Drosophila Sprouty (dSpry) was genetically identi®ed as a novel antagonist of ®broblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGFmediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DN11 and mSpry4, both de®cient in c-Cbl binding, to instigate these effects. hSpry2 interacts speci®cally with the c-Cbl RING ®nger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by speci®cally intercepting c-Cbl-mediated effects on receptor down-regulation.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogenactivated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.

Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated Spry expression levels in various cancers and found that Spry1 and Spry2 were downregulated consistently in breast cancers. Such prevalent patterns of downregulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. Spry1 and 2 were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mutant of Spry, hSpry2Y55F resulted in enhanced cell proliferation in vitro. The hSpry2Y55F stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An in vivo nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2 Y55F stable cells. The consistent down-regulation of Spry1 and 2 in breast cancer and the experimental evidence using a dominant-negative hSpry2 Y55F indicate that Spry proteins may actively maintain tissue integrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development.