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.
L: Melanoma, the deadliest form of skin cancer, affects nearly 80,000 individuals in the United States each year. The proposed study focuses on the role of a receptor known as the melanocortin 1 receptor (MC1R) that is important in normal pigmentation. Inherited defects in MC1R affect millions of Americans who are at elevated risk from ultraviolet light damage and mutations and as a result have a high risk for melanoma. By discovering how MC1R protects against melanoma formation, our goal is to develop therapies to prevent melanoma.
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