NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal focal adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called focal adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.     

The structural basis of localization and signaling by the focal adhesion targeting domain

The localization of focal adhesion kinase (FAK) to sites of integrin clustering initiates downstream signaling. The C-terminal focal adhesion targeting (FAT) domain causes this localization by interacting with talin and paxillin. FAT also mediates signaling through Grb2 via phosphorylated Y925. We report two crystal structures of the FAT domain. Large rearrangements of the structure are indicated to allow phosphorylation of Y925 and subsequent interaction with Grb2. Sequence homology and structural compatibility suggest a FAT-like fold for the C-terminal domains of CAS, Efs/Sin, and HEF1. A structure-based alignment including these proteins and the vinculin tail domain reveals a conserved region that could play a role in focal adhesion targeting. Previously postulated "paxillin binding subdomains" may contribute to structural integrity rather than directly to paxillin binding.     

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.     

New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate

Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.     

Structural features of the focal adhesion kinase-paxillin complex give insight into the dynamics of focal adhesion assembly

The C-terminal region of focal adhesion kinase (FAK) consists of a right-turn, elongated, four-helix bundle termed the focal adhesion targeting (FAT) domain. The structure of this domain is maintained by hydrophobic interactions, and this domain is also the proposed binding site for the focal adhesion protein paxillin. Paxillin contains five well-conserved LD motifs, which have been implicated in the binding of many focal adhesion proteins. In this study we determined that LD4 binds specifically to only a single site between the H2 and H3 helices of the FAT domain and that the C-terminal end of LD4 is oriented toward the H2-H3 loop. Comparisons of chemical-shift perturbations in NMR spectra of the FAT domain in complex with the binding region of paxillin and the FAT domain bound to both the LD2 and LD4 motifs allowed us to construct a model of FAK-paxillin binding and suggest a possible mechanism of focal adhesion disassembly.     

Structural insight into the mechanisms of targeting and signaling of focal adhesion kinase

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domain interacts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a "right-turn" elongated bundle; the globular fold is mainly maintained by hydrophobic interactions. The bound peptide further stabilizes the structure. Certain signaling events such as phosphorylation and molecule interplay may induce opening of the helix bundle. Such conformational change is proposed to precede departure of FAK from focal adhesions, which starts focal adhesion turnover.     

Crystal structures of free and ligand-bound focal adhesion targeting domain of Pyk2

Focal adhesion targeting (FAT) domains target the non-receptor tyrosine kinases FAK and Pyk2 to cellular focal adhesion areas, where the signaling molecule paxillin is also located. Here, we report the crystal structures of the Pyk2 FAT domain alone or in complex with paxillin LD4 peptides. The overall structure of Pyk2-FAT is an antiparallel four-helix bundle with an up-down, up-down, right-handed topology. In the LD4-bound FAT complex, two paxillin LD4 peptides interact with two opposite sides of Pyk2-FAT, at the surfaces of the α1α4 and α2α3 helices of each FAT molecule. We also demonstrate that, while paxillin is phosphorylated by Pyk2, complex formation between Pyk2 and paxillin does not depend on Pyk2 tyrosine kinase activity. These experiments reveal the structural basis underlying the selectivity of paxillin LD4 binding to the Pyk2 FAT domain and provide insights about the molecular details which influence the different behavior of these two closely-related kinases.     

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.     

Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion-dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion-targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion-dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.     

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.     

The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.

Focal adhesion kinase (FAK) is a tyrosine kinase found in focal adhesions, intracellular signaling complexes that are formed following engagement of the extracellular matrix by integrins. The C-terminal 'focal adhesion targeting' (FAT) region is necessary and sufficient for localizing FAK to focal adhesions. We have determined the crystal structure of FAT and show that it forms a four-helix bundle that resembles those found in two other proteins involved in cell adhesion, alpha-catenin and vinculin. The binding of FAT to the focal adhesion protein, paxillin, requires the integrity of the helical bundle, whereas binding to another focal adhesion protein, talin, does not. We show by mutagenesis that paxillin binding involves two hydrophobic patches on opposite faces of the bundle and propose a model in which two LD motifs of paxillin adopt amphipathic helices that augment the hydrophobic core of FAT, creating a six-helix bundle.     

