Multisite phosphorylation of nuclear interaction partner of ALK (NIPA) at G₂/M involves cyclin B1/Cdk1

Nuclear interaction partner of ALK (NIPA) is an F-box-containing protein that defines a nuclear skp1 cullin F-box (SCF)-type ubiquitin E3 ligase (SCF^NIPA) implicated in the regulation of mitotic entry. The SCF ^NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase, whereas phosphorylation of NIPA in late G₂ phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G₂/M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCF^NIPA complex, resulting in reduced levels of mitotic cyclin B1. These data are compatible with a process of sequential NIPA phosphorylation where cyclin B1/Cdk1 amplifies phosphorylation of NIPA once an initial phosphorylation event has dissociated the SCF ^NIPA complex. Thus, cyclin B1/Cdk1 may contribute to the regulation of its own abundance in early mitosis.