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.

Summary Toll-Like Receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/23. Herein we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/23 with an unknown target and has been evaluated in clinical trials for patients with Crohn’s disease or rheumatoid arthritis. Using a chemical genetics approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/23p40 expression. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR signaling.

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 ...

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.Paxillin is a 68-kDa focal adhesion-associated protein that functions as a scaffolding protein assembling signaling molecules into complex downstream of integrins (6, 34). It plays an important role in regulating cell spreading and migration. The paxillin knockout mouse exhibits embryonic lethality, which suggests that paxillin plays an essential role in development (18). Paxillin contains five LD motifs in the N-terminal half of the molecule. These peptide motifs mediate protein-protein interactions and bind a number of proteins, including focal adhesion kinase (FAK) and vinculin (6,40). Four LIM domains are found in the C-terminal half of paxillin, two of which are required for the discrete localization of paxillin to focal adhesions (3).Multiple stimuli induce phosphorylation of paxillin, including growth factors, integrin-dependent cell adhesion to extracellular matrix, and other ligands (6, 34). Two major tyrosine phosphorylation sites, Y31 and Y118, have been identified in the N-terminal half of paxillin (35). Phosphorylation of these sites modulates docking of SH2 domain-containing proteins, such as CRK, and is important for regulation of cell motility (32, 43). In addition to tyrosine phosphorylation sites, serine and threonine phosphorylation sites have been identified in paxillin. Serine residues 188 and 190 are phosphorylated following integrin ligation (1). Threonines 398 and 403 in LIM2 and serines 457 and 481 in LIM3 are phosphorylated following cell adhesion and stimulation with angiotensin II (4, 5). Phosphorylation of these LIM domain residues regulates focal adhesion localization ...

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.