Force-induced Unfolding of the Focal Adhesion Targeting Domain and the Influence of Paxillin Binding

Membrane-bound integrin receptors are linked to intracellular signaling pathways through focal adhesion kinase (FAK). FAK tends to colocalize with integrin receptors at focal adhesions through its C-terminal focal adhesion targeting (FAT) domain. Through recruitment and binding of intracellular proteins, FAs transduce signals between the intracellular and extracellular regions that regulate a variety of cellular processes including cell migration, proliferation, apoptosis and detachment from the ECM. The mechanism of signaling through the cell is of interest, especially the transmission of mechanical forces and subsequent transduction into biological signals. One hypothesis relates mechanotransduction to conformational changes in intracellular proteins in the force transmission pathway, connecting the extracellular matrix with the cytoskeleton through FAs. To assess this hypothesis, we performed steered molecular dynamics simulations to mechanically unfold FAT and monitor how force-induced changes in the molecular conformation of FAT affect its binding to paxillin.     

pHocal adhesion kinase regulation is on a FERM foundation

Increases in intracellular pH (pHi) occur upon integrin receptor binding to matrix proteins and in tumor cells. In this issue, Choi et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308034) show that pHi increase activates FAK by causing deprotonation of histidine 58 in its FERM (band 4.1, ezrin, radixin, moesin) homology domain, which exposes a region important for FAK autophosphorylation. This model of FAK activation could contribute to motility of tumor cells by promoting focal adhesion turnover.Integrins are transmembrane receptors for extracellular matrix that mediate physical cell attachment and also control cell shape, growth, and survival. Integrin signals are generated by the recruitment and activation of protein tyrosine kinases (PTKs) such as Src, Abl, Syk, and FAK that initiate protein phosphorylation signaling cascades. These sites of integrin signal initiation and cell attachment are generally termed focal adhesions. Despite identification of adhesion protein constituents, our understanding of the molecular mechanisms of PTK activation at focal adhesions remains rudimentary. FAK, Src, and Abl signaling contribute to tumor growth and metastasis, and small molecule drugs targeting these PTKs have been approved or are undergoing clinical trials. However, for FAK we really do not fully understand the contributing factors that lead to its elevated activation, and although FAK is an amplified gene in cancer, mutations that increase FAK activation are uncommon.In this issue, Choi et al. (2013) elucidate a novel connection between increased intracellular pH (pHi) and FAK activation. In the early 1990s, transient pHi elevation upon matrix binding was one of the first integrin-associated signals identified (Schwartz et al., 1991). Pharmacological inhibitors pointed to the importance of sodium–proton antiporters in mediating increased pHi. NHE-1 (sodium-hydrogen antiporter 1) is part of a larger family (NHE1-9) and a ubiquitously expressed transmembrane protein that actively extrudes protons from inside the cell to counter balance acidity and maintain cytosolic pHi (Malo and Fliegel, 2006). NHE-I can be found at focal adhesions (Grinstein et al., 1993) and can connect to the actin cytoskeleton via binding to ezrin (Denker et al., 2000). NHE-1 point mutations disrupting either ion translocation or its binding to ezrin prevent cell migration (Denker and Barber, 2002). NHE-1 overexpression in cancer cells elevates pHi and tumor progression (Webb et al., 2011).Also in the early 1990s, FAK was the first PTK shown to localize to focal adhesions and to be activated by integrins (Parsons, 2003). FAK and the closely related proline-rich tyrosine kinase 2 (Pyk2) share a common domain structure of an N-terminal FERM domain and an ∼40 amino acid linker domain containing an autophosphorylation site (Y397 in FAK) that serves as a Src homology 2 (SH2) binding site for Src-family PTKs. The FAK linker is followed by a central kinase domain, a scaffolding region containing two proline-rich motifs that are SH3 domain binding sites, and a C-terminal focal adhesion–targeting (FAT) domain (Fig. 1 A). It is the FAK FAT domain that binds to integrin-associated proteins (paxillin and talin) and facilitates FAK phosphorylation at Y397 via protein clustering (Toutant et al., 2002). However, the importance of the results of Choi et al. (2013) lies in the role of the FAK FERM domain in the intramolecular regulation of FAK Y397 autophosphorylation.Open in a separate windowFigure 1.Overview of FAK structure and activation. (A) FAK schematic. Depicted is the FAK N-terminal FERM domain comprised of three lobes (F1, F2, and F3), a linker domain, central kinase domain, and a C-terminal focal adhesion–targeting (FAT) domain. Shown are histidine (H) residue 58 and tyrosine (Y) residues 194, 397, 576/577, and 925, and proline-rich domains (Pro-1, Pro-2, and Pro-3) that are sites for SH3 domain binding. FERM plus linker (F+L), FERM-linker-kinase (F+L+K), and linker-kinase (L+K) are constructs used by Choi et al. (2013). (B) Model of growth factor–stimulated FAK activation. Upon binding of the FAK FERM F2 lobe to c-Met receptor and/or phosphatidylinositol 4,5-P₂ (PIP2) lipid, FAK undergoes conformational changes and Y194 phosphorylation, leading to FAK Y397 autophosphorylation in the linker region. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation. HGF, hepatocyte growth factor.FERM domains are typically comprised of three lobes (F1, F2, and F3) grouped in a cloverleaf-like structure (Frame et al., 2010). In an inactive conformation, the FAK FERM F2 lobe binds to and blocks the FAK kinase domain active site. The FAK FERM F1 lobe binds to and sequesters FAK Y397 in the linker region (Fig. 1 B). Point mutations of FAK in the F1 lobe, F2 lobe, or within the kinase domain can weaken these inhibitory intramolecular binding interactions and result in elevated FAK Y397 phosphorylation (Lietha et al., 2007). It has been hypothesized that the normal sequence of events for FAK activation starts with the binding of some “activating” factor to the FAK F2 lobe that would trigger FERM lobe displacement and allow FAK cis or trans auto-phosphorylation of Y397 (Fig. 1 B). Subsequent full FAK activation occurs via SH2 domain binding of Src to phosphorylated Y397, resulting in Src-mediated phosphorylation within the FAK kinase domain at Y576 and Y577 to promote catalytic activation and phosphorylation within the FAT domain at Y925 to