Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 control antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. B and C. HCT 116 cells were transfected with an empty vector or wild-type SHP2 (SHP2-WT-HA) or dominant-negative SHP2 mutant (SHP2-C459S-HA) expression constructs, as indicated. Forty-eight hours after transfection, the cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 (A) or anti-HA (B) antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. The position of the exogenously expressed SHP2 constructs that migrated more slowly, due to a fused HA-Tag, are indicated by an arrowhead. The immunoglobulin heavy chains (IgH) are indicated by asterisks (*). The results shown are representative of at least four independent experiments.

Article Snippet: However, the use of the specific SHP2 inhibitor NSC-87877 (Tocris Bioscience) did not provide any conclusive result and the question regarding whether CDCP1 internalization is regulated by the activity of SHP2 or by its association and potential recruitment of other molecules remains open.

Techniques: Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Expressing, Construct

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: HCT 116 cells were plated at (A) 0.2x10 6 cells/ml (non confluent) or (B) 1.4x10 6 cells/ml (confluent) for 48h. The cells were left untreated or were incubated with 25μM PerVO3 or 5μg/ml anti-CDCP1 antibody, as indicated, for 15 minutes. The cells were lysed and anti-CDCP1 antibodies were used for immunoprecipitation (IP), as indicated and as described in the experimental procedures section. Control immunoprecipitations were performed with irrelevant mouse immunoglobulins (Ig). Total cell lysates and immunoprecipitates were analyzed by western blotting with anti-pTyr, anti-CDCP1 and anti-SHP2 antibodies. The results shown are representative of at least three independent experiments. The position of endogenous SHP2 is indicated by an arrowhead. The asterisks (*) indicate the position of the Ig Heavy chains used for the immunoprecipitation or the triggering of CDCP1.

Article Snippet: However, the use of the specific SHP2 inhibitor NSC-87877 (Tocris Bioscience) did not provide any conclusive result and the question regarding whether CDCP1 internalization is regulated by the activity of SHP2 or by its association and potential recruitment of other molecules remains open.

Techniques: Incubation, Immunoprecipitation, Control, Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO 3 ), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated ( , compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.

Article Snippet: However, the use of the specific SHP2 inhibitor NSC-87877 (Tocris Bioscience) did not provide any conclusive result and the question regarding whether CDCP1 internalization is regulated by the activity of SHP2 or by its association and potential recruitment of other molecules remains open.

Techniques: Stable Transfection, Expressing, Mutagenesis, Affinity Purification, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. HeLa cells were transfected with a CDCP1-myc expression construct and were left untreated or were treated, 48h after transfection, with 25μM pervanadate (PerVO 3 ). The cells were lysed and CDCP1 was immunoprecipitated with an anti-Myc antibody (myc). Mouse immunoglobulins were used as a negative control, as indicated. The immune complexes were resolved by SDS-PAGE and transferred on membranes. An overlay assay was performed, as described in the experimental procedures, with a purified SHP2 trapping mutant protein fused to GST (GST-DACS, upper panel). The membrane was then stripped and reprobed with antibodies against phosphorylated tyrosine residues (middle panel) and CDCP1 (bottom panel). In HeLa cells, CDCP1 was detected as a doublet, only the upper band of which (arrowhead) was found to be phosphorylated and to interact with SHP2. B. HeLa cells were transfected with a construct encoding the wild-type CDCP1 (WT), or a CDCP1 protein with substitution of the Y734 or Y743 tyrosine residues (Y734F or Y743F, respectively) or both (DM). All these constructs were Myc-tagged. The cells were treated and lysed as described above. The intensities of the signals were analyzed and were normalized on the signal obtained for the CDCP1 blot (lower panel). The normalized value is considered to be 100% in CDCP1-WT transfected cells treated with PerVO 3 (lane 3). The values are expressed as fold increase or decrease. The data shown are representative of at least five independent experiments.

