Journal: The Journal of biological chemistry

Article Title: Epidermal growth factor induces coupling of protein-tyrosine phosphatase 1D to GRB2 via the COOH-terminal SH3 domain of GRB2.

doi: 10.1074/jbc.271.35.20981

Figure Lengend Snippet: FIG. 1. EGF induces coupling of PTP1D to GRB2 via the COOH-terminal SH3 domain of GRB2. A, serum-starved MCF-10A cells were stimulated with 30 ng/ml EGF for 2 min at 37 °C, whole cell lysates were generated as described under “Materials and Methods,” and specific proteins were immunoprecipitated (IP) with either of two antibodies to PTP1D (Ab1 or Ab2) or with an antibody to GRB2. Im- munoprecipitates were fractionated by SDS-PAGE in an 8–16% gradi- ent SDS-PAGE gel and analyzed by Western blotting for PTP1D (aPTP1D) and for GRB2 (aGRB2). Serum-starved MCF-10A cells (B) and NR6-WT fibroblasts (C) were stimulated with either 30 ng/ml EGF (E) or PDGF-BB (P) for 2 min at 37 °C, and cell lysates were generated. One mg of glutathione S-transferase (GST) or GST fusion protein bound to glutathione-agarose was added to 0.5 mg of cell lysate and incubated for 12 h at 4 °C, washed, and analyzed by SDS-PAGE in an 8% acryl- amide gel. The upper and lower parts of the transfer membrane were probed for Sos (aSos) and PTP1D (aPTP1D), respectively. GRB2, NT- SH3, SH2, and CT-SH3 refer to the full-length, amino-terminal SH3 domain, SH2 domain, and carboxyl-terminal SH3 domain of GRB2 fused to GST, respectively.

Article Snippet: The glutathione S-transferase fusion proteins and antiPTP1D-Ab2 and anti-GRB2 polyclonal antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Generated, Immunoprecipitation, SDS Page, Western Blot, Incubation, Membrane

Journal: The Journal of biological chemistry

Article Title: Epidermal growth factor induces coupling of protein-tyrosine phosphatase 1D to GRB2 via the COOH-terminal SH3 domain of GRB2.

doi: 10.1074/jbc.271.35.20981

Figure Lengend Snippet: FIG. 2. EGF-stimulated linkage of PTP1D to GRB2 is independ- ent of Sos and the PTP1D SH2 domains cannot mediate the coupling. Cell lysates from EGF-stimulated MCF-10A cells and PDGF-BB-stimulated NR6-WT fibroblasts (A) were generated and probed with GRB2-GST fusion protein as described in the legend to Fig. 1 in the presence of 0, 5, 25, 100, or 250 mM N10 peptide. N10 peptide corresponds to the sequence of residues 1149–1158 in human Sos (27). Binding of Sos and PTP1D to the GRB2-GST was analyzed by Western blotting with specific antibodies against these proteins (aSos and aPTP1D). In B, lysates from cells treated with EGF (E) or PDGF-BB (P) were probed with a GST fusion protein corresponding to both SH2 domains of PTP1D (PTP1D-SH2-GST). Binding of phosphotyrosine- containing proteins (aPY) or GRB2 (aGRB2) to the fusion protein was analyzed by Western blotting in a 8–16% gradient SDS-PAGE gel. The numbers on the left denote the position in the gel and size in kilodaltons of molecular mass marker proteins.

Article Snippet: The glutathione S-transferase fusion proteins and antiPTP1D-Ab2 and anti-GRB2 polyclonal antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Generated, Sequencing, Binding Assay, Western Blot, SDS Page, Marker

Journal: The Journal of biological chemistry

Article Title: Epidermal growth factor induces coupling of protein-tyrosine phosphatase 1D to GRB2 via the COOH-terminal SH3 domain of GRB2.

