Structural Basis for the Autoinhibition of Focal Adhesion Kinase

SummaryAppropriate 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

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

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

Focal Adhesion Remodeling Is Crucial for Glucose-Stimulated Insulin Secretion and Involves Activation of Focal Adhesion Kinase and Paxillin

Actin cytoskeleton remodeling is known to be involved in glucose-stimulated insulin secretion (GSIS). We have observed glucose-stimulated changes at the β-cell basal membrane similar to focal adhesion remodeling in cell migration. This led us to study the role of two key focal adhesion proteins, focal adhesion kinase (FAK) and paxillin, in GSIS

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

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

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

Nudel and FAK as Antagonizing Strength Modulators of Nascent Adhesions through Paxillin

Competition for binding to the cellular protein paxillin by the proteins Nudel and focal adhesion kinase is important for the proper regulation of cell adhesion and migration

Engineered allosteric activation of kinases in living cells

Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. We describe here a new approach to confer allosteric regulation specifically on the catalytic activity of kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity, but does not affect other protein interactions. Catalytic activity is restored by addition of rapamycin or non-immunosuppresive analogs (Fig. 1A). We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells, thereby demonstrating a novel role for FAK in regulation of membrane dynamics. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. Successful regulation of Src and p38 suggest that modification of this highly conserved site will be applicable to other kinases