RKTG对Gβγ的空间调控作用以及RKTG/p53基因剔除小鼠的功能分析
文献类型：学位论文
| 作者 | 蒋玉辉 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-05-17 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 陈雁 |
| 关键词 | RKTG G protein βγ 亚基 GRK AKT p53 |
| 其他题名 | Spatial regulation of Gβγ by RKTG and function analysis of RKTG/p53 knockout mice |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | RKTG (能将Raf激酶锚定到高尔基体的蛋白)是一个认知不久的定位于高尔基体的膜蛋白。之前的研究结果表明RKTG是一个潜在的抑癌基因。RKTG基因剔除小鼠生存和发育正常，没有明显的表型。然而，在药物诱导的皮肤肿瘤模型中，RKTG基因剔除后明显促进了小鼠皮肤肿瘤的生成。 GPCR受到配体刺激后，将信号传递给G蛋白三聚体: Gα， Gβ， 和Gγ亚基，继而，Gα与Gβγ亚基分离。虽然Gα 对下游信号通路传递很重要，但Gβγ亚基本身也可以介导一系列细胞内生理反应，比如说GRK介导的GPCR去敏感化以及AKT的活化。在这里，我们首先发现报道了Gβγ亚基在细胞房室化位置改变的空间调控。RKTG通过其N端与Gβγ发生结合,将Gβγ锚定至高尔基体.过表达RKTG干扰了Gβγ与GRK2结合,改变了配体刺激后GRK2在细胞内的分布,以及β-2AR的磷酸化水平，从而改变了β-2AR的脱敏反应。此外, 过表达RKTG抑制了Gβγ过表达以及配体刺激导致的AKT磷酸化水平。而RKTG敲减后增强了Gβγ介导的AKT激活。 同时，RKTG敲减也改变了GRK2在配体刺激后内吞的趋势以及Gβ在配体刺激后向高尔基体的转运。 接下来，我们在RKTG剔除小鼠胚胎成纤维细胞中发现，溶血磷酯酸 (LPA)但不是表皮生长因子 (EGF)能够进一步增强AKT以及GSK3β的磷酸化，同时伴随着p53，p53丝氨酸15磷酸化以及p53靶基因p21，p16的上调。p53基因是一个被研究很深入的抑癌基因。 p53杂合缺失小鼠在12个月左右开始发生肉瘤。我们繁殖了RKTG/p53双剔除小鼠，发现在RKTG纯合p53杂合缺失小鼠中在7个月内有大约25%的小鼠有自发性皮肤肿瘤发生。在肿瘤皮肤部位可以观察到高度增殖以及上皮向间质细胞转化现象，另外还可以观察到p53杂合丢失的现象。在p53突变的A431皮肤肿瘤细胞系中，敲减掉RKTG导致LPA刺激的AKT和GSK3β磷酸化进一步增强， 同时β-catenin量也有增强，细胞出现了EMT现象。而EMT现象可以被过表达的野生型p53回复。在HepG2这种含有野生型p53的上皮细胞系中，当RKTG和p53同时敲减时，可以导致细胞在LPA刺激下发生最大程度的EMT。总而言之，这些结果表明RKTG作为一个抑癌基因能够协同p53抵抗肿瘤的发生，同时说明p53能起到一个EMT关卡的作用。因而p53与RKTG的共同丢失能推动EMT以及肿瘤的发生。 |
| 索取号 | D2011-001 |
| 英文摘要 | RKTG (Raf kinase trapping to Golgi apparatus) is a newly characterized membrane protein specifically localized at the Golgi apparatus. Previous studies indicated that RKTG is a potential tumor suppressor gene. Interestingly, RKTG-deficient mice are free of tumors, although they are prone to form skin cancer on carcinogen administration. Upon ligand binding, G-protein-coupled receptors (GPCRs) impart the signal to heterotrimeric G proteins composed of α, β, and γ subunits, leading to dissociation of the Gα subunit from the Gβγ subunit. While the Gα subunit is imperative for downstream signaling, the Gβγ subunit, in its own right, mediates a variety of cellular responses such as GPCR desensitization via recruiting GRK to the plasma membrane and AKT stimulation. Firstly, we report a mode of spatial regulation of the Gβγ subunit through alteration in subcellular compartmentation. The N terminus of RKTG interacts with Gβ and tethers Gβ to the Golgi apparatus. Overexpression of RKTG impedes theinteraction of Gβ with GRK2, abrogates the ligand-inducedchange of subcellular distribution of GRK2, reduces isoproterenol-stimulatedphosphorylation of the β2-adrenergic receptor (β2AR),and alters β2AR desensitization. In addition, RKTG inhibitsGβ- and ligand-mediated AKT phosphorylation that is enhancedin cells with downregulation of RKTG. Silencing of RKTG alsoalters GRK2 internalization and compromises ligand-induced Gβtranslocation to the Golgi apparatus. Next, we found in RKTG-null mouse embryonic fibroblasts, lypophosphatidic acid (LPA) but not EGF could stimulate hyperphosphorylation of AKT and GSK3β, accompanied by increases in phosphorylation of p53 at Ser15 and accumulation of p53 as well as its target genes p21 and p16. p53 is a well-characterized tumor suppressor gene and p53-heterozygous mice develop sarcoma and other tumors starting from 12 months of age. Next, we developed the RKTG/p53 double knockout mouse, and found spontaneous skin cancer-like tumors in ~25% of RKTG-nullizygous and p53-heterozygous mice within 7 months of age. Hyperplasia and epithelial-mesenchymal transition (EMT) were observed in the tumor-overlaying epidermis, in which loss of heterozygosity of p53 occurred and EMT features emerged. In p53-mutated A431 epithelial carcinoma cells, knockdown of RKTG led to enhancement of LPA-stimulated AKT and GSK3β phosphorylation, together with increased accumulation of β-catenin and appearance of EMT features that were antagonized by p53 overexpression. In HepG2 epithelial cells, LPA-stimulated AKT phosphorylation and EMT features reached maximum when both RKTG and p53 were simultaneously silenced. In summary, these results not only indicate that RKTG has an in vivo tumor suppressor function to cooperate with of p53 in tumorigenesis, but also suggest that p53 has a EMT checkpoint function and the loss of this function can combine with loss of RKTG to drive EMT and tumor progression。 |
| 语种 | 中文 |
| 源URL | [http://202.127.25.144/handle/331004/114]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_信号转导与营养相关疾病研究组 |
| 推荐引用方式 GB/T 7714 | 蒋玉辉. RKTG对Gβγ的空间调控作用以及RKTG/p53基因剔除小鼠的功能分析[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2011. |
入库方式： OAI收割
来源：上海营养与健康研究所
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