Article Snippet: However, the use of the specific SHP2 inhibitor NSC-87877 (Tocris Bioscience) did not provide any conclusive result and the question regarding whether CDCP1 internalization is regulated by the activity of SHP2 or by its association and potential recruitment of other molecules remains open.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Negative Control, SDS Page, Overlay Assay, Purification, Mutagenesis, Membrane

Journal: PLoS ONE

Article Title: The Tyrosine Phosphatase SHP2 Associates with CUB Domain-Containing Protein-1 (CDCP1), Regulating Its Expression at the Cell Surface in a Phosphorylation-Dependent Manner

doi: 10.1371/journal.pone.0123472

Figure Lengend Snippet: A. HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. B. Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10 –4 in non-parametric Student's t tests. The data shown are representative of at least three independent experiments performed in triplicate.

Article Snippet: However, the use of the specific SHP2 inhibitor NSC-87877 (Tocris Bioscience) did not provide any conclusive result and the question regarding whether CDCP1 internalization is regulated by the activity of SHP2 or by its association and potential recruitment of other molecules remains open.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Construct, shRNA, Western Blot, Incubation, Flow Cytometry, Membrane

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Knockdown of Grb2 and Shp2 reveal that the level of WT FGFR2 phosphorylation is controlled by Grb2. (A) Total HEK293T cell lysates were immunoblotted with anti-pFGFR antibody (top), and reprobed for total FGFR (middle) and Grb2 (bottom). Anti-pFGFR2 antibody is specific for A loop residues Y653 and Y654. (B) Cell lysates of overnight serum-staved stable A431 cells containing control shRNA (Ci), Grb2-shRNA (Grb2i), or Shp2-shRNA (Shp2i) were analyzed for FGFR2 phosphorylation as above. Only the nonstimulated state is shown (i.e., each lane is duplicated). Numbers on pFGFR2 panel are normalized intensity pFGFR2/total FGFR2. (C) Analysis of FGFR2 phosphorylation in Rat-1 fibroblast cells with control shRNA (Ci) and Grb2-shRNA (Grb2i) as above. Only the nonstimulated state was investigated (i.e., each lane is duplicated). (D) Inhibition of Shp2 in Grb2 knockdown cells restores basal receptor phosphorylation. Serum-starved WT-Ci and WT-Grb2i cells were incubated with 50 µM NSC87877 for 4 h and the resultant cell lysates were analyzed by Western blotting with anti-pFGFR2 antibody, Shp2 pY542-specific antibody, and anti-Grb2 antibody. The immunoblot was stripped and reprobed for total FGFR2 and Shp2 as the loading control. The numbers on the pFGFR2 panel represent normalized intensity pFGFR2/total FGFR2.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Phospho-proteomics, Control, shRNA, Inhibition, Incubation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Grb2 inhibits the interaction of Shp2 with FGFR2. (A) FLIM analysis of the FRET between the FGFR2-GFP and RFP-Shp2. In the control (WT-Ci) serum-starved cells no interaction between FGFR2 and Shp2 was observed in the basal state. The mean FRET lifetime is ∼2.0 ns (line in right-hand panel), which corresponds to the mean lifetime for isolated GFP. No apparent interaction between FGFR2 and Shp2 in WT-Grb2i cells (B), or WT-Ci cells transfected with the RFP-tagged substrate-trapping C459S Shp2 mutant (C). Interaction between FGFR2 and Shp2 is observed in the Grb2i the substrate-trapping C459S mutant (D). Stimulating cells that contain WT Shp2 or C459S mutant Shp2 with FGF9 results in clear binding between FGFR2 and Shp2 after 15 min (E and F), respectively. Bar, 10 µm.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Control, Isolation, Transfection, Mutagenesis, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Shp2 phosphorylation by FGFR2 is inhibited by Grb2. (A) Serum-starved HEK293T cells were incubated with 30 µM FGFR inhibitor (SU5402) for 2 h and then either stimulated with 10 ng/ml FGF9 for 15 min or left untreated. Cell lysates were prepared and analyzed by Western blotting with the indicated antibody. Anti-pFGFR and anti-Y542 on Shp2 antibodies were used to evaluate phosphorylation of proteins. The immunoblot was stripped and reprobed with a pan-antibody to determine total protein level. (B) Comparison of ligand-stimulated Shp2 phosphorylation between A431-Ci and A431-Grb2i cells in nonstimulated and on stimulation by FGF2 or FGF9 for 1 h. Shp2 phosphorylation was detected with anti-pY542 antibody (top). The immunoblot was reprobed for total Shp2 as a loading control (middle) and with Grb2 (bottom). (C) Densitometric quantification of basal state Shp2 phosphorylation levels in A431 cells in control shRNA (A431-Ci) and Grb2-shRNA (A431-Grb2i). Error bars represent SD, n = 7.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Phospho-proteomics, Incubation, Western Blot, Comparison, Control, shRNA

