Aurora-A在细胞中的功能以及与肿瘤的关系

Aurora蛋白激酶家族,在细胞的有丝分裂期发挥着重要作用。目前分为3种即Aurora-A、Au-rora-B、Aurora-C。其中Aurora-A定位于中心体和纺锤体,主要参与中心体的成熟、分离和纺锤体形成。此外Aurora-A可以参与p53通路的调节,细胞凋亡和分裂之间平衡的调节等。Aurora-A高表达可引起中心体异常扩增,异倍体产生。在肿瘤组织中的研究显示它在多种肿瘤乳腺癌、卵巢癌、结肠癌、胃癌等组织中存在着高表达,并且与某些肿瘤的预后相关。Aurora-A可能通过多种机制参与了肿瘤的发生和发展。     

CyclinE-CDK2分子活性调节机制

Cyc linE-CDK2是细胞周期G1/S期的转折中的关键调控因子,其分子活性受各种细胞因子、磷酸化/去磷酸化修饰、泛素介导的蛋白降解途径及细胞周期依赖性蛋白激酶抑制物(cyc lin-dependent k inase in-h ib itor,CK Is)等多种方式构成的精细调控网络调节。其活性失调将导致细胞增殖紊乱甚至肿瘤的发生。认识Cyc linE-CDK2分子活性调节机制有助于理解它在肿瘤形成中的作用。     

有丝分裂激酶Aurora-A的功能及其与肿瘤发生的关系

有丝分裂激酶Aurora-A具有调节中心体分离、成熟以及纺锤体装配的功能，并且在调节细胞周期G2-M期转变以及checkpoint（监控点）方面发挥重要的作用。近年来研究证实Aurora-A过表达与中心体异常、非整倍体、细胞转化以及肿瘤发生方面存在很大程度的相关性，并通过对抑癌基因p53以及癌基因c-Myc等的调节促进肿瘤的发生。     

Aurora-A在细胞中的功能以及与肿瘤的关系

Aurora蛋白激酶家族，在细胞的有丝分裂期发挥着重要作用。目前分为3种即Aurora-A、Aurom-B、Aurora-C。其中Aurora-A定位于中心体和纺锤体，主要参与中心体的成熟、分离和纺锤体形成。此外Aurora-A可以参与p53通路的调节，细胞凋亡和分裂之间平衡的调节等。Aurom-A高表达可引起中心体异常扩增，异倍体产生。在肿瘤组织中的研究显示它在多种肿瘤乳腺癌、卵巢癌、结肠癌、胃癌等组织中存在着高表达，并且与某些肿瘤的预后相关。Aurom-A可能通过多种机制参与了肿瘤的发生和发展。     

Bim介导细胞凋亡的调控及临床意义

Bim是Bcl-2家族成员，只具有一个BH3结构域，可以通过拮抗Bcl-2等抗凋亡因子的作用或直接与Bax等相互作用并共同转位到线粒体膜上引起细胞色素C释放而诱导凋亡。敲除Bim的小鼠表现出自身免疫病，提示Bim参与免疫系统的自我稳态调节。Bim的转录可以被FoxO3、Myb、E2F及TGF-beta等因子调控；而JNK对其某些特定位点的磷酸化对调节Bim的活性起着重要作用；同时，Erk1/2可以使Bim特定位点磷酸化后被蛋白酶体降解。Bim与自身免疫病、退行性疾病以及肿瘤的发生发展及治疗都有密切的关系，因而，Bim在细胞凋亡中的基础研究必然为临床治疗提供理论基础与线索。     

蛋白质泛素化降解途径

泛素系统(UPS)广泛存在于真核生物中，是精细的特异性的蛋白质降解系统。它由泛素、26S蛋白酶体、多种酶（如E1、E2、E3、去泛素酶）构成。在泛素系统中，泛素（Ubiquitin Ub）是一种序列保守的小分子蛋白，蛋白质与泛素结合后，被蛋白酶体以ATP依赖的方式降解。E1、E2酶分别称为泛素活化酶和泛素载体酶，使泛素通过Ub-腺苷酸中间产物形成E2-Ub巯基酯。泛素连接酶E3负责连接泛素和特异性的底物，这样泛素化的底物可以被26S蛋白酶体降解为若干肽段。泛素系统通过特异性的降解蛋白质，调节细胞分化、免疫反应，参与转录、离子通道、分泌的调控及神经元网络、细胞器的形成等等，泛素系统还与人类某些疾病有关。本文对泛素系统的成员、作用机制和功能的研究进展作了介绍。     

ERK通过MDM2介导的降解作用抑制FOXO3a而促进肿瘤发生

RAS-ERK途径在细胞分化、细胞增殖和肿瘤发展中起着极其重要的作用。Yang等证明了ERK通过直接干扰和磷酸化FOXO3a丝氨酸残基而下调FOXO3a，继而促使细胞增殖和肿瘤发生。他们提出一个新的信号途径：ERK经由MDM2介导的泛素-蛋白酶体途径的交互作用和降解作用调节肿瘤抑制基因FOXO3a。     