promote Grb2 SH2 binding (Schaller, 2010). Recently, growth factor receptor phosphorylation of FAK at Y194 within the FERM F2 lobe was shown to promote FAK activation (Fig. 1 B; Chen et al., 2011). Conformational changes triggered by FAK kinase activity also regulate FAK FERM–mediated binding to targets such as VE-cadherin (Chen et al., 2012). Outside of FAK phosphorylation at Y194, and a potential role for lipid binding to FAK FERM (Cai et al., 2008), additional “initiators” of FERM conformational changes remain undefined.Choi et al. (2013) provide new and important insights in FERM-mediated FAK activation by changes in pHi. By using a chimeric paxillin construct fused to the pH biosensor pHluorin and to mCherry, Choi et al. (2013) showed that pHi increases within peripheral adhesions in mouse embryo fibroblasts (MEFs) spreading on fibronectin (FN) at 30 to 50 min after plating. Stable NHE-1 knockdown resulted in the lowering of pHi and the inhibition of FAK but not Src activation at 30 and 60 min during MEF spreading on FN. Interestingly, NHE-1 knockdown or pharmacological NHE-1 inhibition resulted in MEFs with a rounded morphology and an increased number of small focal adhesions. This adhesion and spreading phenotype is similar to that of FAK-null MEFs (Sieg et al., 1999).To determine if there was a connection between increased pHi and FAK activation, Choi et al. (2013) performed recombinant FAK in vitro phosphorylation assays. The FAK FERM-linker-kinase (F+L+K) fragment but not the linker-kinase (L+K) (Fig. 1 A) exhibited increased Y397 phosphorylation as a function of pH (pH 7.5 > pH 6.5). Intriguingly, regulation of FAK Y397 phosphorylation by pH was dependent upon the presence of the FAK FERM domain, which was shown by using the FERM-linker (F+L) as a substrate in trans (Fig. 1 A). These results support the hypothesis that exposure of the FAK linker region for phosphorylation is pH dependent (Fig. 2). Changes in amino acid protonation can be considered a post-translational modification, as ionic interactions contribute to secondary and tertiary protein structure (Schönichen et al., 2013). The pK_a of histidine is ∼6.5, and variations from this value depend upon the local protein environment. The challenge is to determine which sites functionally serve as pH sensors in vivo.Open in a separate windowFigure 2.Simplified model of FAK activation via histidine 58 (H58) deprotonation. The FAK FERM F1 lobe sequesters FAK Y397 in the linker region keeping FAK in an inactive and closed conformation. Integrin engagement at focal adhesions results in transient and local increases of pHi through NHE-1 activity. Changes in pHi result in H58 deprotonation within the FERM F1 lobe, leading to FAK conformational changes that expose the FAK linker region and enabling FAK Y397 autophosphorylation. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation.The FAK FERM domain contains seven histidine residues, three of which (H41, H58, and H75) are within the FERM F1 lobe. Mutation of these sites individually to alanine revealed that H58A mutation selectively enhanced F+L and F+L+K Y397 phosphorylation at pH 6.5. This approach allowed Choi et al. (2013) to conclude that H58 deprotonation confers some type of change within the F+L region to facilitate Y397 autophosphorylation (Fig. 2). Using the crystal structure of FAK F+L+K as a template, molecular dynamic simulations with H58 (neutral or positively charged) revealed conformational differences within residues of the linker region around Y397 despite the lack of direct binding to H58. Although Choi et al. (2013) did not identify key partner electrostatic interactions that contribute to conformational changes upon H58 deprotonation, recent studies by (Ritt et al. (2013) proposed that E466 within the FAK kinase domain may be important for this regulation within full-length FAK. However, this does not explain results from Choi et al. (2013) for the pH dependence of Y397 phosphorylation within F+L.Lastly, full-length H58A FAK exhibited elevated Y397 phosphorylation upon re-expression in FAK-null MEFs in combination with NHE-1 shRNA knockdown. H58A FAK promoted spreading and adhesion changes in both control and NHE-1 shRNA FAK-null MEFs, whereas wild-type FAK did not rescue FAK-null phenotypes in the absence of NHE-1 expression. Choi et al. (2013) note that pH-regulated FAK Y397 phosphorylation required combined integrin stimulation and elevated pH to activate FAK. In tumor cells, NHE-1 inhibition prevents elevated FAK Y397 phosphorylation and recent studies show that FAK can phosphorylate cortactin to promote adhesion turnover (Tomar et al., 2012). Cortactin tyrosine phosphorylation facilitates the recruitment of NHE-1 to tumor cell invadopodia (Magalhaes et al., 2011), leading to the pH-dependent release of actin-depolymerizing factor cofilin from cortactin (Frantz et al., 2008). As FAK activity promotes ovarian and breast tumor metastasis (Walsh et al., 2010; Ward et al., 2013), it is possible that FAK may serve as a pH-dependent sensor to initiate cell spreading.In the control of cell motility, NHE-1 is postulated to create pH nanodomains at focal adhesions to control protein–protein interactions (Ludwig et al., 2013). A simplistic model is that integrin clustering facilitates rapid FAK recruitment to focal adhesion where increases in pH trigger FERM conformational changes, release of the FAK linker region, and allow for FAK Y397 phosphorylation in cis or trans (Fig. 2). Adhesion turnover is increased at alkaline pHi, consistent with leading edge cell spreading and extension. At nascent adhesions FAK recruits talin (Lawson et al., 2012), and at alkaline pH FAK signaling activity may be enhanced over talin binding to filamentous actin needed for adhesion maturation (Srivastava et al., 2008). However, as pHi falls, pH sensor residues within the talin rod domain confer enhanced actin binding and this may be part of a signaling switch to promote a cycle of focal adhesion maturation. Additionally, pHi changes may alter phosphorylation site specificity by Src within the FAK FAT domain (Cable et al., 2012). Thus, pHi can affect FAK activity and FAK phosphorylation. Moreover, the FAK-related Pyk2 PTK is activated by acidic pHi within cells of the kidney (Li et al., 2004). Although the molecular mechanism is not known as to how Pyk2 is regulated by acidity, clearly there is much more to discover about the role of pHi changes at adhesions and invadopodia and how this may alter PTK activation in the control of cell movement and invasion.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Arf GTPase-activating Protein AGAP2 Regulates Focal Adhesion Kinase Activity and Focal Adhesion Remodeling