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Shp2 dephosphorylates Grb2. (A) Wild-type or FGFR2 stably transfected HEK293T were starved overnight, then stimulated using 10 ng/ml FGF9 for either 15 or 60 min. Cells were lysed in the presence of protease and phosphatase inhibitors. 50 µg of total cell lysate were used for immunoblotting studies. Phosphorylation of FGFR2 was examined using anti-pFGFR2 (first panel). To examine the Grb2 phosphorylation states in the absence or presence of FGFR2 expression, 1 mg of total cell lysates were used for immunoprecipitation using an anti-Grb2 antibody, and probed with an anti-Grb2 antibody. The immunoprecipitated Grb2 from FGFR2-overexpressing cells show multiple bands (both serum starved and FGF9 stimulated), suggesting the high molecular weight species is tyrosine-phosphorylated Grb2, which is only phosphorylated in the presence of FGFR2. (B) Recombinant Grb2 C-SH3 mutants (Y160F, left; Y209F, right) were expressed and purified from E. coli and incubated with pure FGFR2 cytoplasmic domain in a 1:1 molar ratio in the presence of ATP and MgCl 2 at room temperature for 1 h. Recombinant GST-fused pShp2 was obtained via the same protocol. A general anti-pY antibody was used to examine the phosphorylation state of FGFR2-phosphorylated Shp2 (lanes 4, 6, 10, and 12; panel 1) and Grb2 C-SH3 domains (lanes 2 and 8; panel 4). A specific anti-pY542 Shp2 antibody was also used to confirm that Y542 of Shp2 is phosphorylated. A pool of both proteins was dephosphorylated by mixing phosphatase (either pShp2 or Shp2) with phosphorylated protein substrates (either pGrb2 C-SH3 Y160F or phospho-Grb2 C-SH3 Y209F) at 4°C overnight. The anti-pY blot shows only the pGrb2 C-SH3 Y160F can be dephosphorylated by both pShp2 and Shp2 (lanes 5 and 6; panel 4). However, the phosphorylation state of pGrb2 C-SH3 Y209F is not affected by Shp2, suggesting that the Y209 is the target of Shp2. A total Shp2 antibody (panel 3) and total Grb2 antibody (panel 5) were used to confirm equal protein loading. (C) HEK293T cells were cotransfected with FGFR2-GFP and Grb2-strep-tag. After 48 h cells were starved for 4 h and incubated with either FGFR-specific inhibitor (50 µM SU5402) or Shp2-specific inhibitor (100 µM NSC87877) for 1 h. Cell lysates were subjected to affinity purification using strep-tactin agarose beads and immunoblotted with anti-pY antibody (top) followed by anti-Grb2 antibody (bottom).

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Stable Transfection, Transfection, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation, High Molecular Weight, Recombinant, Purification, Incubation, Strep-tag, Affinity Purification

