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

Abstract

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).

SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells

“…Our data suggest that, in N1 cells, SHIP2 controls PI(4,5)P2 levels, which could control focal adhesions and/or possibly IQGAP1 activity. PI(4,5)P2 is an upstream activator of pFAK Y397 in focal adhesions and has been reported to change cell attachment and spreading (Goni et al, 2014). We observed that pFAK Y397 is actually upregulated in SHIP2-deficient cells as compared to control cells.…”

“…It activates focal adhesion kinase (FAK, also known as PTK2) by a combination of clustering and conformational changes that promote efficient autophosphorylation (Goni et al, 2014). Consistent with SHIP2-mediated control of PI(4,5)P2 levels, western blotting showed that FAK phosphorylated at Y397 ( pFAK Y397) was upregulated in N1shSHIP2 cells as compared to control cells, with no change in the total levels of FAK (Fig.…”

Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization

“…As discussed above, FAK activation depends on PIP 2 binding, which allows its autophosphorylation (32,33). It seems plausible that the ␥-Pcdh lipid binding motif competes locally with FAK for binding to phospholipids.…”

“…FAK activation depends on its FERM domain binding to PIP 2 , which opens up its structure, exposing its kinase domain and allowing autophosphorylation (32,33). It could be that the ␥-Pcdh lipid binding motif competes locally with FAK for binding to phospholipids, which might reduce the activation of ␥-Pcdh-associated FAK.…”

Addressing the Functional Determinants of FAK during Ciliogenesis in Multiciliated Cells

“…It has been implicated in a plethora of interactions that regulate the activity of FAK and downstream signaling. Such interactions include binding to growth factor receptors and phosphatidylinositol 4,5-biphosphate (PIP 2 ) (2,(12)(13)(14)(15). Moreover, the FERM domain has been shown to interact with the Arp2/3 complex and N-WASP, providing a link between integrin activation and remodeling of the actin cytoskeleton, which is critical for cell spreading and migration, whereas it has also been proven to be responsible for the interaction of FAK with the ERM protein ezrin (16 -18).…”