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. 2002 Apr 1;157(1):149-60.
doi: 10.1083/jcb.200109079. Epub 2002 Apr 1.

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

Brian P Eliceiri  1 , Xose S Puente, John D Hood, Dwayne G Stupack, David D Schlaepfer, Xiaozhu Z Huang, Dean Sheppard, David A Cheresh
Affiliations

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling

Brian P Eliceiri et al. J Cell Biol. .

Abstract

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin alpha(v)beta5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with beta1 and beta3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/alpha(v)beta5 signaling complex. Moreover, formation of this FAK/alpha(v)beta5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin beta5, but not integrin beta3, have a reduced VP response to VEGF. This FAK/alpha(v)beta5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin alpha(v)beta5 into a FAK-containing signaling complex during growth factor-mediated biological responses.

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Figures

Figure 1.
Figure 1.
VEGF promotes FAK phosphorylation and translocation in endothelial cells. (A) Lysates of VEGF-stimulated primary HUVECs (20 ng/ml; 5 min), mouse brain and lung brain (2 μg/animal, 5 min) were prepared as described in Materials and methods and subjected to immunoblotting with anti-phosphotyrosine antibodies specific for aa 397, 407, 576, 577, 861, or 925 within FAK. The sensitivity and specificity of the phosphospecific antibodies were characterized in various tissues as described in the Materials and methods. These immunoblots are representative of three different experiments. (B) Translocation of endogenous FAK in VEGF-stimulated HUVECs (20 ng/ml; 5–60 min) to focal adhesions was determined by indirect immunofluorescence with an anti-FAK antibody in representative micrographs, as described in Materials and methods. Bar, 5 μm. (C) Representative FAK activity in lysates of VEGF-stimulated HUVECs (20 ng/ml; 5–60 min) was measured by immune complex in triplicate in vitro kinase assays as described in Materials and methods (P < 0.05). (D) Lysates of VEGF-stimulated HUVECs (20 ng/ml; 2–60 min) were subjected to immunoblotting with an anti-phosphotyrosine antibody specific for aa 397, 861, an anti-phospho Erk antibody, or an anti-FAK antibody. Each of these panels are representative of triplicate experiments.
Figure 2.
Figure 2.
VEGF-induced assembly of FAK with integrin αvβ5 in endothelial cells. (A) FAK association with integrin αvβ5 or αvβ3 in HUVECs with or without VEGF (20 ng/ml; 5 min) was measured by immunoprecipitation from whole cell lysates with anti-αvβ5 or anti-αvβ3 monoclonal antibodies, respectively. These immune complexes were immunoblotted and detected with an anti-FAK antibody. Immunoblotting with an anti-phospho Erk antibody reveals the activation of the MAP kinase pathway in these VEGF-stimulated endothelial cells. The bottom panel shows the association of FAK with αvβ5 after stimulation with basic fibroblast growth factor. (B) The capacity for cytosolic proteins other than FAK to associate with αvβ5 was determined by immunoblotting αvβ5 immunoprecipitates from VEGF-stimulated HUVEC lysates with anti-p130Cas, paxillin, phosphorylated pan PKC, or phosphotyrosine antibodies. No background bands in the molecular weight range of FAK were detected by FAK immunoblotting of mock immunoprecipitations performed with anti-αvβ5 in the absence of cell lysate (shown above) or with control antibodies (i.e., anti-VEGFR2) in the presence of lysate (unpublished data).
Figure 3.
Figure 3.
Src kinase regulates VEGF-induced assembly of a FAK/αvβ5 complex in cultured endothelial cells and in the chick embryo during angiogenesis. (A) The Src kinase family inhibitor, PP1 (Hanke et al., 1996) was used to inhibit (10 μM; 2 min pretreatment) VEGF-induced assembly of the FAK/αvβ5 complex in HUVECs (20 ng/ml; 5 min). Retroviral gene delivery of GFP or a dominant negative Src, kinase-deleted Src (Src 251) was used to suppress Src kinase activity in VEGF-stimulated HUVECs as described in Materials and methods. (B) Induction of a FAK/αvβ5 complex in the CAM was measured by stimulation of 10-d-old CAMs with VEGF (1 μg/ml; 5 min) as previously described (Eliceiri et al., 1999). Retroviral gene delivery of Src 251 or control GFP, was used to suppress Src kinase activity in angiogenic blood vessels as previously described (Eliceiri et al., 1999). Lysates of these CAMs were subjected to immunoprecipitation with an anti-αvβ5 antibody and the immunoprecipitates immunoblotted with an anti-FAK antibody. These immunoblots were representative of at least three separate experiments.
Figure 4.
Figure 4.
