Thyroid hormone signaling

甲状腺激素信号传导

• 批准号: 8149107
• 负责人: David Armstrong
• 金额: $ 51.28万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149107
• 关键词: Signal Transduction; Thyroid Hormones

We have continued to investigate the mechanism of PI3K stimulation by TRbeta and the consequences of PI3K signaling for the physiological effects of thyroid hormone. We have used a fluorescent PIP3 binding domain from the Akt protein kinase coupled with cyan and yellow fluorecent proteins to detect PIP3 production by fluorescence resonance energy transfer (FRET) in live cells in real time. Fret signals are blocked by inhibition of TRbeta with the nuclear receptor antagonist,1-850, and by wortmannin, an active site inhibitor of PI3K. PIP3 production is also blocked completely by 10 min preexposure to 100 nM TCDD, an environmental toxicant. We have used immunoprecipitation to show that TRbeta associates with the regulatory p85 subunit of PI3K in the absence of ligand but dissociates in the presence of thyroid hormone. We have also observed rapid thyroid hormone-dependent phosphorylation of the Akt protein kinase and recruitment of Rac to the plasma membrane confirming that thyroid hormone stimulates PI3K-dependent effectors. Finally we have discovered that the Src family kinase, Lyn, is part of the signaling complex and identified its binding site with mass spectrometry. Other proteins interact with p85 through two Src homology (SH2) domains which recognize phosphotyrosine. TRbeta but not TRalpha contains a consensus SH2-binding domain with high affinity for p85. Mutating the tyrosine in that consensus site to phenylalanine prevents reconstitution of Kv11.1 regulation by thyroid hormone in CHO cells but not activation of a heterologous transcription of a luciferase gene driven by a thyroid hormone receptor response element (TRE). A second tyrosine that is responsible for Lyn binding has been identified and is also required for PI3K stimulation. We have created a transgenic mouse line in which a Phe has been substituted for the Tyr. We have also discovered that dioxin (TCDD) blocks thyroid hormone signaling through PI3, and we have shown that the Ser/Thr protein phosphatase, PP5, which we previously identified as a Rac effector downstream of thyroid hormone signaling (Gentile et al 2006) protects neurons from the oxidative stress of exposure to amyloid beta. Thus thyroid hormone could contribute to human protectin from Alzheimer's disease.

我们继续研究TRbeta刺激PI 3 K的机制以及PI 3 K信号传导对甲状腺激素生理效应的影响。我们已经使用来自Akt蛋白激酶的荧光PIP 3结合结构域与青色和黄色荧光蛋白偶联，通过荧光共振能量转移（FRET）在活细胞中真实的时间检测PIP 3的产生。通过用核受体拮抗剂1-850抑制TRbeta和通过PI 3 K的活性位点抑制剂渥曼青霉素阻断Fret信号。PIP 3的生产也完全阻断了10分钟的预暴露于100 nM的TCDD，一种环境毒物。我们已经使用免疫沉淀显示，TRbeta协会与PI 3 K的调节p85亚基在配体的情况下，但在甲状腺激素的存在下解离。我们还观察到Akt蛋白激酶的快速甲状腺激素依赖性磷酸化和Rac向质膜的募集，证实甲状腺激素刺激PI 3 K依赖性效应物。最后，我们发现Src家族激酶林恩是信号复合物的一部分，并通过质谱鉴定了其结合位点。其他蛋白质通过识别磷酸酪氨酸的两个Src同源（SH 2）结构域与p85相互作用。TRbeta而不是TRalpha含有对p85具有高亲和力的共有SH 2结合结构域。将该共有位点中的酪氨酸突变为苯丙氨酸可阻止CHO细胞中甲状腺激素对Kv11.1调节的重建，但不会激活甲状腺激素受体反应元件（TRE）驱动的荧光素酶基因的异源转录。已经鉴定了负责林恩结合的第二种酪氨酸，并且也是PI 3 K刺激所需的。我们已经创建了一个转基因小鼠系，其中Phe已取代Tyr。我们还发现二恶英（TCDD）通过PI 3阻断甲状腺激素信号传导，并且我们已经表明，我们先前确定为甲状腺激素信号传导下游的Rac效应物的Ser/Thr蛋白磷酸酶PP 5（Gentile et al 2006）保护神经元免受暴露于淀粉样蛋白β的氧化应激。因此，甲状腺激素可能有助于保护人类免受阿尔茨海默病。