胞内接头蛋白TRAFs家族

肿瘤坏死因子（tumor necrosis factor,TNF）受体相关因子（receptor-associated factor,TRAF）家族是一类胞内接头蛋白,能介导TNF受体和Toll-like／IL-1受体超家族成员与多种下游信号通路包括NF—κB（nuclear factor κB）和JNK（Jun N-terminal kinase）的信号传导。TRAF蛋白家族参与调控细胞增殖、分化乃至凋亡等生理过程。由于其在信号通路中的关键作用,TRAFS蛋白的失调与多种疾病的发生相关。本文结合最新的研究进展对TRAFs蛋白家族在信号传导通路中的地位进行介绍,并探讨了TRAFs及其相关蛋白在临床上可能的应用前景。     

TNF-α信号传导通路的分子机理

肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）是一种具有多效生物学效应的细胞因子.TNF的生物学效应都是通过细胞表面的2种TNF受体（TNFR）引发,其信号传导通路主要包括caspase家族介导的细胞凋亡、衔接蛋白TRAF介导的转录因子NF-κB和JNK蛋白激酶的活化.TNFR1和TNFR2的生物学功能不是独立的,许多生物学活性由二者共同完成.3条信号传导通路之间及各通路内部含有各种调节机制,使TNF的各种生物学功能协调发挥出来.本文评述了3条信号传导通路最新进展、关键激酶的研究状况及其在整个信号网络中的作用机理,如IKK的激活以及重要的信号转导分子RIP、TRAF2、TRUSS的结构、相互作用的方式等     

鼻咽癌中EB病毒LMP1激活TRAFs的初步研究

在EB病毒潜伏膜蛋白LMP1介导的信号传导通路中,TRAFs作为LMP1活化的第一位信号分子,可能扮演着重要的分子开关角色。令人关注的是,在上皮性肿瘤NPC的发生中,EB病毒LMP1能否激活重要的TRAFs信号分子?究竟激活何种TRAFs信号分子,激活的机制何在?将LMP1cDNA导入LMP1表达阴性的HNE2中,建立稳定表达LMP1的鼻咽癌细胞系HNE2-LMP1。以此为材料,应用差异RT-PCR和Western blotting法证实,无论在RNA水平,还是蛋白水平上,TRAF1在HNE2-LMP1中表达较HNE2强,而TRAF2及TRAF3在HNE2-LMP1与HNE2细胞中表达无明显差异;进一步用免疫共沉淀-Western blotting证实LMP1可使TRAF1、TRAF2、TRAF3磷酸化而被活化。这些结果提示在鼻咽癌中,LMP1可能诱导TRAF1表达,而对TRAF2及TRAF3并不影响,但LMP1可磷酸化TRAF1、TRAF2、TRAF3而使其功能性活化。q     

鼻咽癌中EB病毒LMP1激活TRAFs的初步研究

在EB病毒潜伏膜蛋白LMP1介导的信号传导通路中,TRAFs作为LMP1活化的第一位信号分子,可能扮演着重要的分子开关角色,令人关注的是,在上皮性肿瘤NPC的发生中,EB病毒LMP1能否激活重要的TRAFs信号分子？究竟激活何种TRAFs信号分子,激活的机制何在？将LMP1 cDNA导入LMP1表达阴性的HNE2中,建立稳定表达LMP1的劓咽癌细胞系：HNE2－LMP1。以此为材料,应用差异RT－PCR和Western blotting法证实,无论在RNA水平,还是蛋白水平上,TRAF1在HNE2－LMP1中表达较HNE2强,而TRAF2及TRAF3在HNE2－LMP1与HNE2细胞中表达无明显差异;进一步用免疫共沉淀－Western blotting证实LMP1可使TRAF1、TRAF2、TRAF3磷酸化耐被活化,这些结果提示在鼻咽癌中,LMP1可能诱导TRAF1表达,而对TRAF2及TRAF3并不影响,但LMP1可磷酸化TRAF1、TRAF2、TRAF3面使其功能性活化。     

肿瘤坏死因子受体作用因子3(TRAF3)结构及生物学功能

目前在哺乳动物中发现6个肿瘤坏死因子受体作用因子(TNF receptor associated factors,TRAFs)家族成员,它们主要参与TNF受体家族信号通路.这些TRAF成员在C末端有螺旋卷曲结构和保守的TRAF结构域.TRAF3是TRAF家族中功能最为多样化的成员之一.1996年对TRAF3基因敲除小鼠进行研究发现,小鼠在出生早期死亡,这阻碍了TRAF3的生物学功能进一步研究,另一方面也证实了TRAF3在出生后发育以及维持正常的免疫系统功能方面有着重要生物学功能.10年后研究发现,TRAF3的缺失能够导致非经典NF-κB信号通路激活,这使得TRAF3在该信号通路中的功能得到了进一步的阐述.最近研究表明,TRAF3不仅能够负向调节NF-κB和MAPK信号通路,还能够正向调节Ⅰ型干扰素的产生.通过研究还发现,TRAF3可能存在着负向调节钙调蛋白磷酸酶活性的新功能.因此,研究TRAF3在免疫信号通路中的作用以及与之相关的病毒疾病具有重要的意义.     

