Article |
Address correspondence to Brian P. Eliceiri, IMM-24, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-9317. Fax: (858) 784-8926. E-mail: eliceiri{at}scripps.edu
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Abstract |
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Key Words: VEGF; vascular permeability; Src; tyrosine kinase; integrin
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Introduction |
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Angiogenesis requires the coordination of growth factor receptors and integrins (Brooks et al., 1994; Friedlander et al., 1995), leading to the activation of downstream signals in endothelial cells (Eliceiri et al., 1998; Short et al., 1998). Two pathways of growth factorinduced angiogenesis have been identified in which basic FGF (bFGF) induces angiogenesis dependent on integrin vß3 ligation, whereas VEGF induces angiogenesis dependent on the ligation of integrin
vß5 (Friedlander et al., 1995). The mechanisms underlying the selective coordination of inputs from growth factors and the extracellular matrix (Plopper et al., 1995; Miyamoto et al., 1996; Giancotti and Ruoslahti, 1999), such as the VEGF pathway with integrin
vß5, remains poorly understood. For example, whereas
vß5-deficient mice develop normally (Huang et al., 2000), the ligation state of integrin
vß5 and Src kinase activity in normal animals are critical during VEGF-induced angiogenesis in vivo (Friedlander et al., 1995; Eliceiri et al., 1999).
Recent work from several laboratories indicates that Src and focal adhesion kinase (FAK) are activated by growth factor receptors and/or after integrin-mediated cell adhesion (Parsons and Parsons, 1997; Schlaepfer and Hunter, 1998). Src and FAK also associate with the cytoplasmic domain of growth factor receptors (Ralston and Bishop, 1985; Gould and Hunter, 1988; Kypta et al., 1990; Sieg et al., 2000), and after integrin-mediated cell adhesion, FAK can recruit Src to focal adhesions leading to Erk activation (Courtneidge et al., 1993; Aplin et al., 1998; Schlaepfer and Hunter, 1998; Wary et al., 1998). In addition to Src, several adapter and signaling molecules can associate with FAK (Cobb et al., 1994; Schlaepfer et al., 1994), including p130Cas (Polte and Hanks, 1995), paxillin (Turner and Miller, 1994), PI 3-kinase (Chen and Guan, 1994), and Grb2 (Schlaepfer et al., 1994). However, the coordination of inputs from growth factor receptors leading to the selective recruitment or activation of specific integrins in vivo remains poorly understood. To investigate the mechanism by which the VEGF pathway coordinates with integrin vß5 and Src kinase, an in vivo angiogenesis model was used with a defined growth factor input, (i.e., VEGF), and a known requirement for a specific integrin, i.e.,
vß5. Although we have previously shown an Src-requirement for VEGF-mediated vascular responses (Eliceiri et al., 1999; Paul et al., 2001), experiments were designed to determine whether Src and its substrate, FAK, could functionally regulate
vß5 during the VEGF-mediated response in intact blood vessels.
Evidence is provided that VEGF and other growth factors activate Src kinase, which induces the phosphorylation of tyrosine 861 (Y861) within the FAK COOH terminus, facilitating the association of FAK with integrin vß5 both in vivo and in vitro. Src deficiency or blockade of Src activity inhibits the formation of a VEGF-induced FAK/
vß5 complex. In contrast, both ß1 and ß3 integrins were found to couple to FAK in the absence of growth factor stimulation. The physiological relevance of this pathway is underscored by the finding that mice lacking the integrin ß5 subunit, or mice deficient in Src, have reduced VEGF-induced VP, suggesting a critical role for integrin
vß5, together with Src kinase activity, in regulating VEGF-induced vascular responses in vivo.
