Growth factor signaling by receptor tyrosine kinases regulates several cell fates, such as proliferation and differentiation. Sef was genetically identified as a negative regulator of fibroblast growth factor (FGF) signaling. Using bioinformatic methods and rapid amplification of cDNA ends-PCR, we isolated both the mouse and the human Sef genes, which encoded the Sef protein and Sef-S isoform that was generated through alternative splicing. We provide evidence that the Sef gene products were located mainly on the cell membrane. Co-immunoprecipitation and immunostaining experiments indicate that hSef interacts with FGFR1 and FGFR2 but not FGFR3. Our results demonstrated that stably expressed hSef strongly inhibits FGF2-or nerve growth factorinduced PC-12 cell differentiation. The intracellular domain of hSef is necessary for the inhibitory effect on FGF2-induced PC-12 cell differentiation. Furthermore, our data suggested Sef exerted the negative effect on FGF2-induced PC-12 cell differentiation through the prevention of Ras-mitogen-activated protein kinase signaling, possibly functioning upstream of the Ras molecule. These findings suggest that Sef may play an important role in the regulation of PC-12 cell differentiation.Cell growth and differentiation mediated by receptor tyrosine kinases are regulated by many extracellular signals, most of which activate the Ras-MAPK 1 kinase cascade (1-4). Ras directly interacts with Raf, then activated Raf phosphorylates and activates MEK, which phosphorylates and activates MAPK kinases including ERK1 and ERK2 (5-7). Excessive or inappropriate growth factor signaling by receptor tyrosine kinases has been implicated in the progression of several cancers and disorders of developmental processes. Thus, the strength and the duration of this signaling must be controlled strictly in normal biological processes. A negative feedback loop is one of the mechanisms that provides an effective way to terminate, limit, or modulate receptor tyrosine kinase signaling (8, 9). In typical negative feedback regulation, the signaling molecule induces the expression of its own negative regulator such that signaling is inhibited once a threshold is reached (10 -12).Recently, many novel negative regulators of growth factor signaling were described using genetic screening methods. Sprouty was identified in Drosophila melanogaster as an inhibitor of FGF signaling during tracheal development (13). However, how this gene functions remains unknown. During D. melanogaster eye development, Sprouty seems to inhibit the activation of MAPK upstream of Ras (14). In contrast, during wing development, Sprouty was reported to inhibit MAPK downstream of Ras (15). D. melanogaster has only one Sprouty protein, whereas mammals have at least four isoforms (16 -19). Sef (similar expression of FGF genes) is another novel negative regulator of receptor tyrosine kinase signaling that was first isolated from a zebrafish embryo library through in situ hybridization (20, 21). Sef was reported to be syn-expressed with FGF8, ...

Sef is a transmembrane protein inhibiting FGF signaling. To determine the correlation of Sef with human diseases, Sef expression patterns were observed in cell lines and human cancer tissues. Western blot using anti-hSef antibodies showed that hSef, when expressed in Cos7 cells gave a molecular mass of 100 KD as compared with 80 KD in an in vitro translation assay suggesting occurrence of glycosylation at the potential N-linked glycosylation sites in the extracellular domain. Northern blot showed that hSef was mainly expressed in human kidney and testis. RT-PCR analysis showed a widely spread expression pattern in several cell lines.Immunohistochemical analysis revealed a high expression level of hSef in kidney, testis, and the corresponding carcinoma tissues. Results demonstrated that Sef might be up-regulated in the cancer tissues suggesting a possible role of Sef in pathophysiology of human diseases.