细菌效应蛋白对宿主细胞Rho小G蛋白信号通路的调节作用

病原体细菌通过自身分泌系统分泌效应蛋白并注入宿主体内,修饰宿主的信号转导系统,破坏宿主细胞中天然免疫有关信号通路,发挥毒性作用使宿主产生疾病。吞噬作用在天然免疫系统中发挥重要作用,这个过程涉及肌动蛋白细胞骨架的重排。Rho(Ras homolog family)小G蛋白家族成员作为细胞骨架结构的重要调控蛋白可调节这一过程,其相关信号通路成为细菌效应蛋白的作用靶点。细菌效应蛋白可以模仿Rho的调节因子破坏信号通路,可以通过剪切Rho C-端的尾部结构使其从细胞膜解离并失去活性,可以直接模仿Rho发挥调控功能,可以影响Rho上游的调控事件影响其活性,也可通过对Rho进行直接的翻译后修饰使其失活,形成有利于细菌生存、繁殖、毒力释放的环境。由此导致的Rho信号通路功能紊乱使宿主产生智力缺陷、免疫功能障碍、癌症等多种疾病。     

小G蛋白ROP的研究进展

小G蛋白(small GTPases)是近年来研究细胞信号转导过程的热点问题,包括Ras、Rab、Rho、Arf和Ran5个亚家族,其中ROP蛋白是Rho家族成员,为植物特有,在调控细胞生长、发育及调节植物对环境响应等各方面起重要作用.对ROP蛋白的活性调节和功能进行了重点介绍.     

Rho GTPase对细胞骨架及信号通路的多重影响

Rho GTPase最基本的功能是结合和水解鸟嘌呤核苷酸，目前已从晡乳动物中分离出16种不同的Rho GTPases，其中以Rho、Rac和Cdc42最为人们关注。研究发现Rho GTPases参与基因转录、细胞周期进程的调控及多条信号通路的调节，与细胞凋亡、肿瘤侵润及细胞骨架构成关系密切。现普遍认为Rho GTPases是调节细胞功能的一类重要蛋白分子，越来越多的Rho家族成员及其调控的蛋白数量逐渐被发现和认识。     

RhoA的泛素化降解机制及功能

Rho小G蛋白(Ras homology frowth-related,Rho G)家族作为分子开关(molecular switch)在GTP结合的激活形式和GDP结合的非激活形式之间转换,发挥着重要的生物学功能,细胞内Rho小G蛋白的含量可由泛素–蛋白酶体系统(ubiquitin-proteasome system,UPS)降解途径来调控。Rho A(Ras homolog gene family member A,Rho A)是Rho小G蛋白家族成员,其功能涉及细胞极性、细胞迁移、细胞周期调控、神经系统发育等,通过UPS途径对该蛋白在细胞内的含量进行调控,可保证细胞的相关正常生理功能。在Rho A泛素化降解过程中,不同的泛素连接酶(ubiquintin ligases,E3)发挥了重要的作用。该文将简单介绍UPS的过程和Rho A蛋白质的结构、功能,详细论述Rho A泛素化降解过程的分子机制和生物学功能。     

Rho小G蛋白相关激酶的结构与功能

Rho小G蛋白家族是Ras超家族成员之一,人类Rho小G蛋白包括20个成员,研究最清楚的有RhoA、Rac1和Cdc42。Rho小G蛋白参与了诸如细胞骨架调节、细胞移动、细胞增殖、细胞周期调控等重要的生物学过程。在这些生物学过程的调节中,Rho小G蛋白的下游效应蛋白质如蛋白激酶（p21-activated kinase,PAK）、ROCK（Rho-kinase）、PKN（protein kinase novel）和MRCK（myotonin-related Cdc42-binding kinase）发挥了不可或缺的作用。迄今研究发现,PAK可调节细胞骨架动力学和细胞运动,另外,PAK通过MAPK（mitogen-activated protein kinases）参与转录、细胞凋亡和幸存通路及细胞周期进程;ROCK与肌动蛋白应力纤维介导黏附复合物的形成及与细胞周期进程的调节有关;哺乳动物的PKN与RhoA/B/C相互作用介导细胞骨架调节;MRCK与细胞骨架重排、细胞核转动、微管组织中心再定位、细胞移动和癌细胞侵袭等有关。该文简要介绍Rho小G蛋白下游激酶PAK、ROCK、PKN和MRCK的结构及其在细胞骨架调节中的功能,重点总结它们在真核细胞周期调控中的作用,尤其是在癌细胞周期进程中所发挥的作用,为寻找癌症治疗的新靶点提供理论依据。     

