Role of FAK in S1P-regulated endothelial permeability

Abstract

The vascular endothelium serves as a semi-selective barrier between the circulating contents of the blood and the tissues through which they flow. Disruption of this barrier results in significant organ dysfunction during devastating inflammatory syndromes such as sepsis and acute lung injury (ALI). Sphingosine 1-phosphate (S1P) is an endogenous lipid regulator of endothelial permeability that produces potent barrier enhancement via actin and junctional protein rearrangement and resultant cytoskeletal changes. A key effector protein in this S1P response is focal adhesion kinase (FAK), a highly conserved cytoplasmic tyrosine kinase involved in the engagement of integrins and assembly of focal adhesions (FA) through the catalysis of multiple downstream signals. After stimulation by S1P, endothelial FAK undergoes specific tyrosine phosphorylation that results in activation of the kinase and dynamic interactions with other effector molecules to improve the endothelial barrier. FAK participates in peripheral actin cytoskeletal rearrangement as well as cell-matrix (FA) and cell-cell (adherens junction) junctional complex strengthening that combine to decrease vascular permeability. This review summarizes the current knowledge of the role of FAK in mediating enhanced endothelial barrier function by S1P.

Figure 1. S1P regulates endothelial cell structure to enhance barrier function

Ligation of the S1PR1 Gi protein coupled receptor by S1P rapidly (within 1-5 min) activates Rac and recruits signaling molecules and cytoskeletal effectors such as c-Abl, cortactin, and nmMLCK to lipid rafts (or CEMs). Tyrosine phosphorylation of these molecules is observed both in lipid rafts and at the EC periphery in association with cortical actin and lamellipodia formation. This activated complex likely interacts with Arp 2/3 machinery to produce lamellipodia protrusion at the cell periphery, which serves to increase overlap between adjacent EC. The initiation and precise sequence of events responsible for these protein movements are unclear, but within 5 min after S1P stimulation these proteins are found simultaneously distributed in lipid rafts, cortical actin structures, and peripheral membrane ruffling/lamellipodia (indicated by the bi-directional circle). S1P also induces adherens junction (AJ) and tight junction (TJ) assembly that serve to further strengthening the endothelial barrier. Multiple other signaling and cytoskeletal effector molecules participate in this process as reviewed elsewhere (Wang and Dudek 2009). MLCK, nonmuscle myosin light chain kinase; VE-cad, vascular endothelial cadherin; ZO-1, zona occluden protein-1.

Figure 2. Differential phosphorylation of FAK by S1P and thrombin

Depicted are the principal structural domains of FAK and the location of three critical tyrosine phosphorylation sites [adapted from (Schaller 2010)]. Barrier protective S1P induces the specific phosphorylation of the FAK-activating site tyrosine 576 in a Src-dependent manner. Barrier disruptive thrombin induces Src-dependent phosphorylation of tyrosine 576 and tyrosine 925 as well as auto-phosphorylation of tyrosine 397 by FAK (Shikata, Birukov et al. 2003). FERM, four-point-one, ezrin, radixin, moesin binding domain; FAT, focal adhesion targeting domain; Y, tyrosine residue.

Figure 3. FAK is differentially redistributed by S1P and thrombin

Shown are representative merged images of HPAEC immunostained for F-actin (red) and FAK (green) after stimulation with vehicle, thrombin (100 nM, 10 min), or S1P (0.5 μM, 10 min). Thrombin induces FAK localization to the ends of actin stress fibers (yellow, middle panel) while S1P stimulated cells demonstrate a localization of FAK to the cortical actin ring (yellow, right panel). Bar = 10 μm. Reproduced with permission (Shikata, Birukov et al. 2003).

Figure 4. FAK in the S1P barrier-enhancing response

S1P binding to the S1PR1 Gi protein coupled receptor activates Rac and recruits multiple proteins to membraneassociated lipid rafts, including FAK, which exhibits increased tyrosine phosphorylation and presumed activation at this site. Src-dependent phosphorylation of FAK on Y576 also occurs in the cytosol and in existing FA. This phosphorylation releases bound Arp3 from the FAK FERM domain so that it can participate in Arp2/3-catalyzed actin polymerization at peripheral EC membrane ruffling/lamellipodia sites. Src-dependent phosphorylation of FAK on Y576 also transiently increases its association with GIT1 during FA disassembly and rearrangement. FAK, paxillin, and GIT2 then translocate to the EC periphery where they form new focal contacts associated with the increased cortical actin ring produced by S1P. S1P also induces increased peripheral association of FAK, paxillin, VE-cadherin, and β-catenin to strengthen cell-cell AJ linkages. Pax, Paxillin; GIT-1, G protein-coupled receptor kinase interactor-1; GIT-2, G protein-coupled receptor kinase interactor-2 (PKL); Vin, Vinculin; FA, focal adhesion; AJ, adherens junction; VE-cad, Vascular endothelial cadherin.