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Interaction between Grb2 and Shp2. CFP-Grb2 and RFP-Shp2 colocalization and direct interaction measurement using FLIM in A431 cells. (A) Control lifetime measurement for CFP alone. (B) Interaction of CFP-Grb2 with RFP-tagged wild-type Shp2 (RFP- WT Shp2) at basal and after 20 ng/ml FGF9 stimulation. (C) Co-localization and direct interaction of Y542F mutant Shp2 with CFP-Grb2 at basal and after FGF9 stimulation. (D) Constitutive interaction of the C459S substrate-trapping Shp2 mutant with Grb2. A left-shifted peak relative to the line drawn along 2.2 ns indicates a binding. A peak centered on the 2.2 ns line indicates nonbinding. Bar, 20 µM.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Control, Mutagenesis, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: In vitro demonstration of catalytic cycling of FGFR2 and Shp2 in the presence of Grb2. (A) Schematic of interactions performed in vitro to demonstrate catalytic activity of FGFR2 and Shp2 on Grb2. Mixing FGFR2 cyto (blue) with Grb2 (red) promotes the formation of a heterotetrameric complex . Addition of ATP and MgCl 2 to this results in phosphorylation of FGFR2 and Grb2 (green circle). Addition of Shp2 (orange) results in dephosphorylation of FGFR2 and Grb2 (blue line). The heterotetrameric complex is recovered under these conditions. (B) Fluorescence lifetime measurement between GFP-FGFR2 cyto and RFP-Grb2 as a function of time. The first point corresponds to the fluorescence lifetime for isolated GFP-FGFR2 (black arrow). On addition of Grb2 (red arrow) a heterotetrameric complex between Grb2 and FGFR2 forms. This results in FRET between the GFP and RFP and the concomitant reduction in fluorescence lifetime. On addition of ATP/Mg 2+ (purple arrow) up-regulation of the RTK ensues and Y209 on Grb2 becomes phosphorylated and the FGFR2–Grb2 complex dissociates. The lifetime increases, reflecting reduction in complex concentration and the accumulation of pGrb2. After 80 min Shp2 was added (orange arrow). At this point clear reassociation of Grb2 and FGFR2 is observed as Grb2 is dephosphorylated in the presence of Shp2 and consequently the fluorescence lifetime decreases (blue line on graph). Replacing WT Shp2 with the Y542F (red line) or C459S (green line) mutant results in no immediate reduction in lifetime, confirming that the FGFR2–Grb2 complex is not rescued by adding these compromised phosphatases. (C) Measurement of FRET between GFP-FGFR2 cyto (Cyto) and RFP-Grb2 in solution using FLIM. Cyto alone is GFP-FGFR2 cyto and represents the background false-positive percentage FRET readout. Cyto+Grb2 is the population of molecules undergoing FRET when RFP-Grb2 is present. Cyto+Grb2+ATP is the population of GFP-FGFR2 cyto undergoing FRET with RFP-Grb2 when the FGFR2 kinase was activated. Shp2 30 min and 18 h represent the reestablishment of GFP-FGFR2 cyto /RFP-Grb2 complex in the presence of wild-type (blue line), Y542F (red line), and C459S (green line) mutant Shp2 as a function of time.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: In Vitro, Activity Assay, Phospho-proteomics, De-Phosphorylation Assay, Fluorescence, Isolation, Concentration Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Grb2 inhibits Shp2 activity toward FGFR2. (A) WT-Ci and WT-Grb2i cells were incubated with serum-free media with or without 50 µM Shp2 inhibitor NSC87877 overnight, and then either stimulated with 10 ng/ml FGF9 for 15 min or left untreated. Total cell lysates were analyzed by Western blotting with the indicated antibody. (B and C) Densitometric quantification of bands from experiments as described in A. Histogram values correspond to normalized bands for pFGFR2 against total FGFR2 (B) and pErk against total Erk (C) of three independent experiments. Error bars represent SD. (D) A431-Ci and A431-Grb2i cells were serum starved with 50 µM Shp2 inhibitor overnight, then either stimulated with FGF9 or left untreated. 50 µg total cell lysates were immunoblotted with indicated antibody. (E) Densitometric quantification of bands from experiments as described in D, where the ratio of pERK/total Erk is plotted from three independent experiments. Error bars represent SD. The A431-Ci FGF9/NSC87877 ratio was fixed as 1.0 for each experiment. Arrows highlight the comparison of the level of pErk in the control cells after FGF-ligand stimulation and the recovery of this level in the Grb2 knockdown cells only when stimulated in the presence of Shp2 inhibitor. (F) Shp2 inhibition does not affect EGF-stimulated MAP kinase response in A431 cells. The experimental procedure is as above except 50 ng/ml EGF was used to stimulate cells for 5 min.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Activity Assay, Incubation, Western Blot, Comparison, Control, Knockdown, Inhibition