p21活化激酶的生物学活性及其与肿瘤的关系

p21活化激酶(p21-activatedkinase,PAK),为一类进化上保守的丝氨酸/苏氨酸蛋白激酶。PAK在许多组织中广泛表达,作为小G蛋白Rho家族Cdc42和Rac1的下游靶蛋白,可以被生长因子及其他胞外信号通过GTP酶依赖的信号通路或非GTP酶依赖的信号通路活化,发挥多种生物学效应。PAK作为一种重要的生物学调节因子,在哺乳动物一系列细胞功能中具有重要作用,如:细胞运动、细胞生存、细胞周期、血管生成、基因转录调节及癌细胞的侵袭转移。通过对PAK家族成员信号转导机制的研究,为癌症治疗提供分子靶标。     

Rho小G蛋白信号通路在癌细胞生物学中的多样功能

癌症的产生是由于细胞正常行为的多个方面发生改变,例如基因突变的积累、失去控制的细胞增殖、细胞的异常迁移和侵染、染色体的不稳定性等。Rho小G蛋白相关信号通路涉及癌症发展进程的多个方面,例如细胞周期进程、细胞极性的调控、细胞骨架重排、细胞与细胞或细胞与基质相互作用调控的细胞迁移和侵染等。该文总结了近年来Rho小G蛋白在癌症的发生发展过程中相关作用的研究进展,重点阐述其家族成员在癌细胞的增殖、存活、侵染、转移等过程中的作用,并对以Rho小G蛋白信号通路作为癌症治疗靶点的研究进展进行概括总结。     

Rho小G蛋白介导的细胞周期调控

Rho小G蛋白作为一个信号分子家族具有多样化的功能, 可以调节细胞骨架重排 、细胞迁移、细胞极性、基因表达、细胞周期调控等. Rho小G蛋白家族对细胞周期 调控的研究主要集中在其对于有丝分裂期细胞的调节作用,包括调节有丝分裂期前 期细胞趋圆化、后期染色体排列及收缩环的收缩作用.近期的研究显示,Rho小G蛋白及其效应分子对于细胞周期G1、S、G2期的调控主要是通过影响细胞周期的正调控因子细胞周期蛋白D1 (cyclin D1) 和负调控因子细胞周期蛋白依赖型激酶相互作用蛋白1及细胞周期蛋白依赖型激酶抑制蛋白27 (p21cip1/p27kip1) 进行的.本文总结了Rho小G蛋白及其效应分子在细胞周期调控,尤其是对G1/S期调控的研究进展,并简要阐述了Rho小G蛋白介导的细胞周期调控异常与癌症发生的关系.     

RHO蛋白家族与细胞极性

细胞的极性形成对细胞发育、分化及其功能的发挥起着举足轻重的作用,细胞极性的丧失与肿瘤的发生发展密切相关.小G蛋白Rho家族是肌动蛋白细胞骨架重新组装的主要调节因子之一,在协调细胞极性化和正常的形态形成过程中起重要作用.现就Rho蛋白家族与细胞极性及二者的关系作一综述.     

转录因子家族DP蛋白与肿瘤关系的研究进展

E2F是一个重要的转录因子家族,通过调节靶基因的转录活性在细胞周期、DNA复制、生长、分化、凋亡等多种细胞进程中发挥关键的作用.转录因子家族DP与E2F结合形成E2F/DP异二聚体,对E2F的DNA结合亲和力和转录激活功能起到增强的作用,是生理状态下E2F必不可少的组成部分.DP蛋白家族有三个成员,即DP-1、DP-2和DP-3,其中DP-1具有三个异构体,DP-2具有四个异构体.DP蛋白家族成员以及异构体间存在结构和功能的差异,能通过组织特异性表达和显性负性方式在家族内部构成调节系统,对生理功能进行精细调节.E2F-1兼具抑癌和促癌双重作用,已得到人们的广泛关注.DP蛋白作为E2F的结合伴侣对其功能起到重要的调节作用,加之以DP-3在肿瘤细胞中的特异性表达,提示DP蛋白与肿瘤间存在密切的关系.本文就此将近年来的研究成果进行总结,并提出进一步研究的设想.     

