Abstract Background: Prostate cancer (PC) metastasis drives PC-related mortality and morbidity, yet the molecular mechanisms underlying this lethal process remains incompletely understood. Previously, we demonstrated that RIPK2 is critical for PC progression and metastasis (Nat. Commun., 2022). However, the regulatory mechanisms controlling RIPK2 protein levels in PC cells remain unclear. This study aims to address this knowledge gap by investigating the regulation of RIPK2 ubiquitination and degradation in PC cells, with a particular focus on CDK2, which we identified as a key mediator of RIPK2 regulation of the c-Myc oncoprotein. Methods: PC cells—22Rv1 (androgen receptor-positive) and PC3 (androgen receptor-negative)—were treated with two clinically evaluated, CDK2-selective inhibitors, INX-315 and PF-07104091. Changes in RIPK2 protein levels were measured by western blotting. RIPK2 stability was assessed using cycloheximide (CHX) chase assays, and proteasomal degradation was evaluated with MG132 treatment. K48-linked ubiquitination of RIPK2 was examined by immunoprecipitation followed by western blotting with K48-linked ubiquitin antibodies. Cell cycle synchronization was achieved by serum starvation or treatment with Ro-3306, hydroxyurea, thymidine, or monastrol. Proximity ligation assays (PLA) were used to assess protein-protein interactions between RIPK2 and CDK2, its E3 ligase ZNRF4, or deubiquitinases YOD1, OTUB2, and OTUB5. Statistical analyses were performed using unpaired, two-tailed Student’s t-tests. Results: CDK2 inhibition significantly reduced RIPK2 protein half-life in a proteasome-dependent manner and increased RIPK2 K48-linked ubiquitination. RIPK2 protein abundance peaks in the G2 phase, lagging behind the S-phase peak of active CDK2, its stabilizing kinase. Proximity ligation assays showed that RIPK2 associates with CDK2, ZNRF4, YOD1, and OTUB5 primarily in the cytoplasm. Notably, CDK2 inhibition disrupted RIPK2 association with YOD1 but not ZNRF4 in a dose-dependent manner, implicating YOD1 in CDK2-mediated control of RIPK2 stability. Together, these findings identify CDK2 as a key regulator of RIPK2 ubiquitination and stability in PC cells. Conclusion: Kinase-active CDK2 inhibits the K48-linked ubiquitination of RIPK2 and protects it from proteasomal degradation. This protection is potentially mediated by regulating the association between RIPK2 and its deubiquitinase YOD1 in PC cells. The findings support further investigation into the molecular mechanisms by which CDK2 stabilizes RIPK2 in PC and other cancer types. *Corresponding Author: Wei.Yang@stonybrook.edu Citation Format: Lili M. Guerra. CDK2 controls RIPK2 ubiquitination and stability in prostate cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1907.
Guerra et al. (Fri,) studied this question.