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© The Rockefeller University Press,
0021-9525/2002/4/149 $5.00
The Journal of Cell Biology, Volume 157, Number 1, April 1, 2002 149-160
Article |
Src-mediated coupling of focal adhesion kinase to integrin 
vß5 in vascular endothelial growth factor signaling
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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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
vß5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with ß1 and ß3 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/vß5 signaling complex. Moreover, formation of this FAK/vß5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin ß5, but not integrin ß3, have a reduced VP response to VEGF. This FAK/vß5 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 vß5 into a FAK-containing signaling complex during growth factor–mediated biological responses.
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 factor–induced 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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vß5 ligation (Friedlander et al., 1995) contribute to VEGF-mediated angiogenesis and/or VP. Based on these findings, we considered the role of FAK in VEGF-mediated vascular responses, as integrins as well as Src kinase(s) influence FAK phosphorylation and activation, leading to downstream signaling (for review see Aplin et al., 1998). To gain a molecular understanding of this phenomenon, experiments were designed to determine which Src phosphorylation sites within FAK were phosphorylated after VEGF stimulation. As an initial approach, lysates of VEGF-stimulated primary human endothelial cells (HUVECs) or VEGF-treated mouse tissues were immunoblotted with a panel of phosphotyrosine-specific antibodies. These antibodies were directed to the tyrosine-phosphorylated state of amino acids (aa) 397, 407, 576, 577, 861, or 925 within FAK, to detect the known substrate sites for Src. The profile of VEGF-induced tyrosine phosphorylation in cultured HUVECs was compared with lysates of mouse lung and brain tissues exposed to VEGF (5 min) (Fig. 1 A). VEGF-induced robust tyrosine phosphorylation of aa 397 and 861 on FAK within cultured endothelial cells as well as in intact mouse tissues (Fig. 1 A). Other tyrosines within FAK were phosphorylated to a minimal degree or below the detection limit.
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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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vß5 complex after VEGF stimulation in cultured endothelial cells or on the chorioallantoic membrane of 10 1-d-old chick embryos during angiogenesisvß5 disrupts VEGF-mediated signaling and angiogenesis, yet it fails to influence bFGF-mediated angiogenesis (Friedlander et al., 1995). However, inhibition of vß5 has no effect on VEGF-stimulated Src kinase activity (unpublished data). These findings suggest that Src functions upstream of integrin vß5. Therefore, we considered whether Src might be required for assembly of the FAK/vß5 complex in VEGF-stimulated endothelial cells or tissues. To test this possibility, we employed pharmacological or genetic approaches to suppress Src kinase activity in cultured endothelial cells in vitro or intact blood vessels in vivo. Pharmacological inhibition of Src kinase with PP1 (Hanke et al., 1996) or retroviral delivery of kinase-deleted Src (aa 1–251, Src 251) suppressed VEGF-induced levels of the FAK/vß5 complex in HUVECs (Fig. 3 A). To determine whether VEGF could induce a FAK/vß5 complex in blood vessels in vivo, chick chorioallantoic membranes (CAMs) were stimulated with VEGF and analyzed for the presence of a FAK/vß5 complex. Lysates from VEGF-stimulated contained elevated levels of the FAK/vß5 complex, compared with unstimulated controls (Fig. 3 B, top). The formation of the VEGF-induced FAK/vß5 complex was disrupted by exposing these CAMs to an avian-specific retrovirus (RCAS) expressing Src 251 (Fig. 3 B, bottom), providing genetic evidence for a Src requirement for the VEGF-induced assembly of the FAK/vß5 complex in vivo.
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vß5 complex is suppressed in src-/- micevß5 association, mice lacking pp60c-src were injected with VEGF, and the injected tissues analyzed for FAK phosphorylation and assembly of the FAK/vß5 complex. Lysates were prepared from src-/- or control mouse tissues (dermis) stimulated with VEGF or saline and subjected to immunoblotting with phospho-specific antibodies directed to aa 397 or 861. VEGF induced an increase in FAK 397 and 861 phosphorylation in control mice, whereas only a minimal level of FAK phosphorylation was detected in src-/- mice (Fig. 4 A), suggesting that FAK is an important substrate for Src after VEGF stimulation in vivo. Although VEGF treatment increased the level of FAK associated with integrin ß5 in src+/- control animals (threefold), the formation of this complex was significantly suppressed in src- /- mice (Fig. 4 B) (1.1-fold). These results provide genetic evidence in mice to corroborate the finding that the VEGF-induced phosphorylation of FAK and the association of phosphorylated FAK with vß5 depend on VEGF-mediated Src kinase activity.
