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Santa Cruz Biotechnology egfr phosphosite

Effect of GRB2 expression in EGF-stimulated COS1 cells. A, diagram of major constructs used for this study: tdEOS-tagged GRB2 SH2, FL WT GRB2, and a chimera of GRB2 SH3 domains and the CRK SH2 domain (GCG). B, GRB2-mediated enhancement of <t>EGFR</t> phosphorylation is SH2-dependent. Representative immunoblot of lysates from COS1 cells transfected with empty vector (ev) or overexpressing GRB2, tdEOS–GRB2 SH2, or GCG constructs before and after stimulation with 2.5 ng/ml EGF. R86K = R86K mutant that cannot bind pTyr sites. Data from three or four biological replicates are shown in bar graph below (error bars, standard error of the mean (S.E.)). Asterisks indicate phosphorylation increases that were statistically significant (paired Student's t test, p < 0.05) when compared with their empty vector control, i.e. EV or EV + EGF. n = 3 for K86R SH2 mutant constructs; n = 4 for other constructs. C, far-Western blotting and immunoblotting of lysates from COS1 cells transfected with GRB2 constructs. In labels on right, “GRB2 FW” and “CRK FW” indicate far-Western blotting with GRB2 and CRK SH2 domains. Anti-tubulin serves as loading control.

Egfr Phosphosite, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 2216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation"

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.794412

Figure Legend Snippet: Effect of GRB2 expression in EGF-stimulated COS1 cells. A, diagram of major constructs used for this study: tdEOS-tagged GRB2 SH2, FL WT GRB2, and a chimera of GRB2 SH3 domains and the CRK SH2 domain (GCG). B, GRB2-mediated enhancement of EGFR phosphorylation is SH2-dependent. Representative immunoblot of lysates from COS1 cells transfected with empty vector (ev) or overexpressing GRB2, tdEOS–GRB2 SH2, or GCG constructs before and after stimulation with 2.5 ng/ml EGF. R86K = R86K mutant that cannot bind pTyr sites. Data from three or four biological replicates are shown in bar graph below (error bars, standard error of the mean (S.E.)). Asterisks indicate phosphorylation increases that were statistically significant (paired Student's t test, p < 0.05) when compared with their empty vector control, i.e. EV or EV + EGF. n = 3 for K86R SH2 mutant constructs; n = 4 for other constructs. C, far-Western blotting and immunoblotting of lysates from COS1 cells transfected with GRB2 constructs. In labels on right, “GRB2 FW” and “CRK FW” indicate far-Western blotting with GRB2 and CRK SH2 domains. Anti-tubulin serves as loading control.

Techniques Used: Expressing, Construct, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Far Western Blot

Effect of GRB2 expression and EGF stimulation on GRB2-mediated enhancement. A, representative immunoblot of EGFR tyrosine phosphorylation in cells transfected with empty vector (ev) or increasing amounts of GRB2 construct. GRB2 expression levels were determined using a GRB2 SH2 standard and a GRB2 SH2-specific antibody. Densitometric quantification and fitting of EGFR phosphorylation data from three biological replicates are shown on the right. B, effect of increasing EGF stimulation on enhancement of total EGFR pTyr and EGFR pTyr-1068 (a GRB2 SH2-binding site) in cells overexpressing WT GRB2 or the inactive R86K mutant. Densitometric quantification of the data for total pTyr–EGFR is shown to the right. Error bars represent S.E. for three biological replicates. The increase in phosphorylation associated with GRB2 SH2 expression was significant at all EGF concentrations (paired Student's t test, p < 0.05, *), even though the relative effect size decreases with EGF concentration.

Figure Legend Snippet: Effect of GRB2 expression and EGF stimulation on GRB2-mediated enhancement. A, representative immunoblot of EGFR tyrosine phosphorylation in cells transfected with empty vector (ev) or increasing amounts of GRB2 construct. GRB2 expression levels were determined using a GRB2 SH2 standard and a GRB2 SH2-specific antibody. Densitometric quantification and fitting of EGFR phosphorylation data from three biological replicates are shown on the right. B, effect of increasing EGF stimulation on enhancement of total EGFR pTyr and EGFR pTyr-1068 (a GRB2 SH2-binding site) in cells overexpressing WT GRB2 or the inactive R86K mutant. Densitometric quantification of the data for total pTyr–EGFR is shown to the right. Error bars represent S.E. for three biological replicates. The increase in phosphorylation associated with GRB2 SH2 expression was significant at all EGF concentrations (paired Student's t test, p < 0.05, *), even though the relative effect size decreases with EGF concentration.

