Daniel Lietha

Structural Basis for the Autoinhibition of Focal Adhesion Kinase

Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.

The FERM domain: organizing the structure and function of FAK

Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural work reveals that regulatory protein partners convert auto-inhibited FAK into its active state by binding to its FERM domain. Further, the identity of FAK FERM domain-interacting proteins yields clues as to how FAK coordinates diverse cellular responses, including cell adhesion, polarization, migration, survival and death, and suggests that FERM domains might mediate information transfer between the cell cortex and nucleus. Importantly, the FAK FERM domain might act as a paradigm for the actions of other FERM domain-containing proteins.

Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells

ABSTRACT Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

Crystal Structures of the FAK Kinase in Complex with TAE226 and Related Bis-Anilino Pyrimidine Inhibitors Reveal a Helical DFG Conformation

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase required for cell migration, proliferation and survival. FAK overexpression has been documented in diverse human cancers and is associated with a poor clinical outcome. Recently, a novel bis-anilino pyrimidine inhibitor, TAE226, was reported to efficiently inhibit FAK signaling, arrest tumor growth and invasion and prolong the life of mice with glioma or ovarian tumor implants. Here we describe the crystal structures of the FAK kinase bound to TAE226 and three related bis-anilino pyrimidine compounds. TAE226 induces a conformation of the N-terminal portion of the kinase activation loop that is only observed in FAK, but is distinct from the conformation in both the active and inactive states of the kinase. This conformation appears to require a glycine immediately N-terminal to the “DFG motif”, which adopts a helical conformation stabilized by interactions with TAE226. The presence of a glycine residue in this position contributes to the specificity of TAE226 and related compounds for FAK. Our work highlights the fact that kinases can access conformational space that is not necessarily utilized for their native catalytic regulation, and that such conformations can explain and be exploited for inhibitor specificity.

Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes

Significance Nonreceptor tyrosine kinases are major players in cell signaling. Among them, focal adhesion kinase (FAK) is the key integrator of signals from growth factors and cell adhesion. In cancer, FAK is frequently overexpressed, and by promoting adhesion to the tumor stroma and ECM, FAK provides important signals for tumor invasion and metastasis. Although autoinhibitory mechanisms have previously been described and the players involved in FAK regulation are largely known, on a mechanistic level, FAK activation is currently not understood. Here, we present a multidisciplinary approach demonstrating a multistep mechanism resulting in FAK activation. This mechanistic insight enables the design of alternative strategies for the discovery of potential anticancer drugs that inhibit both catalytic and scaffolding functions of FAK with high specificity. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase (NRTK) with key roles in integrating growth and cell matrix adhesion signals, and FAK is a major driver of invasion and metastasis in cancer. Cell adhesion via integrin receptors is well known to trigger FAK signaling, and many of the players involved are known; however, mechanistically, FAK activation is not understood. Here, using a multidisciplinary approach, including biochemical, biophysical, structural, computational, and cell biology approaches, we provide a detailed view of a multistep activation mechanism of FAK initiated by phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Interestingly, the mechanism differs from canonical NRTK activation and is tailored to the dual catalytic and scaffolding function of FAK. We find PI(4,5)P2 induces clustering of FAK on the lipid bilayer by binding a basic region in the regulatory 4.1, ezrin, radixin, moesin homology (FERM) domain. In these clusters, PI(4,5)P2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P2 does not release autoinhibitory interactions; rather, Src phosphorylation of the activation loop in FAK results in release of the FERM/kinase tether and full catalytic activation. We propose that PI(4,5)P2 and its generation in focal adhesions by the enzyme phosphatidylinositol 4-phosphate 5-kinase type Iγ are important in linking integrin signaling to FAK activation.

Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension.

Synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK inhibitors with anti-angiogenic activity.

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226.

Design, Synthesis, and Evaluation of Novel Imidazo[1,2-a][1,3,5]triazines and Their Derivatives as Focal Adhesion Kinase Inhibitors with Antitumor Activity

A series of triazinic inhibitors of focal adhesion kinase (FAK) have been recently shown to exert antiangiogenic activity against HUVEC cells and anticancer efficacy against several cancer cell lines. We report herein that we further explored the heterocyclic core of these inhibitors by a fused imidazole ring with the triazine to provide imidazo[1,2-a][1,3,5]triazines. Importantly, these new compounds displayed 10(-7)-10(-8) M IC50 values, and the best inhibitor showed IC50 value of 50 nM against FAK enzymatic activity. Several inhibitors potently inhibited the proliferation of a panel of cancer cell lines expressing high levels of FAK. Apoptosis analysis in U87-MG and HCT-116 cell lines suggested that these compounds delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation demonstrated that these compounds strongly inhibited cell-matrix adhesion, migration, and invasion of U87-MG cells.

Allosteric Regulation of Focal Adhesion Kinase by PIP2 and ATP

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation.

Inhibition of both focal adhesion kinase and fibroblast growth factor receptor 2 pathways induces anti-tumor and anti-angiogenic activities

FAK and FGFR2 signaling pathways play important roles in cancer development, progression and tumor angiogenesis. PHM16 is a novel ATP competitive inhibitor of FAK and FGFR2. To evaluate the therapeutic efficacy of this agent, we examined its anti-angiogenic effect in HUVEC and its anti-tumor effect in different cancer cell lines. We showed PHM16 inhibited endothelial cell viability, adherence and tube formation along with the added ability to induce endothelial cell apoptosis. This compound significantly delayed tumor cell growth. Together, these data showed that inhibition of both FAK and FGFR2 signaling pathways can enhance anti-tumor and anti-angiogenic activities.