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Results |
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VEGF induces FAK phosphorylation and formation of a FAK/vß5 complex in cultured endothelial cells
Ligation of integrin vß5 has been shown to be essential for VEGF-induced angiogenesis (Friedlander et al., 1995), although the mechanisms underlying the recruitment of intracellular signaling proteins to integrins in vivo remains poorly understood. For example, an autonomously expressed form of FAK lacking kinase activity, FAK-related non-kinase (Schaller et al., 1993), suppresses VEGF-induced angiogenesis (unpublished data), suggesting that FAK may have an essential role in VEGF-mediated vascular responses. Whereas data in Fig. 1 demonstrates that VEGF stimulation leads to the phosphorylation of FAK on aa 397 and 861 (Fig. 1 A) and its localization in focal contacts (Fig. 1 B), the capacity for phosphorylated FAK to coordinate with integrins in blood vessels is unknown. Therefore, lysates of starved or VEGF-stimulated HUVECs were subjected to immunoprecipitation with anti-integrin antibodies. These immunoprecipitates were then probed for the presence of FAK. VEGF induced a FAK/
vß5 complex in endothelial cells (Fig. 2 A) that was associated with increased FAK phosphorylation (Fig. 1) and kinase activity (Fig. 1 C). Unlike that seen with
vß5,
vß3 showed a constitutive association with FAK that did not increase in response to VEGF (Fig. 2 A). Other angiogenic growth factors such as bFGF do not appear to promote FAK/
vß5 coupling (Fig. 2 A, bottom). The specificity of the FAK/
vß5 complex was supported by blotting for other candidate focal adhesion proteins. For example, these
vß5 immunoprecipitates were probed for paxillin, p130Cas, or PKC, which can bind FAK/integrin complexes (Fig. 2 B). Immunoblotting with an anti-phosphotyrosine antibody did not reveal a significant population of additional tyrosine-phosphorylated proteins other than a 125-kD protein, most likely FAK, in the
vß5 immunoprecipitations. Although we did not detect other proteins associated with FAK/
vß5 complexes, this may be due to the brief VEGF stimulation (5 min) used in this experiment.
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Phosphorylation of the COOH-terminal FAK tyrosine 861 regulates assembly with integrin ß5 in vitro
Previous findings have shown that the membrane proximal region of the ß integrin cytoplasmic tail can bind FAK in vitro (Schaller et al., 1995), a region that is conserved between ß1, ß3, and ß5 integrins. In support of this, we show that integrins vß3 and ß1 (Fig. 5 D) have a constitutive baseline association with FAK, whereas only integrin
vß5 supports increased assembly of a FAK/integrin complex in response to VEGF and other growth factors (Figs. 2 A, 3 B, and 5). Furthermore, the co-immunoprecipitation analysis of HA-FAK/
vß5 in cultured cells suggests that the tyrosine phosphorylation of a specific aa, Y861 in the FAK COOH terminus, is important for the FAK/
vß5 complex (Fig. 5). Therefore, to further characterize the mechanism of the Src-mediated FAK/
vß5 interaction, in vitro binding studies were performed using NH2- or COOH-terminal domains of FAK and various full-length or truncated fusion proteins of ß5 and ß3 cytoplasmic tails. NH2-terminal (FAK NT; aa 1410) and COOH-terminal (FAK CT; aa 852-1052) fragments of FAK were subjected to in vitro phosphorylation with active Src and allowed to bind to fusion proteins derived from integrin ß5 or ß3 cytoplasmic tails. Src failed to phosphorylate FAK NT in vitro (unpublished data), and therefore was not used in subsequent in vitro binding assays. However, Src induced tyrosine phosphorylation of the FAK CT in vitro as detected with phosphotyrosine antibodies to aa 861 and 925 (Fig. 6 C). Mock-treated or phosphorylated FAK CT protein was incubated with the full-length cytoplasmic tails of integrin ß5 (glutathione S-transferase [GST]: aa 716772) or ß3 (GST: aa 716762) (Fig. 6 A). Integrin-bound FAK was captured with glutathione-Sepharose and analyzed by immunoblotting with an anti-FAK antibody. As expected from our previous results (Fig. 2), FAK was constitutively associated with the full-length ß3 cytoplasmic tail. Unexpectedly, some level of constitutive association was detected in complex with full-length ß5. However, this may be anticipated, as ß1, ß3, and ß5 integrin cytoplasmic tails share considerable sequence homology, particularly at the membrane-proximal domain, including the sequence (KLL[V/I]TIHDR[R/K]EFAKF] (Fig. 6 A, ). Therefore, to determine the contribution of the sequences unique to the cytoplasmic tails of the ß3 and ß5 subunits, fusion proteins were prepared lacking the common membrane proximal sequence. Binding assays of mock-treated or phosphorylated FAK CT with these truncated ß3 or ß5 cytoplasmic tails revealed that Src-phosphorylated FAK CT bound selectively to the ß5 tail compared with the ß3 cytoplasmic tail, whereas nonphosphorylated FAK CT failed to bind either ß3 or ß5 cytoplasmic tails. To determine whether the phosphorylation of tyrosine 861 within the FAK CT by Src was required for the interaction of FAK with ß5, a point mutant of the FAK CT (Y861F) was evaluated. Although the Src-phosphorylated FAK CT bound integrin ß5, the mutant FAK CT (Y861) failed to bind integrin ß5 (Fig. 6 C) even though it was phosphorylated on aa 925 as detected by immunoblot analysis (Fig. 6 C). These in vitro binding data with integrin tails lacking the membrane proximal domain are consistent with the data from intact cells in which VEGF-induced an increase in FAK/
vß5 but not FAK/
vß3 complexes. Although the molecular basis of the interactions of many proteins which associate with integrin tails remains poorly understood, our findings suggest that the membrane proximal domain of integrin tails may contribute to the formation of a baseline of the FAK/integrin complex in vivo and in vitro.