TRAF4的结构与生物学功能

TRAF(TNF receptor associated factor)家族蛋白是一类具有相同C末端保守结构域的细胞内接头蛋白,能够与包括TNF受体在内的多种受体蛋白相互作用传递信号并因此得名,目前已经发现了7种TRAF家族蛋白。TRAF4是TRAF家族蛋白中最古老的成员之一,最早在乳腺癌的转移淋巴结中发现,在多种实体肿瘤组织中存在高表达和亚细胞定位的异常。与其他TRAF家族蛋白主要参与免疫和炎症反应不同,TRAF4在免疫中的作用非常有限,目前其已知功能主要体现在胚胎发育、细胞极性、凋亡以及活性氧生成调节等方面。     

β-Arrestins参与GPCRs信号通路的分子机制

β-抑制蛋白(β arrestins)是一类在β肾上腺素受体激酶（βARK)提纯过程中发现的重要支架蛋白和信号调控因子;G蛋白偶联受体（GPCRs）为7次跨膜受体,在细胞信号转导中发挥关键作用,是很多临床药物的作用靶点. β-抑制蛋白作为衔接蛋白,调控GPCRs相关的信号通路,介导GPCRs的脱敏、内化、循环、复敏等生理过程,影响多种疾病的进程. 本文总结了β-抑制蛋白参与GPCRs信号通路的研究进展,侧重阐明了其中的分子机制,以期为开发新一代调控GPCRs功能活性的相关药物提供理论基础.     

泛素化在TRAF2介导的NF-κB信号通路中的作用

NF-κB（核因子κ增强子结合蛋白）是核转录因子家族成员,具有调节免疫、炎症和细胞存活的功能.它可被TRAF2（tumor necrosis factor receptor associated factor 2,肿瘤坏死因子受体相关因子2）等相关因子活化.TRAF2包含了N-端的环指结构域和C-端的高度保守结构域.它通过与肿瘤坏死因子受体超家族成员相互作用,介导了下游信号通路.而TRAF2的泛素化在过程中是关键的,鞘磷脂作为TRAF2的泛素化连接酶辅助因子,在TRAF2介导的NF-κB信号通路中发挥重要作用.     

封面故事

<正>肿瘤坏死因子受体超家族(TNFRSF)的成员是机体非常重要的调节分子,参与细胞的生长、分化、凋亡与存活等过程。已有研究表明,TNFRSF家族成员可以通过在细胞质膜和内吞体上结合不同的接头分子,启动截然不同的下游信号通路。例如,TNF结合细胞膜表面的TNFR1,能够招募TRADD,RIP1和TRAF2组成信号复合物Ⅰ,激活经典的NF-κB通路;而内化的TNF-TNFR1复合物则能够招募TRADD、FADD和Caspase-8组成信号复合物Ⅱ,即招募死亡诱导信号复合物介导细胞凋亡。死亡受体6(DR6)属于肿瘤坏死因子受体超家族的一员,它在免疫系统和神经系统中发挥着重要功     

Rho蛋白和细胞骨架的关系

Rho蛋白作为细胞信号转导的分子开关之一,在细胞骨架动态变化中发挥着极其重要的作用。Rho蛋白对细胞骨架动态变化的调节是一个复杂的信号传递过程,涉及到Rho蛋白介导的信号通路中不同效应物间和Rho蛋白介导的多条信号通路间的相互作用。在Rho蛋白介导的信号通路中,上游调控因子、Rho蛋白、效应物在细胞中的正确定位对信号传递有着决定性的作用。     

TRAF molecules in cell signaling and in human diseases

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally identified as signaling adaptors that bind directly to the cytoplasmic regions of receptors of the TNF-R superfamily. The past decade has witnessed rapid expansion of receptor families identified to employ TRAFs for signaling. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), T cell receptor, IL-1 receptor family, IL-17 receptors, IFN receptors and TGFβ receptors. In addition to their role as adaptor proteins, most TRAFs also act as E3 ubiquitin ligases to activate downstream signaling events. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Compelling evidence obtained from germ-line and cell-specific TRAF-deficient mice demonstrates that each TRAF plays indispensable and non-redundant physiological roles, regulating innate and adaptive immunity, embryonic development, tissue homeostasis, stress response, and bone metabolism. Notably, mounting evidence implicates TRAFs in the pathogenesis of human diseases such as cancers and autoimmune diseases, which has sparked new appreciation and interest in TRAF research. This review presents an overview of the current knowledge of TRAFs, with an emphasis on recent findings concerning TRAF molecules in signaling and in human diseases.     