doi: 10.1074/jbc.271.35.20981

Figure Lengend Snippet: FIG. 3. Phosphotyrosine analysis of PTP1D in EGF and PDGF- stimulated cells. In A, serum-starved MCF-10A and NR6-WT cells were treated with 0, 30, or 150 ng/ml EGF or PDGF-BB for 2 min at 37 °C, and cell lysates were generated as described in the legend to Fig. 1. PTP1D was immunoprecipitated (IP) from 1 mg of cell lysate using anti-PTP1D antibodies (Ab2) and analyzed by Western blotting for phosphotyrosine (aPY). The aPY blots were probed with a mixture of PY20 and 4G10 anti-phosphotyrosine monoclonal antibodies. To verify that constant amounts of PTP1D were immunoprecipitated in all in- stances, an aliquot of each immunoprecipitate was analyzed for PTP1D (aPTP1D). The numbers on the left denote the position in the gel and size in kilodaltons of molecular mass marker proteins. In B, serum- starved MCF-10A cells were treated with EGF (E), and cell lysates (1 mg) were generated and probed with 2 mg of either full-length GRB2- GST fusion protein (GRB2) or the carboxyl-terminal SH3 domain of GRB2 fused to GST (CT-SH3) bound to glutathione-agarose as de- scribed in the legend to Fig. 1. The glutathione-agarose resin was boiled in SDS-PAGE sample buffer, and the sample was split and analyzed in parallel in 8% SDS-PAGE gels. After transfer of the proteins to the membrane, the membrane was sliced and analyzed for the presence of Sos (aSos) and PTP1D (aPTP1D) (lanes 1, 2, 5, and 6) or phosphoty- rosine (aPY) (lanes 3, 4, 7, and 8). Western blotting analysis using antibodies specific for the EGF receptor (EGFR) and SHC confirmed the presence of these proteins only in samples which were probed with the full-length GRB2-GST.

Article Snippet: The glutathione S-transferase fusion proteins and antiPTP1D-Ab2 and anti-GRB2 polyclonal antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Generated, Immunoprecipitation, Western Blot, Bioprocessing, Marker, SDS Page, Membrane

Journal: Cell reports

Article Title: FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma

doi: 10.1016/j.celrep.2021.109905

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Gab1 Y659 , Cell Signaling Technology , Cat# 12745; RRID:AB_2798014.

Techniques: Recombinant, Reverse Transcription, Staining, Software, Sequencing

Journal: Cell reports

Article Title: FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma

doi: 10.1016/j.celrep.2021.109905

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Gab1 Y659 , Cell Signaling Technology , Cat# 12745; RRID:AB_2798014.

Techniques: Recombinant, Reverse Transcription, Staining, Software, Sequencing

Journal: Scientific reports

Article Title: Suppression of Spry1 inhibits triple-negative breast cancer malignancy by decreasing EGF/EGFR mediated mesenchymal phenotype.

doi: 10.1038/srep23216

Figure Lengend Snippet: Figure 5. Suppressing Spry1 results in degradation of EGF activated EGFR, and decrease of EGF induced EGFR/Grb2/Gab1/Shp2 cascade complexes formation and downstream signaling, and Snail and Slug induction. (A) Time course analysis of Snail, Slug induction, and EGFR protein level upon EGF stimulation. Immunoblotting shows that EGF stimulation induced Snail and Slug expression in NT cells but not in S1kd cells. In NT cells EGF did not change EGFR levels within 6 h, but in S1kd and S4kd cells EGF induced a decrease of EGFR at 2 h and 6 h. The results are representative from at least three independent experiments. The blots were first used for probing pEGFR, Slug or Snail, and then stripped off for reprobing EGFR or Tubulin. (B) Quantification to show the trend of EGFR degradation in S1kd cells. (C) Immunoprecipitation of EGFR and blotting with Grb2 and EGFR shows decreased EGFR/Grb2 complex formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-EGFR overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The same blot was cut between 37 kD and 50 kD markers, and separately probed for EGFR (~185 kD) or Grb2 (~25 kD). (D) Quantification of EGF induced EGFR/Grb2 complex formation from three independent experiments. (E) Immunoprecipitation of Gab1 and blotting with Grb2, Shp2 and Gab1 shows decreased Gab/Grb2 and Gab1/Shp2 complexes formation in S1kd cells compared to NT and S4kd cells. Lysates were incubated with rabbit anti-Gab1 overnight, then protein A/G for 1 hr. Beads bound proteins were washed and separated on 8% SDS-PAGE. The blot was cut between 37 kD and 50 kD, and between 75 kD and 100 kD, and probed for Grb2 (~25 kD), Shp2 (~72 kD) or Gab1 (~110 kD). (F) Quantification of EGFR/Grb2, Gab1/Grb2, Gab1/Shp2 complexes from three independent experiments. (G) Representative immunoblotting assay shows decreased EGF mediated pAkt level in S1kd cells compared to NT and S4kd cells. (H) Quantification of pAkt/Akt from at least three independent experiments. *p < 0.05, **p < 0.01 relative to controls; white *heavy chain.