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Discussion |
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An important finding of this study is that VEGF via Src induces the site-specific tyrosine phosphorylation of FAK on Y861, leading to the formation of a complex between FAK and vß5 in both cultured endothelial cells in vitro and blood vessels in vivo, and in EGF-stimulated epithelial cells. These findings are consistent with the emerging role of aa 861 in mediating cell migration in tumor (Slack et al., 2001) and endothelial cells (Abu-Ghazaleh et al., 2001). In this study we have shown that Src deficiency or blockade of Src activity suppresses FAK phosphorylation at aa 861, and thereby reduces VEGF-induced FAK/
vß5 complex formation. These findings indicate that VEGF-induced Src activity and the phosphorylation of Y861 in FAK contribute to the formation of a FAK/
vß5 complex. Although baseline levels of FAK associate with integrins ß1, ß3, and ß5, only the ß5 integrin supports increased levels of FAK/integrin complexes after VEGF stimulation. Our data suggests that this interaction depends on a region within the COOH-terminal half of the ß5 cytoplasmic tail that contains an aa sequence distinct from that of ß1 or ß3. Direct genetic evidence for a role for integrin
vß5 in the VEGF pathway is demonstrated in mice lacking integrin ß5, which, like src-/- mice, have a defective VEGF-mediated VP response. In contrast, mice lacking integrin ß3 have a normal VEGF-induced VP response. In combination with the biochemistry from endothelial cell immunoprecipitations and the in vitro binding assays, the lack of VEGF-mediated VP from Src or ß5 knockout mice suggests that the VEGF-induced formation of the FAK/
vß5 complex may be an important mechanism for coordinating growth factordependent integrin signaling during VEGF-mediated VP.
Previous studies from our laboratory demonstrate that SFKs (Eliceiri et al., 1999) and integrin vß5 (Friedlander et al., 1995) are required for VEGF-induced angiogenesis and VP. In contrast, bFGF-induced angiogenesis depends on the ligation of integrin
vß3 (Friedlander et al., 1995), and is independent of Src kinase activity (Eliceiri et al., 1999). Several other signaling molecules, such as PKC or eNOS, selectively contribute to the VEGF pathway (Friedlander et al., 1995; Ziche et al., 1997), suggesting that at least some of the upstream components of the VEGF and bFGF signaling pathways are distinct.
In addition to the role of VEGF as a mitogen and a VP factor, a functional role for VEGF in inducing edema and tissue damage has been identified after cerebral ischemia (van Bruggen et al., 1999). Direct genetic evidence for the pathophysiological relevance of integrin vß5 in the VEGF pathway is provided by the observation of a reduction in neuronal damage in ß5-deficient mice after cerebral ischemia (Fig. 7 C). We have previously shown that Src deficiency or blockade of Src activity prevents VEGF-mediated VP, thereby reducing neuronal damage after stroke (Paul et al., 2001). In combination with the reduction in VEGF-induced VP (Fig. 7) and neuronal damage in ß5-/- mice, these results suggest a link between integrin
vß5 and the Src-dependent VEGF vascular response in vivo.
Evidence from several cell models indicates that integrin vß5 mediates cell biological processes that require costimulation with growth factors. For example,
vß5-mediated cell adhesion, migration/invasion requires prestimulation with growth factors (Klemke et al., 1994; Brooks et al., 1997; Doerr and Jones, 1996; Lewis et al., 1996). In contrast,
vß3-mediated cell migration/invasion in these cells are independent of growth factor stimulation. These studies suggest that in contrast to
vß3, integrin
vß5 may require an upstream priming signal from an activated growth factor receptor leading to Src kinase activation for biological function of the integrin
vß5 and downstream signaling. The capacity for HEK-293 epithelial cells to form an Src-dependent FAK/
vß5 complex in response to EGF and our results with VEGF-stimulated endothelial cells suggests that this pathway may have a general significance for a wide range of cell types in response to specific growth factors.
