结肠腺瘤性息肉病蛋白通过与SMAP/KAP3相互作用与微管结合

结肠腺瘤性息肉病基因(adenomatous polyposis coli,APC)的突变导致家族性结肠息肉腺瘤病和散发性结肠癌,APC基因编码一个具有多个结构域、多种磷酸化状态的大分子蛋白质.APC蛋白可通过C段直接或间接与微管结合,同时还可以通过中段与微管结合,但其结合的机制目前还不清楚.为进一步研究APC与其他蛋白质的相互作用,利用酵母双杂交技术运用APC中段（1 500 bp～4 800 bp）构建诱饵质粒,筛选人胎脑cDNA文库,得到一个与APC相互作用的蛋白SMAP/KAP3,SMAP/KAP3是驱动蛋白KIF3A/3B的相关蛋白.通过免疫共沉淀和双色免疫荧光共定位的方法,证实了APC与SMAP/KAP3在体内的相互作用,提示APC可能通过SMAP/KAP3-KIF3A/B参与沿微管的运动.     

玉米根细胞中类整合素蛋白与α-微管蛋白的共定位及其可能的相互作用

采用间接免疫荧光标记法对玉米根细胞中的类整合素蛋白和细胞骨架主要组分之一的α-微管蛋白进行了荧光定位。结果表明：类整合素蛋白主要分布在质膜上。与对照相比,用与类整合素蛋白特异结合的5肽GRGDS处理后,质膜上类整合素的分布更为均匀,微管的排列密度降低,而用不与类整合素蛋白特异结合的GRGDS类似物SDGRG处理则对类整合素蛋白分布和微管蛋白的排列均无明显影响。微管蛋白解聚剂或稳定剂处理改变类整合素在质膜上的分布。这些结果表明类整合素蛋白与微管蛋白间有复杂的相互作用。     

植物微管和微丝骨架研究的进展

从以上叙述的资料中可以看出,近年来在植物微管蛋白的分离及其化学性质、微管的组织中心、微管的异质性、微丝的分布,以及微管和微丝骨架的功能及基因调节等方面的研究取得不少新的进展;特别是从植物中直接分离微管蛋白取得成功、以及微管蛋白异型、微管冷稳定性与植物抗寒性的关系及微丝分布广泛性等的发现,对植物细胞骨架的进一步研究具有重要意义。     

糖原合酶激酶3β和腺瘤性结肠息肉病蛋白在气道上皮细胞机械损伤修复过程的时空分布

为了探讨糖原合酶激酶3β（glycogen synthase kinase 3β,GSK3β）和腺瘤性结肠息肉病（adenomatous polyposis coli, APC）蛋白在气道上皮细胞（airway epithelial cells,AECs）损伤和修复中的作用,我们采用机械划线损伤的方法建立体外气道上皮损伤修复模型,采用Western blot、免疫荧光双标共聚焦成像和免疫沉淀的方法观察损伤修复过程中APC蛋白和GSK3β在AECs中表达及分布的动态变化。结果显示：（1）用Western blot方法观察到划线损伤0．5 h后即有GSK3β磷酸化增强（P〈 0．05）,6 h达到高峰（P〈0．05）,持续到12 h（P〈0．05）,24 h开始下降,而GSK3β总量大致保持一致。（2）在免疫荧光双标共聚焦成像实验中,划线损伤0 h组APC蛋白主要表达于胞浆,而划线损伤6 h后APC蛋白主要聚集于损伤前沿区的迁移活跃细胞。（3）免疫共沉淀的实验结果显示,划线损伤0 h时GSK3B和APC蛋白能共同沉淀,但在划线损伤6 h之后,两者发生了分离。以上结果表明：划线损伤后AECs立即启动修复过程,此时GSK3B的活性被抑制,促使APC蛋白游离出来;游离出来的APC蛋白则与微管正极结合,增加了微管的稳定性,从而调节细胞骨架运动,促进气道上皮的损伤修复。     

6s微管蛋白的提纯及其抗体的制备

一、前言细胞骨架中主要结构组分之一是微管系统。现已知与组成微管有关的蛋白质有两大类:微管蛋白和微管伴随蛋白(MAPs),前者中包括α-微管蛋白和β-微管蛋白,其二聚体称为6s微管蛋白,而后者则包括高分子微管伴随蛋白(HMW)和分子量较小的tau蛋白,近年来对这些蛋白质的性质、提纯和其抗体的制备等研究都有相当大的进展。方法学上利用免疫荧光和免疫酶标促进了对细胞的微管系统及细胞骨架整体的了解。我们曾对组成微管的蛋白做过一些工作。本文报道我们在以前工作的     