Regulation of TRAF2 signaling by self-induced degradation

Receptors belonging to the tumor necrosis factor receptor (TNF-R) family utilize cytoplasmic adapter proteins called TNF-R-associated factors (TRAFs) as key elements in their signaling pathways. However, it is not yet clear how individual TRAFs regulate signaling by this large and growing receptor family. Signaling via the TNF-R family member CD40 has recently been shown to result in recruitment of TRAF2 to plasma membrane detergent-resistant microdomains (lipid rafts) as well as to subsequently initiate TRAF2 degradation. As TRAF2 associates with most members of the TNF-R family, we wished to determine how this degradation occurs. We show here that CD40-mediated TRAF2 degradation requires the zinc-binding RING domain of TRAF2 and is preceded by TRAF2 ubiquitination, suggesting that the TRAF2 RING may promote ubiquitination although the RING itself is not a target of ubiquitination. Several approaches show that ubiquitination and proteasomal activity are integral to TRAF2 degradation, and inhibition of this process potentiates CD40 signaling.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Caspase-cleaved TRAF1 negatively regulates the antiapoptotic signals of TRAF2 during TNF-induced cell death

Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic pathways. Previous studies have suggested that TNF receptor-associated factor (TRAF) 2 can mediate crucial antiapoptotic signals during TNF stimulation. However, it is unclear how the antiapoptotic signals via TRAF2 in TNF-R1 signaling is regulated. Here we show that TRAF1 is cleaved by caspase-8 into two fragments during apoptosis induced by TNF. Overexpression of the C-terminal cleavage product, TRAF1-c, increased TNF-induced cell death of hybridoma T cells. Importantly, we demonstrate that the cleavage product of TRAF1 coimmunoprecipitates with TRAF2 that is released from the TNF-R1 complex in response to prolonged TNF treatment. These results indicate that caspase-dependent cleavage of TRAF1 generates TRAF1-c fragments that are able to bind TRAF2, and then sequester TRAF2 from the TNF-R1 complex, rendering cells, at least in part, sensitive to TNF.     

Cell Fate Decisions Regulated by K63 Ubiquitination of Tumor Necrosis Factor Receptor 1

Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-κB, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding.     

Tumor necrosis factor receptor-associated factor family protein 2 is a key mediator of the epidermal growth factor-induced ribosomal S6 kinase 2/cAMP-responsive element-binding protein/Fos protein signaling pathway

TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces ribosomal S6 kinase 2 (RSK2) ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation. In addition, TRAF2 is overexpressed in colon cancer and required for colon cancer development, suggesting that TRAF2 might be a potential molecular target for cancer prevention and treatment.     

Tumor necrosis factor receptor-associated factor (TRAF) 1 regulates CD40-induced TRAF2-mediated NF-kappaB activation

To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-kappaB activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-kappaB activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/CD40 ligand-induced NF-kappaB activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2)2-TRAF1 versus TRAF2-(TRAF1)2) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling.     

Mass spectrometric analysis of tumor necrosis factor receptor-associated factor 1 ubiquitination mediated by cellular inhibitor of apoptosis 2

Signaling complexes formed on tumor necrosis factor receptor 2 (TNF-R2) contain adaptor proteins TNF-R-associated factors (TRAFs) 1 and 2, and cellular inhibitors of apoptosis (cIAPs) 1 and 2 which function as regulators of programmed cell death. TRAF2, cIAP1 and cIAP2 all have RING finger domains known to possess E3 ubiquitin ligase activity, implying that ubiquitination may play an important role in the TNF signaling pathway. In this report, we have shown that cIAP2 specifically mediated ubiquitination and proteasome-dependent degradation of TRAF1. To identify the sites for cIAP2-mediated ubiquitination of TRAF1, we used high pressure liquid chromatography coupled with tandem mass spectrometry. Lys185 and Lys193 of TRAF1 were found to be modified with ubiquitin chains. Mutation of Lys185 and Lys193 to Arg almost completely blocked cIAP2-mediated ubiquitination of TRAF1, indicating that they are the major, if not the only, sites of TRAF1 ubiquitination. Our data suggest that cIAP2 may regulate the turnover of TRAF1 by adding polyubiquitin chains on Lys185 or Lys193 following its recruitment to TNF-R signaling complexes.