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.
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.
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)P 2 ]. 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)P 2 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)P 2 induces a partially open FAK conformation where the autophosphorylation site is exposed, facilitating efficient autophosphorylation and subsequent Src recruitment. However, PI(4,5)P 2 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)P 2 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.cell signaling | phosphoinositides C ell attachment to the ECM is mediated via integrin transmembrane receptors on the cell surface. Integrin engagement to ECM components results in activation and clustering of integrins. In response to integrin activation, a large number of proteins are recruited to their cytoplasmic tails, resulting in the formation of focal adhesions (FAs) (1). On the one side, FAs are anchoring points for actomyosin stress fibers, which allow tension forces to build up when contracting fibers exert their pulling force via FAs against the ECM. On the other hand, integrin activation and the generation of tension trigger intricate signaling cascades. A central signaling component in FAs is the nonreceptor tyrosine kinase (NRTK) focal adhesion kinase (FAK).
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 kinase (FAK) is an essential non-receptor tyrosine kinase, since FAK-null mice exhibit embryonic lethality (21). In endothelial cells, FAK is required for the proper development of the vasculature (5, 51), and in neurons FAK regulates netrin-mediated axon outgrowth and dendrite formation (41,45). In addition to these roles in development, FAK is also implicated in the pathology of disease. FAK expression is required in cardiomyocytes to promote hypertrophy and fibrosis in response to cardiac stress and potentially plays a role in the development of heart disease (18, 42). Overexpression of FAK is observed in many types of cancer (22), and experiments using animal models have implicated FAK in tumor formation and metastasis in a number of neoplasms, including cancer of the brain, breast, and skin (38,39,58).Despite the importance of FAK in controlling multiple developmental and pathological events, the molecular mechanism of regulation remains incompletely elucidated. Like many kinases posttranslation modification, particularly phosphorylation, is a major regulatory mechanism. Tyrosine 397 is the major site of autophosphorylation, and mutation of this site abrogates the biological activity of FAK (47). This site primarily serves a scaffolding function, providing a docking site for a number of proteins containing SH2 domains, including Src and phosphatidylinositol 3-kinase (47). The interaction with FAK occupies both the SH2 and SH3 domains of Src preventing intramolecular inhibitory interactions resulting in stabilization of Src in its active conformation (54). Complex formation also serves to direct Src to its substrates, which include FAK itself. Activated Src phosphorylates FAK on multiple sites, including two tyrosine residues in the activation loop, Y576 and Y577, which function ...
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