c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks

Mol Biol Cell. 2012 May;23(10):1943-54. doi: 10.1091/mbc.E11-10-0829. Epub 2012 Mar 28.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Cell Line, Tumor
  • DNA Damage
  • DNA Replication*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / metabolism*
  • Humans
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • Molecular Sequence Data
  • Phosphorylation
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational*
  • Protein Transport
  • Serine / metabolism
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases


  • DNA-Binding Proteins
  • Proliferating Cell Nuclear Antigen
  • RAD18 protein, human
  • Serine
  • UBE2A protein, human
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases
  • JNK Mitogen-Activated Protein Kinases
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Rad30 protein