Journal: The Journal of Cell Biology

Article Title: Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity

doi: 10.1083/jcb.201204106

Figure Lengend Snippet: Schematic diagram of cycle of enzymatic activity under the control of Grb2 in the absence of extracellular stimulation. (A) FGFR2 (blue) is stabilized in a basally phosphorylated state in the form of a heterotetramer in which a dimer of Grb2 recruits two receptor molecules. In this complex the receptors are able to autophosphorylate the activation loop tyrosines. The partially phosphorylated, nonsignaling state is represented by inclusion of the green circle with dashed border. (B) Active Shp2 is able to dephopshorylate FGFR2. This phosphatase activity is inhibited by Grb2 (red oval) when it is bound to the receptor. (C) Basally activated FGFR2 is able to phosphorylate Shp2 phosphatase (orange). The phosphorylation of Shp2 is represented by a solid green line. On phosphorylation of Y542 Shp2 is enhanced. This catalytic activity is inhibited in the presence of Grb2 bound to FGFR2. The phosphorylated Shp2 is represented by inclusion of the green circle. (D) Grb2 is phosphorylated by FGFR2. In this phosphorylated state Grb2 is no longer able to bind to the receptor and hence its inhibitory properties are lost. (E) Shp2 is able to dephosphorylate Grb2. This restores the adaptor protein to a state competent of binding FGFR2. Key: straight lines between proteins represent the change from one state of that protein to another. Green lines, phosphorylation. Blue lines, dephosphorylation. Green dashed line, autophosphorylation. Red lines, inhibition. Curved arrows, enzymatic activity, e.g., blue line from Shp2 intercepting dephosphorylation blue line between pFGFR and FGFR indicates that Shp2 is the active enzyme for that change of state.

Article Snippet: Antibodies against FGFR2 (C-17), Shp2 (C-18), Grb2 (C-23) and phosphotyrosine (pY99), as well as Shp2 inhibitor NSC87877 and FGFR2 inhibitor SU5402, shRNA, and also lentivirus particles for control (catalog no. sc-108080), Grb2 (catalog no. sc-29335-v), and Shp2 (catalog no. sc-36488-v) were all purchased from Santa Cruz Biotechnology, Inc.

Techniques: Activity Assay, Control, Activation Assay, Phospho-proteomics, Binding Assay, De-Phosphorylation Assay, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 1. Schematic representation of the domain structures of SHP2, Spry2, SPRED1, and SPRED2. FL hSHP2, full-length human SHP2, SH2, and PTP. FLhSpry2,full-lengthhumanSpry2,CRD.FLmSPRED1,full-lengthmouseSPRED1 and SPRED2, Ena-vasodilator-stimulated phosphoprotein homology-1 domain (EVH1), KBD, and CRD. These constructs were used in the subsequent figures.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Construct

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 2. Spry2 binds to the C-terminal tail of SHP2. A, HEK293 cells were transfected with the indicated plasmids (FLAG-Sprys, FGFR1) or the pXJ40 vector control. 24 h post-transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-SHP2. Immunoprecipitates were separated in SDS-PAGE and immunoblotted(IB)withtheantibodiesindicatedontheleft.Wholecelllysates(WCL)wereimmunoblottedtoverifyequalproteinexpressionlevels.B,deletion mutants of SHP2 were co-expressed with Spry2 and FGFR1 on lanes 7–12. Lysates were immunoprecipitated with anti-FLAG, and blots were tested with the antibodiesindicatedontheleft.C,FLAG-Spry2andHA-SHP2mutantswereco-expressedinHEK293cells.Lysateswereimmunoprecipitatedwithanti-FLAGand immunoblotted with anti-HA to probe for interactions with SHP2. Arrow indicates the immunoglobulin light chain.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 3. SPRED1 and SPRED2 interact with SHP2 through via amino acids 135–157. A, endogenous SHP2 was immunoprecipitated in cells overexpressing FLAG-taggedSPRED1andSPRED2withorwithoutFGFR1overexpressed.LysateswereprocessedasdescribedinFig.2A.Immunoblotswereincubatedwithanti-FLAG to prove for interactions with SHP2. B and C, FLAG-SPRED1 and FLAG-SPRED2, respectively, full-length or deletion mutants were transfected in HEK293 cells, and endogenous SHP2 was immunoprecipitated with anti-SHP2. Immunoprecipitates were separated in SDS-PAGE. D and E, HEK293 lysates expressing SPRED1 mutants as indicated on the panels were immunoprecipitated with anti-SHP2 to pull down endogenous SHP2. Immunoprecipitates were separated in SDS-PAGE and probed with anti-FLAG to detect binding. Arrowheads indicate the immunoglobulin heavy chain. IP, immunoprecipitation; IB, immunoblot; WCL, whole cell lysate.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Immunoprecipitation, Transfection, SDS Page, Expressing, Binding Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 4. SPRED1 and SPRED2 bind to the PTP domain of SHP2. A, FLAG- SPRED1 was co-expressed with HA-SHP2 full-length or deletion mutants. Lysates were immunoprecipitated with anti-FLAG and evaluated by Western