Article Snippet: For immunoprecipitation, lysates were incubated with rabbit antibodies against EGFR, Gab1, c-Cbl (Cell Signaling Technology), Grb2, Shp2 (BD Bioscience) or Spry1 (New England Peptide LLC , rabbit polyclonal antibody produced by immunizing animals with a synthetic peptide corresponding to human Spry1 aa143–156, the sequence of the peptide is Ac-CRPVPGHRSERAIRT-amide), then precipitated with protein A/G plus agarose beads (Santa Cruz Technology).

Techniques: Western Blot, Expressing, Immunoprecipitation, Incubation, SDS Page

Journal: Oncology Reports

Article Title: MicroRNA-218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2

doi: 10.3892/or.2020.7759

Figure Lengend Snippet: miR-218 targets GAB2 by binding to its 3′UTR. (A) The GAB2 3′UTR regions containing the wild-type (WT) or mutant (MUT) binding site and the sequence complementarity between miR-218 and the GAB2 3′UTR are shown. (B) The relative luciferase activity was analyzed after transfection with the WT or MUT 3′UTR reporter plasmids in LV-NC or LV-miR-218 293T cells. Overexpression of miR-218 suppressed the luciferase activity of the wild-type reporter (55%). **P<0.01 compared to the MUT reporter. (C) Real-time PCR was used to analyze the expression level of GAB2 in ACHN, 786O and 769P cells transfected with LV-NC or LV-miR-218. **P<0.01 compared to the LV-NC group. (D) GAB2 protein level was analyzed by western blot analysis. The results are representative of three independent experiments. The values were presented as the mean ± standard deviation (SD). GAB2, GRB2-associated binding protein 2; UTR, untranslated region.

Article Snippet: The primary antibodies were GAB2 (Proteintech, 22549-1-AP, dilution 1:50), VEGFA (Abcam, ab1316, dilution 1:50), proliferating cell nuclear antigen (PCNA) (Proteintech, 10205-2-AP, dilution 1:200), and CD31 (Abcam, ab28364, dilution 1:50).

Techniques: Binding Assay, Mutagenesis, Sequencing, Luciferase, Activity Assay, Transfection, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Journal: Oncology Reports

Article Title: MicroRNA-218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2

doi: 10.3892/or.2020.7759

Figure Lengend Snippet: GAB2 plays a crucial role in RCC angiogenesis. (A and B) Real-time PCR and western blot analysis were used to confirm the knockdown of GAB2 in ACHN and 786O cells transfected with LV-shGAB2 both at the mRNA and protein level. GAB2 knockdown decreased VEGFA at the protein level. ***P<0.001 and ****P<0.0001 compared to the shNC group. (C) GAB2 knockdown decreased the recruitment of HUVECs in a co-cultured system. Cancer cells that were cultured in the bottom of a 24-well plate were used to recruit HUVECs. ****P<0.0001 compared to the shNC group. (D) GAB2 knockdown decreased the recruitment of HUVECs through the conditioned medium (CM) collected from the ACHN, 786O/LV-shGAB2, and ACHN, 786O/LV-shNC cells. The migrated cells in six random fields per well were counted (magnification, ×200). ****P<0.0001 compared to the shNC group. (E) GAB2 knockdown reduced the tube formation of HUVECs diluted in SFM or CMs. The representative images of tube-like structures are shown, and the tube numbers in the whole field were counted (magnification, ×100). ****P<0.0001 compared to the shNC group. (F) The concentration of secreted VEGFA protein in the CMs was determined by ELISA. The values were presented as the mean ± standard deviation (SD). GAB2, GRB2-associated binding protein 2; RCC, renal cell carcinoma; HUVECs, human umbilical vein endothelial cells; SFM, serum-free medium; CM, conditioned medium; VEGFA, vascular endothelial growth factor A.