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vß5 complex formation in vivo and in cultured endothelial cells (Figs. 2–4). Although both tyrosines 397 and 861 within FAK are prominently phosphorylated after VEGF stimulation, it remains unclear whether these sites are involved in the formation of the FAK/vß5 complex by phosphorylation of either of these sites. In addition, it is important to determine whether FAK/vß5 complexes can form in other cell types in response to other growth factors. To address these questions and to determine the functional requirement for tyrosines 397 and 861 in the assembly of the FAK/vß5 complex, epitope-tagged (hemagglutinin [HA]) full-length FAK constructs were expressed at similar levels in human epithelial cells (HEK-293) (Fig. 5 A). HA-tagged wild-type FAK (HA-FAK), or mutants of aa 397 (HA-FAK Y397F) or 861 (HA-FAK Y861F) were examined for their capacity to associate with endogenous vß5 in these cells. Like the endothelial cell response to VEGF, epithelial cells such as HEK-293 formed a FAK/vß5 complex in response to EGF, which was blocked by the Src inhibitor, PP1 (unpublished data). Lysates of EGF-stimulated HEK-293 cells expressing FAK constructs were subjected to immunoprecipitation with anti-vß5, and the immunoprecipitates were blotted with anti-HA to detect FAK. Wild-type FAK or the Y397F FAK mutant were readily detected in a complex with vß5, however the Y861F FAK mutant failed to form a complex with vß5 (Fig. 5 B). Immunoblotting of whole cell lysates with an anti-HA antibody revealed equivalent expression levels of each of these tagged FAK constructs (Fig. 5 B). These findings reveal that the tyrosine at position 861 is critical for the formation of FAK/vß5 complex, and that this complex may form in other growth factor–stimulated cell types.
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vß5 complexes in VEGF-treated endothelial cells, HA-tagged wild-type FAK or aY861F mutant FAK were expressed in HUVECs. After VEGF stimulation, theY861F FAK mutant failed to associate with vß5, whereas the WT FAK construct formed a complex with vß5 (Fig. 5 C), consistent with the VEGF-induced coupling of vß5 with endogenous FAK in these cells (Fig. 2 A). These results suggest that the VEGF-induced tyrosine phosphorylation of aa 861 is important in the formation of the FAK/vß5 complex in the endothelium.
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 1–410) 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 716–772) or ß3 (GST: aa 716–762) (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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vß5 interactions in the VEGF pathway, the functional requirement for integrin vß5 in a VEGF-mediated vascular response remained unknown. Previous studies have demonstrated that VEGF-mediated endothelial responses depend on Src kinase activity (Eliceiri et al., 1999) and integrin vß5, but not vß3 (Friedlander et al., 1995). In support of this, mice lacking a single Src family member such as pp60c-src fail to undergo VEGF-induced VP, whereas general suppression of Src with kinase-deleted Src blocks VEGF- but not bFGF-mediated angiogenesis (Eliceiri et al., 1999). Therefore, we reasoned that if Src and vß5 were both downstream of VEGF and on a common signaling pathway, one might predict that mice lacking vß5 would have a phenotype similar to that of src-/- mice. Control mice or those lacking integrin ß5 or ß3 were intradermally injected with VEGF and evaluated for VEGF-mediated VP. The ß5-deficient mice had a significant decrease in VEGF-induced VP compared with control littermates (Fig. 7 A) (P < 0.05), which paralleled the loss of VP observed in src-/- mice (Eliceiri et al., 1999; Paul et al., 2001). Importantly, mice lacking ß3 (Hodivala-Dilke et al., 1999) showed control levels of VP (Fig. 7 A), which is consistent with our previous findings that VEGF-dependent vascular responses depend primarily on vß5 (Friedlander et al., 1995). To corroborate these findings, control mice or mice lacking ß5 were subjected to a stereotactic brain injection of saline or VEGF into the brain which is known to compromise the blood brain barrier (Fig. 7 B) (Eliceiri et al., 1999). The decrease in Evan's blue extravasation in cerebral blood vessels of ß5-/- mice after VEGF administration suggests that there is a requirement for integrin ß5 in the VEGF-mediated breakdown of the blood–brain barrier. Furthermore, the decrease in VEGF-induced VP in these ß5-deficient mice was concomitant with a decrease in brain damage after cerebral ischemia (Fig. 7 C). Together, these results demonstrate an important role for integrin vß5 in VEGF-mediated endothelial responses in vivo that appears identical to that seen in mice lacking pp60c-src.
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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 factor–dependent 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 factor–dependent 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 factor–dependent biological processes and may be applicable to various cell types in vivo.
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Materials and methods |
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vß3 (LM609) or vß5 (P1F6) used for integrin immunoprecipitations from human or chick tissues. Rabbit polyclonal anti-ß5 antibodies used to immunoprecipitate mouse integrin vß5 were from either Dr. M. Hemler (Harvard University, Boston, MA) (Ramaswamy and Hemler, 1990) or Chemicon International. The phospho-specific MAP kinase antibody was from New England Biolabs, and the phospho-FAK antibodies directed to tyrosines 397, 407, 576, 577, 861, or 925 were from Biosource. The specificity of these site-specific anti-phosphotyrosine antibodies targeting FAK was confirmed by immunoblotting various FAK mutants expressed in vitro, in cultured cells, and/or based on previous findings with these reagents (Sieg et al., 2000). RCAS (A)-GFP, and Src 251 were gifts of Dr. P. Schwartzberg (National Institutes of Health, Bethesda, MD) and H. Varmus (Sloan-Kettering, New York, NY). pLNCX–FAK-related non-kinase constructs were a gift of Dr. T. Parsons (University of Virginia, Charlottesville, VA). HUVECs were obtained from Biowhittaker. VEGF was from Peprotech, and bFGF was a gift of Dr. J. Abraham (Scios, Mountain View, CA). Protein A/G was from Pierce Chemical Co., and glutathione-Sepharose was from Amersham Pharmacia Biotech. All other reagents and media were from Sigma-Aldrich.
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 5–10 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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References |
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