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 5. SHP2 dephosphorylates SPRED1 and SPRED2 but not Spry2. A, HEK293 cells were transfected with FLAG-Spry2 and increasing concentrations of SHP2 with or without FGFR1 activation. Lysates were immunoprecipitated with anti-FLAG, and immunoblots were probed with anti-PY20 to detect tyrosine phosphorylation levels. B, cell lysates co-transfected with FLAG-SPRED1 and SHP2 were treated as mentioned in A. C, cell lysates co-transfected with FLAG- SPRED2 and SHP2 were treated as mentioned in A. Numbers next to the asterisk indicate quantification results of tyrosine phosphorylation levels on the PY20 blot. SHP2 band on the anti-SHP2 blots (A–C) on lanes 1 and 2 represent endogenous SHP2 detected by the antibody. IP, immunoprecipitation; IB, immunoblot; WCL, whole cell lysate.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Transfection, Activation Assay, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE7.SHP2phosphataseinhibitionrescuestyrosinephosphorylationofSPRED.A,HEK293cellsweretransfectedwithFLAG-SPRED1andHA-SHP2.24h post-transfection, cells were treated with SHP2 inhibitor NSC-87877 for 2 h (lanes 7–9). Lysates were immunoprecipitated with anti-FLAG, subjected to SDS-PAGE, and immunoblotted as indicated. B, FLAG-SPRED2 and HA-SHP2 overexpressed in HEK293 cells were treated with NSC-87877 (lanes 5–7) for 2 h before harvesting. Lysates were treated as mentioned in A. Numbers next to the asterisk indicate quantification of tyrosine phosphorylation levels on the PY20 blot. IP, immunoprecipitation; IB, immunoblot; WCL, whole cell lysate.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Transfection, Immunoprecipitation, SDS Page, Phospho-proteomics, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

doi: 10.1074/jbc.m110.212662

Figure Lengend Snippet: FIGURE 8. SHP2dephosphorylatesTyr-420onSPRED1.A,HEK293cellsweretransfectedwiththeindicatedplasmids(FLAG-SPREDandRasV12)orthepXJ40vector control. Whole cell lysates (WCL) were analyzed by Western blotting with the indicated antibodies on the left. B, endogenous SHP2 was immunoprecipitated, with anti-SHP2, in cells overexpressing FLAG-SPRED1 WT or respective mutants. Immunoprecipitates were resolved by SDS-PAGE. C, FLAG-SPRED1Y377F was co-trans- fected with increasing concentrations of HA-SHP2 (0.5–3 g), and lysates were immunoprecipitated with anti-FLAG. Immunoblots were probed with the antibodies indicatedontheleft.D,cellsoverexpressingFLAG-SPRED1Y420FandHA-SHP2weretreatedasmentionedinA.Numbersnexttotheasteriskindicatequantificationof the tyrosine phosphorylation levels on the PY20 blot. The arrowhead indicates the light band at 50 kDa corresponding to the immunoglobulin heavy chain.

Article Snippet: Glutathione-Sepharose 4Bwas fromGEHealthcare, and the SHP2 inhibitor NSC-87877 was from TOCRIS Bioscience (Bristol, UK).

Techniques: Control, Western Blot, Immunoprecipitation, SDS Page, Phospho-proteomics