NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.

Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity.

We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation.

We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.