Vav proteins, masters of the world of cytoskeleton organization

Vav proteins are evolutionarily conserved from nematodes to mammals and play a pivotal role in many aspects of cellular signaling, coupling cell surface receptors to various effectors functions. In mammals, there are three family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed. Vav proteins contain multiple domains that enable their function in various fashions. The participation of the Vav proteins in several processes that require cytoskeletal reorganization, such as the formation of the immunological synapse (IS), phagocytosis, platelet aggregation, spreading, and transformation will be discussed in this review. We will also cover how the Vav proteins succeed in controlling these processes by their function as guanine nucleotide exchange factors (GEFs) for the Rho/Rac family of GTPases. The contribution of the Vav proteins in a GEF-independent manner to the organization of the cytoskeleton will also be deliberated. The scope of this review is to highlight the numerous roles of the Vav signal transducer proteins in actin organization.     

Vav GEFs regulate macrophage morphology and adhesion-induced Rac and Rho activation

The Vav family of proteins have the potential to act as both signalling adapters and GEFs for Rho GTPases. They have therefore been proposed as regulators of the cytoskeleton in various cell types. We have used macrophages from mice deficient in all three Vav isoforms to determine how their function affects cell morphology and migration. Macrophages lacking Vav proteins adopt an elongated morphology and have enhanced migratory persistence in culture. To investigate the pathways through which Vav proteins exert their effects we analysed the responses of macrophages to the chemoattractant CSF-1 and to adhesion. We found that morphological and signalling responses of macrophages to CSF-1 did not require Vav proteins. In contrast, adhesion-induced cell spreading, RhoA and Rac1 activation and cell signalling were all dependent on Vav proteins. We propose that Vav proteins affect macrophage morphology and motile behaviour by coupling adhesion receptors to Rac1 and RhoA activity and regulating adhesion signalling events such as paxillin and ERK1/2 phosphorylation by acting as adapters.     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Vav transformation requires activation of multiple GTPases and regulation of gene expression

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.     

Vav1 and Vav2 play different roles in macrophage migration and cytoskeletal organization

Vav family proteins act as guanine nucleotide exchange factors for Rho family proteins, which are known to orchestrate cytoskeletal changes and cell migration in response to extracellular stimuli. Using mice deficient for Vav1, Vav2 and/or Vav3, overlapping and isoform-specific functions of the three Vav proteins have been described in various hematopoietic cell types, but their roles in regulating cell morphology and migration have not been studied in detail. To investigate whether Vav isoforms have redundant or unique functions in regulating adhesion and migration, we investigated the properties of Vav1-deficient and Vav2-deficient macrophages. Both Vav1-deficient and Vav2-deficient cells have a smaller adhesive area; yet, only Vav1-deficient cells have a reduced migration speed, which coincides with a lower level of microtubules. Vav2-deficient macrophages display a high level of constitutive membrane ruffling, but neither Vav1 nor Vav2 is required for colony stimulating factor-1-induced membrane ruffling and cell spreading. Our results suggest that the migration speed of macrophages is regulated independently of spread area or membrane ruffling and that Vav1 is selectively required to maintain a normal migration speed.     

Vav GEFs are required for beta2 integrin-dependent functions of neutrophils

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.     

Critical role of the pleckstrin homology and cysteine-rich domains in Vav signaling and transforming activity

Vav family proteins are members of the Dbl family of guanine nucleotide exchange factors and activators of Rho family small GTPases. In addition to the Dbl homology (DH) domain important for guanine nucleotide exchange factor catalytic function, all Dbl family proteins contain an adjacent pleckstrin homology (PH) domain that serves to regulate DH domain activity. Although the role of the PH domain in Vav function has been evaluated extensively, its precise role and whether it serves a distinct role in different Vav proteins remain unresolved. Additionally, the precise role of an adjacent cysteine-rich domain (CRD) in regulating DH domain function is also unclear. In this study, we evaluated the contribution of these putative protein-protein or protein-lipid interaction domains to Vav signaling and transforming activity. In contrast to previous observations, we found that the PH domain is critical for Vav transforming activity. Similarly, the CRD was also essential and served a function distinct from that of the PH domain. Although mutation of either domain reduced Vav membrane association, addition of plasma membrane targeting sequences to either the CRD or PH domain mutant proteins did not restore Vav transforming activity. This result contrasts with other Dbl family proteins, where a membrane targeting sequence alone was sufficient to restore the loss of function caused by mutation of the PH domain. Furthermore, green fluorescent protein fusion proteins containing the PH domain or CRD, or both, failed to target to the plasma membrane, suggesting that these two domains also serve regulatory functions independent of promoting membrane localization. Finally, we found that phosphatidylinositol 3-kinase activation may promote Vav membrane association via phosphatidylinositol 3,4,5-triphosphate binding to the PH domain.