Oncogene (2001) 20, 3959 ± 3968 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling S Giorgetti-Peraldi*,1, J Murdaca1, JC Mas1 and E Van Obberghen1 1INSERM U145, IFR 50, Faculte de MeÂdecine, avenue de Valombrose, 06107 Nice Cedex 2 France Vascular endothelial growth factor (VEGF) is an expressed only on endothelial cells, Flt-1 (Fms like important regulator of vasculogenesis and angiogenesis. tyrosine kinase) and Flk/KDR (Kinase insert domain Activation of VEGF receptors leads to the recruitment of containing receptor). Both receptors are structurally SH2 containing proteins which link the receptors to the related to the PDGF (Platelet Derived Growth Factor) activation of signaling pathways. Here we report that receptor family (Neufeld et al., 1999). Knock-out Grb10, an adapter protein of which the biological role studies have shown that these receptors have dierent remains unknown, is tyrosine phosphorylated in response biological functions. Indeed, in Flk null mice, to VEGF in endothelial cells (HUVEC) and in 293 cells endothelial and hematopoietic cell development is expressing the VEGF receptor KDR. An intact SH2 impaired. In Flt-1 knock-out mice, there is an apparent domain is required for Grb10 tyrosine phosphorylation in overgrowth of endothelial cells, and blood vessels are response to VEGF, and this phosphorylation is mediated disorganized (Fong et al., 1995; Shalaby et al., 1995). in part through the activation of Src. In HUVEC, VEGF These results and other studies performed by activating increases Grb10 mRNA level. Expression of Grb10 in separately only one receptor suggest that KDR is HUVEC or in KDR expressing 293 cells results in an involved in endothelial cell proliferation and survival, increase in the amount and in the tyrosine phosphoryla- while Flt-1 seems to be required for chemotaxis and tion of KDR. In 293 cells, this is correlated with the procoagulant activity. Although both receptors are activation of signaling molecules, such as MAP kinase. capable of autophosphorylation in vitro (Waltenberger By expressing mutants of Grb10, we found that the et al., 1994), only KDR autophosphorylates in vivo. positive action of Grb10 is independent of its SH2 The deletion of the tyrosine kinase domain of Flt-1 domain. Moreover, these Grb10 eects on KDR seem to does not impair blood vessel formation in homozygous be speci®c since Grb10 has no eect on the insulin mice, suggesting that the kinase domain of Flt-1 is not receptor, and Grb2, another adapter protein, does not required for its action on angiogenesis (Hiratsuka et mimic the eect of Grb10 on KDR. In conclusion, we al., 1998). propose that VEGF up-regulates Grb10 level, which in A series of steps in the VEGF signaling pathway turn increases KDR molecules, suggesting that Grb10 have been elucidated. Activation of VEGF receptors could be involved in a positive feedback loop in VEGF promotes tyrosine phosphorylation of several proteins, signaling. Oncogene (2001) 20, 3959 ± 3968. and recruitment of SH2-containing molecules (Walten- berger et al., 1994). Stimulation of KDR leads to PI-3- Keywords: Grb10; VEGF receptor; KDR; angiogenesis kinase and PKB activation (Gerber et al., 1998; Wu et al., 2000), to MAP kinase activation through PLCg and PKC-dependent pathway (Takahashi et al., 1999), Introduction to nitric oxide synthesis and to FAK tyrosine phosphorylation (Abedi and Zachary, 1997; Shen et Vascular Endothelial Growth Factor (VEGF) is al., 1999). However, many players in VEGF signaling involved in endothelial cell proliferation, microvascular have still to be discovered. Tyrosine kinase receptors permeability, vasodilatation and angiogenesis. Angio- control signaling pathways through association of SH2 genesis, the sprouting of new capillaries from existing containing proteins and tyrosine phosphorylated pro- vasculature, is necessary for vascular development, teins. wound healing, organ regeneration, and has been Grb10 protein contains several interaction motifs, involved in disease states such as diabetic retinopathy, such as a N-terminal proline-rich sequence, a central rheumatoid arthritis and tumor growth (Carmeliet, PH (Pleckstrin homology) domain, a BPS domain 2000; Ferrara and Davis, 1997; Risau, 1997). VEGF (Between PH and SH2) and a C-terminal SH2 domain binds to two speci®c tyrosine kinase receptors (Frantz et al., 1997; He et al., 1998; Ooi et al., 1995). Although Grb10 interacts with tyrosine kinase recep- tors such as PDGF, EGF, insulin and IGF-I receptors, its function as an adapter molecule remains to be *Correspondence: S Giorgetti-Peraldi Received 23 November 2000; revised 6 April 2001; accepted 9 April discovered (He et al., 1998; Stein et al., 1996). Indeed, 2001 Grb10 binds to signaling molecules such as Raf1, Involvement of Grb10 in VEGF signaling pathway S Giorgetti-Peraldi 3960 MEK1, BCR-Abl, Jak2, NEDD4 (Nantel et al., 1998; the conserved arginine 462 by a lysine. This mutant is Bai et al., 1998; Moutoussamy