title: Phosphorylation of clustered serine residues in the N-terminus of BPS domain negatively regulates formation of the complex between human Grb14 and insulin receptor.
abstract: Growth factor receptor-bound protein 14 (Grb14) is a negative regulator of insulin receptor (IR) and is involved in a negative feedback mechanism of insulin signaling. Grb14 associates with IR and inhibits its tyrosine kinase activity through the between pleckstrin homology and Src homology-2 (BPS) domain. We previously reported that the pharmacological inhibition and knockdown of glycogen synthase kinase-3 (GSK-3) facilitates the insulin-induced complex formation of human Grb14 (hGrb14) and IR, suggesting that GSK-3 suppresses hGrb14 recruitment to IR. This study further investigated a functional phosphorylation of the serine residues in hGrb14 BPS domain, identified as putative GSK-3 targets to verify an effect of GSK-3 on the hGrb14-IR complex formation. In vitro kinase assay using the motif-derived peptides showed that the serine residues located in N-terminal (Ser358, Ser362 and Ser366) and C-terminal (Ser419 and Ser423) regions of the BPS domain were phosphorylated by GSK-3. Co-immunoprecipitation and yeast two-hybrid (Y2H) experiments suggested that the negative charges genetically introduced on the Ser358, Ser362 and Ser366 suppressed the association of hGrb14 to IR. Surface plasmon resonance experiment gave Kd values of 8 nM for recombinant hGrb14 with respect to the interaction with IR β-subunit, and this affinity was lost after the replacements of the Ser358, Ser362 and Ser366 with glutamic acid residues. Y2H experiment with the BPS domain alone; however, did not show any difference owing to the same mutations. It is therefore evident that the N-terminus of the BPS domain plays an important role in the regulation of hGrb14-IR complex formation through phosphorylation, in addition to other domains.
authors.affiliation: Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.|Department of Chemistry.
authors.forename: Junichi
authors.initials: J
authors.lastname: Taira
authors.affiliation: Division of Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kida
authors.affiliation: Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Inatomi
authors.affiliation: Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Komatsu
authors.affiliation: Department of Chemistry.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Higashimoto
authors.affiliation: Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, Fukuoka 820-8502, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakamoto
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006801:Humans; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C099655:GRB14 protein, human; D012694:Serine; D011972:Receptor, Insulin
keywords: BPS domain; GSK-3; Grb14; insulin receptor