Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. In this report, we describe the purification, cloning, and characterization of a novel protein, designated FRS2, that is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. We find that FRS2 is myristylated and that this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. Finally, we demonstrate that FRS2 is closely related and probably indentical to SNT, the long-sought target of FGF and NGF receptors.

The family of fibroblast growth factors (FGFs) consists of at least 10 different growth factors that control cellular processes such as growth, differentiation, and cell migration (reviewed in reference 2). FGFs induce their biological responses by binding to and activating a family of cell surface receptors with intrinsic protein tyrosine kinase activity (reviewed in reference 12). By contrast to other growth factors such as platelet-derived growth factor (PDGF) or epidermal growth factor, acidic FGF (aFGF; also called FGF1) binds to the FGF receptor (FGFR1) monovalently, and FGFR dimerization and activation are mediated by multivalent interactions between heparin sulfate proteoglycans and FGF (reviewed in reference 26).Upon activation, receptor tyrosine kinases undergo rapid autophosphorylation on numerous tyrosine residues. Autophosphorylation sites located within the catalytic domain are crucial for stimulation of kinase activity, while autophosphorylation sites located in other regions are usually involved in the recruitment of cellular target proteins (21). FGFR1 (encoded by flg) contains at least seven autophosphorylation sites. Two are located in the catalytic domain (Y653 and Y654) and are essential for kinase activation (17). One phosphorylation site in the C-terminal tail (Y766) functions as a high-affinity binding site for the SH2 domain of phospholipase C-␥ (19). Phosphorylation of Y766 is essential for phosphatidylinositol hydrolysis but not for FGF-induced DNA synthesis in myoblasts or differentiation of PC12 cells, indicating that these biological responses are mediated by different FGF-dependent signaling pathways (18,22). Interestingly, elimination of all known tyrosine autophosphorylation sites on FGFR1 by site-directed mutagenesis (except the two sites in the catalytic domain) does not impair FGF-induced mitogen-activated protein (MAP) kinase activation, mitogenesis, or PC12 cell differentiation (17).The Ras/MAP kinase signaling pathway plays an important role in signaling via FGF receptors (1,20). It is well established that the adapter protein Grb2 (6, 16) links receptor tyrosine kinases with the Ras signaling pathway by binding to the guanine nucleotide-releasing factor Sos through its SH3 domains and to tyrosine-phosphorylated receptors or docking molecules via its SH2 domain (25). We have recently identified a lipid-anchored docking protein, termed FRS2, that links FGFR molecules with the Ras/MAP kinase signaling pathway (14). We demonstrated that FRS2 is tyrosine phosphorylated and forms a complex with Grb2 and Sos in response to FGF stimulation (14). In this report, we demonstrate that in addition to the direct interactions with Grb2, tyrosine-phosphorylated FRS2 forms a complex with the SH2 domain-containing protein tyrosine phosphatase Shp2. This interaction results in tyrosine phosphorylation of Shp2 and complex formation between Shp2 and Grb2. Moreover, an FRS2 mutant impaired in Grb2 and Shp2 binding induces weak and transient MAP kinase response and fails to induce neuronal diffe...

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The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras͞mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2␣ leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2␣:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S͞T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras͞mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

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Galectin-8 belongs to the family of tandem-repeat type galectins. It consists as several isoforms, each made of two domains of approximately 140 amino-acids, both having a carbohydrate recognition domain (CRD). These domains are joined by a 'link peptide' of variable length. The human galectin-8 gene covers 33 kbp of genomic DNA. It is localized on chromosome 1 (1q42.11) and contains 11 exons. The gene produces by alternative splicing 14 different transcripts, altogether encoding 6 proteins. Galectin-8, like other galectins, is a secreted protein. Upon secretion galectin-8 acts as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell surface integrin receptors. Complex formation between galectin-8 and integrins involves sugar-protein interactions and triggers integrin-mediated signaling cascades such as Tyr phosphorylation of FAK and paxillin. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Such a mechanism allows local signals emitted by secreted galectin-8 to specify territories available for cell adhesion and migration. Due to its dual effects on the adhesive properties of cells and its association with fibronectin, galectin-8 might be considered as a novel type of a matricellular protein. Galectin-8 levels of expression positively correlate with certain human neoplasms, prostate cancer being the best example studied thus far. The overexpressed lectin might give these neoplasms some growth and metastasis related advantages due to its ability to modulate cell adhesion and cellular growth. Hence, galectin-8 may modulate cell-matrix interactions and regulate cellular functions in a variety of physiological and pathological conditions.

Galectins, a family of mammalian lectins with specificity to ␤-galactosides, are involved in growthregulatory mechanisms and cell adhesion. A relationship is assumed to exist between the levels of expression of galectins and the level of malignancy in human gliomas. A comparative study of this aspect in the same series of clinical samples is required to prove this hypothesis. Using computerassisted microscopy, we quantitatively characterized by immunohistochemistry the levels of expression of galectins-1, -3 and -8 in 116 human astrocytic tumors of grades I to IV. Extent of transcription of galectins-1, -3, and -8 genes was investigated in 8 human glioblastoma cell lines by means of RT-PCR techniques. Three of these cell lines were grafted into the brains of nude mice in order to characterize in vivo the galectins-1, -3 and -8 expression in relation to the patterns of the tumor invasion of the brain. The role of galectin-1, -3 and -8 in tumor astrocyte migration was quantitatively determined in vitro by means of computer-assisted phase-contrast videomicroscopy. The data indicate that the levels of galectin-1 and galectin-3 expression significantly change during the progression of malignancy in human astrocytic tumors, while that of galectin-8 remains unchanged. These three galectins are involved in tumor astrocyte invasion of the brain parenchyma since their levels of expression are higher in the invasive parts of xenografted glioblastomas than in their less invasive parts. Galectin-3, galectin-1, and to a lesser extent galectin-8, markedly stimulate glioblastoma cell migration in vitro. Since bands for the transcripts of human galectins-2, -4 and -9 were apparently less frequent and intense in the 8 human glioblastoma cell lines, this system provides an excellent model to assign defined roles to individual galectins and delineate overlapping and distinct functional aspects.