Taira Junichi, Kida Yutaka, Inatomi Kohei, Komatsu Hideyuki, Higashimoto Yuichiro, Sakamoto Hiroshi
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.
J Biochem. 2017 Aug 1;162(2):113-122. doi: 10.1093/jb/mvx007.
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.
生长因子受体结合蛋白14（Grb14）是胰岛素受体（IR）的负调节因子，参与胰岛素信号的负反馈机制。Grb14与IR结合，并通过普列克底物蛋白同源结构域和Src同源结构域2之间（BPS）的结构域抑制其酪氨酸激酶活性。我们之前报道，糖原合酶激酶-3（GSK-3）的药理学抑制和敲低促进了胰岛素诱导的人Grb14（hGrb14）与IR的复合物形成，提示GSK-3抑制hGrb14募集到IR。本研究进一步探究hGrb14 BPS结构域中丝氨酸残基的功能性磷酸化，将其鉴定为推测的GSK-3作用靶点，以验证GSK-3对hGrb14-IR复合物形成的影响。使用基序衍生肽进行的体外激酶分析表明，位于BPS结构域N端（Ser358、Ser362和Ser366）和C端（Ser419和Ser423）区域的丝氨酸残基被GSK-3磷酸化。免疫共沉淀和酵母双杂交（Y2H）实验表明，在Ser358、Ser362和Ser366上通过基因手段引入的负电荷抑制了hGrb14与IR的结合。表面等离子体共振实验得出重组hGrb14与IRβ亚基相互作用的解离常数（Kd）值为8 nM，在用谷氨酸残基替换Ser358、Ser362和Ser366后，这种亲和力丧失。然而，单独使用BPS结构域进行的Y2H实验未显示因相同突变而产生的任何差异。因此，很明显，除其他结构域外，BPS结构域的N端在通过磷酸化调节hGrb14-IR复合物形成中起重要作用。
