Mechanism of Focal Adhesion Kinase Mechanosensing

Abstract

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.

Fig 1. Mechanical activation of FK-FAK.

A) Domain organization of FAK and its relative position at the cell periphery in the cytosol. The kinase domain (orange) contains the lobes N and C and the FERM domain (blue) consists of lobes F1–3, with F2 binding to PIP₂ lipids (red) concentrated at the inner leaflet (IL) of the membrane (grey). The major phosphorylation site Tyr576/577 (magenta star), located at the activation loop (green) in the kinase domain, is autoinhibited by the FERM domain. The autophosphorylation site Tyr397 (grey star) is positioned in the loop connecting the kinase and FERM domains (grey). The FAT domain (violet) is not considered in our study. B) Stretching force applied to the basic patch in FERM and the kinase C-terminal residue (red), in form of virtual springs, induces FERM-kinase dissociation. Representative structures of FAK in its initial autoinhibited conformation (1), after dissociation of the kinase C-lobe from the FERM F2 lobe (2), and after Tyr576/577 release and partial C-terminal unfolding (3) are shown. Color-code and orientation of the protein as in A. C) Cumulative number of dissociation events as a function of the distance between the pulled elements at the moment of dissociation (D_e−e). This indicates the extent of unfolding prior dissociation. Two events were monitored: dissociation of the the FERM F2 lobe from the kinase C-lobe, F2-C (transition from (1) to (2) in B), and separation of FERM domain from the Tyr576–577 phosphorylation site, F-YY (transition from (2) to (3) in B). Total number of simulations (83) is indicated with the dashed line.

Fig 2. Mechanical activation of FAK bound to the membrane.

A) Force was applied to the C-terminus of FK-FAK, in vertical or diagonal direction with respect to the membrane, with a counter-force acting on the membrane, leading to the release of autoinhibition (left to right transition). FK-FAK is shown as in Fig 1B, PIP₂ lipids in the membrane (here at 15%) in cyan/red and POPE lipids in grey. B-C) Number of contacts N between the FERM F2-lobe (F2) and the kinase C-lobe (C) compared to the number of contacts between both lobes and the membrane (mem), at 15% (B) and 1% (C) PIP₂ concentration. Number of contacts between lobes was defined as the number of atoms in one of the lobes closer than 0.6 nm to at least one atom of the other lobe. Upper panels show results for diagonal pulling while lower panels for vertical pulling. Densities of N (for a pulling velocity of 0.03 nm/ns) are shown as a grey gradient, with a polynomial fit to the data shown as a solid black line. The labels i, a, b, and u correspond to the inactive, active, bound and unbound states of FK-FAK, respectively, sketched at the right side.

Fig 3. Mechanism of FK-FAK mechanical activation.

A) Interfacial area between the F2- and C-lobe (grey) and average force exerted by the two springs (blue) as a function of the distance between springs, D_spring. Results from six independent FPMD simulations are shown: (1–3) without the membrane pulling at V = 0.006, 0.006 and 0.014 nm/ns, respectively, and (4 and 5) pulling diagonally away from the membrane at V = 0.03 and 0.05 nm/ns, respectively. The interfacial area drops from initial values of 3–4.5 nm² to intermediate values of 1.5–2.8 nm². Afterwards it decreases to zero. Rupture force (highest force peak) always corresponded to the first drop in the interfacial area (red line). The peak force associated to the second drop in the area is highlighted with the green line. B) Distribution of interface areas reflecting the two states of FK-FAK during its force-induced opening (highlighted with arrows). All FPMD simulations were considered to compute the distribution. C) Residues involved in the rupture steps are highlighted as sticks. FERM F2- and Kinase C-lobe are shown in surface representation. Rupture steps are associated to the disruption of hydrophobic interactions (green); salt bridges (blue) and other electrostatic interactions (magenta), and interactions with other partners (cyan). Residues were identified by TRFDA (S5 Fig). They are listed in S2 Table.

Fig 4. FAK mechano-signaling.

A) Rupture force F as a function of the loading rate $\dot{F}$, where F is defined as the maximal force observed during FK-FAK activation (arrow in the inset). Light grey dots represent individual rupture forces F observed in our membrane-free FPMD simulations. Dark grey dots represent their averages (for each loading rate $\dot{F}$). The solid line shows the mean rupture force 〈F〉 predicted by the BSK model [45] for ΔG = 28.5 k _B T, x _b = 0.86 nm, and D = 6.6 × 10⁶ nm²/s. A fit with the HS [44] model yields similar model parameters (not shown). Dashed lines show the variation of the rupture forces predicted by the BSK model (2 standard deviations, see S1 Text for a detailed analysis). Pulling membrane-bound FK-FAK diagonally yielded similarly large rupture forces (green dots). Vertical pulling resulted in significantly lower rupture forces (pink dots), as this direction promotes the less resistant zipper-like dissociation mechanism described in S4B and S4C Fig) Time at which 50% of inactive FAK (B) and GDP-bound Ras protein (C) are consumed, under varying external force. Times obtained for three sets of parameters (1 to 3) corresponding to the three fits presented in S6 Fig.