Trends in Cell Biology
Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling
Introduction
Sprouty (Spry) was identified in Drosophila in genetic screens for regulators of tracheal [1] and eye [2] development. These and other elegant studies established Drosophila Spry as a general inhibitor of RTK-mediated Ras signaling and initiated exploration of the function of the vertebrate orthologs (Box 1). The vertebrate Spry proteins share sequence similarity to Drosophila Spry in the cysteine-rich C-terminus but differ among each other, and dramatically so from the fly protein, in the N-terminus.
Spry proteins specifically inhibit Ras–ERK MAPK signaling by RTKs, leaving the phosphoinositide 3-kinase (PI3K) and other MAPK pathways unaffected 3, 4. The point at which Spry blocks ERK MAPK activation remains controversial, and the evidence to date suggests the existence of multiple mechanisms that depend on the cellular context and/or the identity of the RTK. Spry proteins have been reported to interact with several components of RTK signaling pathways, but, in most cases, how these associations modulate signaling is unclear (Table 1). Epistasis studies in Drosophila indicated that, during eye development, Spry inhibits signaling downstream of the epidermal growth factor receptor (EGFR) and upstream of Ras [1], whereas, during wing and ovary development, Spry functions at the level of Raf [5]. In mouse fibroblasts, mSpry2 acts downstream of the fibroblast growth factor receptor (FGFR) and upstream of Ras [3], while, in 293T cells, it blocks FGFR and β2 adrenergic receptor signaling at the level of Raf [4]. mSpry4 interferes with vascular endothelial growth factor (VEGF)-induced, Ras-independent Raf activation [6]. However, others found that hSpry4 represses insulin receptor and EGFR-induced ERK signaling upstream of, or parallel to, Ras [7]. This finding agrees with a study in endothelial cells where mSpry4 inhibits FGF and VEGF signaling by uncoupling RTK ligation from Ras activation [8].
Studies on Xenopus Spry1 and mSpry2 suggested that Spry antagonizes FGF-induced ERK MAPK activation by competing with the Shp2 tyrosine phosphatase and the FRS2 adaptor protein for binding to the Grb2–SOS complex [9]. In this model, the Spry–Grb2 association is mediated by the binding of the Grb2 Src-homology 2 (SH2) domain to Spry2 Tyr55, and agrees with an earlier study where FGF increased Spry2–Grb2 association at the expense of the Grb2–FRS2 complex [10]. However, others found that Spry2 bound Grb2–SOS in a constitutive manner independent of Spry2 tyrosine phosphorylation 3, 11, and did not affect Grb2–SOS interaction with FRS2 [3]. Importantly, while Spry1 does not associate with Grb2, both Spry1 and Spry2 potently inhibit RTK signaling [3]. Furthermore, interference of FRS2 interaction with Grb2–SOS does not explain how Spry inhibits signaling by the platelet-derived growth factor receptor (PDGFR) [3], which does not signal through FRS2 and might directly recruit Grb2–SOS independently of Shp2. Loss-of-function experiments using primary cells isolated from gene-targeted animals or RNA interference (RNAi) techniques will be required to accurately position the site(s) of Spry action.
Section snippets
Regulation of Sprouty activity
As a negative regulator, Spry itself is subject to tight control on multiple levels. Specifically, this occurs through differential localization, posttranslational modification and regulation of proteins levels. A fraction of Spry1 and Spry2 is constitutively associated with the plasma membrane, likely due to palmitoylation of C-terminal cysteine residues and association with caveolin-1 [12]. Indeed, mutation of the cysteine-rich domain of Spry2 disrupts membrane localization and abrogates Spry
Sprouty and EGF signaling
Spry proteins are generally considered to be inhibitors of RTK signaling, whose effects target the Ras–ERK MAPK cascade. Several studies have challenged this paradigm, demonstrating that mammalian Spry proteins can augment EGF-mediated ERK MAPK signaling in a cell type-dependent manner 6, 12, 20, 21, 30, 31. This agonistic effect of Spry is strictly dependent on c-CBL, which interacts with the activated EGFR, promoting receptor degradation and signal termination [32]. Spry, bound to c-CBL,
Regulation of Sprouty gene expression
In Drosophila, expression of Spry is dependent on RTK signaling. For example, overexpression of Branchless, the Drosophila FGF ortholog, or its downstream effector, the Pointed Ets-domain transcription factor (Pnt), induces high levels of Spry [1]. Furthermore, Spry expression is augmented by a constitutively active EGFR and blocked by a dominant-negative EGFR allele 2, 5, 36. RTK-mediated signaling pathways also induce expression of the vertebrate Spry proteins, most notably through the
Sprouty functions in embryonic development
In Spry mutant flies, ectopic secondary branching of the tracheal system is induced owing to a change in tracheal cell fate [1]. The extra branches are associated with expanded expression domains of downstream effectors of the Breathless FGFR pathway that regulate the later branching events. Additionally, overexpression of Drosophila Spry yields decreased numbers of photoreceptors, which resemble EGFR loss-of-function phenotypes, while eliminating Spry expression results in excess recruitment
Sprouty loss-of-function studies in the mouse
The first studies of the targeted disruption of Spry genes in the mouse confirm the ability of these genes to regulate branching morphogenesis and interfere with FGF signaling during development 55, 56, 57. Basson et al. found that newborn mice lacking the Spry1 gene [55] exhibit kidney and urinary tract defects that resemble the human syndrome ‘congenital anomalies of the kidney and urinary tract’ (CAKUT) [58]. This phenotype arises as a result of defects in the morphogenesis of the ureteric
Sprouty as a tumor-suppressor gene
Aberrant expression and activation of RTKs are common features of malignancy. As Spry is a negative regulator of growth factor-mediated signaling, several groups began investigating the role of Spry in human cancers and discovered that the expression of Spry1 and Spry2 is significantly downregulated in breast (79% and 96%, respectively) and prostate (40% for Spry1) cancer specimens, as well as melanoma cell lines 64, 65, 66, 67.
The process by which Spry is downregulated might be variable and
Concluding remarks
The Spry proteins are key negative regulators that limit the strength, duration and range of activation of RTKs, allowing for controlled growth and differentiation. It is clear that specific Spry proteins are crucial for organ development by limiting RTK signaling, and emerging evidence suggests that loss of Spry could contribute to malignancy. How Spry prevents activation of Ras and Raf has yet to be completely explained. Further studies of the partner proteins of Spry, and of the ability of
Acknowledgements
We thank Gail Martin for permission to adapt Figure 1 from Shim et al. and for insightful discussions, and Steve McKnight for sharing unpublished data. We apologize to those colleagues whose contributions we were unable to include owing to limitations on reference citations. Supported by NIH Grants CA59998 and DK62345 (J.D.L.), the Revson Foundation for Biomedical Research (J.M.M.) and the Wellcome Trust (M.A.B.).
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