Article Snippet: The primary antibodies were GAB2 (Proteintech, 22549-1-AP, dilution 1:50), VEGFA (Abcam, ab1316, dilution 1:50), proliferating cell nuclear antigen (PCNA) (Proteintech, 10205-2-AP, dilution 1:200), and CD31 (Abcam, ab28364, dilution 1:50).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Transfection, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay

Journal: Oncology Reports

Article Title: MicroRNA-218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2

doi: 10.3892/or.2020.7759

Figure Lengend Snippet: miR-218 regulates the PI3K/AKT/mTOR signaling pathway by targeting GAB2. (A) The protein level of phosphorylated (p)-AKT at Ser-473, p-mTOR and HIF1α were analyzed in miR-218-overexpressing RCC cells by western blot analysis. GAPDH was used as an internal control. (B) The protein level of p-AKT at Ser-473, p-mTOR, HIF1α, and VEGFA were analyzed in GAB2-knockdown RCC cells by western blot analysis. GAPDH was used as an internal control. These data are representative of three independent experiments. GAB2, GRB2-associated binding protein 2; RCC, renal cell carcinoma; mTOR, mammalian target of rapamycin; HIF1α, hypoxia inducible factor 1 subunit α; VEGFA, vascular endothelial growth factor A.

Article Snippet: The primary antibodies were GAB2 (Proteintech, 22549-1-AP, dilution 1:50), VEGFA (Abcam, ab1316, dilution 1:50), proliferating cell nuclear antigen (PCNA) (Proteintech, 10205-2-AP, dilution 1:200), and CD31 (Abcam, ab28364, dilution 1:50).

Techniques: Western Blot, Control, Knockdown, Binding Assay

Journal: Oncology Reports

Article Title: MicroRNA-218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2

doi: 10.3892/or.2020.7759

Figure Lengend Snippet: miR-218 inhibits tumor growth and angiogenesis in vivo . (A) Subcutaneous xenografts of 786O/LV-miR-218 and 786O/LV-NC subclones were harvested at four weeks after inoculation. (B) The tumor weights between the two groups. The values are presented as mean ± standard deviation (SD). ****P<0.0001. (C-F) Expression of CD31, GAB2, PCNA and VEGFA were analyzed in paraffin-fixed tumor sections from 786O/LV-miR-218 and 786O/LV-NC xenografts by immunohistochemistry. Representative images are shown at magnification ×200 (CD31) or ×400 (GAB2, PCNA and VEGFA). (G) The 786O/LV-NC cells, but not the 786O /LV-miR-218 cells, induced the angiogenesis in rabbit cornea (n=3). Neonatal vessels invaded the cornea, connecting the tumor (yellow arrow) and the cornea limbal vascular plexus. GAB2, GRB2-associated binding protein 2; RCC, renal cell carcinoma; PCNA, proliferating cell nuclear antigen; VEGFA, vascular endothelial growth factor A.

Article Snippet: The primary antibodies were GAB2 (Proteintech, 22549-1-AP, dilution 1:50), VEGFA (Abcam, ab1316, dilution 1:50), proliferating cell nuclear antigen (PCNA) (Proteintech, 10205-2-AP, dilution 1:200), and CD31 (Abcam, ab28364, dilution 1:50).

