JNK-mediated RAD18 phosphorylation promotes colorectal cancer progression through K48-linked ubiquitination and degradation of SFN

Abstract Colorectal cancer (CRC) remains a major cause of cancer-related mortality, highlighting the need to further elucidate the molecular mechanisms underlying its progression. RAD18, an E3 ubiquitin ligase with established roles in DNA damage tolerance and tumor progression, is aberrantly expressed in multiple malignancies; however, the substrate-specific mechanisms underlying its tumor-promoting function in CRC remain incompletely understood. In this study, we investigated the functional and molecular roles of RAD18 in CRC using public datasets, clinical specimens, CRC cell lines, patient-derived organoids, and xenograft models. Protein interactions and ubiquitination were examined using co-immunoprecipitation, mass spectrometry, cycloheximide-chase, and ubiquitination assays, together with mutant constructs and functional rescue experiments. RAD18 was significantly upregulated in CRC and was associated with poor prognosis. RAD18 promoted CRC cell proliferation and cell-cycle progression, whereas its depletion induced G2/M arrest. Mechanistically, RAD18 interacted with SFN through its RING domain and catalyzed K48-linked ubiquitination of SFN at lysine 122 (K122), leading to its proteasomal degradation. JNK-mediated phosphorylation of RAD18 at serine 409 (S409) enhanced the RAD18–SFN interaction and subsequent SFN ubiquitination. Functional analyses using the phosphorylation-deficient RAD18-S409A mutant and the ubiquitination-resistant SFN-K122R mutant further supported a critical role for phosphorylation-regulated SFN degradation in the proliferative and cell-cycle effects of RAD18. Collectively, these findings identify a JNK–RAD18–SFN regulatory axis in which RAD18-mediated SFN degradation attenuates SFN-dependent G2/M checkpoint control, facilitates G2/M progression, and promotes CRC growth.