N-Terminal Basic Helix and S120 Phosphorylation Cooperatively Regulate Nuclear Localization of UBE2A/B in Mechanotransduction

Cells are continuously exposed to mechanical stimuli arising from the extracellular matrix, neighboring cells, and tissue architecture. Through mechanotransduction, these physical cues are converted into intracellular signaling events that regulate cellular behavior and fate. In our previous study, we showed that UBE2A/B localize predominantly in the cytosol when cells are cultured on soft substrates or at high density but translocate to the nucleus on stiff substrates or at low density. This mechanosensitive translocation promotes H2B monoubiquitination and regulates the expression of genes involved in cell growth. However, the mechanism underlying UBE2A/B nuclear localization remains unclear. In the present study, we used mutagenesis to identify regulatory post-translational modifications and define a putative nuclear localization signal (NLS) within UBE2A/B. The phospho-mimetic mutant S120D enhanced UBE2A nuclear localization, whereas substitution of three arginine residues within the predicted NLS (3RA) reduced nuclear localization under low-density conditions. Pharmacological inhibition of cyclin-dependent kinases (CDKs), which are known to phosphorylate S120, suppressed both exogenous and endogenous UBE2A/B nuclear localization and decreased H2B monoubiquitination. Using pull-down assays, we demonstrated that XPO4, a bidirectional nuclear transport receptor, interacts with S120D UBE2A/B but not with wild-type UBE2A/B. Structural modeling suggested that XPO4 interacts with the N-terminal basic helix and phosphorylated S120 residue of UBE2A/B, consistent with our mutational analyses. Moreover, the 3RA mutation abolished the enhanced nuclear localization conferred by S120D. Analysis of public RNA-seq data identified extensive mechanosensitive regulatory programs controlling CDK activity, which align with the nuclear translocation dynamics of UBE2A/B. Together, these findings demonstrate that UBE2A/B nuclear localization is governed by coordinated regulation of its N-terminal basic helix and S120 phosphorylation and identify XPO4 as a potential mediator of its nuclear import.