Analysis of the Functional Roles of a Novel G-alpha Nucl

新型 G-α 核的功能作用分析

• 批准号: 7313461
• 负责人: JOHN H KEHRL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7313461
• 关键词: Analysis; Functional; Roles; Novel; alpha

Proper chromosomal segregation by microtubule dynamics at the mitotic spindle is required for cell division. Aberrations cause severe developmental abnormalities and can contribute to cancer. Genetic studies using model organisms and biochemical studies have demonstrated the existence of a nucleotide cycle for G alpha subunits that is required for proper mitotic spindle function during cell division. This cycle employs highly conserved G?? regulatory proteins including GoLoco motif containing proteins, which are nucleotide dissociation inhibitors (GDI); Regulator of G-protein Signaling (RGS) domain containing proteins, which are GTPase activating proteins (GAP); and Resistance to inhibitors of cholinesterase 8 (Ric-8), a guanine nucleotide exchange factor (GNEF) for G. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. In mammals, the GoLoco motif-containing protein LGN, G??i, and the microtubule-binding nuclear mitotic apparatus protein (NuMA) regulate microtubule pulling forces during cell division. In vitro, Ric-8 dissociates G alpha i-GDP/LGN/NuMA complexes catalytically, releasing G alpha i-GTP, NuMA, from LGN. Our studies have focused on Ric8 and two RGS proteins, RGS14, and RGS3. We have found that Ric-8 is expressed by most human cells and at high levels in lymphocytes as demonstrated by immunoblotting with a Ric-8 antibody. In interphase cells Ric-8 localizes in the cytosol, in centrosomes, and at unknown sites within the nucleus. Expression of a Ric-8-green fluorescent fusion protein (Ric-8GFP) demonstrated a similar expression patter, but in addition showed heightened expression in lamillepodia. During prophase Ric-8 is recruited to the plasma membrane and to the kinetochore, the site of microtubule attachment to the centomeric region of chromosomes. Gi alpha 2 co-localized with Ric-8 at both the plasma membrane and in the kinetochore. Cells expressing the Ric-8GFP underwent mitotic arrest. Reduction of Ric-8 expression by a Ric-8 mRNA targeted shRNA in HeLa cells resulted in cells with multiple spindles and spindle misalignment leading to multi-nucleation. The decreased Ric-8 expression also led to a loss of NuMA from the plasma membrane and reduced Gai2 at the kinetochore. These results suggest that Ric-8 and Ga GDP/GTP exchange are required for proper chromosome segregation during mitosis. Unique among RGS and GDI proteins, RGS14 and RGS12 contain both an RGS domain and a GoLoco motif. Both the RGS domain and the GoLoco motif of RGS14 target members of the G??i subclass. RGS14 also possesses two Raf-like Ras binding domains. RGS14 associates with centrosomes and microtubules, and loss of Rgs14 expression in mice is catastrophic resulting in failure of zygotes to progress to the 2-cell stage. We have co-localized RGS14 with Gi alpha 1 ?nand Gi alpha 2, but not Gi alpha 3, in centrosomes and with all three Gi alpha subunits in the midbody during cytokinesis. Fluorescence resonance energy transfer analysis confirmed a direct interaction between RGS14 and Gi alpha 1 in centrosomes. Supporting the importance of Gi alpha and their regulators at these sites, cells treated with pertussis toxin, which blocks GTP/GDP exchange of Gi alpha, exhibit cytokinesis defects, bi-nucleation, and G1 cell cycle arrest while expression of a GTPase-deficient G??i1, but not a wild type version, causes mitotic failure. These results suggest that the GTP cycle of the Gi?? subunits modulated by their regulators is necessary for normal cell cycle progression and consistent with the above Ric-8 data. To further our studies of Rgs14, mice in which Rgs14 can be conditionally deleted are being developed. Chimeric mice obtained following injection of correctly targeted embryonic stem (ES) cells into blastocytes are being evaluated for germline transmission of the targeted Rgs14 allele. C. elegans RGS7 functions in early cell divisions and RGS7 mutants show hyper-asymmetric movement of mitotic spindles. Among the mammalian RGS proteins, RGS3 most closely resembles C. elegans RGS7. Numerous RGS3 isoforms exist due to extensive alternative mRNA splicing. One isoform of RGS termed PDZ-RGS3 was originally identified as a binding partner of beta-ephrin and implicated in beta-ephrin reverse signaling in neurons. In addition to its C-terminal RGS domain and N-terminal PDZ domain, PDZ-RGS3 also possesses a C2A domain and an unknown domain that bears some resemblance to a Lim domain. PDZ domains bind the C-termini of membrane proteins and have been widely implicated in forming sub-membrane scaffolds to cluster molecules at the cell surface. Recently, we have shown the involvement of PDZ-RGS3 in the regulation of microtubule dynamics and cytokinesis. PDZ-RGS3-GFP localizes predominantly in the cytosol of interphase cells, but is recruited to the midbody in late telophase. Elevating the expression level of PDZ-RGS3 leads to a profound defect in cytokinesis. Using RNA interference to deplete PDZ-RGS3 in cells, a combination of confocal and video time-lapse microscopy revealed disruption of microtubule organization, spindle elongation, and nuclear separation leading to a block in mitotic exit and cytokinesis. Mass spectroscopy analysis of proteins co-precipitating with the PDZ domain of PDZ-RGS3 identified septin proteins. Confirming this interaction PDZ-RGS3 co-immunoprecipitated and co-localized with the septin Nedd5. Reducing PDZ-RGS3 expression led to a failure of Nedd5 to accumulate in the middle zone/midbody. In addition to septin recruitment, PDZ-RGS3 co-localized with the aurora B kinase at the spindle midzone in late anaphase and the midbody in late telophase. Aurora B kinase is a key enzyme involved in the regulation of normal chromosome segregation during mitosis and cytokinesis. The association of PDZ-RGS3 with aurora B kinase was