Taira Junichi, Higashimoto Yuichiro
Department of Chemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan
Department of Chemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
J Biochem. 2014 Jun;155(6):353-60. doi: 10.1093/jb/mvu011. Epub 2014 Feb 17.
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.
生长因子受体结合蛋白14（Grb14）通过PH和SH2之间的结构域（BPS）与胰岛素受体（IR）相互作用。Grb14-IR复合物的形成由胰岛素刺激引发，这种结合事件会导致胰岛素信号传导受到抑制。因此，Grb14被视为胰岛素信号转导的内源性抑制剂；然而，尚无研究描述在没有胰岛素刺激的情况下Grb14-IR复合物形成被抑制的机制。在本研究中，在Grb14的BPS结构域（人Grb14的Ser(358)、Ser(362)和Ser(366)）内鉴定出糖原合酶激酶3（GSK-3）的多个磷酸化基序。GSK-3的药理学抑制以及敲低促进了Grb14与IR之间的复合物形成，这表明GSK-3活性在调节Grb14-IR结合中发挥作用。磷酸化肽段的原位邻近连接分析和体外激酶分析表明，BPS结构域中的丝氨酸残基将是GSK-3的底物。激酶分析还表明，磷酸化的丝氨酸370（人Grb14中）是GSK-3对Ser(358)、Ser(362)和Ser(366)进行磷酸化所必需的。用丙氨酸取代丝氨酸也促进了Grb14-IR的结合。我们还观察到，Hep G2细胞中内源性Grb14的Ser(366)被磷酸化，并且这种磷酸化受到胰岛素以及GSK-3抑制剂处理的影响。