Techniques Used: Expressing, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Construct, Binding Assay, Mutagenesis, Concentration Assay

Site-specific increases in EGFR phosphotyrosine in SH2-overexpressing cells. A, representative anti-pTyr and phosphosite-specific anti-pTyr–EGFR immunoblots from COS1 cells transfected with empty vector (ev), tdEOS–GRB2 SH2, FL GRB2 SH2, or GCG. EGF + pervanadate (200 ng/ml EGF, 100 μm pervanadate, 40 min) was used as a maximally phosphorylated standard and run at 1:10 dilution on the same membranes. Antibodies are indicated to the left; for phosphospecific antibodies, numbers indicate residue number of phosphosite recognized. Percent maximal phosphorylation for each site and total pTyr–EGFR are shown on the panels to right (n = 3 biological replicates, error bars = S.E.). Expression of GRB2 FL and SH2 constructs resulted in statistically significant increases in the total phosphorylation of EGFR as well as the phosphorylation of multiple EGFR phosphotyrosines, including the GRB2-binding site pTyr-1068 (paired Student's t test, p < 0.05, *). An inter-site comparison of fold increase in phosphorylation revealed a statistically significant increase in phosphorylation of pTyr-1068 associated with GRB2 FL expression in non-stimulated cells (ANOVA, Tukey's pairwise comparison, p < 0.05, **). B, relative increase in abundance of EGFR phosphopeptides detected by quantitative mass spectrometry in COS1 lysates from cells expressing indicated SH2 constructs. C, relative increase in abundance of p130CAS phosphopeptides detected by MS as in B. Error bars for B and C represent S.E. for three biological replicates.

Figure Legend Snippet: Site-specific increases in EGFR phosphotyrosine in SH2-overexpressing cells. A, representative anti-pTyr and phosphosite-specific anti-pTyr–EGFR immunoblots from COS1 cells transfected with empty vector (ev), tdEOS–GRB2 SH2, FL GRB2 SH2, or GCG. EGF + pervanadate (200 ng/ml EGF, 100 μm pervanadate, 40 min) was used as a maximally phosphorylated standard and run at 1:10 dilution on the same membranes. Antibodies are indicated to the left; for phosphospecific antibodies, numbers indicate residue number of phosphosite recognized. Percent maximal phosphorylation for each site and total pTyr–EGFR are shown on the panels to right (n = 3 biological replicates, error bars = S.E.). Expression of GRB2 FL and SH2 constructs resulted in statistically significant increases in the total phosphorylation of EGFR as well as the phosphorylation of multiple EGFR phosphotyrosines, including the GRB2-binding site pTyr-1068 (paired Student's t test, p < 0.05, *). An inter-site comparison of fold increase in phosphorylation revealed a statistically significant increase in phosphorylation of pTyr-1068 associated with GRB2 FL expression in non-stimulated cells (ANOVA, Tukey's pairwise comparison, p < 0.05, **). B, relative increase in abundance of EGFR phosphopeptides detected by quantitative mass spectrometry in COS1 lysates from cells expressing indicated SH2 constructs. C, relative increase in abundance of p130CAS phosphopeptides detected by MS as in B. Error bars for B and C represent S.E. for three biological replicates.

Techniques Used: Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Residue, Expressing, Construct, Binding Assay, Comparison, Mass Spectrometry

GRB2 specifically enhances its canonical binding motifs in a concentration-dependent manner. A, representative EGFR pTyr-specific immunoblots from COS1 cells expressing an increasing amount of GRB2. For phosphospecific antibodies, residue number and pTyr motifs of phosphosite recognized are indicated on the right. Lysates used were the same as for Fig. 2A, and pTyr, GRB2, and tubulin control blots are duplicated here. B, quantification of EGFR pTyr site-specific phosphorylation following EGF stimulation plotted against overexpressed GRB2 concentration (three biological replicates). Curves represent a polynomial fit of the combined data (R2 values for all curves are >0.84). Data for the two canonical GRB2-binding sites (Tyr-1068 and Tyr-1086) are bolded.

Figure Legend Snippet: GRB2 specifically enhances its canonical binding motifs in a concentration-dependent manner. A, representative EGFR pTyr-specific immunoblots from COS1 cells expressing an increasing amount of GRB2. For phosphospecific antibodies, residue number and pTyr motifs of phosphosite recognized are indicated on the right. Lysates used were the same as for Fig. 2A, and pTyr, GRB2, and tubulin control blots are duplicated here. B, quantification of EGFR pTyr site-specific phosphorylation following EGF stimulation plotted against overexpressed GRB2 concentration (three biological replicates). Curves represent a polynomial fit of the combined data (R2 values for all curves are >0.84). Data for the two canonical GRB2-binding sites (Tyr-1068 and Tyr-1086) are bolded.

Techniques Used: Binding Assay, Concentration Assay, Western Blot, Expressing, Residue, Phospho-proteomics, Control

Model parameters All parameters were defined using experimental data unless otherwise noted below. Cell volumes were approximated from measurements of trypsinized cells. EGF-binding constants were taken from the literature. COS1 cell phosphorylation and dephosphorylation rates were measured using pTyr standard as shown in Fig. 6 , B – D . Measurements of in vivo EGFR phosphorylation rates and approximations of EGFR expression in COS1 cells were used to calculate the EGFR k f values. Phosphatase V max and K m values were obtained by fitting the quantitative EGFR dephosphorylation data to the Michaelis-Menten function. GRB2 concentrations were calculated via immunoblotting using a purified GRB2 standard run on the same membrane. GRB2 binding constants were determined using data from previously published work and by fitting experimental data to the model (see ).