Techniques: In Vivo, Standard Deviation, Expressing, Immunohistochemistry, Binding Assay

Journal: Oncology Reports

Article Title: MicroRNA-218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2

doi: 10.3892/or.2020.7759

Figure Lengend Snippet: Schematic representation of the roles of miR-218 on the GAB2/PI3K/AKT/mTOR/HIF-1α/VEGFA pathway in renal cell carcinoma angiogenesis. GAB2, GRB2-associated binding protein 2; RCC, renal cell carcinoma; mTOR, mammalian target of rapamycin; HIF1α, hypoxia inducible factor 1 subunit α; VEGFA, vascular endothelial growth factor A; PI3K, phosphoinositide 3-kinase.

Article Snippet: The primary antibodies were GAB2 (Proteintech, 22549-1-AP, dilution 1:50), VEGFA (Abcam, ab1316, dilution 1:50), proliferating cell nuclear antigen (PCNA) (Proteintech, 10205-2-AP, dilution 1:200), and CD31 (Abcam, ab28364, dilution 1:50).

Techniques: Binding Assay

Journal: Communications Biology

Article Title: The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation

doi: 10.1038/s42003-022-03985-7

Figure Lengend Snippet: a , b A representative western blot and summarised densitometry demonstrating that transfection with exogenous RFP-tagged Grb3-3 significantly reduces phosphorylation of ERK in response to ligand stimulation of receptor tyrosine kinases (RTKs) in HEK293T cells stably transfected with fibroblast growth factor receptor 2 (FGFR2). This experiment demonstrates reduced MAPK activity in the presence of exogenous transfected Grb3-3. α-tubulin is shown as a loading control. See Supplementary Fig. for example uncropped blot. n = 3. c , d A representative western blot and summarised densitometry demonstrating reduced phosphorylation of ERK (i) in Caco-2 cells in the presence of exogenous transfected Grb3-3, despite persistent phosphorylation of EGFR (ii). α-tubulin is shown as a loading control. See Supplementary Fig. for example uncropped blot. n = 3. e , f Cell proliferation BrdU incorporation assays demonstrating that in both HEK293T and Caco-2 cells, transfection with a Grb2 mutant characterised by impaired SH2 domain-mediated binding (Grb2 R82L ) or Grb3-3 each resulted in a reduction in proliferation. n = 24. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤0.0001.

Article Snippet: Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074).

Techniques: Western Blot, Transfection, Phospho-proteomics, Stable Transfection, Activity Assay, Control, BrdU Incorporation Assay, Mutagenesis, Binding Assay

Journal: Communications Biology

Article Title: The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation

doi: 10.1038/s42003-022-03985-7

Figure Lengend Snippet: a Grb3-3 constitutively binds Sos through the N-terminal SH3 domain. A representative western blot from HEK293T cells stably overexpressing fibroblast growth factor receptor 2 (FGFR2) transfected with RFP-tagged full-length Grb3-3, the unique Grb3-3 SH2 domain (SH2Δ 40 ) or truncated Grb3-3 moieties comprising of the Grb3-3 N-terminal SH3 and SH2 domain (NSH3SH2Δ 40 ), the Grb3-3 C-terminal SH3 domain and SH2 domain (SH2Δ 40 CSH3), in both basal conditions and following FGFR2 stimulation with fibroblast growth factor 9 (FGF9). RFP-tagged proteins were purified and co-immunoprecipitation of endogenous Sos assessed by western blot. n = 3. Sos and RFP loading control identified with specific antibody run on same blot: see Supplementary Fig. . b In order to identify the site with which Grb3-3 binds Sos, eight proline-rich sequences derived from the C-terminus, CDC25 catalytic domain and the pleckstrin homology (PH) domain of Sos were synthesised (Supplementary Table ). Binding to His-tagged Grb2 and Grb3-3 was assessed by pulldown assay, with demonstrated the binding of three peptides derived from the c-terminus of Sos (Sos-CTail-5, Sos-CTail-6, Sos-CTail-8) to both Grb2 and Grb3-3 (Supplementary Fig. ). The proline-rich regions within these sequences are highlighted in red. c MST isotherms for the interactions between three proline-rich peptides derived from the C-terminus of Sos and the Grb3-3 N-terminal SH3 and SH2 domain (NSH3SH2Δ 40 ; top row) and the Grb3-3 C-terminal SH3 and SH2 domain (SH2Δ 40 CSH3; bottom row). Fitted K d values are shown individually on each plot and demonstrate that Grb3-3 binding to proline-rich peptides in the C-terminus of Sos was predominantly through the NSH3 domain. n = 3 technical replicated and n = 1 biological replicate.

Article Snippet: Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074).

Techniques: Western Blot, Stable Transfection, Transfection, Purification, Immunoprecipitation, Control, Derivative Assay, Binding Assay

Journal: Communications Biology

Article Title: The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation

doi: 10.1038/s42003-022-03985-7

Figure Lengend Snippet: a A representative western blot showing immunoprecipitation of by RFP-tagged Grb2 of Sos is reduced in the presence of Grb3-3 in HEK293T cells stably expressing FGFR2 in basal conditions and following FGFR2 stimulation by fibroblast growth factor 9 (FGF9). HEK293T cells were transfected with RFP only (Ctrl) or RFP-tagged Grb2 and Strep -tagged Grb3-3 (Strep Grb3-3) or Strep alone. RFP-tagged proteins were purified and immunoprecipitation of endogenous Sos assessed by Western blot. n = 3. RFP-Grb2 and RFP from same blot: See Supplementary Fig. . MST measurements confirming competition between Grb2 and Grb3-3 for binding to Sos. Unlabelled Sos C-terminal proline-rich peptide Sos-CTail-5 (10 nM-1 mM) was titrated into a fixed concentration (100 nM) of labelled Grb2 ( b ) in the absence or ( c ) with pre-incubation with 200 μM Grb3-3. n = 3 technical replicates and n = 1 biological replicate. d , e A representative western blot and summarising densitometry demonstrating significantly greater Sos cytoplasmic localisation in the presence of exogenous transfected RFP-tagged Grb3-3 (RFP Grb3-3) compared with RFP-tagged Grb2 (RFP Grb2) and RFP control (RFP). Cytoplasmic and plasma membrane fractions were separated by ultracentrifugation followed by immunoblotting for β-actin (cytoplasmic marker), E-cadherin (membrane marker) and Sos. Band intensity was quantified and normalised to β-actin for the cytoplasmic fraction and E-cadherin for the membrane fraction using ImageJ software ( n = 3; * p ≤ 0.05). See Supplementary Fig. for uncropped blots. f A schematic representation of the locations for primer annealing (black arrows) used for the splice-sensitive qRT-PCR assay designed to individually detect Grb2 and Grb3-3 transcripts. The Grb2 forward primer annealed to a sequence unique to exon 4, whereas the Grb3-3 forward primer annealed to a sequence spanning the exon 3-exon 5 junction formed by exon skipping. g A chart showing the relative concentration of Grb3-3 mRNA as a proportion of total Grb3-3 mRNA and Grb2 mRNA in samples derived from 13 matched colorectal cancer and surrounding normal colonic tissue samples, as determined using a splice-sensitive qRT-PCR assay. n = 13.

Article Snippet: Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074).

Techniques: Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Transfection, Purification, Binding Assay, Concentration Assay, Incubation, Control, Clinical Proteomics, Membrane, Marker, Software, Quantitative RT-PCR, Sequencing, Derivative Assay

