Journal: The Journal of Experimental Medicine

Article Title: VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd

doi: 10.1084/jem.20111343

Figure Lengend Snippet: TSAd interactions with VEGFR2 and Src. (A) Immobilized peptides corresponding to tyrosine phosphorylation sites in VEGFR2 and TSAd were probed using TSAd GST-SH2 (left) or c-Src GST-SH2 (right) domain fusion proteins. The phosphotyrosine position in VEGFR2 or TSAd is indicated to the left and the peptide sequence to the right in the figure. The figure shows representative results from three independent experiments. Line indicates the splice of two parts of the same filter aligned to one strip. (B) Immobilized peptides corresponding to proline-rich stretches in TSAd were probed with c-Src GST-SH3 domain fusion protein. Peptide sequences are shown to the right. The figure shows representative results from three independent experiments. Line indicates the splice of two parts of the same filter aligned to one strip. (C) Schematic representation of VEGFR2, TSAd, and c-Src molecular interactions.

Article Snippet: Peptide array membranes were probed with GST-tagged SH2 domains (2–40 μg/ml) and blotted with mouse anti-GST antibody (Santa Cruz Biotechnology, Inc.) and anti–mouse HRP secondary antibody followed by chemiluminescent detection.

Techniques: Phospho-proteomics, Sequencing, Stripping Membranes

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Phosphorylation of Tyr 1510 of IQGAP1 creates a docking site for SH2 domains. ( A ). A peptide array containing 124 different GST-tagged recombinant SH2 protein domains, each spotted in duplicate, was generated as detailed in Materials and Methods . GST alone was spotted on the array as the negative control. Biotinylated peptides comprising residues 1502–1518 of IQGAP1 with phosphorylated Tyr 1510 (pTyr 1510 ) were labeled with fluorescent streptavidin and incubated with the array for 16 h at 4 °C. After washing, fluorescence from the bound peptides (green dots) was detected. The red and orange circles delineate the duplicates for the SH2 domains of Abl1 and Abl2, respectively. ( B ). Similar analysis was conducted with the same IQGAP1 peptide, except Tyr 1510 was not phosphorylated. ( C ). The peptide array was probed with anti-GST antibody to show the positions and loading of the GST-SH2 domains and control GST.

Article Snippet: To generate the array, the SH2 domains were cloned into the pGEX-4T1 vector and synthesized by the Biomatik Corporation (Kitchener, Canada).

Techniques: Phospho-proteomics, Peptide Microarray, Recombinant, Generated, Negative Control, Labeling, Incubation, Fluorescence, Control

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: The SH2 domains of Abl1 and Abl2 bind directly to tyrosine-phosphorylated IQGAP1. ( A ). Quantification of the fluorescence intensity of the IQGAP1 peptides with unphosphorylated (Tyr 1510 , pale green bars) or phosphorylated (pTyr 1510 , dark green bars) Tyr 1510 bound to the SH2 domains of Abl1 or Abl2 on the SH2 array. Binding to GST is the control. Data represent the mean fluorescence from two duplicates. a.u., arbitrary units. ( B ). H1993 cells were incubated with 100 nM crizotinib (criz, + ) or vehicle DMSO (−). After 24 h, cells were lysed and equal amounts of protein lysate were incubated with the purified GST-SH2 domains of Abl1 or Abl2 bound to glutathione-Sepharose. Control pull-downs were carried out with GST-glutathione-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by Western blotting. The membrane was probed with anti-IQGAP1 (IQ1) and anti-GST antibodies. Input designates unfractionated cell lysates. Both panels are from the same membrane. Blots are representative of two independent experiments. The full blots of the two replicates are shown in . ( C ). The IQGAP1 bands observed after pull-down were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of IQGAP1 in DMSO-treated cells was set as 1. Data are presented as the means of two independent replicates. ( D ). Purified GST-IQGAP1 (IQ1) on glutathione-Sepharose was incubated with purified active MET in the presence (+ATP) or absence (−ATP) of ATP. After washing, beads were incubated with 2 μg of purified Abl1 (left panel) or Abl2 (right panel). Control pull-downs were carried out with GST-Sepharose. After washing, proteins attached to the beads were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting. The membrane was probed with anti-IQGAP1 (IQ1), anti-phosphotyrosine (pTyr), and anti-Abl1 or anti-Abl2 antibodies. The overlap between IQGAP1 (red) and pTyr (green) signals is visible in the merged image (yellow). Input designates pure Abl1 or Abl2 not subjected to pull-down. The blots are representative of three independent experiments. The full blots of the three replicates are shown in . ( E ). The Abl1 and Abl2 bands observed after pull-down by GST-IQGAP1 were quantified using Image Studio 2.0 (LI-COR Biosciences). The intensity of Abl1 and Abl2 signals observed with non-phosphorylated IQGAP1 (−ATP) was set as 1. Data are the means ± SD of three independent experiments. Statistical analyses were performed with unpaired t -tests (*, p ≤ 0.05).

