Abstract
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.
Highlights
Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that associates with and is activated by integrins in cells at substratum contact sites termed focal adhesions (FAs) [7, 8]
We find that FAK activity controls p190A tyrosine phosphorylation linked to decreased RhoA GTPase activity initiated by integrin binding to fibronectin
Mice—FAK R454 knock-in mutation was generated by homologous recombination (InGenious Targeting Laboratory, Stony Brook, NY) with the cloning strategy and methods shown in supplemental Fig. 1
Summary
Mice—FAK R454 knock-in mutation was generated by homologous recombination (InGenious Targeting Laboratory, Stony Brook, NY) with the cloning strategy and methods shown in supplemental Fig. 1. Vascular and primary capillary plexus structures were visualized by rat anti-mouse CD31 staining (1:100 for 4 h) and detected by incubation with fluorescein-conjugated goat antirat IgG. Cell Migration—Millicell serum chemotaxis assays were performed as described [23], and data points represent enumerations of three migration chambers from at least two independent experiments. Integrin-stimulated signaling assays were performed by serum starvation (0.5% fetal bovine serum) of cells overnight, limited trypsin treatment (0.06% with 2 mM EDTA) to collect cells, inactivation of trypsin with soybean trypsin inhibitor (0.25 mg/ml), and resuspension of cells in migration medium (Dulbecco’s modified Eagle’s medium with 0.5% bovine serum albumin). Golgi Reorientation-Polarity Assay—Golgi reorientation analyses were performed in a scratch wound assay as described [26]. Analyses were performed on 100 cells/experiment to determine the percentage of cells with reoriented Golgi
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