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• W1996667568 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 Spry2Δ50-60 (Δ50-60), 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. 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 Spry2Δ50-60 (Δ50-60), 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. The Ras/ERK 3The abbreviations used are: ERK, extracellular signal-regulated kinase; Spry2, Sprouty2; FGF, fibroblast growth factor; FGFR, FGF receptor; bFGF, basic FGF; PP2A, protein phosphatase 2A; TKB, tyrosine kinase binding domain; aa, amino acids; HA, hemagglutinin; GST, glutathione S-transferase; WT, wild type; OA, okadaic acid; MS/MS, tandem mass spectrometry; WCL, whole cell lysates; KSR, kinase suppressor of Ras.3The abbreviations used are: ERK, extracellular signal-regulated kinase; Spry2, Sprouty2; FGF, fibroblast growth factor; FGFR, FGF receptor; bFGF, basic FGF; PP2A, protein phosphatase 2A; TKB, tyrosine kinase binding domain; aa, amino acids; HA, hemagglutinin; GST, glutathione S-transferase; WT, wild type; OA, okadaic acid; MS/MS, tandem mass spectrometry; WCL, whole cell lysates; KSR, kinase suppressor of Ras. pathway is central to many physiological processes, and several of its key components, for example, members of the epidermal growth factor receptor family, Ras and Raf, have been shown to be deregulated in different cancers (1Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar, 2Yarden Y. Eur. J. Cancer. 2001; 37: S3-S8Abstract Full Text Full Text PDF PubMed Google Scholar). Understanding the mechanism of action of proteins that are inhibitory to the central pathway is therefore important from both a biochemical and a pharmacological point of view. A Drosophila Sprouty (dSpry) protein was discovered in a screen designed to detect genes/proteins involved in trachea formation (3Hacohen N. Kramer S. Sutherland D. Hiromi Y. Krasnow M.A. Cell. 1998; 92: 253-263Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar) and was demonstrated to inhibit the Ras/ERK pathway by an unspecified mechanism (4Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Four mammalian Spry isoforms were subsequently discovered, and they share a conserved C-terminal Cys-rich domain as well as a conserved tyrosine-containing sequence in the N-terminal half. Of the mammalian isoforms, Spry2 has been the focus of the majority of the experiments (5Kim H.J. Bar-Sagi D. Nat. Rev. Mol. Cell. Biol. 2004; 6: 441-450Crossref Scopus (301) Google Scholar). A number of reports detail the Ras/ERK inhibitory activity of several of the mammalian Sprys with most evidence accumulating for Spry2 (6Hanafusa H. Torii S. Yasunaga T. Nishida E. Nat Cell Biol. 2002; 4: 850-858Crossref PubMed Scopus (436) Google Scholar, 7Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (235) Google Scholar, 8Yusoff P. Lao D.H. Ong S.H. Wong E.S. Lim J. Lo T.L. Leong H.F. Fong C.W. Guy G.R. J. Biol. Chem. 2002; 277: 3195-3201Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 9Tefft D. Lee M. Smith S. Crowe D.L. Bellusci S. Warburton D. Am. J. Physiol. 2002; 283: L700-L706Crossref PubMed Scopus (100) Google Scholar, 10Mason J.M. Morrison D.J. Bassit B. Dimri M. Band H. Licht J.D. Gross I. Mol. Biol. Cell. 2004; 15: 2176-2188Crossref PubMed Scopus (103) Google Scholar). Several mechanisms have been proposed for the mode of action (6Hanafusa H. Torii S. Yasunaga T. Nishida E. Nat Cell Biol. 2002; 4: 850-858Crossref PubMed Scopus (436) Google Scholar, 11Sasaki A. Taketomi T. Kato R. Saeki K. Nonami A. Sasaki M. Kuriyama M. Saito N. Shibuya M. Yoshimura A. Nat. Cell Biol. 2003; 5: 427-432Crossref PubMed Scopus (201) Google Scholar), but none of these are universally accepted. Clues as to the physiological role a protein plays can be deduced by identifying bona fide binding proteins. The best characterized Spry2-binding proteins are c-Cbl, an ubiquitin-protein isopeptide ligase (E3) and scaffold protein that binds via its TKB domain to the phosphorylated Tyr-55 of Spry2 (12Fong C.W. Leong H.F. Wong E.S. Lim J. Yusoff P. Guy G.R. J. Biol. Chem. 2003; 278: 33456-33464Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 13Hall A.B. Jura