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　　全国优秀博士学位论文中英文摘要

CUEDC2通过抑制IKK复合体磷酸化下调NF-kB通路

　　作者姓名：李慧艳

　　论文题目：CUEDC2通过抑制IKK复合体磷酸化下调NF-kB通路

　　作者简介：李慧艳，女，1978年02月出生，2004年09月师从于军事医学科学院张学敏教授，于2007年07月获博士学位。

　　中文摘要

　　CUEDC2(CUE Domain Containing 2)目前是一个功能未知的蛋白质，通过生物信息学分析发现，CUEDC2 包含一个CUE结构域。CUE是一个非常小的中度保守的结构域，大约包含40个氨基酸，预测它能和泛素结合，既能识别单泛素化又能识别多泛素化，目前发现它存在各种真核蛋白质中。最近, 我们实验室发现CUEDC2能够和孕激素受体结合，促进孕激素诱导的受体的泛素化和降解(张佩景等， EMBO J.)，并抑制乳腺癌细胞的生长，这就为CUEDC2在乳腺癌增殖过程中的作用提供了重要的线索。本文为进一步揭示CUEDC2的功能，在应用酵母双杂交技术筛选与CUEDC2相互作用蛋白质的基础上，对其功能尤其是在NF-kB通路中的作用进行了更为深入的研究。

　　IKK (IkBa kinase )是一个很大的蛋白复合体，大小约为700-900kDa，包含两个激酶亚单位 IKKa (IKK1) 和 IKKb (IKK2)，和一个调节亚单位NEMO (NF-kB essential modifier)或IKKg。 IKKa和 IKKb都属于丝/苏氨酸激酶。IKKb是IKK蛋白激酶复合体中起催化作用的主要亚基，在经典通路中IkBs的磷酸化主要是由IKKb介导的。Delhase 等在1999年发现IKKb的激活环区的Ser177和Ser181两个磷酸化位点对于TNFa和IL-1诱导的IKK的激活是最关键的。而被激活的IKKb在其C端的丝氨酸簇可以发生自身磷酸化而降低自身活性。IKK复合体的活性调节涉及多种复杂的机制，尤其是在NF-kB信号转导通路调节过程中起着非常重要的作用。

　　NF-kB是细胞因子诱导的主要转录因子，其激活后可诱导表达一些抗凋亡的基因，对于大多数细胞的存活至关重要。在大多数的静息细胞中，NF-kB蛋白质通过与抑制蛋白家族IkB结合而存在于胞质中。 NF-kB的激活可以通过多种方式。但是大多数情况下都要先激活IKK, 降解 IkB，最后激活NF-kB。NF-kB通路经典的激活方式，比如细胞外刺激如肿瘤坏死因子(TNF)和白介素1(IL-1)可以激活IKK复合体，进而磷酸化IkB蛋白，磷酸化的IkB蛋白再被泛素蛋白酶体降解，使NF-kB得以释放，这一过程使NF-kB蛋白质入核并与特异的DNA序列结合，从而启动NF-kB下游靶基因的转录。由此可见IKK是NF-kB活性调节的关键中心环节。

　　GADD34蛋白(生长抑制和DNA损伤蛋白34)最初被发现在各种应激刺激和DNA损伤，如紫外线照射和蛋白质错误折叠中被诱导表达。现在知道GADD34是蛋白磷酸酶 1(PP1)全酶的一个调节亚基，并且在TGFb I 型受体去磷酸化过程中发挥作用。PP1是一种蛋白磷酸酶，属于丝/苏氨酸磷酸酶，即可以引起丝/苏氨酸的去磷酸化作用。目前已知的蛋白磷酸酶家族包括很多亚型，比如PP2A、PP2B、PP2C等等，这些亚型又包括很多亚基，即调节亚基和催化亚基等。调节亚基主要是识别底物的作用，而真正起去磷酸化作用的是催化亚基，正常情况下，只有调节亚基和催化亚基共同存在时，蛋白磷酸酶才能发挥作用。

　　NF-kB通路激活过程中，首先TNF结合TNFR-I(TNF I型受体)导致TRADD、RIP、TRAF2等招募至受体复合体。其他的招募蛋白，如TRAF2能够招募IKK复合体至TNFR-I复合体,RIP在IKK的激活中起非常重要的作用。过表达这些蛋白能够激活NF-kB信号通路，我们结果发现CUEDC2能够有效的抑制IKKb、 TRADD、 TRAF2以及RIP过表达诱导的NF-kB的转录活性。CUEDC2对IKKb持续激活型 (SS/EE)以及p65过表达引起的NF-kB转录活性没有抑制作用。根据我们的实验结果，我们认为CUEDC2对NF-kB信号通路的抑制作用在 IKK水平。另外，我们证明了CUEDC2能够抑制TNFa刺激的IKKa及IKKb的磷酸化，并且在体外激酶实验中验证了CUEDC2能够抑制IKKb 的激酶活性。这些数据为CUEDC2抑制NF-kB信号通路提供了重要的线索。

