SUMMARY Endothelial cells (ECs) form cell-cell adhesive junctional structures maintaining vascular integrity. This barrier is dynamically regulated by vascular endothelial growth factor (VEGF) receptor signaling. We created an inducible knockin mouse model to study the contribution of the integrin-associated focal adhesion tyrosine kinase (FAK) signaling on vascular function. Here we show that genetic or pharmacological FAK inhibition in ECs prevents VEGF-stimulated permeability downstream of VEGF receptor or Src tyrosine kinase activation in vivo. VEGF promotes tension-independent FAK activation, rapid FAK localization to cell-cell junctions, binding of the FAK FERM domain to the vascular endothelial cadherin (VE-cadherin) cytoplasmic tail, and direct FAK phosphorylation of β-catenin at tyrosine-142 (Y142) facilitating VE-cadherin-β-catenin dissociation and EC junctional breakdown. Kinase inhibited FAK is in a closed conformation that prevents VE-cadherin association and limits VEGF-stimulated β-catenin Y142 phosphorylation. Our studies establish a role for FAK as an essential signaling switch within ECs regulating adherens junction dynamics.

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development.

Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC–specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor–induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKC␣ activity, and it can directly interact with the phosphoinositide PIP 2 . We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKC␣␤␥ show that in the absence of phosphatidylserine and diolein, PIP 2 increased the extent of autophosphorylation of PKC␣␤␥ and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKC␣␤␥ by PIP 2 , resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP 2 and PKC␣, localize them to forming focal adhesions, and potentiate PKC␣ activity there.The control of cellular adhesion status is complex, involving several signaling mechanisms (1-4). Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 plays important roles in the organization of the actin cytoskeleton. PIP 2 may control actin polymerization by regulating the binding of actin-binding proteins such as profilin and gelsolin to actin (5, 6). PIP 2 may also interact with ␣-actinin and vinculin (7) and regulate their association with the cytoskeleton (8). The level of PIP 2 decreases upon detachment of cells from the substratum and increases upon reattachment to fibronectin (1). The difference in the levels of PIP 2 is probably due to different rates of phosphorylation of phosphatidyl 4-phosphate to PIP 2 by phosphatidylinositol 4-phosphate 5-kinase. Phosphatidylinositol 4-phosphate 5-kinase is stimulated 3-4-fold by adhesion of cells to fibronectin (1), probably through interactions with the small GTP-binding proteins Rac and Rho, the latter of which has also been implicated in the regulation of assembly of actin stress fibers and focal adhesions (9 -13).PIP 2 may enter several different pathways in signal transduction. It can be hydrolyzed by phospholipase C␥ to generate two intracellular messengers: inositol 1,4,5-triphosphate, which mobilizes Ca 2ϩ , and diacylglycerol, which is a physiological activator of protein kinase C (PKC). It can be further phosphorylated by phosphatidylinositol 3-kinase to generate phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), which has been proposed to regulate numerous activities including cytoskeletal organization (14) and vesicle trafficking (15). PIP 2 c...

Tumor cells can grow in an anchorage-independent manner. This is mediated in part through survival signals that bypass normal growth restraints controlled by integrin cell surface receptors. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that associates with integrins and modulates various cellular processes including growth, survival, and migration. As increased FAK expression and tyrosine phosphorylation are associated with tumor progression, inhibitors of FAK are being tested for anti-tumor effects. Here, we analyze PND-1186, a substituted pyridine reversible inhibitor of FAK activity with a 50% inhibitory concentration (IC50) of 1.5 nM in vitro. PND-1186 has an IC50 of ~100 nM in breast carcinoma cells as determined by anti-phospho-specific immunoblotting to FAK Tyr-397. PND-1186 did not alter c-Src or p130Cas tyrosine phosphorylation in adherent cells, yet functioned to restrain cell movement. Whereas 1.0 µM PND-1186 (>5-fold above IC50) had limited effects on cell proliferation, under non-adherent conditions or when grown as spheroids or colonies in soft agar, 0.1 µM PND-1186 blocked FAK and p130Cas tyrosine phosphorylation, promoted caspase-3 activation, and triggered cell apoptosis. PND-1186 inhibited 4T1 breast carcinoma subcutaneous tumor growth correlated with elevated tumor cell apoptosis and caspase 3 activation. Addition of PND-1186 to the drinking water of mice was well tolerated and inhibited ascites-associated ovarian carcinoma tumor growth associated with the inhibition of FAK tyrosine phosphorylation. Our results with low-level PND-1186 treatment support the conclusion that FAK activity selectively promotes tumor cell survival in three-dimensional environments.

An alternative linkage is shown whereby FAK brings talin to nascent adhesions independent of talin binding to β1 integrins.

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK R454/R454 embryos. In contrast to the inability of embryonic FAK ؊/؊ cells to proliferate ex vivo, primary FAK R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.