N. DaSilva J. Jang Y.J. Gong D. Bar-Sagi D. Curr. Biol. 2003; 13: 308-314Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 14Hu J. Hubbard S.R. J. Biol. Chem. 2005; 280: 18943-18949Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and the adaptor protein Grb2 (10Mason J.M. Morrison D.J. Bassit B. Dimri M. Band H. Licht J.D. Gross I. Mol. Biol. Cell. 2004; 15: 2176-2188Crossref PubMed Scopus (103) Google Scholar, 15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The mode of binding of Grb2 to Spry2 is somewhat controversial with evidence being presented that the SH2 domain of Grb2 binds to Tyr-55, or conversely, that the N-terminal SH3 domain of Grb2 binds to a PXXPXR motif on the C terminus of Spry2 (6Hanafusa H. Torii S. Yasunaga T. Nishida E. Nat Cell Biol. 2002; 4: 850-858Crossref PubMed Scopus (436) Google Scholar, 15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The PXXPXR motif is cryptic in unstimulated cells and is revealed upon activation of FGFRs (15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The question that then arises is as follows. What is the mechanism that causes the necessary change in conformation of Spry2? Changes in phosphorylation are the most likely means by which a protein can alter its tertiary structure. Evidence has been presented that Tyr residues other than Tyr-55 impact on the physiological function of Spry2 (16Rubin C. Zwang Y. Vaisman N. Ron D. Yarden Y. J. Biol. Chem. 2005; 280: 9735-9744Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, in comparison with Tyr phosphorylation, there has been less work done on investigating the effect of Ser or Thr phosphorylation of the Spry isoforms. A previous study demonstrated that Spry1 and Spry2 were phosphorylated on Ser residues in unstimulated cells (7Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (235) Google Scholar). We had noted in a previous study that endogenous Spry2 partitions into two major bands when run on SDS-PAGE and that stimulation with FGF favored the faster migrating band (15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The partitioning of Spry isoforms had been reported earlier together with the demonstration that treatment of cell lysates with a phosphatase resulted in the “loss” of the slower migrating band when analyzed on SDS-PAGE with subsequent Western blotting (7Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (235) Google Scholar). Taking this evidence into account, we postulated that a Ser/Thr phosphatase could be involved in the activation of Spry2 via selective dephosphorylation of Ser residues. Four major classes of protein phosphatases have been described, including PP1, PP2A, PP2B (calcineurin), and PP2C. PP2A is widely expressed in mammalian cells and regulates signaling pathways by a mechanism of phosphorylation/dephosphorylation with a variety of substrates including core components of the Ras/ERK pathway such as RAF, KSR1, and ERK (18Janssens V. Goris J. Biochem. J. 2001; 353: 417-433Crossref PubMed Scopus (1500) Google Scholar, 19Janssens V. Goris J. Van Hoof C. Curr. Opin. Genet. Dev. 2004; 15: 34-41Crossref Scopus (361) Google Scholar, 20Dougherty M.K. Muller J. Ritt D.A. Zhou M. Zhou X.Z. Copeland T.D. Conrads T.P. Veenstra T.D. Lu K.P. Morrison D.K. Mol. Cell. 2005; 17: 215-225Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The predominant form of PP2A in cells is a heterotrimeric holoenzyme. The core enzyme consists of the 36-kDa catalytic subunit (PP2A-C) and a 65-kDa regulatory subunit (PP2A-A or PR65). In addition, there are a number of regulatory subunits (B subunits) that bind to the core enzyme and confer substrate specificity to its dephosphorylating activity (17Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (623) Google Scholar). With the long term goal of determining the mechanism of action of Spry2 in inhibiting the Ras/ERK pathway, we sought to locate and characterize binding proteins and their sites of activity that contribute to the conformational changes necessary to activate the protein. We chose to do this with respect to FGFR activation primarily due to evidence that suggests that Spry function is predominantly associated with the FGF