title: Functional importance of amino-terminal domain of Shc for interaction with insulin and epidermal growth factor receptors in phosphorylation-independent manner.
abstract: Shc has two distinct domains, amino-terminal and SH2 domain, which can interact with activated growth factor receptors. Shc interacts with insulin receptor via Shc-amino-terminal (N) domain, whereas Shc associates with epidermal growth factor (EGF) receptor through both Shc-N and -SH2 domains. In accordance with the different functional roles between insulin and EGF receptors, EGF stimulated tyrosine phosphorylation of Shc faster than insulin. To clarify the functional importance of three distinct Shc domains on insulin and EGF signaling, we microinjected glutathione S-transferase (GST) fusion proteins containing the amino terminus plus collagen homology domain (NCH), collagen homology domain (CH), and Src homology 2 domain (SH2) into Rat1 fibroblasts expressing insulin receptors (HIRc). Bromodeoxyuridine (BrdUrd) incorporation into newly synthesized DNA was subsequently studied to assess the importance of the three distinct domains of Shc. Microinjection of the NCH-GST fusion protein inhibited BrdUrd incorporation induced by both EGF and insulin, whereas microinjection of the SH2-GST fusion protein inhibited EGF, but not insulin stimulation of DNA synthesis. Neither EGF- nor insulin-induced BrdUrd incorporation was inhibited by the CH-GST fusion protein. Following EGF or insulin stimulation, Shc is phosphorylated on single Tyr-317 residue serving as a docking site for Grb2. Microinjection of Shc-N+CH GST fusion protein with Tyr-317 --> Phe replacement (Y317F) also inhibited insulin stimulation of DNA synthesis. Next, we stably overexpressed wild-type Shc or Y317F mutant Shc into HIRc cells. Insulin-induced tyrosine phosphorylation of IRS-1 was compared among the transfected cell lines, since IRS-1 and Shc could competitively interact with insulin receptor. Insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in both WT-Shc and Y317F-Shc cells compared with that in HIRc cells. Furthermore, overexpression of the Shc-SH2 domain or Shc-N+CH domain with Y317F mutation interfered with EGF-stimulated endogenous Shc phosphorylation. These results suggest that the amino terminus domain of Shc is functionally important in insulin- and EGF-induced cell cycle progression and that the phosphorylation of Shc Tyr-317 residue is independent of Shc interaction with these receptors.
authors.affiliation: First Department of Medicine, Toyama Medical and Pharmaceutical University, Toyama, 930-01 Japan.
authors.forename: T
authors.initials: T
authors.lastname: Sasaoka
authors.forename: H
authors.initials: H
authors.lastname: Ishihara
authors.forename: T
authors.initials: T
authors.lastname: Sawa
authors.forename: M
authors.initials: M
authors.lastname: Ishiki
authors.forename: H
authors.initials: H
authors.lastname: Morioka
authors.forename: T
authors.initials: T
authors.lastname: Imamura
authors.forename: I
authors.initials: I
authors.lastname: Usui
authors.forename: Y
authors.initials: Y
authors.lastname: Takata
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D004815:Epidermal Growth Factor / Q000502:physiology; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013329:Structure-Activity Relationship; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin