Phosphorylation of Ephexin4 at Ser-41 contributes to chromosome alignment via RhoG activation in cell division

Ephexin proteins are guanine nucleotide exchange factors for the Rho GTPases. We reported that Ephexin4 regulates M-phase progression downstream of phosphorylated EphA2, a receptor-type tyrosine kinase, through RhoG activation; however, the regulation of Ephexin4 during M phase remains unknown. In this study, a novel Ephexin4 phosphorylation site was identified at Ser41, exclusively in M phase. Ephexin4 knockdown prolonged the duration of M phase by activating the spindle assembly checkpoint, at which BubR1 was localized at the kinetochores of the misaligned chromosomes. This delay was alleviated by re-expression of wild-type, but not S41A Ephexin4. The Ephexin4 knockdown caused chromosome misalignment and reduced the RhoG localization to the plasma membrane. These phenotypes were rescued by re-expression of wild type and phospho-mimic S41E mutant, but not the S41A mutant. Consistently, S41E mutant enhanced active RhoG levels, even in the interphase. Regardless of the Ephexin4 knockdown, active RhoG-G12V was localized at the plasma membrane. Furthermore, Ephexin4 knockdown exacerbated vincristine-induced chromosome misalignment, which was prevented by re-expressing the wild-type but not S41A Ephexin4. Overexpression of wild type and S41E mutant, but not S41A mutant, resulted in an increased number of Madin-Darby canine kidney cysts with cells inside the lumen, indicating disruption of epithelial morphogenesis by deregulating Ephexin4/RhoG signaling in cell division. Our results suggest that Ephexin4 undergoes phosphorylation at Ser41 in cell division, and the phosphorylation is required for chromosome alignment through RhoG activation. Combined with mitosis-targeting agents, inhibition of Ephexin4 phosphorylation may represent a novel strategy for cancer chemotherapy.