in command and control of cell motility.
Nat. Rev. Mol. Cell. Biol.
6
,
56
-68.
Parsons, J. T. (
2003
). Focal adhesion kinase: the first ten years.
J. Cell Sci.
116
,
1409
-1416.
Parsons, J. T., Martin, K. H., Slack, J. K., Taylor, J. M. and Weed, S. A. (
2000
). Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement.
Oncogene
19
,
5606
-5613.
Polte, T. R. and Hanks, S. K. (
1995
). Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.
Proc. Natl. Acad. Sci. USA
92
,
10678
-10682.
Poullet, P., Gautreau, A., Kadare, G., Girault, J. A., Louvard, D. and Arpin, M. (
2001
). Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.
J. Biol. Chem.
276
,
37686
-37691.
Richardson, A., Malik, R. K., Hildebrand, J. D. and Parsons, J. T. (
1997a
). Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation.
Mol. Cell. Biol.
17
,
6906
-6914.
Richardson, A., Shannon, J. D., Adams, R. B., Schaller, M. D. and Parsons, J. (
1997b
). Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A.
Biochem. J.
324
,
141
-149.
Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R. and Parsons, J. T. (
1994
). Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.
Mol. Cell. Biol.
14
,
1680
-1688.
Schlaepfer, D. D. and Hunter, T. (
1996
). Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.
Mol. Cell. Biol.
16
,
5623
-5633.
Schlaepfer, D. D. and Mitra, S. K. (
2004
). Multiple connections link FAK to cell motility and invasion.
Curr. Opin. Genet. Dev.
14
,
92
-101.
Schlaepfer, D. D., Hanks, S. K., Hunter, T. and van der Geer, P. (
1994
). Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.
Nature
372
,
786
-791.
Sieg, D. J., Hauck, C. R., Ilic, D., Klingbeil, C. K., Schaefer, E., Damsky, C. H. and Schlaepfer, D. D. (
2000
). FAK integrates growth-factor and integrin signals to promote cell migration.
Nat. Cell Biol.
2
,
249
-256.
Toutant, M., Costa, A., Studler, J. M., Kadare, G., Carnaud, M. and Girault, J. A. (
2002
). Alternative splicing controls the mechanisms of FAK autophosphorylation.
Mol. Cell. Biol.
22
,
7731
-7743.
Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T. and Horwitz, A. F. (
2004
). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.
Nat. Cell Biol.
6
,
154
-161.
Xie, Z. and Tsai, L. H. (
2004
). Cdk5 phosphorylation of FAK regulates centrosome-associated miocrotubules and neuronal migration.
Cell Cycle
3
,
108
-110.