Abstract

Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA’s associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA’s associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.

Highlights

  • DNA damage was dependent on protein kinase A (PKA)-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key nucleotide excision repair (NER) factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage

  • We found that cAMP signaling enhanced levels of ATR-pS435 in cisplatin-damaged chromatin in MC1RWT-expressing cells treated with MSH compared

  • We previously reported that a key molecular event linking melanocortin 1 receptor (MC1R) signaling to NER is cAMP-induced activation of PKA, which phosphorylates ATR on the S435 residue and leads to recruitment of the NER factor XPA and co-localization of the ATR-pS435-XPA complex to sites of UV photodamage to enhance NER19

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Summary

D Results

MC1R/cAMP signaling enhances the repair of cisplatin-induced DNA damage. E whether cAMP signaling affected repair of DNA damage caused by platinum-based chemotherapeutics, we measured DNA repair kinetics in HEK293 cells pre-treated with either vehicle or forskolin, an adenylyl cyclase activator that induces a strong cAMP signal independently of MC1R status. We determined whether MC1R signaling influenced repair efficiency of cisplatin-mediated DNA damage. MSH treatment of cells expressing functional MC1R (PHM and HEK293-MC1R-WT) resulted in significantly increased repair efficiency compared to vehicle (Table 1 and Fig. S1). The presence of MC1R did not appear to impact cellular sensitivity to cisplatin in HEK293 cells (Fig. S2) Together, these data strongly support an integral role for MC1R/cAMP to accelerate clearance of platinum DNA adducts. ATR-pS435 generation occurred in response to MC1R signaling as demonstrated by robust induction of MC1R-intact cells treated with either MSH or forskolin (Table 2 and Fig. S3). MC1R-dependence of ATR-pS435 was demonstrated by observing the lack of induction by MSH

E HEK293-MC1R-WT RTICL HEK293-MC1R-R151C
Findings
C Vehicle I Forskolin
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