Transforming growth factor beta signaling pathways

描述（由申请人提供）：我们的长期目标是了解调节disabled-2（Dab 2）蛋白表达水平的分子机制。Dab 2是与网格蛋白介导的内吞作用相关的细胞质衔接分子。它最初被鉴定为在卵巢癌中下调的蛋白质，并且已经确定其表达水平对细胞增殖、分化和癌症发展具有显著影响。由于其在卵巢癌、乳腺癌、前列腺癌和胰腺癌细胞中的表达水平降低，已被指定为推定的肿瘤抑制因子。我们已经获得的初步数据表明，除了转录调控，细胞Dab 2的表达水平也保持通过转录后机制，涉及转录特异性的翻译控制和泛素介导的蛋白酶体降解。在两种细胞分化系统中，即小鼠乳腺上皮（NMuMG）细胞中TGF β介导的上皮至间充质转分化模型和F9畸胎瘤细胞中维甲酸（RA）诱导的内脏内胚层分化模型，我们提供的数据表明，单独对Dab 2 mRNA的转录诱导不能解释诱导的Dab 2蛋白表达水平。我们证明了在基础条件下，Dab 2表达水平通过Dab 2 3 '-非翻译区（3'-UTR）中所含序列元件驱动的机制被顺序沉默。来自TGF β处理的NMuMG或RA处理的F9细胞的胞质提取物可以克服这种3 '-UTR介导的沉默，导致Dab 2蛋白表达增加。我们进一步表明，一旦合成，Dab 2水平通过GSK-3 β依赖性磷酸化和泛素介导的降解途径进行调节。这些结果表明Dab 2的表达是高度调节的，并支持其表达水平是其增殖和分化功能的基础的假设。在这个提议中，我们希望进一步探索Dab 2表达水平调节的分子机制，并确定这些机制的失调是否可能是其在癌症中异常表达的基础。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms regulating disabled-2 (Dab2) protein expression levels. Dab2 is a cytoplasmic adaptor molecule that is associated with clathrin mediated endocytosis. It was originally identified as a protein down-regulated in ovarian carcinoma and it has since been established that its expression levels have significant impact on cellular proliferation, differentiation, and cancer development. It has been designated as a putative tumor suppressor due to its decreased expression levels in ovarian, breast, prostate and pancreatic carcinoma cells. We have obtained preliminary data suggesting that, in addition to transcriptional regulation, cellular Dab2 expression levels are also maintained through post-transcriptional mechanisms involving transcript-specific translational control and ubiquitin-mediated proteasomal degradation. In two cellular differentiation systems, a TGFbeta-mediated epithelial to mesenchymal transdifferentiation model in mouse mammary epithelial (NMuMG) cells and a retinoic acid (RA)-induced visceral endoderm differentiation model in F9 teratocarcinoma cells, we present data suggesting that transcriptional induction of Dab2 mRNA alone can not account for induced Dab2 protein expression levels. We demonstrate that under basal .conditions, Dab2 expression levels are translationally silenced through a mechanism driven by sequence elements contained in the Dab2 3'-untranslated region (3'-UTR). Cytosolic extracts from TGFbeta-treated NMuMG or RA-treated F9 cells can overcome this 3'- UTR-mediated silencing, resulting in increased Dab2 protein expression. We further show that once synthesized, Dab2 levels are regulated through a GSK-3beta-dependent phosphorylation and ubiquitin-mediated degradation pathway. These results demonstrate that Dab2 expression is highly regulated and support the hypothesis that its expression levels underlie its proliferate and differentiative functions. In this proposal, we wish to further explore the molecular mechanisms through which Dab2 expression levels are regulated and determine whether dysregulation of these mechanisms might underlie its aberrant expression in cancer.