et al., 1998; Morrione et unable to associate with KDR in yeast two hybrid. All al., 1999), but the consequences of these associations the results show that Grb10 binds to KDR only have not been elucidated. The role of Grb10 in cellular through its SH2 domain. In each condition, the signaling is controversial. According to some authors, amount of expressed proteins was identical as revealed microinjection of the Grb10 SH2 domain decreases by Western blot (data not shown). We conclude that insulin- and IGF-I-mediated mitogenesis (Morrione et association of Grb10 to KDR is dependent on the al., 1997), while in other reports, Grb10 has a positive tyrosine kinase activity of the receptor, and requires a role in PDGF, IGF-I and insulin-induced mitogenesis functional SH2 domain of Grb10. (Wang et al., 1999). In this study, we show that Grb10 is tyrosine Grb10 is tyrosine phosphorylated in response to VEGF phosphorylated in response to VEGF in HUVEC and in 293 cells expressing KDR. Moreover, VEGF We studied whether Grb10 is tyrosine phosphorylated increases Grb10 mRNA in HUVEC. In 293 cells and in response to VEGF. Human endothelial cells in HUVEC, overexpression of Grb10 induces an (HUVEC) were stimulated with VEGF and tyrosine increase in the amount and in the tyrosine phosphor- phosphorylation of endogenous Grb10 was assessed ylation of KDR, coupled to an increase in the VEGF- after immunoprecipitation with an antibody to Grb10. induced MAP kinase activity. We show that Grb10 As shown in Figure 2a, VEGF treatment induces a interacts directly with VEGF receptor KDR, and that three-fold increase in Grb10 tyrosine phosphorylation. the SH2 domain of Grb10 mediates this association. To study the mechanism of this phosphorylation, we Since VEGF stimulates the expression of Grb10 expressed epitope-tagged Grb10-HA and KDR-Myc in mRNA, and Grb10 increases KDR molecules, we 293 cells (that lack endogenous VEGF receptors). Cells would like to suggest that a positive feed-back loop were stimulated with VEGF, and Grb10-HA was could exist. immunoprecipitated with an antibody to the HA epitope followed by an antiphosphotyrosine immuno- blot. As shown in Figure 2b, VEGF-induced activation of KDR stimulates Grb10 tyrosine phosphorylation. Results Grb10 phosphorylation is abolished when a SH2 domain de®cient mutant of Grb10 is expressed KDR interacts with Grb10 SH2 domain (Grb10 R462K) (Figure 2c). Although Grb10 binds to several tyrosine kinase Src is known to phosphorylate Grb10 on tyrosine receptors, its biological role remains unknown. To (Langlais et al., 2000). Moreover, Src kinases are elucidate the function of Grb10, we investigated its involved in VEGF signaling pathway (Eliceiri et al., implication in VEGF signaling. 1999). To study whether phosphorylation of Grb10 in VEGF binds to two tyrosine kinase receptors, Flt-1 response to VEGF could be due to Src, we treated and KDR. Since it has been shown that only KDR HUVEC with the Src inhibitor, PP2 prior to VEGF autophosphorylates in endothelial cells, we determined stimulation. We observed that PP2 treatment of whether KDR interacts with Grb10 using the yeast two HUVEC did not aect the VEGF-induced tyrosine hybrid system. KDR cDNA corresponding to the phosphorylation of KDR (Figure 3a). Grb10 was cytoplasmic domain of the receptor was subcloned in immunoprecipitated and analysed by antiphosphotyr- frame with the binding domain of LexA. Grb10 cDNA osine immunoblot (Figure 3b). PP2 treatment of was subcloned in frame with the activation domain of HUVEC induces a 40% decrease in the VEGF induced Gal-4. Yeasts were co-transformed by the two hybrids, tyrosine phosphorylation of Grb10. However, the and the association between the proteins was measured tyrosine phosphorylation of Grb10 is not totally by the transactivation of a reporter gene, LacZ. Results blocked suggesting that both Src and KDR are in Figure 1a, show that pLex-KDR interacts with involved in the tyrosine phosphorylation of Grb10 pAct-Grb10 as compared to a negative control, Raf. after VEGF stimulation. In conclusion, Grb10 is This interaction is dependent on the tyrosine kinase tyrosine phosphorylated in response to VEGF. This activity of the receptor, since the generation of a phosphorylation seems to be due to both an action of kinase-dead mutant (KD) of KDR by replacement of KDR and Src. lysine 868 by alanine, abolishes the interaction of KDR with Grb10 in the yeast two hybrid system. Grb10 mRNA is increased by VEGF To localize the binding site on Grb10, we generated several mutants of pACT-Grb10 (Figure 1b). As Since Grb10 seems to be involved in VEGF signaling, represented, Grb10 possesses a proline-rich region, a we investigated whether VEGF could modulate Grb10 PH domain, a BPS region and a SH2 domain. All the expression. Endothelial cells (HUVEC) were treated deletion mutants of Grb10 were tested for their abilities with VEGF for 2, 4 and 6 h, RNA were extracted, and to bind to pLex-KDR (Figure 1c).