A Src Homology 3-binding Sequence on the C Terminus of Sprouty2 Is Necessary for Inhibition of the Ras/ERK Pathway Downstream of Fibroblast Growth Factor Receptor Stimulation

Abstract

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.

Targeted Online Liquid Chromatography Electron Capture Dissociation Mass Spectrometry for the Localization of Sites of in Vivo Phosphorylation in Human Sprouty2

“…30,33 Alterations in Sprouty2 phosphorylation, observed as a shift in SDS PAGE migration, have been implicated in Sprouty2 activity. [33][34][35] Precise localization of the sites of Sprouty2 phosphorylation will allow the role of phosphorylation in Sprouty2 function to be assessed further.…”

Sprouty2 expression controls endothelial monolayer integrity and quiescence

“…A subgroup of SPRY2 translocates to the plasma membrane [10] and interferes with the RTK cascade on several levels. Under certain conditions, SPRY binding to GRB2 is sufficient to inhibit FGF-induced activation of MAPK, while in others GRB2 association is not required [131,132]. In addition, SPRY binding to SOS1 and potentially to Raf1 inhibits FGF-stimulated MAPK activation [8,133].…”

Sprouty2 Interacts with Protein Kinase Cδ and Disrupts Phosphorylation of Protein Kinase D1

“…Far-Western Analysis-Mammalian proteins were immunoprecipitated and transferred onto polyvinylidene difluoride membranes as described previously (5). Briefly, membranes were blocked in 5% skim milk overnight and subsequently overlaid with GST-tagged Spry2 protein produced in Escherichia coli DH5␣.…”

“…Human PKD1 was purchased from Addgene (deposited by Alex Toker, plasmid 10808) and subsequently subcloned into pXJ40-HA vector. FGFR1, FLAGtagged Spry1, Spry2, and Spry4 have been described previously (5,7). Point mutants and truncations of Spry2 were generated using Pfu DNA polymerase.…”

“…Subsequent studies revealed that Spry expression was induced by the Ras/ERK signaling pathway and acts as a negative feedback inhibitor of that same pathway (4). Various studies have alluded to points of action of Spry2 on the Ras/ERK pathway, including upstream of Ras, by disrupting the Grb2-Sos interaction (5,6) or at the level of Raf (7). Therefore, it appears that there may be multiple points at which Spry2 inhibits Ras/ERK signaling.…”