Thyroid hormone signaling

我们继续研究TRbeta刺激PI3K的机制以及PI3K信号对甲状腺激素生理效应的影响。我们使用Akt蛋白激酶的PIP3荧光结合域与青色和黄色荧光蛋白偶联，通过荧光共振能量转移（FRET）实时检测活细胞中PIP3的产生。Fret信号被核受体拮抗剂1-850和wortmannin （PI3K的活性位点抑制剂）抑制TRbeta阻断。在环境毒物TCDD（一种100 nM的环境毒物）中，预先暴露10分钟，可以完全阻断PIP3的生成。我们使用免疫沉淀法证明TRbeta在没有配体的情况下与PI3K的调节p85亚基结合，但在甲状腺激素存在时解离。我们还观察到甲状腺激素依赖性Akt蛋白激酶的快速磷酸化和Rac向质膜的募集，证实甲状腺激素刺激pi3k依赖性效应物。最后，我们发现Src家族激酶Lyn是信号复合体的一部分，并通过质谱法确定了其结合位点。其他蛋白质通过两个Src同源（SH2）结构域与p85相互作用，这些结构域识别磷酸酪氨酸。TRbeta而非TRalpha含有一致的sh2结合域，对p85具有高亲和力。将该共识位点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的调节重建，但不会激活由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。已经确定了负责Lyn结合的第二种酪氨酸，它也是PI3K刺激所必需的。我们已经创建了一个转基因小鼠系，其中一个Phe已经取代了Tyr。我们还发现，二恶英（TCDD）通过PI3阻断甲状腺激素信号传导，我们已经证明，丝氨酸/苏氨酸蛋白磷酸酶PP5，我们之前确定为甲状腺激素信号传导下游的Rac效应物（Gentile et al 2006）保护神经元免受暴露于β淀粉样蛋白的氧化应激。因此，甲状腺激素可能有助于人类预防阿尔茨海默病。

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.