pathway (21Chambers D. Mason I. Mech. Dev. 2000; 91: 361-364Crossref PubMed Scopus (109) Google Scholar). In this study, we identify a role for PP2A in the activation of Spry2. Wild type full-length constructs of the different isoforms of Spry, FGFR1, ERK2, HA-Cbl TKB have been described previously (8Yusoff P. Lao D.H. Ong S.H. Wong E.S. Lim J. Lo T.L. Leong H.F. Fong C.W. Guy G.R. J. Biol. Chem. 2002; 277: 3195-3201Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 23Wong E.S. Fong C.W. Lim J. Yusoff P. Low B.C. Langdon W.Y. Guy G.R. EMBO J. 2002; 21: 4796-4808Crossref PubMed Scopus (199) Google Scholar). The Spry2 Δ50-60 mutant was generated using standard polymerase chain reaction and molecular cloning techniques. Point mutants of Spry2 were generated by site-directed mutagenesis using the proofreading Pfu DNA polymerase from Promega (Madison, WI). The HA-tagged PP2A-C and EE-tagged PP2A-A were kindly provided by Assoc. Prof. E. Sontag (University of Texas Southwestern Medical Center, Dallas, TX). GST-PP2A-A was purchased from Abnova (Abnova Corp., Taipei City, Taiwan). Mouse and rabbit anti-FLAG, rabbit anti-HA, agarose-conjugated anti-FLAG M2 beads, rabbit anti-Sprouty2 (N-terminal), Cy3-conjugated mouse anti-β-tubulin were from Sigma-Aldrich. In addition, affinity-purified rabbit polyclonal antibodies against aa 66-80 of Spry2 were also raised (Bio-Genes, Berlin, Germany). Mouse anti-PP2A-A, rabbit anti-Cbl, anti-Grb2, and anti-FGFR1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-phosphoTyr (PY20), mouse anti-ERK2, panERK, c-Cbl, and Grb2 were from BD Transduction Laboratories, mouse anti-phospho ERK1/2, anti-PP2A-C, and anti-PP2A-A subunits were from Cell Signaling Technology (Beverly, MA). Rabbit anti-Spry was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Texas Red-conjugated AffiniPure rabbit anti-mouse IgG and fluorescein isothiocyanate-conjugated AffiniPure goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa Fluor 647 goat anti-mouse IgG-Cy5 was purchased from Molecular Probes Inc. (Eugene, OR). Okadaic acid was purchased from Upstate Biotechnology and reconstituted in Me2SO. The human Spry2 biotinylated peptides (48-61 aa) RKKRRQRRRIRNTNEYTEGPTV(KBtnX) and the phoshopeptide RKKRRQRRRIRNTNEpYTEGPTV(KBtnX) were custom made by Sigma-Genosys. All cell lines used in this study were purchased from ATCC (Manassas, VA). Human embryonic kidney 293T and PC12 cells were cultured, maintained, and treated as described previously (23Wong E.S. Fong C.W. Lim J. Yusoff P. Low B.C. Langdon W.Y. Guy G.R. EMBO J. 2002; 21: 4796-4808Crossref PubMed Scopus (199) Google Scholar). For acute stimulation, bFGF (Sigma-Aldrich) was used at various concentrations and different time points as described in the text. 293T cells transfected with Spry2 constructs were treated with various doses of okadaic acid for 18 h. Cell lysates were prepared and analyzed for immunoblotting as described previously (8Yusoff P. Lao D.H. Ong S.H. Wong E.S. Lim J. Lo T.L. Leong H.F. Fong C.W. Guy G.R. J. Biol. Chem. 2002; 277: 3195-3201Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Immunoprecipitation and Western blot analyses were carried out as described previously (Ref. 8Yusoff P. Lao D.H. Ong S.H. Wong E.S. Lim J. Lo T.L. Leong H.F. Fong C.W. Guy G.R. J. Biol. Chem. 2002; 277: 3195-3201Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar or Ref. 24Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) for the PP2A experiments. The sequential precipitation with anti-PP2A-A was completed four times on lysates from FGFR1-activated Spry2-transfected cells. Four subsequent immunoprecipitations were then performed on the PP2A-A-depleted lysates using anti-c-Cbl. The precipitated proteins were then resolved on SDS-PAGE and analyzed using immunoblotting protocols. Immunofluorescence in PC12 differentiation experiments were performed as described previously (15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Lim J. Wong E.S. Ong S.H. Yusoff P. Low B.C. Guy G.R. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Wong E.S. Fong C.W. Lim J. Yusoff P. Low B.C. Langdon W.Y. Guy G.R. EMBO J. 2002; 21: 4796-4808Crossref PubMed Scopus (199) Google Scholar). For the neurite outgrowth assay, PC12 cells were transfected with the various Spry constructs. 