Phosphorylation-Mediated Regulation of FBXO31 Stability Under Cellular Homeostasis.

Abstract

The S-phase kinase-associated protein 1 (Skp1)-Cullin-F-box protein E3 ligase adaptor F-box-only protein 31 (FBXO31) regulates genomic stability and cell signaling in normal, genotoxic, and tumor cells by recognizing and ubiquitinating multiple downstream substrates. The stability and role of FBXO31 may be regulated by specific residual modification. In this study, five FBXO31 phosphorylation sites are identified in HEK293T cells using biochemical and biological techniques. Liquid chromatography-tandem mass spectrometry identifies phosphorylated residues, including threonine-28 and -37 and serine-33, -400, and -523. The PyMOL crystal structure reveals the location of these residues on FBXO31 and assesses whether they interact with the reported kinases. Western blotting and fluorescence-activated cell sorting demonstrate that the phosphorylation of Thr-37 and Ser-523 contributes to FBXO31 protein stabilization, which is further confirmed by cycloheximide experiments. The regulatory roles of Thr-37 and Ser-523 in FBXO31 stability are associated with variations in phosphorylation levels and degradation pathways. These results demonstrate that phosphorylation regulates FBXO31 turnover, and phosphorylation at Thr-37 or Ser-523 may help identify upstream kinases and enhance the understanding of the physiological role of FBXO31.