Data presented here indicate that a FAK/integrin complex can form in an integrin-specific manner depending on the stimulus. Although FAK can bind the membrane distal region of the ß1 integrin tail (Lewis and Schwartz, 1995; Klingbeil et al., 2001), the FAK NH2 terminus binds a conserved membrane proximal ß1 integrin cytoplasmic tail sequence (Schaller et al., 1995). The molecular basis of this constitutive baseline association of FAK with the membrane proximal region of ß integrins remains unknown; however, it is possible that the Src-mediated association of the FAK CT with the truncated ß5 cytoplasmic tail may depend on a ß5-specific distal sequence(s). There are no obvious motifs within the integrin ß5 cytoplasmic tail, such as a phosphotyrosine binding domain that might account for such an interaction, but evidence presented here suggests that Src-mediated tyrosine phosphorylation of the FAK CT at aa 861 can contribute to the FAK/vß5 association. It is conceivable that phosphorylation of aa 861 influences the structure of FAK through intramolecular rearrangement, enabling it to bind the cytoplasmic tail of integrin ß5. This may involve more than one interaction, such that the FAK NT might associate with the membrane proximal region of the ß integrin cytoplasmic tail, as suggested by previous workers (Schaller et al., 1995), whereas the FAK CT associates selectively with the COOH terminus of the ß5 integrin cytoplasmic tail. Recent findings indicate that the FAK NT may be important for coordinating with growth factors receptors (Sieg et al., 2000), whereas tyrosine phosphorylation of aa 861 in the FAK CT is increased during integrin-mediated cell migration (Abu-Ghazaleh et al., 2001; Slack et al., 2001). Furthermore, our data with different FAK mutants suggest that wild-type FAK interacts with
vß5 through mechanism(s) distinct from Y397F/
vß5 interactions. Not surprisingly, the Y397F mutation of aa 397 influences a wide range of other phosphorylation events, which may complicate the interpretation of this mutant in these assays. Indeed, phosphorylation of Y397 (Wennerberg et al., 2000), Y925 and other sites within FAK may influence the complexity of integrin-associated proteins in vivo, and mediate baseline levels of FAK/integrin interactions. We believe that the design of the in vitro binding assays with the FAK COOH terminus lacking aa 397, facilitates the analysis of the potential role of aa 861 in mediating growth factordependent interactions with the distal portion of the integrin tail. Although our in vitro binding data suggests that the FAK/
vß5 complex forms in the absence of other proteins, it is possible that other focal-adhesion associated proteins (for review see Aplin et al., 1998) can associate with this FAK/
vß5 complex in cells.
Evidence is provided that endothelial cells can coordinate VEGF-induced vascular responses through a specific integrin-mediated signaling mechanism. Although both Src kinase and integrin vß5 are necessary for these VEGF responses in blood vessels, we propose that the Src-mediated association of FAK with
vß5 represents a novel mechanism for the coordination of different integrin and growth factordependent biological processes and may be applicable to various cell types in vivo.
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Materials and methods |
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HUVEC, HEK-293, chick embryo, and mouse treatments
Low-passage (P2-P5) HUVEC were serum starved for 16 h in serum-free media before stimulation with growth factors. Gene delivery of various constructs into HUVECs was performed by retroviral infection using the replication-defective murine Moloney retrovirus pLNCX and amphotropic packaging cells (NX-Ampho, a gift of G. Nolan, Stanford University, Stanford, CA) as described previously (Eliceiri et al., 1999). HEK-293 cells expressing various FAK constructs (HA-tagged full-length wildtype FAK, or mutants Y397F and Y861F) were pooled populations of cells expressing HA-tagged FAK proteins. Both HUVECs and 293 cells were serum starved for 16 h in serum-free media before VEGF or EGF stimulation, respectively. Fertilized chick embryos (McIntyre Farms) were stimulated with growth factors or infected with retroviruses as previously described (Eliceiri et al., 1999). High-titer avian-specific retroviruses used for the transduction of CAM tissue with mutant constructs were prepared as previously described (Eliceiri et al., 1999). 48 h after infection with the retroviruses expressing GFP or mutant Src 251, chick CAMs were stimulated with VEGF for 5 min, and lysates were prepared for analysis.