confirmed by co-immunoprecipitation and FRET analysis. The depletion of endogenous PDZ-RGS3 led to a defect in the spatial orientation of aurora B kinase during telophase. To further our studies of Rgs3, mice with targeted deletion of Rgs3 have been obtained. We have two independent mouse lines each with a targeted deletion of Rgs3. The phenotypes are dramatically different with one line failing to produce viable offspring, while no obvious abnormalities have been noted with the other line. We are back-crossing both lines onto a C57/Bl6 background to help sort out the differences between the two lines.

细胞分裂需要在有丝分裂主轴下通过微管动力学进行适当的染色体分离。畸变会导致严重的发育异常，并可能导致癌症。使用模型生物和生化研究的遗传研究表明，Gα亚基的核苷酸周期存在，这是细胞分裂过程中正确有丝分裂纺锤体功能所必需的。这个周期采用高度保守的G？调节性蛋白质，包括含有蛋白质的戈洛科基序，这些蛋白是核苷酸解离抑制剂（GDI）；含有蛋白质的G蛋白信号传导（RGS）结构域的调节剂，它们是GTPase激活蛋白（GAP）；胆碱酯酶8（RIC-8）的耐药性，G的鸟嘌呤核苷酸交换因子（GNEF）。这些蛋白质相互作用以调节染色体有丝分裂运动过程中的微管拉力。在哺乳动物中，含糖基序的蛋白LGN，G ?? I和微管结合核有丝分裂设备蛋白（NUMA）调节细胞分裂过程中的微管拉力。在体外，RIC-8分离了G alpha I-GDP/LGN/Numa复合物，催化催化，从LGN释放G alpha I-GTP Numa。我们的研究集中在RIC8和两个RGS蛋白RGS14和RGS3上。我们发现，RIC-8由大多数人类细胞表达，在淋巴细胞中高水平表达，通过用RIC-8抗体进行免疫印迹证明。在相间细胞中，RIC-8定位于细胞质，中心体和核内未知位点。 RIC-8-绿色荧光融合蛋白（RIC-8GFP）的表达表现出相似的表达模式，但此外显示在lamillepodia中表达增强。在预言期间，RIC-8被募集到质膜和动力学，这是微管附着位于染色体染色体中的微管区域。 GI Alpha 2与RIC-8在质膜和动力学中均共定位。表达RIC-8GFP的细胞经历了有丝分裂停滞。在HeLa细胞中，RIC-8 mRNA靶向shRNA的RIC-8表达降低导致多个纺锤体和纺锤体未对准的细胞导致多核。降低的RIC-8表达也导致质膜中的NUMA损失，并在动力学上降低了GAI2。这些结果表明，RIC-8和GA GDP/GTP在有丝分裂过程中需要适当的染色体分离所必需。在RGS和GDI蛋白之间，RGS14和RGS12在RGS和GDI蛋白之间唯一，既包含RGS域和Goloco基序。 G ?? I子类的RGS14目标成员的RGS域和Goloco基序。 RGS14还具有两个RAF样RAS结合域。 RGS14与中心体和微管相关，而小鼠中RGS14表达的丧失是灾难性的，导致合子无法发展到2细胞阶段。我们与Gi alpha 1？nand Gi alpha 2共定位了RGS14，但在中心体中没有GI Alpha 3，但在细胞因子期间中体中的所有三个GI Alpha亚基。荧光共振能量传递分析证实了RGS14和GI Alpha 1在中心体中的直接相互作用。 Supporting the importance of Gi alpha and their regulators at these sites, cells treated with pertussis toxin, which blocks GTP/GDP exchange of Gi alpha, exhibit cytokinesis defects, bi-nucleation, and G1 cell cycle arrest while expression of a GTPase-deficient G??i1, but not a wild type version, causes mitotic failure.这些结果表明GI的GTP周期？由其调节剂调节的亚基对于正常的细胞周期进程是必需的，并且与上述RIC-8数据一致。为了进一步研究RGS14的研究，正在开发有条件地删除RGS14的小鼠。在将正确靶向的胚胎茎（ES）细胞注射后获得的嵌合小鼠正在评估靶向RGS14等位基因的生殖线传递。秀丽隐杆线虫RGS7在早期细胞分裂和RGS7突变体中的功能显示出有丝分裂纺锤体的超对称运动。在哺乳动物RGS蛋白中，RGS3最类似于秀丽隐杆线虫RGS7。由于广泛的替代mRNA剪接而存在许多RGS3同工型。一种称为PDZ-RGS3的RGS的同工型最初被鉴定为β-磷蛋白的结合伴侣，并与神经元中的β-磷逆逆向信号有关。除了其C末端RGS域和N末端PDZ结构域外，PDZ-RGS3还具有C2A结构域和一个未知域，与LIM域具有一定的相似之处。 PDZ结构域结合了膜蛋白的C末端，并已广泛与在细胞表面形成亚膜支架与簇分子形成簇分子。最近，我们显示了PDZ-RGS3参与微管动力学和细胞因子的调节。 PDZ-RGS3-GFP主要在相间细胞的细胞质中定位，但在末期晚期招募到中体。升高PDZ-RGS3的表达水平会导致细胞因子的严重缺陷。使用RNA干扰来消耗细胞中的PDZ-RGS3，共焦和视频延时显微镜的组合揭示了微管组织，纺锤体伸长和核分离的破坏，从而导致有丝分裂出口和细胞因子的阻滞。与PDZ-RGS3的PDZ结构域共沉淀的质谱分析已鉴定出SEPTIN蛋白。证实了这种相互作用的PDZ-RGS3共承受沉淀并与Septin Nedd5共定位。降低PDZ-RGS3表达导致NEDD5失败在中间区域/中体中积聚。除SEPTIN招募外，PDZ-RGS3还与Aurora B激酶在后期的纺锤体中区和晚期末期中的中体共定位。 Aurora B激酶是涉及有丝分裂和细胞力学期间正常染色体分离的关键酶。通过共免疫沉淀和FRET分析证实了PDZ-RGS3与Aurora B激酶的关联。内源性PDZ-RGS3的耗竭导致了末期中极光B激酶的空间方向的缺陷。 为了进一步研究RGS3的研究，已经获得了具有靶向缺失RGS3的小鼠。我们有两个独立的鼠标线，每个鼠标均具有RGS3的靶向缺失。表型截然不同，一条线未能产生可行的后代，而另一条线没有明显的异常。我们正在将这两条线划分到C57/BL6背景上，以帮助整理两条线之间的差异。