Focal adhesions are specialized sites of cell attachment to the extracellular matrix where integrin receptors link extracellular matrix to the actin cytoskeleton, and they are constantly remodeled during cell migration. Focal adhesion kinase (FAK) is an important regulator of focal adhesion remodeling. AGAP2 is an Arf GTPase-activating protein that regulates endosomal trafficking and is overexpressed in different human cancers. Here we examined the regulation of the FAK activity and the focal adhesion remodeling by AGAP2. Our results show that FAK binds the pleckstrin homology domain of AGAP2, and the binding is independent of FAK activation following epidermal growth factor receptor stimulation. Overexpression of AGAP2 augments the activity of FAK, and concordantly, the knockdown of AGAP2 expression with RNA interference attenuates the FAK activity stimulated by epidermal growth factor or platelet-derived growth factor receptors. AGAP2 is localized to the focal adhesions, and its overexpression results in dissolution of the focal adhesions, whereas knockdown of its expression stabilizes them. The AGAP2-induced dissolution of the focal adhesions is independent of its GTPase-activating protein activity but may involve its N-terminal G protein-like domain. Our results indicate that AGAP2 regulates the FAK activity and the focal adhesion disassembly during cell migration.Focal adhesions are macromolecular structures that connect actin cytoskeleton to the extracellular matrix and play an important role in cell migration (1). Components of focal adhesions include signaling proteins such as focal adhesion kinase (FAK),³ c-Src, and paxillin, as well as structural proteins such as talin and vinculin (2, 3). Focal adhesions are constantly formed and disassembled (i.e. remodeled) at the leading edge of migrating cells, and they are disassembled at the trailing edge during the cell migration (4, 5). Available evidence demonstrates that the remodeling of focal adhesions is regulated by FAK (6) and Arf-directed GTPase-activating proteins (Arf GAPs) (7).FAK is a member of the Src family nonreceptor tyrosine kinases whose activities are regulated by intra-molecular phosphorylation (8). Autophosphorylation of FAK on tyrosine residue 397 provides docking sites for Src homology 2 domain-containing proteins, including c-Src. After binding to FAK, c-Src phosphorylates FAK on Tyr-576 and Tyr-577 to activate fully the intrinsic kinase activity of FAK (9). Cellular functions of FAK are many and include cell migration, survival, and proliferation; and activation of FAK occurs upon integrin clustering or stimulation of cell surface receptors such as the epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) receptors. FAK activation following integrin clustering results in recruitment of structural and signaling proteins that collectively contribute to the formation of the focal adhesions (10). In FAK null cells, focal adhesions are formed but cannot disassemble (11), suggesting that FAK is required for the focal adhesion disassembly.ADP-ribosylation factors (Arfs) are GTP-binding proteins that lack detectable intrinsic GTPase activities. Therefore, hydrolysis of GTP bound to Arf is mediated by Arf GAPs (12, 13). The AZAP family of Arf GAPs are multidomain proteins that contain a catalytic core of pleckstrin homology (PH), Arf GAP, and ankyrin repeat domains (14), and each subgroup possesses characteristic domain(s). For example, ASAPs have a BAR (Bin, Amphiphysin, Rvs) domain at their N termini and a Src homology 3 domain at their C termini; ARAPs have a Rho GAP domain and five PH domains; and AGAPs have a G protein-like domain (GLD) at their N termini and their PH domains are split, i.e. there is an insert of 80–100 amino acids between the β5 strand and β6 strand. The Arf GAPs regulate membrane trafficking and remodeling of the actin cytoskeleton (7, 15), but the molecular mechanisms underlying the contribution of individual Arf GAPs to membrane trafficking and actin remodeling are being defined. We have reported that AGAP2 binds the clathrin adaptor protein AP-1 and regulates the AP-1/Rab4-dependent endosomal trafficking (16). Studies from other groups have indicated that AGAP2 is overexpressed in different human cancers, including glioblastoma, and that AGAP2 enhances the invasion of glioblastoma cells (17, 18).In this study, we tested the hypothesis that AGAP2 regulates focal adhesion remodeling and cell migration. We find that AGAP2 forms a complex with FAK, increases the FAK activity, and provokes the focal adhesion disassembly that may lead to increased cell migration. Some Arf GAPs have been shown to regulate focal adhesions, and each Arf GAP seems to regulate the focal adhesions by a distinct mechanism. Our results introduce a new way to regulate the focal adhesions by the Arf GAP AGAP2, i.e. through the regulation of FAK activity. These observations support the idea that various Arf GAPs function coordinately to provide temporal and spatial regulation of the focal adhesions during cell migration.     

