Abstract
In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50-60. We show that human Spry2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Delta50-60 (Delta50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.
Highlights
MARCH 23, 2007 VOLUME 282 NUMBER 12 bers of the epidermal growth factor receptor family, Ras and Raf, have been shown to be deregulated in different cancers [1, 2]
A Distal Motif on Spry2 Likely Controls the Presentation of the C-terminal PXXPXR Sequence—We have recently shown that a PXXPXR motif on the C terminus of Spry2 is necessary for the ERK inhibitory activity of the protein in the context of FGF receptor (FGFR) signaling [15]
In recent experiments [15], we have demonstrated that, in the context of fibroblast growth factor (FGF) signaling, Spry2 causes the most profound inhibition of ERK phosphorylation as compared with other Spry isoforms, and this inhibitory function correlates with binding to Grb2 but not c-Cbl
Summary
Wild type full-length constructs of the different isoforms of Spry, FGFR1, ERK2, HA-Cbl TKB have been described previously [8, 23]. Mouse anti-PP2A-A, rabbit anti-Cbl, anti-Grb, and anti-FGFR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Four subsequent immunoprecipitations were performed on the PP2A-A-depleted lysates using anti-c-Cbl. The precipitated proteins were resolved on SDS-PAGE and analyzed using immunoblotting protocols. Cell lysates were prepared and analyzed for immunoblotting as described previously [8]
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