Stress- and mitosis-dependent phosphorylation of NIFK on Ser247.

NIFK is a nucleolar protein involved in ribosome biogenesis that interacts with Ki-67 (also known as MKI67) and is recruited to the perichromosomal layer during mitosis. NIFK expression is elevated in multiple cancers, and its expression correlates with poor prognosis. Although NIFK is highly phosphorylated, the regulation and functional significance of NIFK phosphorylation is not fully understood. Here, we identify Ser247 as a stress- and mitosis-responsive phosphorylation site on NIFK. Stress induces Ser247 phosphorylation through p38 mitogen-activated protein kinases (p38 MAPKs) and MSK1 (also known as RPS6KA5). Independently of stress, Ser247 is phosphorylated during mitosis by Aurora B (AURKB). In vitro assays confirm that MSK1 and Aurora B directly phosphorylate Ser247. During mitosis, Ser247 phosphorylation acts as a priming event enabling extensive multisite phosphorylation of NIFK. Mutation of Ser247 (S247A) abolishes this phosphorylation cascade but does not impair recruitment of NIFK to mitotic chromosomes. Nonetheless, cells with a NIFK S247A mutation display a delay in mitotic entry. Whether S247A mutation affects the ribosome biogenesis function of NIFK is not clear. These findings reveal that Ser247 is a hub integrating stress and mitotic cues, and establish it as a priming site for mitotic hyperphosphorylation of NIFK.