　　在细胞因子诱导的NF-kB通路激活中，IkB 激酶 IKKa和IKKb的激活是关键的步骤。因此，对IKK复合体磷酸化的精确调控是NF-kB通路信号传导的重要组成部分。我们通过研究发现一个未知功能的蛋白CUEDC2能够结合IKKa和IKKb，并且能够通过使IKKa和IKKb去磷酸化而抑制NF-kB信号通路。利用siRNA抑制内源CUEDC2 表达导致TNF诱导的NF-kB转录活性增加，过表达CUEDC2增加了细胞对凋亡信号如TNF诱导的凋亡。我们还发现CUEDC2与蛋白磷酸酶 1(PP1)的调节亚基-GADD34(调节亚基生长抑制和DNA损伤蛋白34)存在相互作用。此外，我们发现CUEDC2对NF-kB信号通路的抑制是由IKK-CUEDC2-GADD34-PP1复合体介导的。siRNA抑制内源CUEDC2的实验证明CUEDC2对于IKK-CUEDC2- GADD34复合体的形成是必须的，并且作为招募蛋白使IKK去磷酸化。目前虽然对NF-kB信号通路中的许多激酶已有研究，但磷酸酶在NF-kB信号通路中发挥的作用并不是十分清楚。我们的研究证明PP1c通过CUEDC2招募至其底物IKK，并使IKK发生去磷酸化。总之，我们发现了一个新的抑制 NF-kB信号通路的蛋白质，对CUEDC2在NF-kB信号通路中的作用提供了重要线索。

　　关键词： CUDEC2, GADD34, PP1, IKK, NF-kB

　　Study on the CUE Domain Containing 2 (CUEDC2) negative regulation of NF-kB activity through inhibition of IkBa kinase phosphorylation

　　Li Huiyan

　　ABSTRACT

　　CUEDC2 is revealed by bioinformatics analysis as a CUE domain-contained protein, whose function is unknown. Identified as ubiquitin binding motifs, the CUE domains are small, moderately-conserved domains of about 40 amino acid residues that are found in a variety of eukaryotic proteins. CUE domains interact with both mono- and poly-ubiquitin, and have a dual role in mono- and poly-ubiquitin recognition as well as in facilitating intramolecular monoubiquitination. Recently, we demonstrate that CUEDC2 interacts with progesterone receptor and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. These provide an important insight into the function of CUEDC2 in breast cancer proliferation. In this study, we performed yeast two-hybrid screens to identify CUEDC2-interacting proteins and preliminary analysis on its biological function in NF-κB signaling, to help us understand its mechanisms.

　　A key event leading connecting extracellular stimuli to the activation of NF-kB is the regulation of IKK activity. It is believed that IKK activity is controlled by the opposing action of kinases and phosphatases. IKK is composed of two highly homologous catalytic subunits, IKKa and IKKb, and a regulatory subunit NF-kB essential modulator (NEMO, also called IKKg). After TNF or IL-1 treatment, IKK is recruited to receptor signaling complexes, where the two catalytic subunits, IKKa and IKKb are activated by phosphorylation. In the case of TNF or IL-1 treatment, IKK is activated promptly, but this elevated activity is reduced to basal amounts shortly after stimulation. Despite extensive study of the mechanisms of IKK activation, the molecular machinery involved in the deactivation of IKK remains poorly understood.

　　The transcription factor NF-kB is a critical regulator of diverse cytokine-mediated cellular responses and plays a key role in cell survival through inducing the expression of antiapoptotic genes. In most cells, the NF-kB proteins are normally inactive because they are sequestered in the cytoplasm by a family of inhibitory proteins known as IkBs. Extracellular stimuli, including cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), lead to the activation of the IkB kinase complex (IKK), which phosphorylates and ubiquitinates IkB proteins and promotes their subsequent degradation by the proteasome. This process allows translocation of NF-kB proteins from the cytoplasm to the nucleus, where they activate expression of NF-kB-regulated genes.

　　The GADD34 (growth arrest and DNA damage) protein was initially reported to be induced by various types of cellular stress and DNA damage such as UV irradiation and unfolded proteins. It is now known that GADD34 is a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme. In recent study, the protein phosphatase family includes several isoforms, such as PP2A, PP2B and PP2C. These phosphatases have their regulatory suunits. The regulatory subunit is necessary for the activation of protein phosphatase.

　　Here, we demonstrate that CUE domain containing 2 (CUEDC2), whose function was previously unknown, interacts with IKKa/b and represses NF-κB signaling by reducing IKK phosphorylation. Cytokine-induced activation of the IkB kinases IKKa and IKKb is a key step in the activation of the NF-kB pathway. Thus, precise control of IKK phosphorylation is a crucial component of NF-kB signaling. Specific knockdown of CUEDC2 by siRNA results in increased TNF-induced NF-kB activation, while enforced expression of CUEDC2 sensitizes cells to apoptotic stimuli such as TNF. Interestingly, we also found that CUEDC2 interacts with GADD34, a regulatory subunit of protein phosphatase 1 (PP1). Moreover, we demonstrated that inhibition of NF-kB activation by CUEDC2 is mediated, at least in part, by an IKK-CUEDC2-PP1 complex that exists endogenously. siRNA silencing of CUEDC2 revealed that it is required for formation of this complex, indicating CUEDC2 acts as an adaptor protein that targets IKK for dephosphorylation. Therefore, we have uncovered a potent inhibitor of NF-kB signaling that controls IKK activity, providing important insight into the function of CUEDC2 in NF-kB signaling.

　　Key words: CUDEC2, GADD34, PP1, IKK, NF-kB

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