UV and p21 Degradation

描述(申请人提供)：p21WAF1/CIP1是肿瘤抑制基因p53的转录靶点。P21蛋白在电离辐射后的细胞周期停滞、某些形式的癌症、自身免疫和干细胞再繁殖中起着至关重要的作用。虽然p21基因转录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。在初步结果中，我们发现紫外线可以诱导p21的降解。我们表明，虽然低剂量的紫外线会导致p21的降解，但高剂量的紫外线不会。紫外线诱导p21降解的能力依赖于泛素和F盒蛋白Skp2。我们的结果表明，紫外线对细胞周期的抑制是p21非依赖性的，而不是经典的电离辐射对p21的依赖。最后，我们发现紫外线照射后不能降解p21会抑制增殖细胞核抗原对染色质的募集和DNA修复合成。在这个提案中，我们将研究哺乳动物细胞对紫外线的三个方面的反应以及p21在这一过程中所起的关键作用。首先，我们将确定在低剂量紫外线照射后，导致p21降解的ATR下游信号蛋白。其次，我们将确定使p21磷酸化的激酶和p21中决定紫外线后Skp2/p21结合的磷酸化位点。我们还将检查是否依赖Skp2的UV诱导的p21降解也依赖于Cks1，并允许我们在体外重建UV诱导的p21泛素化。第三，我们将建立细胞p21不能泛素化的完整动物模型，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.