泛素-蛋白酶体途径及意义

泛素-蛋白酶体途径介导的蛋白降解是机体调节细胞内蛋白水平与功能的一个重要机制。负责执行这个调控过程的组成成分包括泛素及其启动酶系统和蛋白酶体系统。泛素启动酶系统负责活化泛素,并将其结合到待降解的蛋白上,形成靶蛋白多聚泛素链,即泛素化。蛋白酶体系统可以识别已泛素化的蛋白并将其降解。此外,细胞内还有另一类解离泛素链分子的去泛素化蛋白酶形成反向调节。泛素-蛋白酶体途径涉及许多细胞的生理过程,其调节异常与多种疾病的发生有关。     

p34~(cdc2)与真核生物细胞周期调控

cdc2基因的编码产物p34~(cdc2)在真核生物的细胞周期调控中起中心作用,它具有细胞周期中特有的蛋白激酶活性,引起多种蛋白质的磷酸化,从而引起如核膜破裂,染色体形成等细胞分裂事件的发生,推动细胞周期过程。循环素和其它因子直接或间接调节它的活性从而调节细胞周期过程。     

人泛素偶联酶E2研究进展

在真核细胞中,细胞内蛋白质的降解受到精确的调控,其中泛素-蛋白酶体途径能高效选择性降解细胞周期蛋白(cyclin)、P21、E2F等短寿命蛋白质,在细胞周期调控,细胞增殖,信号转导,免疫应答等方面都发挥着重要作用[1].泛素偶联酶E2(UbcH10/Ube2c)在泛素的活化与底物蛋白结合过程中是必不可少的.它与有丝分裂后期促进复合物(anaphase-promoting complex,APC)相互识别,把活化的泛素转移到底物蛋白上,使底物蛋白多泛素化,进而被蛋白酶体水解.近年来研究发现,泛素偶联酶E2与肿瘤细胞的发生发展,增殖分化密切相关,并有望成为肿瘤诊断、治疗的目标靶蛋白之一[2-4].本文就泛素偶联酶E2的生物学特性,作用机制及与肿瘤的关系做一综述.     

The centrosomal protein Lats2 is a phosphorylation target of Aurora-A kinase

Human Lats2, a novel serine/threonine kinase, is a member of the Lats kinase family that includes the Drosophila tumour suppressor lats/warts. Lats1, a counterpart of Lats2, is phosphorylated in mitosis and localized to the mitotic apparatus. However, the regulation, function and intracellular distribution of Lats2 remain unclear. Here, we show that Lats2 is a novel phosphorylation target of Aurora-A kinase. We first showed that the phosphorylated residue of Lats2 is S83 in vitro. Antibody that recognizes this phosphorylated S83 indicated that the phosphorylation also occurs in vivo. We found that Lats2 transiently interacts with Aurora-A, and that Lats2 and Aurora-A co-localize at the centrosomes during the cell cycle. Furthermore, we showed that the inhibition of Aurora-A-induced phosphorylation of S83 on Lats2 partially perturbed its centrosomal localization. On the basis of these observations, we conclude that S83 of Lats2 is a phosphorylation target of Aurora-A and this phosphorylation plays a role of the centrosomal localization of Lats2.     

Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer's disease

In Alzheimer's disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.     

NMDA receptor activity downregulates KCC2 resulting in depolarizing GABAA receptor-mediated currents

KCC2 is a neuron-specific K(+)-Cl(-) co-transporter that maintains a low intracellular Cl(-) concentration that is essential for hyperpolarizing inhibition mediated by GABA(A) receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca(2+) influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.     

AMPK activity is required for the induction of anhydrobiosis in a tardigrade Hypsibius exemplaris,and its potential up‐regulator is PP2A

The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time‐series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP‐activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre‐treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.     

The N-terminal domain of the Aurora-A Phe-31 variant encodes an E3 ubiquitin ligase and mediates ubiquitination of IkappaBalpha

Aurora-A is an important regulator of mitosis and is frequently amplified in human cancer. Ectopic expression of Aurora-A in mammalian cells induces centrosome amplification, genomic instability and transformation. A common genetic variant in Aurora-A (F31I) is preferentially amplified and is associated with the occurrence and the status of colon, oesophageal and breast cancers. Here we demonstrate that the N-terminal domain of Aurora-A Phe-31 variant exhibits an intrinsic ubiquitin ligase activity. Mutation of cysteines 8, 33 and 49 of Aurora-A abolishes the ubiquitin ligase activity of the protein. Aurora-A in a complex with UBE2N/MMS2 catalyses polyubiquitination of IkappaBalpha in vitro and in vivo.     

Protein kinase-A activity in PRKAR1A-mutant cells,and regulation of mitogen-activated protein kinases ERK1/2

Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.