VEGF-induced FAK phosphorylation and formation of FAK/αvβ5 complex is reduced in src −/− mice. (A) Lysates of VEGF-stimulated mouse lungs (2 μg i.v./animal; 5 min) from src−/− or control mice were subjected to immunoblotting with a generic anti-phosphotyrosine antibody, or anti-phosphotyrosine antibodies specific for aa 397 or 861 within FAK, as described in Materials and methods. (B) The dermis of the ears of src+/− or src−/− mice were injected intradermally with VEGF (500 ng) or PBS, and after 5 min, whole tissue lysates were prepared for immunoprecipitation with an anti-β5 antibody, followed by immunoblotting for FAK, an anti-phosphotyrosine antibody specific for tyrosine 861 within FAK. Parallel lysates were subjected to immunoblotting with anti-FAK or β5 antibodies as loading controls. Laser scanning densitometry of the FAK/αvβ5 complex in the top row revealed a threefold increase in FAK/αvβ5 complex after VEGF stimulation in src+/− compared with only a 1.1-fold increase in src−/−mice. These immunoblots were representative of at least three separate experiments.
Figure 5.
Figure 5.
A role for the growth factor–induced tyrosine phosphorylation of the COOH-terminal FAK aa 861 for assembly with integrin β5 in vivo. (A) Various epitope-tagged (HA) FAK constructs (HA wild-type, Y397F, or Y861F) were expressed in HEK-293 cells, as previously described (Sieg et al., 2000). (B) Lysates of EGF-stimulated cells (20 ng/ml; 5 min) expressing these various FAK constructs were subjected to immunoprecipitation with an anti-αvβ5 antibody and immunoblotted with an anti-HA antibody to detect FAK/αvβ5 complexes. Expression levels of each HA-tagged construct was confirmed by HA immunoblotting. (C) Association of HA-WT FAK or HA-Y861 mutant FAK with integrin αvβ5 in VEGF-stimulated HUVECs. Parallel blotting of HUVEC lysates with anti-HA antibody reveals transient expression levels of HA-tagged FAK constructs. (D) Lysates of VEGF or mock-treated HUVECs were analyzed for the VEGF-induced association of FAK with αvβ5, αvβ3, or β1 integrins by immunoprecipitation with anti-αvβ5, αvβ3, or β1 antibodies and immunoblotting with an anti-FAK antibody. These immunoblots were representative from at least three different experiments.
Figure 6.
Figure 6.
Phosphorylation of the COOH-terminal FAK tyrosine 861 regulates assembly with integrin β5 in vitro. (A) Alignment of the cytoplasmic tails of β integrin subunit for integrins β1, β3, and β5. Full-length fusion proteins of β3 and β5 cytoplasmic tails (dashed line), truncated β3 and β5 cytoplasmic tails lacking the membrane-proximal domain (solid line) used in this study, and previously mapped conserved peptide region of β1 integrin (Schaller et al., 1995; circles). Boxed regions represent conserved domains. (B) Associations between Src-phosphorylated FAK COOH terminus (FAK CT*) or mock-phosphorylated FAK CT (FAK CT) with GST fusions of full length β3 or β5 integrin cytoplasmic tails, or truncated β3 or β5 integrin cytoplasmic tails (5 min) were identified by immunoblotting glutathione-Sepharose pulldowns with an anti-FAK antibody, as described in Materials and methods. These blots were representative of at least three different experiments. (C) (upper) Phosphorylated FAK CT or a mutant of tyrosine 861 within the FAK CT (FAK CT Y861F) was incubated with the truncated β5 integrin cytoplasmic tail. (lower) Src-mediated phosphorylation of the FAK CT was determined by immunoblotting with phosphotyrosine specific anti-FAK antibodies recognizing aa 861 or 925.
Figure 7.
Figure 7.
VEGF-induced VP defect in integrin β5–deficient mice. (A) The VEGF-induced VP response in the dermis of β5−/−, β3−/−, or control mice was determined by intradermal injection of VEGF (400 ng) into mice that had been previously injected with EB, a fluorescent VP indicator. The extravasation of EB from the blood vessels was quantitated by fluorimetry (O.D.600), as previously described (Eliceiri et al., 1999). (n = 7) (*, P < 0.05) (B) The VEGF-induced VP response in cerebral blood vessels was identified by the extravasation of EB from β5−/− or control mice that had been stereotactically injected with VEGF or saline, as previously described (Eliceiri et al., 1999; Paul et al., 2001). Laser confocal scanning microscopy was used to visualize the fluorescence of the extravasated EB in representative brain cross sections. Bar, 100 μm. (C) Infarct volumes following cerebral ischemia were determined in TTC-stained coronal sections of β5−/− or control mice, as previously described (Paul et al., 2001). Quantitation of the infarct volumes was measured as previously described (Paul et al., 2001) (P < 0.02) (left). Representative micrographs of TTC-stained brain sections reveal the zone of VEGF-mediated neuronal damage (right). Bar, 2 mm.
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