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

定义 ATR 对 MC1R 增强黑素细胞 DNA 修复的贡献

• 批准号: 9026277
• 负责人: John A D'Orazio
• 金额: $ 33.86万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9026277
• 关键词: A kinase anchoring protein; ATR gene; Accounting; Address; Adenylate Cyclase; Affect; American; Ataxia; Binding; Biological Assay; Cancer Etiology; Cells; Cessation of life; Co-Immunoprecipitations; Complex; Coupled; Coupling; Cutaneous; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Binding; DNA Damage; DNA Repair; Defect; Development; Diagnosis; ERCC1 gene; Event; Funding; Generations; Genetic Polymorphism; Genome; Goals; Gravin; Incidence; Individual; Inherited; Laboratories; Left; Ligation; Link; Maintenance; Malignant Neoplasms; Measures; Mediating; Melanins; Melanocortin 1 Receptor; Melanocyte stimulating hormone; Melanoma Cell; Membrane; Modeling; Molecular; Mus; Mutagenesis; Mutagens; Mutation; Nuclear; Nucleotide Excision Repair; Oligonucleotides; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Pigmentation physiologic function; Population; Positioning Attribute; Post-Translational Protein Processing; Prevention strategy; Preventive therapy; Production; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Proteomics; Public Health; Radiation therapy; Receptor Signaling; Regulation; Repair Complex; Reporting; Resistance; Retrieval; Risk; Risk Factors; Role; Seminal; Signal Transduction; Skin; Skin Cancer; Surface; Surgical incisions; Tissues; UV Radiation Exposure; UV response; Ultraviolet Rays; United States; Woman; Xeroderma Pigmentosum; base; cell type; chemotherapy; defined contribution; design; high risk; loss of function; melanocyte; melanoma; men; mortality; prevent; receptor; repaired; research study; response; therapy development; ultraviolet; ultraviolet damage

 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.

 描述（由申请人提供）：黑色素瘤是最致命的皮肤恶性肿瘤，是癌症死亡的重要原因，通常影响壮年男性和女性。促黑素细胞激素（MSH）-黑皮质素-1受体（MC 1 R）信号传导轴是一种可诱导的皮肤途径，调节黑素细胞中的紫外线（UV）反应。MC 1 R信号的功能丧失多态性，仅在美国就影响数百万人，终生黑色素瘤风险增加一倍以上。通过MC 1 R（一种GS偶联膜受体）的信号传导导致腺苷酸环化酶的激活、cAMP的产生和黑素细胞修复UV损伤的DNA的能力的增强，如果不修复，则导致“UV特征”突变，从而促进黑素细胞进展为黑素瘤。因此，DNA修复反应迟钝的MC 1 R缺陷个体在紫外线暴露后积累了更多的突变，易患黑色素瘤。我们的实验室确定了一个关键的分子途径，连接MC 1 R/cAMP信号传导到核苷酸切除修复（NER），基因组维护途径负责从DNA中去除紫外线损伤的碱基。cAMP依赖性蛋白激酶（PKA）通过MC 1 R信号传导和cAMP生成激活，使S435残基上的共济失调和rad 3相关（ATR）蛋白磷酸化。这种翻译后修饰导致ATR与NER因子着色性干皮病A（XPA）相关联，加速其与核光损伤的相互作用并增强DNA修复。上一个资助周期的令人信服的发现表明，A激酶锚定蛋白12（AKAP 12）整合了PKA-ATR-XPA相互作用。我们推测，通过AKAP调节PKA介导的ATR磷酸化，MC 1 R信号通过增强NER保护黑素细胞免受UV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，并了解它如何影响NER。在第一个目标中提出的实验将使用共定位、激酶测定、邻近连接测定（PLA）、蛋白质组学和UV诱导的小鼠黑素瘤模型来确定AKAP 12如何调节黑素细胞中MC 1 R增强的NER。第二个目标中提出的研究将通过使用免疫共沉淀、PLA、荧光链切割试验和寡核苷酸修复试验（ORA）作为NER的功能性测量，重点研究DNA结合和链切割的机制，确定pS435 ATR如何影响NER。最后，由于我们已经确定失调的pS435信号使细胞对遗传毒性剂高度敏感，第三个目标将定义p-S435 ATR如何在黑素细胞中失活，重点关注PP 2A磷酸酶在pS435 ATR失活中的作用，并确定持续的pS435 ATR如何使黑素瘤细胞对DNA损伤剂敏感。总之，这些研究将确定MC 1 R信号传导如何促进NER和UV抗性，并将作为在高风险MC 1 R缺陷人群中制定合理的黑色素瘤预防策略的平台。