title: c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks.
abstract: The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1-dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage-independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη-PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.
authors.affiliation: National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
authors.forename: Laura R
authors.initials: LR
authors.lastname: Barkley
authors.forename: Komaraiah
authors.initials: K
authors.lastname: Palle
authors.forename: Michael
authors.initials: M
authors.lastname: Durando
authors.forename: Tovah A
authors.initials: TA
authors.lastname: Day
authors.forename: Aditi
authors.initials: A
authors.lastname: Gurkar
authors.forename: Naoko
authors.initials: N
authors.lastname: Kakusho
authors.forename: Jianying
authors.initials: J
authors.lastname: Li
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
authors.forename: Cyrus
authors.initials: C
authors.lastname: Vaziri
mesh_terms: D020816:Amino Acid Motifs; D045744:Cell Line, Tumor; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012694:Serine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D000099270:Y-Family DNA Polymerases
chemical_list: D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D048031:JNK Mitogen-Activated Protein Kinases; D018809:Proliferating Cell Nuclear Antigen; D012694:Serine; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C471545:UBE2A protein, human; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein