Functional analysis of a novel signaling cascade activated by thyroid hormone: Role of PI3 kinase→PKB→mTOR→ZAKI-4αactivation by thyroid hormone

甲状腺激素（TH）是正常发育，生长和代谢所必需的。其作用是通过三碘甲腺原氨酸（T_3）介导的，T_3是TH受体（TRs）的配体。在TH正调控基因的经典模型中，TR首先作为异二聚体或同二聚体结合在位于靶基因启动子区的TH应答元件（TRE）上，在那里它与辅阻遏物相互作用。在配体结合后，TR同源二聚体解离，有利于与类维生素A-X受体（RXR）形成异源二聚体，导致辅阻遏物的释放和辅激活物的募集。这种新的复合物吸引了大量的蛋白质，这些蛋白质使RNA聚合酶II参与靶基因的转录。除了经典的TH作用的“核”模式之外，我们证明TH还通过非基因组机制快速激活PI 3 K-Akt信号通路。结果表明：（1）TH处理可诱导TH磷酸化和活化， 关于我们 e丝氨酸/苏氨酸激酶Akt，哺乳动物雷帕霉素靶蛋白（mTOR）及其底物p70 α<S6K>和2）这些蛋白的磷酸化被PI 3 K抑制剂LY 294002和渥曼青霉素以及PI 3 K的显性负调控亚基（p85 α）消除。这种TH作用非常迅速，不依赖于蛋白质合成，表明它不涉及TR的典型核作用。值得注意的是，这种快速TH作用通过将显性负性TRP（TRG 345 R）引入表达野生型TRβ的细胞中而被废除，表明TRβ的参与。在人皮肤成纤维细胞中，TRβ与PI 3 K的调节亚基p85 a直接相互作用。这种相互作用不依赖于配体结合，而PI 3 K仅在TH存在下才能被激活。我们和其他人确定了几个基因的控制下，这一行动，包括ZAKI-4a，缺氧诱导因子-la，葡萄糖转运蛋白1，磷酸果糖激酶，和单羧酸转运蛋白4。ZAKI-4基因最初被我们发现是位于人类6号染色体上的一个甲状腺激素（TH）反应基因。我们后来发现，三个转录本，α，β1和β2，由单个ZAKI-4基因通过差异剪接产生。β1和β2亚型编码相同的蛋白产物ZAKI-4β，其与ZAKI-4a共享共同的羧基末端区域。ZAKI-4a和ZAKI-4 β都属于一个结构相关的小蛋白家族，它们与钙调磷酸酶（蛋白磷酸酶2B）结合并下调其活性。在人类和啮齿动物中证实，仅同种型的表达上调。通过TH。由于钙调神经磷酸酶参与神经元的可塑性，TH可能通过调节钙调神经磷酸酶的活性而在神经元的可塑性中发挥作用。少

Thyroid hormone (TH) is essential for normal development, growth and metabolism. Its effects are mediated through triiodothyronine (T_3), which acts as a ligand for the TH receptors (TRs). In the classical model of genes positively regulated by TH, the TR first binds as a heterodimer or homodimer on TH response elements (TRE) located in the promoter regions of target genes, where it interacts with corepressors. Upon ligand binding, the TR homodimers are dissociated in favor of heterodimer formation with the retinoid-X receptor (RXR), resulting in release of the corepressors and recruitment of coactivators. This new complex attracts a large number of proteins which engage the RNA polymerase II in the transcription of the targeted gene. In addition to the classical, "nuclear" mode of TH action, we demonstrated that TH also activates PI3K-Akt signaling pathway rapidly through a non-genomic mechanism. It was found that 1) TH treatment induced sequential phosphorylation and activation of th … More e serine/threonine kinase Akt, the mammalian target of rapamycin (mTOR) and its substrate p70^<S6K> and 2) phosphorylation of these proteins was abrogated by both PI3K inhibitors, LY294002 and wortmannin, and by a dominant negative regulatory subunit of PI3K (p85a). This TH action is very rapid and independent of protein synthesis, suggesting that it does not involve the typical nuclear action of TR. It is of note that this rapid TH action was abrogated by the introduction of a dominant negative TRP (TRG345R) into the cells expressing the wild type TRβ, indicating the participation of TRβ. It was demonstrated that TRβ directly interacts with the regulatory subunit of PI3K, p85a in human skin fibroblasts. The interaction is independent of ligand binding, while PI3K can be activated only in the presence of TH. We and others identified several genes under the control of this action, including ZAKI-4a, hypoxia-inducible factor-la, glucose transporter 1, phosphofructokinase, and the monocarboxylate transporter 4. ZAKI-4 gene was originally identified by us as a thyroid hormone (TH) responsive gene which locates on human chromosome 6. We later found that three transcripts, a, β1 and β2, were generated by the single ZAKI-4 gene through differential splicing. The β1 and β2 isoforms encode the same protein product ZAKI-4β, which shares the common carboxyl terminal region with ZAKI-4a. Both ZAKI-4a and β belong to a family of small structurally related proteins which bind to, and down-regulate the activity of calcineurin (protein phosphatase 2B). It was demonstrated in human and rodents that the expression of only a isoform is upregulated. by TH. Since it is known that calcineurin is involved in neuronal plasticity, TH may play the role in neuronal plasticity through regulating calcineurin activity. Less