Article Snippet: To generate the array, the SH2 domains were cloned into the pGEX-4T1 vector and synthesized by the Biomatik Corporation (Kitchener, Canada).

Techniques: Fluorescence, Binding Assay, Control, Incubation, Purification, Western Blot, Membrane, SDS Page

Journal: Cells

Article Title: IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins

doi: 10.3390/cells12030483

Figure Lengend Snippet: Model of IQGAP1 in receptor tyrosine kinase signaling. ( A ). Schematic of IQGAP1 that highlights known post-translational modifications. The five domains are CHD (calponin-homology domain), WW, IQ, GRD (GAP-related domain), and RGCT (RasGAP_C-terminus). Post-translational modifications that have been characterized on IQGAP1 are shown below the modified amino acid. The identified domain to which receptor tyrosine kinases bind is depicted above IQGAP1. Phosphorylation of IQGAP1 on Tyr 1510 , leading to the recruitment of SH2 domains, is also shown. ( B ). Model depicting the identified modes of action of IQGAP1 in receptor tyrosine kinase signaling. Upper panel: constitutive scaffolding by IQGAP1. IQGAP1 binds constitutively to both the growth factor receptor and a downstream signaling protein. (i) Ligand binding activates the receptor. (ii) Scaffolding by IQGAP1 facilitates activation of the signaling protein by the activated receptor, initiating downstream signaling. (iii) Signal transduction from the activated receptor to the effector protein does not occur in the absence of scaffolding by IQGAP1. The mode of action depicted here is for the EGF receptor (EGFR) and B-Raf kinase in activation of the MAPK cascade [ , ]. Lower panel: phosphotyrosine-dependent scaffolding by IQGAP1. (i) Phosphorylation of tyrosine on IQGAP1 by an activated receptor tyrosine kinase (ii) initiates recruitment of selected SH2-containing proteins. The mechanism illustrated here is for the MET receptor tyrosine kinase and the SH2-containing proteins Abl1 and Abl2. In this example, IQGAP1 (iii) impairs MET activation and signaling and (iv) decreases HGF-stimulated signaling of Abl to the adaptor protein CrkL. Therefore, IQGAP1 functions as a rheostat regulating the flux of signaling between activated MET receptors and SH2-containing signaling proteins. (v) Recruitment of SH2-containing proteins does not occur in the absence of receptor tyrosine kinase-catalyzed phosphorylation of IQGAP1. Abbreviations: EGF, epidermal growth factor; EGFR, EGF receptor; HER2, human epidermal growth factor receptor 2; HGF, hepatocyte growth factor; IR, insulin receptor; P, phosphate; pS, serine phosphorylation; pY, tyrosine phosphorylation; SUMO, SUMOylation; Ub, ubiquitination.

Article Snippet: To generate the array, the SH2 domains were cloned into the pGEX-4T1 vector and synthesized by the Biomatik Corporation (Kitchener, Canada).

Techniques: Modification, Phospho-proteomics, Scaffolding, Ligand Binding Assay, Activation Assay, Transduction, Ubiquitin Proteomics