48 h after transfection, the cells were serum-starved for 24 h followed by stimulation with bFGF (50 ng/ml) for 6 days or left in serum-free medium as control. Cell extracts from 293T cells activated with overexpressed FGFR1 or stimulated with bFGF (50 ng/ml) for 30 min and 2 h were prepared in Tris buffer containing 0.2% Triton X-100, 10% glycerol, 1.5 mm MgCl2, 1 mm EGTA, protease inhibitors, and 1 mm Na3VO4, pH 7.5. Free phosphate from samples was removed using a desalting column and tested for phosphate contamination using the malachite green assay (Upstate Biotechnologies) before proceeding with the assay. 100 μg of samples were subjected to the PP2A immunoprecipitation assay using the PP2A assay kit purchased from Upstate Biotechnologies according to the manufacturer's recommendations. Cell lysates of 293T cells transfected with FLAG-Spry2 were harvested in lysis buffer without sodium orthovanadate and subjected to incubation with calf intestinal phosphatase from New England Biolabs (Beverly, MA) for 2 h at 37 °C using bovine serum albumin as a control. Proteolysis—Proteins were separated using SDS-PAGE, and protein bands were excised into small pieces (1 × 1 mm) and transferred to a polypropylene 96-well microtitre plate (Greiner). Gel pieces were soaked in 25 mm Tris-HCl, pH 8.5, containing 50% acetonitrile (EM Science, Gibbstown, NJ) for 24 h followed by a brief rinse with the same solution and drying in a Savant SpeedVac centrifugal concentrator (Holbrook, NY). Enzymatic digestion performed by the addition of 10 μl of 0.02 μg/μl trypsin (Promega) in 25 mm ammonium bicarbonate buffer, pH 8.5 (Sigma-Aldrich). Samples were incubated over-night at 37 °C with shaking. Peptides were extracted with 50% acetonitrile in 0.1% trifluoroacetic acid (Pierce) using a Crest sonicating water bath for 10 min (Crest, Trenton, NJ), dried down in a SpeedVac and resolubilized in a solution of 2% methanol (Fisher) and 1% formic acid (Sigma-Aldrich) and stored at -80 °C until further analysis. Liquid Chromatography-MS/MS—Digested samples were separated using a nano-flow high-performance liquid chromatography system (LC Packings). Each sample of 7 μl was injected and concentrated onto a trap cartridge (μPrecolumn, 300 μm × 5 mm, C18 PepMap 100, LC Packings) in 0.1% formic acid in water at a flow rate of 25 μl/min. After 5 min of washing, the flow was switched in line to a resolving column (75-μm internal diameter Picotip emitter, New Objective, Boston, MA) packed in-house with 10 cm of C18 reversed-phase packing material (Column Engineering, Ontario, CA), and the flow rate decreased to 100 nl/min. A gradient was then developed from 0 to 60% acetonitrile in 0.1% formic acid over 60 min at the same flow rate. Using a liquid junction at the distal end of the column, a voltage of 2300 V was introduced to form a spray at the tip of the column. The spray was directed at the inlet orifice of a quadrupole-time-of-flight hybrid tandem mass spectrometer (QSTAR-XL, Applied Biosystems, Foster City, CA). The mass spectrometer was run in information-dependent acquisition mode to capture and fragment doubly and triply charged mass ions automatically. Selected mass ions with a minimum signal of 8 counts/s were isolated and fragmented with nitrogen gas. The collision energy used was proportional to the mass of the peptide and was calculated during analysis using the Analyst-QS software (Applied Biosystems). Data Base Analysis—All data files obtained were searched against the mammalian subset of the UniProt non-redundant protein data base (European Bioinformatics Institute, Cambridge, UK) using the MS/MS Ion Search mode of the Mascot search engine (Matrix Science, London, UK). Variable modifications were set to include carboxyamidomethyl cysteine, oxidized methionine, and phosphorylation of serine, threonine, and tyrosine residues. A peptide mass tolerance of 200 ppm was set, and an MS/MS tolerance of 0.5 Da was set. All doubly and triply charged spectra were considered. Up to one missed cleavage was allowed. For a protein to be positively identified, at least two peptides had to match the protein with a score equal to or above that shown to be statistically significant (p < 0.05). The in vitro binding was carried out using the PP2A-A-GST, Cbl-TKB-GST, or GST vector with the non-phospho (48-61 aa RKKRRQRRRIRNTNEYTEGPTV(KBtnX)) or phospho-hS2 biotinylated (RKKRRQRRRIRNTNEpYTEGPTV(KBtnX)) peptide. 