Figure Legend Snippet: Model parameters All parameters were defined using experimental data unless otherwise noted below. Cell volumes were approximated from measurements of trypsinized cells. EGF-binding constants were taken from the literature. COS1 cell phosphorylation and dephosphorylation rates were measured using pTyr standard as shown in Fig. 6 , B – D . Measurements of in vivo EGFR phosphorylation rates and approximations of EGFR expression in COS1 cells were used to calculate the EGFR k f values. Phosphatase V max and K m values were obtained by fitting the quantitative EGFR dephosphorylation data to the Michaelis-Menten function. GRB2 concentrations were calculated via immunoblotting using a purified GRB2 standard run on the same membrane. GRB2 binding constants were determined using data from previously published work and by fitting experimental data to the model (see ).

Techniques Used: Phospho-proteomics, De-Phosphorylation Assay, In Vivo, Expressing, Western Blot, Purification, Membrane, Binding Assay, Concentration Assay, Construct, Transfection

$Computational model and parameter determination. A, diagram of the computational model used to quantify the effect of SH2 expression on EGFR phosphorylation. The effect of SH2 domain expression in unstimulated cells utilized the same scheme but used an EGFR kf value obtained in unstimulated cells. B–D, measurement of in vivo phosphorylation and dephosphorylation rates in COS1 cells. B, plot of absolute phosphorylation rate in serum-starved cells treated with pervanadate. C, plot of absolute phosphorylation rate in starved cells treated with pervanadate 5 min after stimulation with 2.5 ng/ml EGF. D, plot of absolute dephosphorylation rate in cells treated with erlotinib 5 min after stimulation with 2.5 ng/ml EGF. Black lines show amount of phosphorylation quantified from experimental data. Red lines show initial rate used to calculate model parameters. pTyr–EGFR concentrations were obtained by comparing signal from anti-pTyr immunoblots of COS1 lysates with a phosphotyrosine standard run on the same blot. E, plot of fitted curves from experimental data for fraction of EGFR pTyr-1068 (blue squares) and total pTyr EGFR (red circles) overlaid on model predictions generated at varying kon values for binding of GRB2 SH2 to EGFR. Plots were created by holding the koff constant at 1 s−1 and varying kon values (black lines).$
Figure Legend Snippet: Computational model and parameter determination. A, diagram of the computational model used to quantify the effect of SH2 expression on EGFR phosphorylation. The effect of SH2 domain expression in unstimulated cells utilized the same scheme but used an EGFR kf value obtained in unstimulated cells. B–D, measurement of in vivo phosphorylation and dephosphorylation rates in COS1 cells. B, plot of absolute phosphorylation rate in serum-starved cells treated with pervanadate. C, plot of absolute phosphorylation rate in starved cells treated with pervanadate 5 min after stimulation with 2.5 ng/ml EGF. D, plot of absolute dephosphorylation rate in cells treated with erlotinib 5 min after stimulation with 2.5 ng/ml EGF. Black lines show amount of phosphorylation quantified from experimental data. Red lines show initial rate used to calculate model parameters. pTyr–EGFR concentrations were obtained by comparing signal from anti-pTyr immunoblots of COS1 lysates with a phosphotyrosine standard run on the same blot. E, plot of fitted curves from experimental data for fraction of EGFR pTyr-1068 (blue squares) and total pTyr EGFR (red circles) overlaid on model predictions generated at varying kon values for binding of GRB2 SH2 to EGFR. Plots were created by holding the koff constant at 1 s−1 and varying kon values (black lines).

Techniques Used: Expressing, Phospho-proteomics, In Vivo, De-Phosphorylation Assay, Western Blot, Generated, Binding Assay

Computational modeling recapitulates experimental data. A, comparison of model predictions (red bars) and experimental data (green bars) on the effect of EGF concentration on GRB2 SH2-mediated EGFR phosphosite enhancement relative to GRB2 SH2 R86K mutant-expressing cells (see Fig. 2B). Error bars represent the S.E. of three biological replicates. For modeling data, GRB2 concentration was set at 5.4 μm. There was no statistical difference between the experimental and model data for the effect of EGF on GRB2-mediated pTyr enhancement and between 0 and 2.5 ng/ml EGF (one-way Student's t test, p > 0.05 “NS”). At higher concentrations of EGF (25 and 250 ng/ml), there was a small but statistically significant difference between experimental data and model predictions (one-way Student's t test, p < 0.05, *). B, model predictions of the relationship between SH2-bound phosphosites (pTyr-SH2, brown) and unbound phosphosites (pTyr, blue) in EGF-treated cells at varying SH2 concentrations. C, model predictions showing the effect of an increasing concentration of a GRB2-binding site competitor (SH2competitor) on the amount of pTyr-bound GRB2. Total concentration of GRB2 was held at 0.24 μm. Dark green bars represent pTyr-GRB2 binding in a system with pTyr flux (i.e. rapid phosphorylation and dephosphorylation; Fig. 6A). Light green bars represent pTyr-GRB2 binding data in a system containing a constant 3.6% pTyr EGFR (equal to the amount of pTyr EGFR present in the flux model without any exogenous SH2 present). D, quantification of phosphorylated ERK1 and ERK2 in COS1 cells expressing empty vector or tdEOS–GRB2 SH2 before and after EGF stimulation, using the same lysates as in Fig. 3A. All values are normalized to empty vector-transfected unstimulated cells and total ERK expression. Error bar represents S.E. from three biological replicates. There was no statistically significant difference between the phosphorylation of pERK1/pERK2 in empty vector and GRB2 SH2-expressing cells, before or after EGF stimulation (paired Student's t test, “NS”).

Figure Legend Snippet: Computational modeling recapitulates experimental data. A, comparison of model predictions (red bars) and experimental data (green bars) on the effect of EGF concentration on GRB2 SH2-mediated EGFR phosphosite enhancement relative to GRB2 SH2 R86K mutant-expressing cells (see Fig. 2B). Error bars represent the S.E. of three biological replicates. For modeling data, GRB2 concentration was set at 5.4 μm. There was no statistical difference between the experimental and model data for the effect of EGF on GRB2-mediated pTyr enhancement and between 0 and 2.5 ng/ml EGF (one-way Student's t test, p > 0.05 “NS”). At higher concentrations of EGF (25 and 250 ng/ml), there was a small but statistically significant difference between experimental data and model predictions (one-way Student's t test, p < 0.05, *). B, model predictions of the relationship between SH2-bound phosphosites (pTyr-SH2, brown) and unbound phosphosites (pTyr, blue) in EGF-treated cells at varying SH2 concentrations. C, model predictions showing the effect of an increasing concentration of a GRB2-binding site competitor (SH2competitor) on the amount of pTyr-bound GRB2. Total concentration of GRB2 was held at 0.24 μm. Dark green bars represent pTyr-GRB2 binding in a system with pTyr flux (i.e. rapid phosphorylation and dephosphorylation; Fig. 6A). Light green bars represent pTyr-GRB2 binding data in a system containing a constant 3.6% pTyr EGFR (equal to the amount of pTyr EGFR present in the flux model without any exogenous SH2 present). D, quantification of phosphorylated ERK1 and ERK2 in COS1 cells expressing empty vector or tdEOS–GRB2 SH2 before and after EGF stimulation, using the same lysates as in Fig. 3A. All values are normalized to empty vector-transfected unstimulated cells and total ERK expression. Error bar represents S.E. from three biological replicates. There was no statistically significant difference between the phosphorylation of pERK1/pERK2 in empty vector and GRB2 SH2-expressing cells, before or after EGF stimulation (paired Student's t test, “NS”).

Techniques Used: Comparison, Concentration Assay, Phospho-proteomics, Mutagenesis, Expressing, Binding Assay, De-Phosphorylation Assay, Plasmid Preparation, Transfection

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Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Effect of GRB2 expression in EGF-stimulated COS1 cells. A, diagram of major constructs used for this study: tdEOS-tagged GRB2 SH2, FL WT GRB2, and a chimera of GRB2 SH3 domains and the CRK SH2 domain (GCG). B, GRB2-mediated enhancement of EGFR phosphorylation is SH2-dependent. Representative immunoblot of lysates from COS1 cells transfected with empty vector (ev) or overexpressing GRB2, tdEOS–GRB2 SH2, or GCG constructs before and after stimulation with 2.5 ng/ml EGF. R86K = R86K mutant that cannot bind pTyr sites. Data from three or four biological replicates are shown in bar graph below (error bars, standard error of the mean (S.E.)). Asterisks indicate phosphorylation increases that were statistically significant (paired Student's t test, p < 0.05) when compared with their empty vector control, i.e. EV or EV + EGF. n = 3 for K86R SH2 mutant constructs; n = 4 for other constructs. C, far-Western blotting and immunoblotting of lysates from COS1 cells transfected with GRB2 constructs. In labels on right, “GRB2 FW” and “CRK FW” indicate far-Western blotting with GRB2 and CRK SH2 domains. Anti-tubulin serves as loading control.

Article Snippet: EGFR phosphosite-specific immunoblots were performed using the following antibodies: pTyr-845 (Santa Cruz Biotechnology, Inc., catalog no. sc-575442); pTyr-974 (Cell Signaling Technology, catalog no. 2641S); pTyr-992 (Cell Signaling Technology, catalog no. 2235P); pTyr-1045 (Cell Signaling Technology, catalog no. 2237P); pTyr-1148 (Cell Signaling Technology, catalog no. 4404S); pTyr-1068 (Cell Signaling Technology, catalog no. 3777P); pTyr-1086 (Cell Signaling Technology, catalog no. 2220S); and pTyr-1173 (Cell Signaling Technology, catalog no. 4407S). pERK1 and pERK2 were detected using rabbit anti-p44/42 pThr-202/pTyr-204 (Cell Signaling Technology, catalog no. 9101S).

Techniques: Expressing, Construct, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Far Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Effect of GRB2 expression and EGF stimulation on GRB2-mediated enhancement. A, representative immunoblot of EGFR tyrosine phosphorylation in cells transfected with empty vector (ev) or increasing amounts of GRB2 construct. GRB2 expression levels were determined using a GRB2 SH2 standard and a GRB2 SH2-specific antibody. Densitometric quantification and fitting of EGFR phosphorylation data from three biological replicates are shown on the right. B, effect of increasing EGF stimulation on enhancement of total EGFR pTyr and EGFR pTyr-1068 (a GRB2 SH2-binding site) in cells overexpressing WT GRB2 or the inactive R86K mutant. Densitometric quantification of the data for total pTyr–EGFR is shown to the right. Error bars represent S.E. for three biological replicates. The increase in phosphorylation associated with GRB2 SH2 expression was significant at all EGF concentrations (paired Student's t test, p < 0.05, *), even though the relative effect size decreases with EGF concentration.

Techniques: Expressing, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Construct, Binding Assay, Mutagenesis, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Site-specific increases in EGFR phosphotyrosine in SH2-overexpressing cells. A, representative anti-pTyr and phosphosite-specific anti-pTyr–EGFR immunoblots from COS1 cells transfected with empty vector (ev), tdEOS–GRB2 SH2, FL GRB2 SH2, or GCG. EGF + pervanadate (200 ng/ml EGF, 100 μm pervanadate, 40 min) was used as a maximally phosphorylated standard and run at 1:10 dilution on the same membranes. Antibodies are indicated to the left; for phosphospecific antibodies, numbers indicate residue number of phosphosite recognized. Percent maximal phosphorylation for each site and total pTyr–EGFR are shown on the panels to right (n = 3 biological replicates, error bars = S.E.). Expression of GRB2 FL and SH2 constructs resulted in statistically significant increases in the total phosphorylation of EGFR as well as the phosphorylation of multiple EGFR phosphotyrosines, including the GRB2-binding site pTyr-1068 (paired Student's t test, p < 0.05, *). An inter-site comparison of fold increase in phosphorylation revealed a statistically significant increase in phosphorylation of pTyr-1068 associated with GRB2 FL expression in non-stimulated cells (ANOVA, Tukey's pairwise comparison, p < 0.05, **). B, relative increase in abundance of EGFR phosphopeptides detected by quantitative mass spectrometry in COS1 lysates from cells expressing indicated SH2 constructs. C, relative increase in abundance of p130CAS phosphopeptides detected by MS as in B. Error bars for B and C represent S.E. for three biological replicates.

Techniques: Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Residue, Expressing, Construct, Binding Assay, Comparison, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: GRB2 specifically enhances its canonical binding motifs in a concentration-dependent manner. A, representative EGFR pTyr-specific immunoblots from COS1 cells expressing an increasing amount of GRB2. For phosphospecific antibodies, residue number and pTyr motifs of phosphosite recognized are indicated on the right. Lysates used were the same as for Fig. 2A, and pTyr, GRB2, and tubulin control blots are duplicated here. B, quantification of EGFR pTyr site-specific phosphorylation following EGF stimulation plotted against overexpressed GRB2 concentration (three biological replicates). Curves represent a polynomial fit of the combined data (R2 values for all curves are >0.84). Data for the two canonical GRB2-binding sites (Tyr-1068 and Tyr-1086) are bolded.

Techniques: Binding Assay, Concentration Assay, Western Blot, Expressing, Residue, Phospho-proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Model parameters All parameters were defined using experimental data unless otherwise noted below. Cell volumes were approximated from measurements of trypsinized cells. EGF-binding constants were taken from the literature. COS1 cell phosphorylation and dephosphorylation rates were measured using pTyr standard as shown in Fig. 6 , B – D . Measurements of in vivo EGFR phosphorylation rates and approximations of EGFR expression in COS1 cells were used to calculate the EGFR k f values. Phosphatase V max and K m values were obtained by fitting the quantitative EGFR dephosphorylation data to the Michaelis-Menten function. GRB2 concentrations were calculated via immunoblotting using a purified GRB2 standard run on the same membrane. GRB2 binding constants were determined using data from previously published work and by fitting experimental data to the model (see ).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, In Vivo, Expressing, Western Blot, Purification, Membrane, Binding Assay, Concentration Assay, Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Computational model and parameter determination. A, diagram of the computational model used to quantify the effect of SH2 expression on EGFR phosphorylation. The effect of SH2 domain expression in unstimulated cells utilized the same scheme but used an EGFR kf value obtained in unstimulated cells. B–D, measurement of in vivo phosphorylation and dephosphorylation rates in COS1 cells. B, plot of absolute phosphorylation rate in serum-starved cells treated with pervanadate. C, plot of absolute phosphorylation rate in starved cells treated with pervanadate 5 min after stimulation with 2.5 ng/ml EGF. D, plot of absolute dephosphorylation rate in cells treated with erlotinib 5 min after stimulation with 2.5 ng/ml EGF. Black lines show amount of phosphorylation quantified from experimental data. Red lines show initial rate used to calculate model parameters. pTyr–EGFR concentrations were obtained by comparing signal from anti-pTyr immunoblots of COS1 lysates with a phosphotyrosine standard run on the same blot. E, plot of fitted curves from experimental data for fraction of EGFR pTyr-1068 (blue squares) and total pTyr EGFR (red circles) overlaid on model predictions generated at varying kon values for binding of GRB2 SH2 to EGFR. Plots were created by holding the koff constant at 1 s−1 and varying kon values (black lines).

