BPS结构域n端丝氨酸簇残基的磷酸化负向调控人Grb14与胰岛素受体复合物的形成

J. Taira, Yutaka Kida, Kohei Inatomi, H. Komatsu, Y. Higashimoto, H. Sakamoto
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引用次数: 4

摘要

生长因子受体结合蛋白14 (Growth factor receptor-bound protein 14, Grb14)是胰岛素受体(insulin receptor, IR)的负调控因子,参与胰岛素信号的负反馈机制。Grb14通过pleckstrin同源性和Src同源性-2 (BPS)结构域与IR结合并抑制其酪氨酸激酶活性。我们之前报道了糖原合成酶激酶3 (GSK-3)的药理抑制和敲低促进胰岛素诱导的人Grb14 (hGrb14)和IR复合物的形成,这表明GSK-3抑制了hGrb14向IR的募集。本研究进一步研究了hGrb14 BPS结构域中丝氨酸残基的功能性磷酸化,这些丝氨酸残基被确定为GSK-3的推测靶点,以验证GSK-3对hGrb14- ir复合物形成的影响。利用基元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)的丝氨酸残基被GSK-3磷酸化。共免疫沉淀和酵母双杂交(Y2H)实验表明,在Ser358、Ser362和Ser366上遗传引入的负电荷抑制了hGrb14与IR的关联。表面等离子体共振实验显示,重组hGrb14与IR β-亚基相互作用的Kd值为8 nM,在用谷氨酸残基取代Ser358、Ser362和Ser366后,这种亲和力丧失。单独使用BPS结构域的Y2H实验;然而,由于相同的突变,没有表现出任何差异。因此很明显,除了其他结构域外,BPS结构域的n端在通过磷酸化调控hGrb14-IR复合物形成中起着重要作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Phosphorylation of clustered serine residues in the N-terminus of BPS domain negatively regulates formation of the complex between human Grb14 and insulin receptor
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.
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