No abstract

The stimulation of vascular endothelial growth factor receptor-2 (VEGFR-2) by tumor-derived VEGF represents a key event in the initiation of angiogenesis. In this work, we report that VEGFR-2 is localized in endothelial caveolae, associated with caveolin-1, and that this complex is rapidly dissociated upon stimulation with VEGF. The kinetics of caveolin-1 dissociation correlated with those of VEGF-dependent VEGFR-2 tyrosine phosphorylation, suggesting that caveolin-1 acts as a negative regulator of VEGF R-2 activity. Interestingly, we observed that in an overexpression system in which VEGFR-2 is constitutively active, caveolin-1 overexpression inhibits VEGFR-2 activity but allows VEGFR-2 to undergo VEGF-dependent activation, suggesting that caveolin-1 can confer ligand dependency to a receptor system. Removal of caveolin and VEGFR-2 from caveolae by cholesterol depletion resulted in an increase in both basal and VEGF-induced phosphorylation of VEGFR-2, but led to the inhibition of VEGF-induced ERK activation and endothelial cell migration, suggesting that localization of VEGFR-2 to these domains is crucial for VEGF-mediated signaling. Dissociation of the VEGFR-2/caveolin-1 complex by VEGF or cyclodextrin led to a PP2-sensitive phosphorylation of caveolin-1 on tyrosine 14, suggesting the participation of Src family kinases in this process. Overall, these results suggest that caveolin-1 plays multiple roles in the VEGF-induced signaling cascade.

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events. INTRODUCTIONEpithelial-mesenchymal transition and cell migration are required during normal embryonic development and during pathological situations such as the dispersal of tumor cells. Hepatocyte growth factor (HGF) is a multifunctional factor that, in addition to promoting epithelial cell growth and survival, has the ability in vitro to stimulate epithelial cell dissociation, motility, invasion, and the endogenous morphogenic program of epithelial cells in three-dimensional matrix or organ culture Nakamura, 1996, 1997;Montesano et al., 1997). Genetic studies have shown that HGF/Met signaling is essential for normal murine embryological development (Birchmeier and Gherardi, 1998). In addition, HGF/Met signaling was also shown to be involved in angiogenesis (Bussolino et al., 1992;Grant et al., 1993) and has been implicated in the dissociation and migration of muscle precursor cells of the dermomyotome, in the guidance and survival of motor neurons, and in the development and survival of sensory neurons (Birchmeier and Gherardi, 1998). HGF and Met were also shown to play a role in pathological conditions, including tissue regeneration (Matsumoto and Nakamura, 1997), tumorigenesis, and metastasis (Jeffers et al., 1996;Bardelli et al., 1997).The conversion from a sessile to a migratory phenotype requires an extensive remodeling of the actin cytoskeleton (Mitchison and Cramer, 1996). Members of the Rho family of small GTP-binding proteins, including Cdc42, Rac, and Rho, are involved in regulating the organization of the actin cytoskeleton and the assembly of associated integrin com-** Corresponding author. E-mail address: morag@lan1.molonc.mcgill.ca. ...

The Gab family of docking proteins is phosphorylated in response to various growth factors and cytokines and serves to recruit multiple signaling proteins. Gab1 acts downstream from the Met-hepatocyte growth factor receptor, and Gab1 overexpression promotes Met-dependent morphogenesis of epithelial cells. Recruitment of Gab1 to Met or epidermal growth factor (EGF) receptors requires a receptor-binding site for the Grb2 adapter protein and a proline-rich domain in Gab1, defined as the Met-binding domain. To determine the requirement for Grb2 in Gab1 recruitment, we have mapped two Grb2 carboxyl-terminal SH3 domain binding sites conserved in Gab1 and related protein Gab2. One corresponds to a canonical Grb2-binding motif, whereas the second, located within the Gab1 Met-binding domain, requires the proline and arginine residues of an atypical PXXXR motif. The PXXXR motif is required but not sufficient for Grb2 binding, whereas an extended motif, PX3RX2KPX7PLD, conserved in Gab proteins as well as the Grb2/Gads-docking protein, Slp-76, efficiently competes binding of Grb2 or Gads adapter proteins. The association of Gab1 with Grb2 is required for Gab1 recruitment to the EGF receptor but not the Met receptor. Hence different mechanisms of Gab1 recruitment may reflect the distinct biological functions for Gab1 downstream from the EGF and Met receptors.Receptor tyrosine kinases regulate diverse biological processes ranging from cell proliferation and survival to cell motility, metabolism, and differentiation. The initiation of receptor signaling involves ligand-induced activation of the catalytic domain of the enzyme and the phosphorylation of specific tyrosine residues on the receptor. These provide binding sites for proteins containing Src homology 2 (SH2) 1 and phosphotyrosine-binding domains that act to transduce signals to the interior of the cell (reviewed in Ref.