Journal: Communications Biology

Article Title: The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation

doi: 10.1038/s42003-022-03985-7

Figure Lengend Snippet: a Schematic overview of the combined coverage of the orthogonal approaches used to identify splicing factors that bind to and therefore regulate inclusion of GRB2 exon 4. Three desthiobiotin labelled oligos (Oligo1, Oligo2, Oligo3) spanning GRB2 exon 4 were synthesised and used as bait in RNA pull downs in lysates obtained from HEK293T cells stably transfected with FGFR2. Bound proteins were identified by mass spectrometry. Separately, the bioinformatics programme SpliceAid2 was used to identify splicing factor binding sites in exon 4, in addition to potential cognate splicing factor proteins. b A Venn diagram illustrating the number of splicing factor proteins bound to each oligonucleotide, cross-referenced against splicing factors with predicted binding sites in the exon 4 sequence as determined by SpliceAid2. Shaded number corresponds to 3 splicing factor proteins predicted by SpliceAid2 but not validated by MS. n = 3. c , d Western blot and related densitometry of pulled down samples showing binding of hnRNPC to RNA oligo 1 and reduced binding to mutant RNA oligo 1. n = 1. A dot blot demonstrating the relevant labelled RNA oligos used in the pulldown is shown below the western blot bands. See Supplementary Fig. for uncropped blots. e Relative mRNA expression of hnRNPC in normal colonic tissue and matched tumour tissue. Green circles represent normal patient colonic tissue, orange circles represent the matched tumour tissue. Black lines connect the patient’s normal and tumour tissue samples. n = 22.

Article Snippet: Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074).

Techniques: Stable Transfection, Transfection, Mass Spectrometry, Binding Assay, Sequencing, Western Blot, Mutagenesis, Dot Blot, Expressing

Journal: Communications Biology

Article Title: The Grb2 splice variant, Grb3-3, is a negative regulator of RAS activation

doi: 10.1038/s42003-022-03985-7

Figure Lengend Snippet: a In Grb3-3 expressing cells, hnRNPC binds to the pentameric poly uracil sequence in GRB2 exon 4, resulting in the skipping of exon 4 leading to the production of Grb3-3. Grb3-3 binds to the proline-rich C-terminus of Sos and inhibits Grb2 binding to phosphorylated sites (red dots) on tyrosine residues of activated RTKs and concomitant membrane recruitment. Sos remains in the cytoplasm which prevents RAS activation. This ultimately limits ERK translocation to the nucleus and hence cell proliferation. b In tumour cells, hnRNPC expression is down-regulated, therefore exon 4 is included producing Grb2. Grb2 binds to Sos and to activated RTKs at the cell surface bringing Sos into close proximity of its substrate RAS. Sos activates downstream signalling via RAS. This results in ERK translocation to the nucleus and transcription of multiple gene leading to cell proliferation.

Article Snippet: Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074).

Techniques: Expressing, Sequencing, Binding Assay, Membrane, Activation Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling *

doi: 10.1074/jbc.M116.764431

Figure Lengend Snippet: Recruitment of the GBA motif of GIV to activated EGFR is sufficient to induce G protein activation. A, diagram depicting how Grb2 fused to the GBA motif (aa 1660–1705) of GIV (Grb2-GBA) is recruited from the cytosol to tyrosine phosphorylated EGFR at the plasma membrane upon activation. Grb2-mediated binding to EGFR brings the GBA motif close to membrane-bound Gi3. B and C, HEK293T cells were transfected with plasmids for all the components required BRET-based G protein activity measurements as described for Fig. 3, Grb2-GBA (WT or FA) and EGFR. EGF (50 ng/μl) was added at the indicated time (arrow in B). BRET results (B) are the averages of three or four independent experiments, and the error bars are the S.E. (shown only at 5-s intervals for clarity). Representative immunoblots of the cells used in these experiments are shown in C.

Article Snippet: Grb2-GBA was generated by replacing the mRFP and FKBP sequences of FKBP-GIV-GBA (1660–1705) with the human Grb2 sequence (amplified from Addgene plasmid catalog no. 70383) preceded by a Myc tag (NheI/NruI sites). pcDNA6A-EGFR was obtained from Addgene (catalog no. 42665).

