Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT⅐Grb2⅐Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2⅐Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway.Normal development requires precise spatial and temporal regulation of signal transduction pathways involved in cell growth and differentiation. Negative control of growth factor response is achieved both by restriction of the incoming signal itself and induction of counter regulatory mechanisms affecting the propagation of the signal. The expression of many inhibitors are induced by the pathway they eventually antagonize, providing the potential for a tight autoregulation (for a review, see Ref. 1). Recently, sprouty (spry) was identified by genetic studies as such an inhibitor (2).Spry was originally described as an antagonist of Breathless FGF 1 receptor signaling during tracheal branching in Drosophila Loss of function mutations of spry led to excessive FGF signaling and ectopic branching, whereas engineered overexpression of spry blocked the branching (2). As other groups reported genetic interactions between spry and several different receptor tyrosine kinases (RTK) in multiple contexts, it became clear that spry was a general inhibitor of RTK signaling during Drosophila development (3-6). Through a data base search, three human genes were identified with sequence similarity to Drosophila spry (2) and a fourth family member was described in the mouse (7). Mammalian spry genes are expressed in highly restricted patterns in the embryo during early development and in many adult tissues (7-9). In most tissues, the different family members appear to be co-regulated and their expression shows a close correlation with known sites of FGF signaling. Mammalian Spry proteins may be key regulators of several developmental processes, including lung branching morphogenesis, midbrain and anterior hindbrain patterning, and limb chondrocyte differentiation (8 -10).Genetic and biochemical analysis performed by Casci et al. (3) suggested that Drosophila Spry negatively regulates the Ras pathway, but the molecular mechani...

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.

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␥-Secretase activity is associated with a presenilin (PS)-containing macromolecular complex. Whether PS contains the active site of ␥-secretase has been controversial. One challenge is to find PS that is engaged in the active ␥-secretase complex at the cell surface, where some substrates appear to be processed. In this study, we developed an intact cell photolabeling technique that allows the direct visualization of active ␥-secretase at the cell surface. We demonstrated that active ␥-secretase is present in the plasma membrane. Moreover, the PS1 heterodimer is specifically photolabeled at the cell surface by a potent inhibitor that binds to only the active ␥-secretase. We also explored the cellular processing sites of ␥-secretase for amyloid precursor protein (APP) and Notch by using small molecular probes. MRL631, a ␥-secretase inhibitor that is unable to penetrate the cell membrane, significantly blocks ␥-secretase-mediated Notch cleavage but has little effect on APP processing. These results indicate that Notch is processed at the cell surface and that the majority of APP is processed by intracellular ␥-secretase. Furthermore, the fact that inhibitors first target ␥-secretase in the plasma membrane for Notch processing, and not for APP, will have important implications for drug development to treat Alzheimer's disease and cancer.intact cell photolabeling ͉ intramembrane protease ͉ presenilin

Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased A␤42/A␤40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect ␥-secretase, a protease that generates the termini of A␤40 and A␤42. Here we have determined the reaction mechanism of ␥-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased A␤42/A␤40 ratio, these mutations each display rather distinct reactivity to ␥-secretase. Intriguingly, we found that the ratio of A␤42/A␤40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of A␤42/A␤40. Moreover, we demonstrated that reduction of ␥-secretase substrate concentration by BACE1 inhibition in cells decreased the A␤42/A␤40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of ␥-secretase substrate, can augment the A␤42/A␤40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the A␤42/A␤40 ratio through partial reduction of ␥-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.␥-Secretase cleaves the amyloid precursor protein (APP) to generate the C termini of ␤-amyloid (A␤) 2 peptides, generally 40 or 42 amino acids in length (A␤40 and A␤42, respectively). A␤ peptides are believed to be a major causative factor in the pathogenesis of Alzheimer disease (AD) (1). A␤42 is more prone to aggregation than A␤40 (2), and therefore biological or environmental factors that promote increased A␤42 production accelerate the pathological cascade leading to AD. Expression of A␤42, rather than A␤40, in Drosophila and mice leads to the formation of A␤ plaques (3, 4). Furthermore, mouse model studies suggest that the ratio of A␤42/A␤40, rather than total amount of A␤, correlates with the load of characteristic AD plaques in the brain (5, 6). Moreover, evidence suggests A␤40 may play a beneficial role in that it antagonizes A␤42 aggregation (5, 6). Therefore, inhibition of ␥-secretase activity that specifically generates A␤42 or reduction of the Ab42/A␤40 ratio would be an appealing strategy for treatment of AD. However, despite intensive studies on ␥-secretase, the mechanism of cleavage specificity for ␥-secretase is still unknown.APP was the first gene found to be linked with inherited AD (7). Each mutation surrounding the ␥-secretase cleavage site appears to alter the production of A␤40 and A␤42. Suzuki et al. (8) demonstrated that mutating APP at Val-46 to Phe or Ile increased the ratio of secreted A␤42 to A␤40 in transfected cells. An increased ratio of A␤42/A␤40 was also observed with other mutations (9 -11). De Jonghe et al. (9) found certain mutations enhanced the stability of the ␥-secretase substrates known as C-terminal fragm...