A Novel Role for RNF126 in the Promotion of G2 Arrest via Interaction With 14-3-3σ.

Purpose: Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint.
Methods and Materials: The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining.
Results: RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR.
Conclusions: RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment.
(Copyright © 2021 Elsevier Inc. All rights reserved.)