Thyroid hormone signaling

我们继续研究TRbeta刺激PI3K的机制以及PI3K信号对甲状腺激素的生理效应的影响。我们使用Akt蛋白激酶的一个荧光PIP3结合域与青色和黄色荧光蛋白偶联，实时检测到活细胞中通过荧光共振能量转移(FRET)产生PIP3。FRET信号被核受体拮抗剂1-850抑制TRbeta和PI3K的活性位点抑制剂wortmannin所阻断。PIP3的产生也被100 NM的环境毒物TCDD预先暴露10分钟完全阻止。我们用免疫沉淀法证明，在没有配体的情况下，TRbeta与PI3K的调节P85亚单位结合，但在甲状腺激素存在的情况下，TRbeta与PI3K的调节P85亚单位结合。我们还观察到了甲状腺激素依赖的Akt蛋白激酶的快速磷酸化和RAC在质膜上的募集，证实了甲状腺激素刺激PI3K依赖的效应分子。最后，我们发现了Src家族的激酶Lyn是信号复合体的一部分，并用质谱仪鉴定了它的结合部位。其他蛋白质通过识别磷酸酪氨酸的两个Src同源(SH2)结构域与P85相互作用。TRbeta但不包含与P85有高亲和力的SH2结合域。将该共有位点的酪氨酸突变为苯丙氨酸可以阻止Kv11.1在CHO细胞中由甲状腺激素调控的重建，但不能阻止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的激活。另一种负责LYN结合的酪氨酸已经被确定，并且也是PI3K刺激所必需的。我们已经创造了一个转基因小鼠品系，其中Phe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，我们已经证明了Ser/Thr蛋白磷酸酶PP5，我们以前发现它是甲状腺激素信号下游的RAC效应因子(Gentile等人，2006)，保护神经元免受淀粉样β蛋白暴露的氧化应激。因此，甲状腺激素可能有助于保护人类免受阿尔茨海默病的侵袭。

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.