title: Phosphorylation of Grb14 BPS domain by GSK-3 correlates with complex forming of Grb14 and insulin receptor.
abstract: Growth factor receptor-bound protein 14 (Grb14) interacts with insulin receptor (IR) through the between PH and SH2 (BPS) domain. Grb14-IR complex formation is initiated by insulin stimulation, and the binding event results in the inhibition of insulin signalling. Thus, Grb14 is regarded as an endogenous suppressor of insulin signal transduction; however, there are no studies describing the mechanism whereby Grb14-IR complex formation is suppressed in the absence of insulin stimulation. In the present study, multiple phosphorylation motifs for glycogen synthase kinase 3 (GSK-3) were identified within the Grb14 BPS domain (Ser(358), Ser(362) and Ser(366) of human Grb14). Pharmacological inhibition as well as knockdown of GSK-3 facilitated complex formation between Grb14 and IR, implicating GSK-3 activity in regulating Grb14-IR binding. In situ proximity ligation assay and in vitro kinase assays of phosphopeptides suggested that serine residues in the BPS domain would be substrates for GSK-3. The kinase assays also indicated phosphoserine 370 (in human Grb14) was required for the phosphorylation of Ser(358), Ser(362) and Ser(366) by GSK-3. Grb14-IR binding was also facilitated by replacement of the serines with Ala. We also observed that Ser(366) of endogenous Grb14 in Hep G2 cell was phosphorylated and the phosphorylation was influenced by treatments with insulin, as well as the GSK-3 inhibitor.
authors.affiliation: Department of Chemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan taira_junichi@med.kurume-u.ac.jp.
authors.forename: Junichi
authors.initials: J
authors.lastname: Taira
authors.affiliation: Department of Chemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Higashimoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D055785:Gene Knockdown Techniques; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C099655:GRB14 protein, human; D012694:Serine; D011972:Receptor, Insulin; D038362:Glycogen Synthase Kinase 3
keywords: CK2; GSK-3; Grb14; IR; insulin signalling