Techniques: Expressing, Phospho-proteomics, In Vivo, De-Phosphorylation Assay, Western Blot, Generated, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation

doi: 10.1074/jbc.M117.794412

Figure Lengend Snippet: Computational modeling recapitulates experimental data. A, comparison of model predictions (red bars) and experimental data (green bars) on the effect of EGF concentration on GRB2 SH2-mediated EGFR phosphosite enhancement relative to GRB2 SH2 R86K mutant-expressing cells (see Fig. 2B). Error bars represent the S.E. of three biological replicates. For modeling data, GRB2 concentration was set at 5.4 μm. There was no statistical difference between the experimental and model data for the effect of EGF on GRB2-mediated pTyr enhancement and between 0 and 2.5 ng/ml EGF (one-way Student's t test, p > 0.05 “NS”). At higher concentrations of EGF (25 and 250 ng/ml), there was a small but statistically significant difference between experimental data and model predictions (one-way Student's t test, p < 0.05, *). B, model predictions of the relationship between SH2-bound phosphosites (pTyr-SH2, brown) and unbound phosphosites (pTyr, blue) in EGF-treated cells at varying SH2 concentrations. C, model predictions showing the effect of an increasing concentration of a GRB2-binding site competitor (SH2competitor) on the amount of pTyr-bound GRB2. Total concentration of GRB2 was held at 0.24 μm. Dark green bars represent pTyr-GRB2 binding in a system with pTyr flux (i.e. rapid phosphorylation and dephosphorylation; Fig. 6A). Light green bars represent pTyr-GRB2 binding data in a system containing a constant 3.6% pTyr EGFR (equal to the amount of pTyr EGFR present in the flux model without any exogenous SH2 present). D, quantification of phosphorylated ERK1 and ERK2 in COS1 cells expressing empty vector or tdEOS–GRB2 SH2 before and after EGF stimulation, using the same lysates as in Fig. 3A. All values are normalized to empty vector-transfected unstimulated cells and total ERK expression. Error bar represents S.E. from three biological replicates. There was no statistically significant difference between the phosphorylation of pERK1/pERK2 in empty vector and GRB2 SH2-expressing cells, before or after EGF stimulation (paired Student's t test, “NS”).

Techniques: Comparison, Concentration Assay, Phospho-proteomics, Mutagenesis, Expressing, Binding Assay, De-Phosphorylation Assay, Plasmid Preparation, Transfection

$( a ) Cell survival rates of PC9 and PC9/gef cells under treatment with various concentrations of gefitinib. ( b ) Phosphorylation profile of EGFR kinase active sites with or without EGF stimulation. Site-specific antibodies against pY1148, pY1173, pY1086 and pY1045 were used for western blot detection. The intensity of phosphorylation sites were normalized to the intensity of total EGFR protein intensity in comparison analysis. ( c ) Experimental workflow of the quantitative proteomic and phosphoproteomic analysis of TKI-sensitive PC9 and TKI-resistant PC9/gef cells under gefitinib treatment (10 μM and 20 μM). The label-free quantitation approach integrated gel-assisted digestion and pH/acid-controlled IMAC for phosphopeptide purification. After IMAC purification, the eluted fraction was used for phosphoproteome analysis, while the flow-through fraction was used for protein-level quantitation. The quantitation of protein and phosphopeptides was performed using Ideal-Q software.$

Journal: Scientific Reports

Article Title: Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

doi: 10.1038/srep44021

Figure Lengend Snippet: ( a ) Cell survival rates of PC9 and PC9/gef cells under treatment with various concentrations of gefitinib. ( b ) Phosphorylation profile of EGFR kinase active sites with or without EGF stimulation. Site-specific antibodies against pY1148, pY1173, pY1086 and pY1045 were used for western blot detection. The intensity of phosphorylation sites were normalized to the intensity of total EGFR protein intensity in comparison analysis. ( c ) Experimental workflow of the quantitative proteomic and phosphoproteomic analysis of TKI-sensitive PC9 and TKI-resistant PC9/gef cells under gefitinib treatment (10 μM and 20 μM). The label-free quantitation approach integrated gel-assisted digestion and pH/acid-controlled IMAC for phosphopeptide purification. After IMAC purification, the eluted fraction was used for phosphoproteome analysis, while the flow-through fraction was used for protein-level quantitation. The quantitation of protein and phosphopeptides was performed using Ideal-Q software.

Article Snippet: The membranes was blocked with blocking buffer (5% skim milk in TBS) for 1 hr, and then incubated with anti-EGFR, anti-EGFR phosphosite-specific antibodies and anti-HMGA1 antibody (all from cell signaling), anti-pSer102 HMGA1 antibody (GeneTex) or anti-CK2 antibody (SANTA CRUZ) by 1:1000 diluted in blocking buffer.

