Defining the Contribution of MC1R Physiological Ligands to ATR Phosphorylation at Ser435, a Predictor of DNA Repair in Melanocytes

Abstract

The melanocortin 1 receptor (MC1R), a GS-coupled receptor that signals through cAMP and PKA, regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions and assess NER function. We report that melanocyte stimulating hormone (α-MSH) or adrenocorticotropic hormone (ACTH) induce ATR-pS435, enhance XPA’s association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human β-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction and reduce DNA repair. Although ASIP and HBD3 each blocked α-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.

Retracted: AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair

“…To further interrogate the role of ATR's kinase activity for AKAP12's ability to associate with UV-damaged DNA, we utilized a technique termed ‘ORiP’ to measure protein–DNA interactions (53) using chromatin fractions isolated from HEK293 cells expressing either wild-type ATR or a kinase dead variant (ATR-KD). The ORiP assay relies on UV exposure or mock treatment of cells, isolation of chromatin fractions, incubation with a biotinylated oligonucleotide construct containing either a non-damaged or UV-damaged fragment, retrieval of the oligonucleotide by streptavidin and identification of bound proteins.…”

“…To test this, in vitro kinase assays using an ATR peptide containing S435 and an ATR-pS435 phosphospecific antibody were performed (53). We tested the ability of HEK293 cells expressing wild-type AKAP12 or AKAP12 ΔPKA to support S435 phosphorylation of ATR.…”

“…A 30-nt oligonucleotide, 5′-CTCGTCAGCATCTTCATCATACAGTCAGTG-3′, was exposed to 10 J/m 2 of UVC, annealed and ligated with two oligonucleotides as previously described (43,44) for AKAP12 binding studies. For XPA and ERCC1-XPF binding a 5′-biotinylated stem-loop substrate (GCCAGCGCTCGG(T) 22 CCGAGCGCTGGC) was utilized (45).…”

Enhancement of UV-induced nucleotide excision repair activity upon forskolin treatment is cell growth-dependent

“…3B). Activation of the PKA pathway triggers NER activity by mediating phosphorylation of ATR at Ser435, which enhances XPA recruitment to sites of UV photolesions (14, 18). To determine whether FSK’s growth-dependent effect on NER activity is regulated by ATR, we treated cells with the ATR-specific inhibitor VE822 before UV irradiation.…”

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment

“…Melanoma cells also have DNA damage checkpoint defects-70% of cutaneous melanoma cell lines demonstrate defective G1 checkpoint arrest (Carson et al, 2012). Finally, some patients predisposed to melanoma have mutations in the MC1R receptor which is known to activate DNA repair mechanisms (Cassidy et al, 2015) by modulating the activity of the ATR kinase (Jarrett et al, 2015; Jarrett et al, 2014). …”