ß5-/- and control ß5+/- mice were generated as previously described (Huang et al., 2000). ß3-/- mice were generated as previously described (Hodivala-Dilke et al., 1999). Src+/- and src-/- mice were generated as previously described (Soriano et al., 1991), and were a gift of Drs. P. Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA), P. Stein (University of Pennsylvania, Philadelphia, PA), and P. Schwartzberg. Systemic intravenous VEGF injections (2 µg/animal in 100 µl), stereotactic brain injections and intradermal ear injections of anesthetized mice was performed with VEGF (500 ng in 5 µl) as previously described (Eliceiri et al., 1999). Statistical analysis of the quantitation of mouse matrigel angiogenesis, VP, and ischemia assays was performed with the Student t test.
Immunoprecipitation, immunoblotting, kinase assays, and immunostaining
For coimmunoprecipitation of FAK with integrin vß5 in HUVECs, lysis was performed in a buffer (HNG) containing 1% Brij (HNG buffer: 50 mM Hepes, pH 7.4, 150 mM NaCl, 10% glycerol) (Berditchevski et al., 1997), and the lysates diluted with one volume of PBS for immunoprecipitation. For the HUVEC in vitro kinase assays and immunoprecipitation of FAK/
vß5 complexes from mouse tissues, lysates were prepared in modified RIPA buffer as described previously (Eliceiri et al., 1999) and diluted with PBS for immunoprecipitation. To detect FAK/
vß5 complexes in CAM tissue and HEK-293 cells, lysates were prepared in HNG buffer with 1.0% TX100 using a motorized grinder as necessary. SDS-PAGE and immunoblotting were performed as previously described (Eliceiri et al., 1999). FAK activity was measured by the ability of immunoprecipitated FAK to phosphorylate poly-Glu-Tyr (4:1) in an in vitro kinase assay. FAK was immunoprecipitated from equivalent amounts of protein from whole cell lysates as described above, subjected to the kinase assay, and the samples were analyzed by 16% SDS-PAGE as previously described (Eliceiri et al., 1998). Immunostaining of serum-starved HUVEC in the presence or absence of VEGF was performed with an anti-FAK antibody (Abedi and Zachary, 1997) and fixed in acetone as previously described (Takahashi et al., 1999).
In vitro binding assay
GST fusion proteins of NH2- and COOH-terminal fragments of FAK and various ß3 and ß5 integrin cytoplasmic tails were prepared in Escherichia coli (BL21[DE3]). The FAK constructs were phosphorylated in vitro with active Src kinase (UBI), and the GST domain removed from the FAK constructs by Factor Xa (Amersham Pharmacia Biotech) cleavage. The integrin tail constructs retained the GST domain to facilitate the pulldown of FAK/integrin complexes after incubation with glutathione-Sepharose after 510 min in PBS on ice. Complexes were resolved by 16% SDS-PAGE and immunoblotted with anti-FAK or phosphospecific Y861 and Y925 antibodies.
In vivo VP models
Extravasation of Evan's Blue (EB) in the dermis after intradermal injection of VEGF was quantitated by extraction with formamide and spectrophotometry of eluted EB dye (Eliceiri et al., 1999). Laser scanning confocal microscopy was used to visualize the VP of cerebral blood vessels by detection of the fluorescence of the EB dye in brain cross sections (Eliceiri et al., 1999; Paul et al., 2001). Cerebral ischemia experiments were performed as previously described (Paul et al., 2001). In brief, permanent occlusion of the middle cerebral artery was performed in anesthetized mice by coagulation using a heating filament (Nawashiro et al., 1997). The brains were removed after 24 h and the infarcts determined by staining 1-mm coronal brain sections with 2% TTC. The infarct was measured from digital images of the sections and the volume calculated by summing the infarcted nonstained areas multiplied by their thickness (Eliasson et al., 1997).
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Footnotes |
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Acknowledgments |
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This work was supported by the American Heart Association (B.P. Eliceiri, 0130209N), a Human Frontier Science Program Fellowship (X.S. Puente), and the National Institutes of Health (J.D. Hood, training grant 1T32CA75924; D.D. Schlaepfer, CA75240 and CA 87038; and D.A. Cheresh, CA50286, CA45726, and CA78045). This is manuscript 13254 of The Scripps Research Institute.
Submitted: 24 September 2001
Revised: 25 February 2002
Accepted: 27 February 2002
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