Techniques: Western Blot, Quantitation Assay, Purification, Software

Sialylation suppresses EGFR dimerization and the EGF-binding ability of sEGFR. (A) EGF-induced dimerization of EGFR. The average MM of sEGFR at various concentrations was measured in the presence of EGF and transformed into percentage of dimerization (n = 3). Data were analyzed by nonlinear curve-fitting using GraphPad Prism software; the Kd for each sample is listed. (B) Dissociation of dimerized sEGFR. Dimerized sEGFR was prepared by incubating EGF and sEGFR at a saturated concentration. The decrement in MM was measured in gradual dilution condition and analyzed with nonlinear curve fitting. The purple trace represents sEGFR; the blue trace represents sEGFR with sialidase treatment. (C) SPR study of sEGFR binding to EGF. The binding constants of sEGFR to immobilized EGF were measured by SPR with various concentrations of sEGFR (n = 4). The calculated kinetics parameters (Kd, Kon, and Koff) of both sEGFR and desialylated sEGFR are shown.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: Sialylation suppresses EGFR dimerization and the EGF-binding ability of sEGFR. (A) EGF-induced dimerization of EGFR. The average MM of sEGFR at various concentrations was measured in the presence of EGF and transformed into percentage of dimerization (n = 3). Data were analyzed by nonlinear curve-fitting using GraphPad Prism software; the Kd for each sample is listed. (B) Dissociation of dimerized sEGFR. Dimerized sEGFR was prepared by incubating EGF and sEGFR at a saturated concentration. The decrement in MM was measured in gradual dilution condition and analyzed with nonlinear curve fitting. The purple trace represents sEGFR; the blue trace represents sEGFR with sialidase treatment. (C) SPR study of sEGFR binding to EGF. The binding constants of sEGFR to immobilized EGF were measured by SPR with various concentrations of sEGFR (n = 4). The calculated kinetics parameters (Kd, Kon, and Koff) of both sEGFR and desialylated sEGFR are shown.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Binding Assay, Transformation Assay, Software, Concentration Assay

Phosphorylation profiling of EGFR. (A) Purified flEGFR and desialylated flEGFR were treated with or without EGF at two concentrations of ATP (0.02 and 0.2 μM). The level of phosphorylation was analyzed by site-specific anti-EGFR phosphotyrosine antibodies (n = 3). (B) Semiquantitative results for the phosphorylation level of flEGFR incubated with 0.2 μM ATP. Relative fold change of phosphotyrosines between flEGFR and desialylated flEGFR was calculated. Error bars represent SD values. P values were calculated by paired t test. *P < 0.05; **P < 0.01.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: Phosphorylation profiling of EGFR. (A) Purified flEGFR and desialylated flEGFR were treated with or without EGF at two concentrations of ATP (0.02 and 0.2 μM). The level of phosphorylation was analyzed by site-specific anti-EGFR phosphotyrosine antibodies (n = 3). (B) Semiquantitative results for the phosphorylation level of flEGFR incubated with 0.2 μM ATP. Relative fold change of phosphotyrosines between flEGFR and desialylated flEGFR was calculated. Error bars represent SD values. P values were calculated by paired t test. *P < 0.05; **P < 0.01.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Phospho-proteomics, Purification, Incubation

Identification of EGFR phosphorylation in the lung cancer cell line CL1-5. The intensities of identified phosphopeptides containing phosphotyrosines (A), phosphothreonines (B), and phosphoserines (C) are shown. The EGFR phosphopeptides derived from EGF-treated or untreated cells were identified by mass spectrometry, and the intensity of phosphopeptides was quantified based on a label-free strategy and normalized with the sum of intensity of the three most abundant EGFR peptides. The relative fold change of each sample was calculated by dividing the intensity of normalized EGFR phosphopeptides from sialidase-treated cells by the intensity of normalized EGFR phosphopeptides of untreated cells. The positive (fold change >0) or negative (fold change <0) effect of desialylation on EGFR phosphorylation is indicated (n = 4). Error bars represent SD values.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: Identification of EGFR phosphorylation in the lung cancer cell line CL1-5. The intensities of identified phosphopeptides containing phosphotyrosines (A), phosphothreonines (B), and phosphoserines (C) are shown. The EGFR phosphopeptides derived from EGF-treated or untreated cells were identified by mass spectrometry, and the intensity of phosphopeptides was quantified based on a label-free strategy and normalized with the sum of intensity of the three most abundant EGFR peptides. The relative fold change of each sample was calculated by dividing the intensity of normalized EGFR phosphopeptides from sialidase-treated cells by the intensity of normalized EGFR phosphopeptides of untreated cells. The positive (fold change >0) or negative (fold change <0) effect of desialylation on EGFR phosphorylation is indicated (n = 4). Error bars represent SD values.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Phospho-proteomics, Derivative Assay, Mass Spectrometry

