ABSTRACT
Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)-specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.
Subject(s)
Cell Survival , Embryo, Mammalian/physiology , Embryonic Development/physiology , Endothelial Cells/physiology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cadherins/genetics , Cadherins/metabolism , Cells, Cultured , Embryo, Mammalian/anatomy & histology , Endothelial Cells/cytology , Female , Focal Adhesion Protein-Tyrosine Kinases/genetics , Gene Knock-In Techniques , Male , Mice , Mice, Knockout , Neovascularization, Physiologic/physiology , PregnancyABSTRACT
Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis.
Subject(s)
Centrosome/enzymology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Mitosis , Phosphoserine/metabolism , Animals , Cells, Cultured , Cytoplasm/enzymology , Dyneins/metabolism , Focal Adhesion Protein-Tyrosine Kinases/deficiency , Focal Adhesion Protein-Tyrosine Kinases/genetics , Gene Expression Regulation, Enzymologic , Mice , Mice, Knockout , Mutation/genetics , Neoplasms/blood supply , Neoplasms/enzymology , Neoplasms/genetics , Neoplasms/pathology , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Spindle Apparatus/enzymologyABSTRACT
Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.
