Nouaille Sébastien, Blanquart Christophe, Zilberfarb Vladimir, Boute Nicolas, Perdereau Dominique, Roix Johan, Burnol Anne-Françoise, Issad Tarik
Département de Biologie Cellulaire, Institut Cochin, Université Paris Descartes, 22 Rue Méchain, UMRCNRS 8104, INSERM U567, Paris 75014, France.
EMBO Rep. 2006 May;7(5):512-8. doi: 10.1038/sj.embor.7400668. Epub 2006 Mar 31.
The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.
利用生物发光共振能量转移（BRET）技术，在活的人胚肾细胞中实时监测胰岛素受体（IR）与Grb14的相互作用动态。我们观察到胰岛素能快速且呈剂量依赖性地刺激这种相互作用。我们还观察到，Grb14能显著降低胰岛素诱导的IR与蛋白酪氨酸磷酸酶1B（PTP1B）之间的BRET，这表明Grb14在活细胞中调节这种相互作用。使用针对IR磷酸化酪氨酸的位点特异性抗体，我们发现Grb14可保护激酶环的三个酪氨酸不被PTP1B去磷酸化，同时促进酪氨酸972的去磷酸化。这导致IRS-1与IR的结合减少，细胞外信号调节激酶途径的激活降低。在人肝源性HuH7细胞中增加Grb14的表达似乎也能特异性降低Y972的磷酸化。因此，我们的研究表明，Grb14可能通过以位点特异性方式控制IR的酪氨酸去磷酸化来调节其信号传导。