Effect of sialylation on tyrosine phosphorylation in EGFR mutants. (A and B) The EGFR mutant proteins EGFR L858R (A) and EGFR L858R/T790M (B) were purified for the in vitro phosphorylation assay. The relative fold change of tyrosine phosphorylation in each phosphopeptide was calculated by dividing the intensity of phosphorylation in sialidase-treated EGFR by the intensity of phosphorylation in untreated EGFR. The positive (fold change >0) or negative (fold change <0) effect of desialylation on EGFR phosphorylation is indicated (n = 3). Error bars represent SD values. Representative Western blots are shown in Fig. S6. (C) Tyrosine phosphorylation (pY1068, pY1086, and pY1173) of H1975 cells treated with STI or sialidase is shown. The relative intensities of phosphosites were normalized to their individual amounts of EGFR.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: Effect of sialylation on tyrosine phosphorylation in EGFR mutants. (A and B) The EGFR mutant proteins EGFR L858R (A) and EGFR L858R/T790M (B) were purified for the in vitro phosphorylation assay. The relative fold change of tyrosine phosphorylation in each phosphopeptide was calculated by dividing the intensity of phosphorylation in sialidase-treated EGFR by the intensity of phosphorylation in untreated EGFR. The positive (fold change >0) or negative (fold change <0) effect of desialylation on EGFR phosphorylation is indicated (n = 3). Error bars represent SD values. Representative Western blots are shown in Fig. S6. (C) Tyrosine phosphorylation (pY1068, pY1086, and pY1173) of H1975 cells treated with STI or sialidase is shown. The relative intensities of phosphosites were normalized to their individual amounts of EGFR.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Phospho-proteomics, Mutagenesis, Purification, In Vitro, Western Blot

Effect of sialylation on gefitinib sensitivity and EGFR phosphorylation in lung cancer cell lines with EGFR mutations. (A) Proliferation of TKI-resistant lung cancer cell lines with or without STI treatment in the presence of gefitinib. The proliferation assay was performed as described in SI Materials and Methods. (B) Levels of sialylation on EGFR in lung cancer cell lines. Sialylation was analyzed by a lectin pull-down experiment with SNA as described in SI Materials and Methods. Error bars represent SD values. S, TKI sensitive; R, TKI resistant. P values were calculated by paired t test. *P < 0.05; **P < 0.01. (C) Profiling of EGFR phosphorylation in lung cancer cell lines. The levels of site-specific phosphorylation of EGFR were detected by immunoblotting with antibodies recognizing specific phosphosites, and the relative phosphorylation was calculated by normalization to the intensity of A549 cells with EGF treatment. Error bars represent SD values. Representative Western blots are shown in Fig. S7C. Cell lines examined were 1, H3255; 2, PC9; 3, H1975; 4, CL97; 5, CL68. S, TKI sensitive; R, TKI resistant.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: Effect of sialylation on gefitinib sensitivity and EGFR phosphorylation in lung cancer cell lines with EGFR mutations. (A) Proliferation of TKI-resistant lung cancer cell lines with or without STI treatment in the presence of gefitinib. The proliferation assay was performed as described in SI Materials and Methods. (B) Levels of sialylation on EGFR in lung cancer cell lines. Sialylation was analyzed by a lectin pull-down experiment with SNA as described in SI Materials and Methods. Error bars represent SD values. S, TKI sensitive; R, TKI resistant. P values were calculated by paired t test. *P < 0.05; **P < 0.01. (C) Profiling of EGFR phosphorylation in lung cancer cell lines. The levels of site-specific phosphorylation of EGFR were detected by immunoblotting with antibodies recognizing specific phosphosites, and the relative phosphorylation was calculated by normalization to the intensity of A549 cells with EGF treatment. Error bars represent SD values. Representative Western blots are shown in Fig. S7C. Cell lines examined were 1, H3255; 2, PC9; 3, H1975; 4, CL97; 5, CL68. S, TKI sensitive; R, TKI resistant.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Phospho-proteomics, Proliferation Assay, Western Blot

TKI-sensitive and -resistant EGFRs and their sialylation and phosphorylation on Y1068, Y1086, and Y1173. Compared with the TKI-sensitive L858R mutant, the TKI-resistant L858R/T790M mutant showed a higher level of phosphorylation at Y1068, Y1086, and Y1173 in the absence of EGF; with EGF, Y1086 showed a higher level of phosphorylation. Note that in the absence of EGF the kinase domain of EGFR L858R and L858R/T790M mutants can dimerize to activate the downstream signaling.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition

doi: 10.1073/pnas.1507329112

Figure Lengend Snippet: TKI-sensitive and -resistant EGFRs and their sialylation and phosphorylation on Y1068, Y1086, and Y1173. Compared with the TKI-sensitive L858R mutant, the TKI-resistant L858R/T790M mutant showed a higher level of phosphorylation at Y1068, Y1086, and Y1173 in the absence of EGF; with EGF, Y1086 showed a higher level of phosphorylation. Note that in the absence of EGF the kinase domain of EGFR L858R and L858R/T790M mutants can dimerize to activate the downstream signaling.

Article Snippet: Samples were separated by SDS/PAGE and subjected to immunoblotting with antibodies specific for EGFR phosphosites (Cell Signaling).

Techniques: Phospho-proteomics, Mutagenesis

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