UV and p21 Degradation

描述（由申请人提供）：p21WAF1/CIP1是肿瘤抑制因子p53的转录靶点。P21蛋白在电离辐射后的细胞周期阻滞、某些形式的癌症、自身免疫和干细胞再生中发挥重要作用。尽管汇聚在p21基因转录上的途径已经得到了很好的研究，但在这里，我们研究了调节p21蛋白稳定性的机制及其对p21功能的影响。初步结果表明，紫外诱导p21降解。我们表明，虽然低剂量的紫外线诱导p21降解，高剂量则不会。UV诱导p21降解的能力依赖于泛素和F box蛋白Skp2。我们的研究结果表明，与传统的电离辐射的p21依赖性阻滞相比，紫外线的细胞周期阻滞与p21无关。最后，我们发现紫外线照射后不能降解p21抑制了PCNA向染色质的募集和DMA修复合成。在本提案中，我们将研究哺乳动物细胞对紫外线反应的三个方面以及p21在这一过程中所起的关键作用。首先，我们将鉴定ATR下游诱导p21在低剂量紫外线下降解的信号蛋白。其次，我们将确定磷酸化p21的激酶和p21中决定Skp2 / p21在紫外线照射后结合的磷酸化位点。我们还将研究Skp2依赖的紫外线诱导的p21降解是否也依赖于Cks1，并允许我们在体外重建紫外线诱导的p21泛素化。第三，我们将生成细胞p21不能泛素化的全动物模型，并确定这种突变的生物学后果。这将包括对细胞周期、检查点信号和皮肤癌的影响。

DESCRIPTION (provided by applicant): p21WAF1/CIP1 is a transcriptional target of the tumor suppressor p53. p21 protein plays an essential role in cell cycle arrest after ionizing radiation, in certain forms of cancer, autoimmunity and stem cell repopulation. Although pathways that converge on p21 gene transcription are well studied, here we examine the mechanisms that regulate p21 protein stability and their effect on p21 function. In preliminary results we show that UV induces p21 degradation. We show that while low doses of UV induce p21 degradation high doses do not. The ability of UV to induce p21 degradation is dependent on ubiquitin and the F box protein Skp2. Our results show that the cell cycle arrest by UV is p21 independent in contrast to the classical p21 dependent arrest by ionizing radiation. Finally we show that failure to degrade p21 after UV irradiation inhibits both the recruitment of PCNA to chromatin and DMA repair synthesis. In this proposal we will study three aspects of the mammalian cellular response to UV and the critical role p21 plays in this process. First, we will identify the signaling proteins downstream of ATR which induce p21 degradation after low doses of UV. Second, we will identify the kinase which phosphorylates p21 and the phosphorylation site in p21 which dictates Skp2 / p21 binding after UV. We will also examine if Skp2 dependent UV induced p21 degradation is also dependent on Cks1 and allow us to reconstitute UV induced p21 ubiquitination in vitro. Third, we will generate whole animal models in which cellular p21 cannot be ubiquitinated and determine the biological consequences of such a mutation. This will include the effect on cell cycle, checkpoint signaling and skin cancer.