Generation of point‐mutant <scp>FAK</scp> knockin mice

Tavora, Bernardo; Batista, Sílvia; Alexopoulou, Annika N.; Kostourou, Vassiliki; Fernandez, Isabelle; Robinson, Stephen D.; Lees, Delphine M.; Serrels, Bryan; Hodivala‐Dilke, Kairbaan

doi:10.1002/dvg.22823

Cited by 8 publications

(16 citation statements)

References 23 publications

(24 reference statements)

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“…Pdgfb‐iCre ert ;FAK fl/fl ;R26FAK WT/WT mice expressed WT FAK, and were used to validate the system. The generation and characterization of these mice, together with proof of chicken FAK knockin, have been described previously .…”

Section: Methodsmentioning

confidence: 99%

“…To further delineate which domains of FAK regulate these phenotypes in vivo , we have recently generated a series of endothelial‐specific inducible mutant FAK knockin mice . In this study, we used the FAK‐K454R mutant, which is KD , and a double mutant (DM) that carries both the KD mutation and a glutamate substitution of Y397 (Y397E), in order to test the requirement for FAK autophosphorylation and Src recruitment.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, after recombination in ECs, the endogenous FAK is replaced by the myc‐tagged FAK, expressed under control of the R26 promoter, at a dose similar to that of endogenous FAK. As the knockin FAK is expressed under control of the R26 promoter, our system does not reflect any changes due to FAK promoter regulation , as other models have .…”

Section: Introductionmentioning

confidence: 99%

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Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Alexopoulou

Lees

Bodrug

et al. 2017

The Journal of Pathology

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Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase‐dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb‐iCreert‐driven endothelial cell (EC)‐specific, tamoxifen‐inducible homozygous mutant mouse lines: FAK wild‐type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT / WT, ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT / WT and ECCre−;FAKWT / WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice, as compared with Cre − littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice. Furthermore, although ECCre+;FAKKD / KD mice showed reduced blood vessel leakage, ECCre+;FAKDM / DM and ECCre−;FAKDM / DM mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD / KD ECs as compared with Cre+;FAKWT / WT cells, with no change in phosphorylation of the known Src targets FAK‐Y577, FAK‐Y861, FAK‐Y925, paxillin‐Y118, p130Cas‐Y410. Cre+;FAKDM / DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE‐cadherin‐pY658 levels in Cre+;FAKKD / KD ECs were rescued in Cre+FAKDM / DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM / DM mice. We show that EC‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Alexopoulou

Lees

Bodrug

et al. 2017

The Journal of Pathology

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“…Here, using inducible EC-specific FAK Y397F/Y397F and FAK Y861F/Y861F mice (24), we have identified the differential in vivo requirements for these motifs in tumor angiogenesis.…”

Section: Introductionmentioning

confidence: 99%

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861

et al. 2019

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Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCreþ;FAK Y397F/Y397F-mutant mice. Conversely, ECCreþ;FAK Y861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased b1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in b1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in VegfaþAng2-or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to VegfaþAng2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. Significance: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

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“…To examine the effect of pericyte-specific FAK mutations in vivo, we developed PdgfrbCre;FAK W/WT mice (FAK WT), PdgfrbCre;FAK Y397F/Y397F (Y397F) mice and PdgfrbCre;FAK Y861F/Y861F (Y861F) mice [44].…”

Section: Micementioning

confidence: 99%

Phosphorylation of pericyte FAK-Y861 affects tumour cell apoptosis and tumour blood vessel regression

Lees¹,

Reynolds²,

Pedrosa³

et al. 2020

Preprint

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Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrbCre+;FAK WT/WT , PdgfrbCre+;FAK Y397F/Y397F and PdgfrbCre+;FAK Y861F/Y861F mice, our data demonstrate that tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrbCre+;FAK Y861F/Y861F but not PdgfrbCre+;FAK Y397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of a pericyte derived 'pericrine' signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

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Generation of point‐mutant FAK knockin mice

Cited by 8 publications

References 23 publications

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium‐specific FAK‐kinase dead mice

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861

Phosphorylation of pericyte FAK-Y861 affects tumour cell apoptosis and tumour blood vessel regression

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