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.

通过有丝分裂纺锤体的微管动力学进行适当的染色体分离是细胞分裂所必需的。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进行的遗传研究已经证明，G α 亚基的核苷酸循环的存在是细胞分裂过程中适当的有丝分裂纺锤体功能所必需的。该循环采用高度保守的 G??调节蛋白，包括含有 GoLoco 基序的蛋白，它们是核苷酸解离抑制剂 (GDI)； G 蛋白信号传导 (RGS) 结构域的调节蛋白，即 GTP 酶激活蛋白 (GAP)；对胆碱酯酶 8 (Ric-8) 抑制剂的抗性，胆碱酯酶 8 (Ric-8) 是 G 的鸟嘌呤核苷酸交换因子 (GNEF)。这些蛋白质相互作用，在染色体有丝分裂运动期间调节微管拉力。在哺乳动物中，含有 GoLoco 基序的蛋白 LGN、G??i 和微管结合核有丝分裂装置蛋白 (NuMA) 调节细胞分裂过程中的微管拉力。在体外，Ric-8 催化解离 G α i-GDP/LGN/NuMA 复合物，从 LGN 中释放 G α i-GTP、NuMA。我们的研究主要集中在 Ric8 和两种 RGS 蛋白 RGS14 和 RGS3。我们发现，Ric-8 在大多数人类细胞中表达，并且在淋巴细胞中表达水平很高，如 Ric-8 抗体免疫印迹所证明的那样。在间期细胞中，Ric-8 定位于细胞质、中心体和细胞核内的未知位点。 Ric-8-绿色荧光融合蛋白（Ric-8GFP）的表达表现出类似的表达模式，但此外在片状伪足中表现出增强的表达。在前期，Ric-8 被募集到质膜和动粒（微管附着到染色体着丝粒区域的位点）。 Gi α2 与 Ric-8 在质膜和着丝粒中共定位。表达 Ric-8GFP 的细胞经历有丝分裂停滞。 HeLa 细胞中 Ric-8 mRNA 靶向 shRNA 减少 Ric-8 表达，导致细胞具有多个纺锤体，纺锤体错位导致多核。 Ric-8 表达的减少还导致质膜上 NuMA 的丢失以及动粒处 Gai2 的减少。这些结果表明，Ric-8 和 Ga GDP/GTP 交换是有丝分裂过程中正确染色体分离所必需的。 RGS14 和 RGS12 在 RGS 和 GDI 蛋白中独一无二，同时包含 RGS 结构域和 GoLoco 基序。 RGS14 的 RGS 结构域和 GoLoco 基序均以 G??i 亚类的成员为目标。 RGS14 还拥有两个 Raf 样 Ras 结合域。 RGS14 与中心体和微管相关，小鼠中 Rgs14 表达的丧失是灾难性的，导致受精卵无法进展到 2 细胞阶段。我们在中心体中将 RGS14 与 Gi α 1 -n 和 Gi α 2 共定位，但不与 Gi α 3 共定位，并在胞质分裂过程中与中体中的所有三个 Gi α 亚基共定位。荧光共振能量转移分析证实了中心体中 RGS14 和 Gi α 1 之间的直接相互作用。百日咳毒素可阻断 Gi α 的 GTP/GDP 交换，经百日咳毒素处理的细胞表现出胞质分裂缺陷、双核和 G1 细胞周期停滞，同时表达 GTPase 缺陷的 G，这支持了 Gi α 及其调节因子在这些位点的重要性。 ??i1，但不是野生型版本，会导致有丝分裂失败。这些结果表明 Gi?? 的 GTP 循环由其调节因子调节的亚基对于正常细胞周期进展是必要的，并且与上述 Ric-8 数据一致。为了进一步研究 Rgs14，我们正在开发可以有条件删除 Rgs14 的小鼠。将正确靶向的胚胎干 (ES) 细胞注射到胚细胞中后获得的嵌合小鼠正在评估靶向 Rgs14 等位基因的种系传递。线虫 RGS7 在早期细胞分裂中发挥作用，RGS7 突变体显示有丝分裂纺锤体的超不对称运动。在哺乳动物 RGS 蛋白中，RGS3 与秀丽隐杆线虫 RGS7 最相似。由于广泛的选择性 mRNA 剪接，存在多种 RGS3 同工型。 RGS 的一种同工型 PDZ-RGS3 最初被鉴定为 β-肝配蛋白的结合伴侣，并与神经元中的 β-肝配蛋白反向信号传导有关。除了 C 端 RGS 结构域和 N 端 PDZ 结构域外，PDZ-RGS3 还拥有一个 C2A 结构域和一个与 Lim 结构域有些相似的未知结构域。 PDZ 结构域结合膜蛋白的 C 末端，并广泛参与形成亚膜支架以在细胞表面聚集分子。最近，我们发现 PDZ-RGS3 参与微管动力学和胞质分裂的调节。 PDZ-RGS3-GFP 主要定位于间期细胞的胞浆中，但在末期晚期被募集至中体。 PDZ-RGS3 表达水平的升高会导致胞质分裂的严重缺陷。使用RNA干扰来消除细胞中的PDZ-RGS3，共聚焦和视频延时显微镜的结合揭示了微管组织的破坏、纺锤体的伸长和核分离，导致有丝分裂退出和胞质分裂受阻。对与 PDZ-RGS3 的 PDZ 结构域共沉淀的蛋白质进行质谱分析，鉴定出 septin 蛋白质。证实了这种相互作用 PDZ-RGS3 与 septin Nedd5 共免疫沉淀并共定位。减少 PDZ-RGS3 表达导致 Nedd5 无法在中间区/中体积累。除了septin招募之外，PDZ-RGS3与极光B激酶在后期后期的纺锤体中区和末期后期的中体共定位。 Aurora B 激酶是参与有丝分裂和胞质分裂过程中正常染色体分离调节的关键酶。通过免疫共沉淀和 FRET 分析证实了 PDZ-RGS3 与极光 B 激酶的关联。内源性 PDZ-RGS3 的消耗导致极光 B 激酶在末期的空间方向缺陷。 为了进一步研究 Rgs3，我们获得了靶向删除 Rgs3 的小鼠。我们有两个独立的小鼠品系，每个品系都靶向删除 Rgs3。表型显着不同，一个品系无法产生可存活的后代，而另一品系则没有发现明显的异常。我们将两条品系回交到 C57/Bl6 背景上，以帮助找出两条品系之间的差异。