title: Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.
abstract: While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.
authors.affiliation: Raymond and Beverly Sackler Laboratory of Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Jadwin
authors.affiliation: Richard D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Dongmyung
authors.initials: D
authors.lastname: Oh
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States.|Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Curran
authors.affiliation: Raymond and Beverly Sackler Laboratory of Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Mari
authors.initials: M
authors.lastname: Ogiue-Ikeda
authors.affiliation: Raymond and Beverly Sackler Laboratory of Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Lin
authors.initials: L
authors.lastname: Jia
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States.|Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States.
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
authors.affiliation: Raymond and Beverly Sackler Laboratory of Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
authors.affiliation: Richard D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Ji
authors.initials: J
authors.lastname: Yu
authors.affiliation: Raymond and Beverly Sackler Laboratory of Molecular Medicine, Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States.|Richard D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, United States.
authors.forename: Bruce J
authors.identifier: http://orcid.org/0000-0002-4580-3187
authors.initials: BJ
authors.lastname: Mayer
mesh_terms: D026481:Blotting, Far-Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry; D061848:Optical Imaging; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases
keywords: SH2 domain; biochemistry; biophysics; human; receptor tyrosine kinase; signal transduction; structural biology