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.     

The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.     

Structural and Mechanistic Insights into the Interaction between Pyk2 and Paxillin LD Motifs

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) subfamily of cytoplasmic tyrosine kinases. The C-terminal Pyk2-focal adhesion targeting (FAT) domain binds to paxillin, an adhesion molecule. Paxillin has five leucine-aspartate (LD) motifs (LD1–LD5). Here, we show that the second LD motif of paxillin, LD2, interacts with Pyk2-FAT, similar to the known Pyk2-FAT/LD4 interaction. Both LD motifs can target two ligand binding sites on Pyk2-FAT. Interestingly, they also share similar binding affinity for Pyk2-FAT with preferential association to one site relative to the other. Nevertheless, the LD2-LD4 region of paxillin (paxillin^133 -290) binds to Pyk2-FAT as a 1:1 complex. However, our data suggest that the Pyk2-FAT and paxillin complex is dynamic and it appears to be a mixture of two distinct conformations of paxillin that almost equally compete for Pyk2-FAT binding. These studies provide insight into the underlying selectivity of paxillin for Pyk2 and FAK that may influence the differing behavior of these two closely related kinases in focal adhesion sites.     

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.     

Molecular recognition of leucine-aspartate repeat (LD) motifs by the focal adhesion targeting homology domain of cerebral cavernous malformation 3 (CCM3)

Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ∼15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.