肿瘤抑制蛋白APC的结构与功能

肿瘤抑制蛋白APC(adenomatous polyposis coli)是一种多功能蛋白,它不仅参与Wnt信号途径,调节β-链蛋白(β-catenin)的降解,同时也调节细胞骨架运动,影响细胞的迁移、黏合和分裂等。APC和其他相关因子之间的平衡对于肠上皮细胞的正常发育是十分重要的,这种平衡一旦被打破可能导致结肠功能的破坏及癌症的发生。该文着重介绍APC蛋白的结构及对细胞生长的影响。     

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。     

微管蛋白的翻译后修饰及功能研究

微管是由α/β微管蛋白(α/βtubulin)聚合形成的管状细胞骨架,在许多生物学过程中起着重要的作用。微管的结构与性质受到多种因素的调控,其中微管蛋白的翻译后修饰是一类重要的调控方式。主要介绍目前已发现的微管蛋白翻译后修饰种类,并讨论这些修饰的生物学功能与作用机制。     

细胞骨架与血糖调节

细胞骨架由微丝、微管和中间丝构成,参与血糖调节这一复杂的生理过程,在胰岛素分泌、胰岛素功能和糖代谢相关酶类的细胞内分布等方面具有重要的作用。本文将从以上三个方面,对细胞骨架与血糖调节的关系加以综述。     

真核细胞伴侣素CCT及其与细胞骨架的关系

CCT(the chaperonin containing tailless complex polypeptide 1)是一种广泛存在于细胞浆中的异型寡聚蛋白,也是迄今为止真核细胞胞浆中发现的唯一伴侣素。目前认为大约15%的哺乳动物蛋白折叠需要CCT的参与,其中研究得最多的是肌动蛋白和微管蛋白。研究发现,CCT的异常会导致细胞骨架蛋白发生改变,甚至影响细胞骨架的形成与解聚。由此推测,一些细胞骨架相关疾病可能与CCT异常有关。     

Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, β-catenin (βcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to βcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds βcat and the affinity is significantly increased. These distinct APC-βcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for βcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.     

The many faces of the tumor suppressor gene APC

Inactivation of the tumor suppressor adenomatous polyposis coli (APC) protein is a critical early step in the development of familial and sporadic colon cancer. Close examination of the function of APC has shown that it is a multifunctional protein involved in a wide variety of processes, including regulation of cell proliferation, cell migration, cell adhesion, cytoskeletal reorganization, and chromosomal stability. Tantalizing clues to the different functions of APC have been provided by the identification of proteins interacting with several discrete motifs within APC. Each of these putative functions could link APC inactivation with tumorigenesis. Here, we will summarize recent findings regarding the diverse role of APC. We will emphasize the interaction of APC with different binding partners, the role of these complex interactions for normal functioning of the cell, and how disruption of these interactions may play a role in tumor development. The rapid progress made recently shows the many faces of APC, leading to a constant reappreciation of this multitasking tumor suppressor protein.     

The Role of the Anaphase-Promoting Complex/Cyclosome in Plant Growth

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that plays a major role in the progression of the eukaryotic cell cycle. This unusual protein complex targets key cell cycle regulators, such as mitotic cyclins and securins, for degradation via the 26S proteasome by ubiquitination, triggering the metaphase-to-anaphase transition and exit from mitosis. Because of its essential role in cell cycle regulation, the APC/C has been extensively studied in mammals and yeasts, but relatively less in plants. Evidence shows that, besides its well-known role in cell cycle regulation, the APC/C also has functions beyond the cell cycle. In metazoans, the APC/C has been implicated in cell differentiation, disease control, basic metabolism and neuronal survival. Recent studies also have shed light on specific functions of the APC/C during plant development. Plant APC/C subunits and activators have been reported to play a role in cellular differentiation, vascular development, shoot branching, female and male gametophyte development and embryogenesis. Here, we discuss our current understanding of the APC/C controlling plant growth.     

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.     

Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C

Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.     

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.     

Activated protein C N-linked glycans modulate cytoprotective signaling function on endothelial cells

Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma β-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.     

Adenomatous polyposis coli is down-regulated by the ubiquitin-proteasome pathway in a process facilitated by Axin

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.