Defining the contribution of ATR to MC1R-enhanced DNA repair in melanocytes

 描述(申请人提供)：黑色素瘤，最致命的皮肤恶性肿瘤，是癌症死亡的重要原因，通常影响到年富力强的男性和女性。黑素细胞刺激素(MSH)-黑素皮质素-1受体(MC1R)信号轴是一种可诱导的皮肤通路，调节黑素细胞对紫外线的反应。MC1R信号的功能缺失多态，仅在美国就影响了数百万人，终身患黑色素瘤的风险增加了一倍以上。通过一种Gs偶联的膜受体MC1R发出信号，导致腺苷环化酶的激活，cAMP的产生，并增强黑素细胞修复紫外线损伤DNA的能力，如果不修复，会导致“紫外线信号”突变，促进黑素细胞向黑色素瘤的进展。因此，DNA修复反应迟钝的MC1R缺陷个体在紫外线照射后积累了更多的突变，容易患上黑色素瘤。我们的实验室发现了一条将MC1R/cAMP信号与核苷酸切除修复(NER)联系起来的关键分子途径，NER是负责从DNA中移除紫外线损伤碱基的基因组维持途径。在MC1R信号和cAMP生成的激活下，cAMP依赖的蛋白激酶(PKA)磷酸化S435残基上的共济失调和Rad3相关(ATR)蛋白。这种翻译后修饰使ATR与NER因子着色性干皮病A(XPA)结合，加速其与核光损伤的相互作用，促进DNA修复。在前一个资金周期中令人信服的发现表明，A-激酶锚定蛋白12(AKAP12)整合了PKA-ATR-XPA相互作用。我们推测，通过AKAP调节的PKA介导的ATR磷酸化，MC1R信号通过增强NER来保护黑素细胞免受紫外线诱变。该项目的总体目标是确定PKA-ATR-XPA DNA修复轴是如何调节的，并了解它是如何影响NER的。第一个目标中提出的实验将通过共定位、蛋白组学和紫外线诱导的小鼠黑色素瘤模型来确定AKAP12如何调节黑素细胞中MC1R增强的NER，方法包括共定位、激酶分析、邻近连接分析(PLA)、蛋白质组学和紫外线诱导小鼠黑色素瘤模型。第二个目标是通过免疫共沉淀、聚乳酸、荧光链切割试验和寡核苷酸回收试验(ORA)作为NER的功能指标，重点研究DNA结合和链切割的机制，以确定pS435 ATR如何影响NER。最后，由于我们已经确定了失调的pS435信号使细胞对遗传毒物深刻敏感，第三个目的将定义p-S435 ATR在黑素细胞中如何失活，重点是PP2A磷酸酶在pS435 ATR失活中的作用，并确定持续的pS435 ATR如何使黑色素瘤细胞对DNA损伤剂敏感。总之，这些研究将确定MC1R信号如何促进NER和紫外线抵抗，并将作为在高危MC1R缺陷人群中开发合理的黑色素瘤预防策略的平台。

 DESCRIPTION (provided by applicant): Melanoma, the most lethal skin malignancy, is a significant cause of cancer mortality, often affecting men and women in their prime. The melanocyte-stimulating hormone (MSH)-melanocortin-1 receptor (MC1R) signaling axis is an inducible cutaneous pathway that regulates ultraviolet (UV) responses in melanocytes. Loss-of- function polymorphisms of MC1R signaling, affecting millions in the United States alone, more than double lifetime melanoma risk . Signaling through the MC1R, a Gs-coupled membrane receptor, leads to activation of adenylyl cyclase, production of cAMP and enhancement of the ability of melanocytes to repair UV-damaged DNA that if left unrepaired causes "UV signature" mutations that fuel progression of melanocytes into melanoma. As a result, MC1R-defective individuals with blunted DNA repair responses accumulate more mutations after UV exposure and are predisposed to melanoma. Our laboratory identified a critical molecular pathway linking MC1R/cAMP signaling to nucleotide excision repair (NER), the genome maintenance pathway responsible for removing UV-damaged bases from DNA. Activated by MC1R signaling and cAMP generation, cAMP-dependent protein kinase (PKA) phosphorylates the ataxia and rad3 related (ATR) protein on the S435 residue. This post-translational modification causes ATR to associate with the NER factor xeroderma pigmentosum A (XPA), accelerating its interaction with nuclear photodamage and enhancing DNA repair. Compelling findings during the previous funding cycle indicate that A-kinase anchoring protein 12 (AKAP12) integrates PKA-ATR-XPA interactions. We hypothesize that through AKAP-regulated PKA-mediated ATR phosphorylation, MC1R signaling protects melanocytes from UV mutagenesis by enhancing NER. The overall goal of this project is to determine how the PKA-ATR-XPA DNA repair axis is regulated and to understand how it impacts NER. Experiments proposed in the first Aim will determine how AKAP12 regulates MC1R-enhanced NER in melanocytes using co-localization, kinase assays, proximity ligation assay (PLA), proteomics and a UV-inducible mouse melanoma model. Studies proposed in the second Aim will identify how pS435 ATR impacts NER by focusing on mechanisms of DNA binding and strand incision using co-immunoprecipitation, PLA, fluorescent strand incision assay and oligonucleotide retrieval assay (ORA) as a functional measure of NER. Finally, since we have determined that dysregulated pS435 signal profoundly sensitizes cells to genotoxic agents, the third aim will define how p-S435 ATR is inactivated in melanocytes, focusing on the role of PP2A phosphatase in pS435 ATR inactivation and determining how persistent pS435 ATR sensitizes melanoma cells to DNA damaging agents. Together, these studies will define how MC1R signaling promotes NER and UV resistance and will serve as a platform for the development of rational melanoma-preventive strategies among high-risk MC1R-defective populations.