Techniques: Activation Assay, Binding Assay, Transfection, Activity Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling *

doi: 10.1074/jbc.M116.764431

Figure Lengend Snippet: Model depicting the parallelism between the mechanisms of activation of Gαi and Ras by their respective cytoplasmic GEFs, GIV, and SOS, upon RTK stimulation. A, under resting conditions, SOS is primarily located in the cytosol along with Grb2, whereas its substrate G protein Ras is constitutively anchored to the plasma membrane, thereby precluding SOS action. Upon RTK stimulation, Grb2-SOS complexes translocate to the plasma membrane via binding of Grb2 SH2 domains to tyrosine phosphorylated EGFR. This change of localization brings SOS in physical proximity to Ras, thereby promoting G protein activation. B, under resting conditions, GIV is primarily located in the cytosol, whereas its substrate G protein Gαi is constitutively anchored to the plasma membrane, thereby precluding GIV action. Upon RTK stimulation, GIV translocates to the plasma membrane via binding of its SH2-like domain to tyrosine phosphorylated EGFR. This change of localization brings GIV in physical proximity to Gαi, thereby promoting G protein activation.

Article Snippet: Grb2-GBA was generated by replacing the mRFP and FKBP sequences of FKBP-GIV-GBA (1660–1705) with the human Grb2 sequence (amplified from Addgene plasmid catalog no. 70383) preceded by a Myc tag (NheI/NruI sites). pcDNA6A-EGFR was obtained from Addgene (catalog no. 42665).

Techniques: Activation Assay, Binding Assay

Journal: Journal of the American Chemical Society

Article Title: High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1

doi: 10.1021/jacs.9b10710

Figure Lengend Snippet: Grb2 and SOS1 PR domain. (A) Full-length human Grb2 (PDB: 1GRI) contains 217 residues and is composed of nSH3 (blue, residues 1–58), SH2 (white, residues 60–153), and cSH3 (green, residues 156–215) domains. The polar, nonpolar, positively charged, and negatively charged residues are colored green, black/white, blue, and red, respectively, in the amino acid sequence of Grb2 (top). Available NMR/crystal structures of nSH3/cSH3 domains with the binding partners are nSH3–VPPPVPPRRR (PDB: 1AZE) and cSH3–APPPRPPKP (PDB: 2W0Z). (B) Candidate nSH3/cSH3 binding sites on SOS1 are denoted as S1–S10 in the PR domain (residues 1014–1333) amino acid sequence, colored the same way as for Grb2.

Article Snippet: Wild-type human Grb2 nSH3/cSH3 plasmids and selected SOS1 PR peptides were commercially obtained from Biomatik Corporation.

Techniques: Sequencing, Binding Assay

Journal: Journal of the American Chemical Society

Article Title: High-Affinity Interactions of the nSH3/cSH3 Domains of Grb2 with the C-Terminal Proline-Rich Domain of SOS1

doi: 10.1021/jacs.9b10710

Figure Lengend Snippet: The four most probable structural models of the Grb2–SOS1 complex. Grb2–SOS1 interactions are suggested by the strong binding affinity of (A) nSH3–S4/cSH3–S10 (mode 1), (B) nSH3–S5/cHS3–S4 (mode 2), (C) nSH3–S5/cHS3–S10 (mode 3), and (D) nSH3–S9/cHS3–S4 (mode 4). The models of Grb2–SOS1 PR are obtained from replica exchange simulation for 100 ns. Grb2 nSH3, SH2, cSH3, and SOS1 PR domain are colored blue, white, green, and yellow, and the binding peptides (S4, S5, S9, and S10) are shown as red tubes. The heat maps of contact frequency show the interactions between nSH3/cSH3 residues (y-axis) and various SOS1 peptides residues (x-axis) during the simulation. The dark brown color indicates highly frequent contacts. The three main interaction regions of nSH3/cSH3 are around residues 10, 35, and 50 for nSH3 and 170, 195, and 205 for cSH3. These maps show correlation between nSH3–SOS1 and cSH3–SOS1 interactions. For mode 2, the unfolded S5 tends to dissociate from nSH3, resulting in unstable Grb2–SOS1 association.

Article Snippet: Wild-type human Grb2 nSH3/cSH3 plasmids and selected SOS1 PR peptides were commercially obtained from Biomatik Corporation.

Techniques: Binding Assay