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PI3K刺激的机理，以及PI3K信号传导对甲状腺激素的生理作用的后果。我们已经从Akt蛋白激酶与青色和黄色荧光蛋白的Akt蛋白激酶中使用了荧光PIP3结合结构域，以实时在活细胞中通过荧光共振能传递（FRET）检测PIP3的产生。通过用核受体拮抗剂（1-850）和PI3K的活性部位抑制剂Wortmannin抑制TRBETA，将FRET信号阻止。 PIP3的产生也完全阻止了10分钟的前环境有毒物质100 nm TCDD。我们已经使用免疫沉淀表明，在没有配体的情况下，Trbeta与PI3K的调节p85亚基相关联，但在存在甲状腺激素的情况下会解离。我们还观察到Akt蛋白激酶的甲状腺激素依赖性依赖性磷酸化的快速磷酸化以及RAC募集到质膜上，证实甲状腺激素刺激了PI3K依赖性效应子。最后，我们发现SRC家族激酶Lyn是信号复合物的一部分，并用质谱鉴定其结合位点。其他蛋白质通过两个识别磷酸酪氨酸的SRC同源性（SH2）结构域与p85相互作用。 TRBETA但不包含一个共识SH2结合域，对P85具有高亲和力。将酪氨酸在该共识位点突变至苯丙氨酸，可防止KV11.1 CHO细胞中甲状腺激素对甲状腺激素调节的重构，而不是激活由甲状腺激素受体受体反应元件（TRE）驱动的荧光素酶基因的异源转录。已经确定了负责LYN结合的第二个酪氨酸，也需要PI3K刺激。我们创建了一个转基因小鼠系，其中PHE已被取代为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.因此，甲状腺激素可能有助于人类免受阿尔茨海默氏病的保护。