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

项目摘要 编码10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）的PTEN是一种肿瘤， 在多种人类癌症中突变的抑制基因。然而，与其他肿瘤抑制因子不同， 基因，即使是小的干扰，在PTEN活性可以促进肿瘤发生，这表明它是高度 监管. PTEN的翻译后调节可以影响蛋白质的稳定性、定位和表达。 构象以及磷酸酶活性，并且在许多情况下，该作用是细胞类型特异性的。 我们的实验室已经观察到，从长3'UTR同种型翻译的PTEN活性显著降低， 在PI3K途径中，PTEN的表达比从短的3'UTR亚型翻译的要高， 3'UTR同种型在组织和细胞类型中不同。拟议研究的目标是 阐明了3'UTR依赖性调节PTEN活性以增强其表达的分子机制。 我们对PTEN失调癌症的疾病机制的理解。该提案有两个 具体目标。目的1研究3'UTR依赖性PTEN的生物学效应 调控由于在主要表达PTEN的细胞中没有观察到PTEN蛋白水平的差异， 与主要表达短3'UTR的细胞相比， 同种型，假设是替代同种型的使用必须影响亚细胞 定位、同源二聚化或磷酸酶活性。排除性shRNA介导 长3'UTR同种型的敲低将用于产生主要表达 短的3'UTR，其将与同等表达两种同种型的对照细胞进行比较。细胞 对细胞系进行内源性PTEN的免疫染色，并比较亚细胞定位。 它们还将用于免疫沉淀主要从短3'UTR翻译的蛋白质， 从两种同种型翻译的蛋白质，以评估二聚化和磷酸酶活性是如何 受替代3'UTR同种型使用的影响。在目标2中，这些细胞系将用于鉴定 与长3'UTR同种型特异性相互作用的蛋白因子。假设是， PTEN的长3 'UTR招募一种与新翻译的PTEN形成复合物的蛋白质， 从而抑制其在PI3K途径中的活性。长3'UTR特异性蛋白质相互作用 伙伴将通过质谱鉴定，目标1中获得的信息将 用于选择候选物以验证它们在调节PTEN蛋白中的作用。shRNA将 用于击倒候选交互伙伴。如果候选人需要3'UTR依赖型 PTEN的调控，然后shRNA介导的敲低该候选物将恢复PTEN的活性。 从长的3'UTR同种型翻译的PTEN的基因转化为从短的3'UTR同种型翻译的PTEN的基因。

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.