3'UTR-dependent regulation of the PTEN tumor suppressor gene

项目摘要 PTEN编码10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)，是一种肿瘤 抑癌基因在多种人类癌症中发生突变。然而，与其他肿瘤抑制因子不同的是， 基因，即使是PTEN活性的微小扰动也能促进肿瘤的发生，这表明它是高度 受监管的。PTEN的翻译后调节可以影响蛋白质的稳定性、定位和 构象以及磷酸酶活性，在许多情况下的影响是特定的细胞类型。 我们的实验室观察到，从长3‘非编码区异构体翻译而来的PTEN活性显著降低 在PI3K途径中，PTEN比PTEN从短的3‘UTR亚型翻译而来，并且表达 3‘非编码区亚型随组织和细胞类型的不同而变化。拟议研究的目标是 阐明3‘端非编码区调控PTEN活性增强的分子机制 我们对PTEN失调癌症发病机制的理解。这项提案有两个 明确的目标。目的1研究3‘非编码区依赖的PTEN的生物学效应 监管。因为没有观察到PTEN蛋白水平在细胞中的差异 表达长3‘非编码区亚型的细胞与主要表达短3’非编码区的细胞相比 异构体，假设是替代的异构体使用一定会影响亚细胞 定位、同源二聚化或磷酸酶活性。独占性shRNA介导 长3‘非编码区亚型的敲除将被用来产生主要表达 短3‘非编码区，这将与两种异构体表达相同的对照细胞进行比较。单元格 对不同株系进行内源性PTEN免疫组织化学染色，并进行亚细胞定位比较。 它们还将用于免疫沉淀主要从短3‘非编码区和 从两种异构体翻译的蛋白质来评估二聚化和磷酸酶活性的程度 受替代的3‘非编码区亚型使用的影响。在目标2中，这些细胞系将被用来鉴定 与长3‘非编码区亚型特异相互作用的蛋白质因子。我们的假设是 PTEN的长3‘非编码区招募了一种与新翻译的PTEN形成复合体的蛋白质， 从而抑制其在PI3K途径中的活性。长3‘非编码区特异的蛋白质相互作用 合作伙伴将通过质谱学确定，在目标1中获得的信息将是 被用来选择候选者，以验证他们在调节PTEN蛋白中的作用。ShRNA将会是 用于击倒候选交互合作伙伴。如果3‘Utr依赖的候选人是必需的 PTEN的调节，然后shRNA介导的敲除该候选将恢复PTEN的活性 从长的3‘非编码区亚型翻译成从短的3’非编码区亚型翻译的PTEN。

Project Summary PTEN, encoding phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is a tumor suppressor gene mutated in a variety of human cancers. However, unlike other tumor suppressor genes, even small perturbations in PTEN activity can promote tumorigenesis suggesting it is highly regulated. Post-translational regulation of PTEN can affect protein stability, localization, and conformation as well as phosphatase activity, and in many cases the effects are cell type specific. Our lab has observed that PTEN translated from the long 3'UTR isoform is significantly less active in the PI3K pathway than PTEN translated from the short 3'UTR isoform, and that the expression of 3'UTR isoforms varies across tissue and cell types. The goal of the proposed study is to elucidate the molecular mechanism of 3'UTR-dependent regulation of PTEN activity to augment our understanding of disease mechanisms in PTEN dysregulated cancers. The proposal has two specific aims. Aim 1 will investigate the biological consequence of 3'UTR-dependent PTEN regulation. Since there is no observed difference in PTEN protein levels in cells that predominantly express the long 3'UTR isoform compared to cells that predominantly express the short 3'UTR isoform, the hypothesis is that alternative isoform usage must effect either subcellular localization, homo-dimerization, or phosphatase activity. Exclusive shRNA-mediated knockdown of the long 3'UTR isoform will be used to generate cells that predominantly express the short 3'UTR, which will be compared to control cells that express both isoforms equally. The cell lines will be immunostained for endogenous PTEN and subcellular localization can be compared. They will also be used to immunoprecipitate protein translated from primarily the short 3'UTR and protein translated from both isoforms to assess how dimerization and phosphatase activity is affected by alternative 3'UTR isoform usage. In Aim 2, these cell lines will be used to identify protein factors that specifically interact with the long 3'UTR isoform. The hypothesis is that the long 3'UTR of PTEN recruits a protein that forms a complex with newly translated PTEN, thus repressing its activity in the PI3K pathway. Long 3'UTR-specific protein interaction partners will be identified by mass spectrometry and the information obtained in Aim 1 will be leveraged to select candidates to validate for their role in regulating PTEN protein. shRNA will be used to knockdown candidate interaction partners. If a candidate is required for 3'UTR-dependent PTEN regulation, then shRNA-mediated knockdown of this candidate will restore activity of PTEN translated from the long 3'UTR isoform to that of PTEN translated from the short 3'UTR isoform.