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（由适用提供）：黑色素瘤是最致命的皮肤恶性肿瘤，是癌症死亡率的重要原因，通常会影响男性和女性。黑素细胞刺激的马酮（MSH）-Melanocortin-1受体（MC1R）信号轴是一种可诱导的皮肤途径，可调节黑素细胞中紫外线（UV）反应。 MC1R信号传导的功能丧失多态性，仅影响美国数百万，超过两倍的寿命黑色素瘤风险。通过MC1R（一种GS耦合的膜受体）通过MC1R发出信号，导致腺苷周期的激活，cAMP的产生以及黑素细胞修复紫外线损伤的DNA的能力，如果没有留下未经修复的紫外线引起的“ UV签名”突变，则使梅拉氏菌进入蜂妈素瘤的进展。结果，具有钝化DNA修复反应的MC1R缺陷个体在暴露紫外线后会积累更多突变，并且易于黑色素瘤。我们的实验室确定了一个关键的分子途径，将MC1R/CAMP信号与核丁基惊喜修复（NER）联系起来，这是负责从DNA中去除紫外线损伤碱基的基因组维持途径。由MC1R信号传导和cAMP产生激活，cAMP依赖性蛋白激酶（PKA）磷酸化了S435住所上的共济失调和RAD3相关（ATR）蛋白。这种翻译后的修饰使ATR与NER因子Xeroderma色素A（XPA）相关联，从而加速了其与核光损伤的相互作用并增强了DNA修复。在先前的融资周期中，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。我们假设通过AKAP调节的PKA介导的ATR辐射化，MC1R信号传导通过增强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如何调节PKA-ATR-XPA DNA修复轴并了解其对NER的影响。在第一个目标中提出的实验将决定AKAP12如何使用共定位，激酶测定，接近结扎测定法（PLA），蛋白质组学和UV-诱导的小鼠黑色素瘤模型来调节黑色素细胞中MC1R增强NER。在第二个目标中提出的研究将通过使用共免疫沉淀，PLA，荧光链切口测定法和寡核苷酸检索测定（ORA）来确定PS435 ATR如何通过关注DNA结合和链切口的机制来影响NER。最后，由于我们已经确定PS435信号的失调使细胞深刻地感知细胞对遗传毒性剂，因此第三个目标将定义在黑色素细胞中PP2A磷酸酶在PS435 ATR失活中的作用的PP2A磷酸酶的作用，并确定PP2A磷酸酶在黑色素形成细胞中的作用，并确定如何耐受PS435 ATRESS SENTES SENTES SENTES SENSMOMAMAMAMAMAMALY SENTRAMAMAS MELANAMA SENTRAMAS MELANomAS搅拌搅拌。总之，这些研究将定义MC1R信号如何促进NER和UV耐药性，并将成为发展高风险MC1R缺陷人群中有理黑色素瘤策略的平台。