55 μm GST protein was incubated with various concentrations of the peptides or buffer for control at 4 °C over-night with rotation. 15 μl of streptavidin-Sepharose beads were added to each tube the following day for 2 h. The complex was then washed three times with the PP2A lysis buffer, and after the final wash, the beads were boiled in 15 μl of 2× Laemmli buffer. The boiled samples were then subjected to SDS-PAGE and analyzed using immunoblotting protocols. 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 FGFR signaling (15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The proline-rich motif is cryptic in unstimulated cells and is revealed upon FGFR stimulation. The question we next wanted to answer was: what was the mechanism that exposed the binding sequence? From our recent work, we had shown that a mutation of Tyr-55 can affect the Grb2 binding to the distal C-terminal sequence. We thought it was possible that some or all of the amino acids that surround Tyr-55 could be involved in regulating the exposure of the C-terminal proline-rich motif. With this in mind, we constructed a mutant by deleting aa 50-60, termed Δ50-60 (Fig. 1A), as well as the indicated point mutants and combinations of these. We investigated the effects of these mutations on Grb2 binding, c-Cbl binding, and ERK inhibition. A representative result is depicted in Fig. 1B. The YR (Y55F,R309A double mutant), Δ50-60, and Δ50-60R (deletion of aa 50-60 and R309A point mutant) mutations show no ability to inhibit ERK phosphorylation, none of them bind c-Cbl as they all lack the phosphorylated Tyr-55 residue, and all show minimal or no binding to Grb2. As shown previously, the Y55F mutant has a reduced binding to Grb2 and a diminished ability to inhibit ERK phosphorylation. An interesting observation here is that the Δ50-60 mutant of Spry2 separates into two obvious protein bands (panels 3 and 7, lanes 9 and 10). We had noted in a number of previous experiments that Spry2 separates on SDS-PAGE as two distinct bands in unstimulated cells (15Lao D.H. Chandramouli S. Yusoff P. Fong C.W. Saw T.W. Tai L.P. Yu C.Y. Leong H.F. Guy G.R. J. Biol. Chem. 2006; 281: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). To investigate the disposition of the two major Spry2 bands on SDS-PAGE, we set up an experiment where 293T cells were left unstimulated or stimulated with overexpressed FGFR1 in the presence of WT Spry2 or Δ50-60. The result of a representative experiment is shown in Fig. 1C. Notably, in the absence of stimulation (lanes 1 and 3), the two Spry2 bands contain essentially equal amounts of protein. Upon FGFR1 stimulation, the cells that contained WT Spry2 showed a significant increase in the faster migrating Spry2 band (lane 2). Conversely, in the lysates from cells with Δ50-60 transfected, there is an increase in the slower migrating band (lane 4). These changes in band migration in SDS-PAGE provided us with a clue that certain covalent modifications are necessary for Spry2 to function as an ERK inhibitor. We therefore set up a series of experiments to investigate the cause of the “bandshifting.” Spry2 Is Dephosphorylated upon FGFR Activation by a Ser/Thr Phosphatase That Targets Conserved Residues—Spry1 and 2 had previously been shown to be phosphorylated predominantly on Ser residues (7Impagnatiello M.A. Weitzer S. Gannon G. Compagni A. Cotten M. Christofori G. J. Cell Biol. 2001; 152: 1087-1098Crossref PubMed Scopus (235) Google Scholar). To investigate whether the Spry2 bandshift was due to phosphorylation/dephosphorylation, lysates from unstimulated cells, expressing either full-length Spry2 (plus and minus FGFR1) or the Spry2 Δ50-60 deletion mutant, were treated with either alkaline phosphatase or an equivalent amount of bovine serum albumin as a control. It can be seen in Fig. 2A (lanes 2 and 8) that treatment with alkaline phosphatase causes the “disappearance” of the slower migrating band in these lanes, indicating that the retardation in SDS-PAGE was most likely due to phosphorylation of specific residues on Spry2. The predominance of the faster migrating band upon FGFR1 activation in Figs. 1C and 2A indicates that a Spry2-targeting phosphatase is activated upon cell stimulation. Since the deletion of aa 50-60 abrogated the accumulation of the faster migrating band, it can be assumed that the activated Ser/Thr phosphatase binds directly or indirectly to Spry2 via this sequence or possibly that the deletion may have caused a conformational change that affects the recruitment of a phosphatase at an adjacent, or less likely, a distal site on Spry2. The next question that arose was: where are the amino acids that are targeted by the phosphatase located? An alignment of the various mammalian Spry proteins indicated that there is an N-terminal, Ser/Thr-rich sequence that is conserved to a relatively high degree in the various mammalian Spry isoforms (Fig. 2B, upper panel). We had shown that Ser/Thr phosphorylation was potentially responsible for the multiple bands of Spry2 seen on SDS-PAGE, and one way of seeing which of these putative phospho-targets contributed to the bandshifting was to perform a series of “alanine scan” point mutations over this sequence and compare the outcome on SDS-PAGE and subsequent immunoblotting. From the data shown in Fig. 2B (middle panels), it can be observed that a loss of the slower migrating band can be seen on Spry2 with point mutations of Ser-110, Ser-112, Ser-115, Ser-118, Ser-121, Thr-124, Ser-125, Ser-127, Ser-128, Ser-130, Ser-131, and E132A. No difference was noted with point mutations of Ser-108, Thr-113, Ser-116, Ser-120, Thr-122, Thr-126, and Ser-129. These data are summarized in Fig. 2B (lower panel); the arrows indicate changes in the upper band, and underlined letters indicate no change. The working hypothesis at this point is that some or all of these conserved Ser/Thr residues would be phosphorylated in unstimulated cells and would be dephosphorylated upon stimulation and there would be a subsequent structural change that exposes the proline-rich C-terminal tail. CK2 is a likely “priming” kinase that phosphorylates residues in resting cells, and its recognition site starts with an acidic Glu or Asp residue that is C-terminal to multiple serines. In Fig. 2B, we included Glu-132 in the point mutational analysis, and this results in a downshifting of Spry2 similar to some of the serine point mutations, possibly implicating CK2 as the kinase that opposes a phosphatase in the phosphorylation/dephosphorylation cycle of these conserved residues. To identify the sites of phosphorylation on Spry2 before and after FGFR1 stimulation, we next employed immunoprecipitation followed by peptide analysis utilizing mass spectrometry. We separated the bands of Spry2 as designated in the Coomassie Blue-stained protein separation blot in Fig. 2C (upper panel). To simplify visualization of the data, in Fig. 2C (lower panel), the table shows the residues revealed to be phosphorylated in several determinations. There are two points to note. Two residues, Ser-112 and Ser-115, are shown to be phosphorylated in unstimulated cells and not to be phosphorylated in FGFR1-stimulated cells. We have shown in the previous experiment that Ser-112 and Ser-115 were two of the residues that, when mutated, caused the slower migrating band to “disappear.” However, we could not detect peptides covering the majority of the conserved Ser/Thr sequence we used for the point mutation analysis despite repeated attempts. This arose due to the technical disadvantage of mass spectrometry in that all of the protein sequence is not usually recovered. We believe, however, that Ser-112 and Ser-115 provide a proof of concept in that the mass spectrometry-derived data agree with the point mutational analysis in that the phosphorylation status of certain residues can alter the gel migratory characteristics of Spry2 and that this potential “switch”" @default.
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