Efficacious inhibition of Importin α/β-mediated nuclear import of human inositol phosphate multikinase.

Human inositol phosphate multikinase (IPMK) is a nucleocytoplasmic shuttling protein involved in multiple signal transduction pathways located both in the nucleus and in the cytoplasm. To efficaciously inhibit the conventional nuclear import of IPMK, we first examined the effect of different inhibitors and cellular stressors on nuclear import of enhanced green fluorescent protein monomer and octamer, both fused with a monopartite nuclear localization signal (NLS), in HeLa and H1299 cells. Most efficacious inhibition of conventional nuclear protein import was observed when using Importazole and hydrogen peroxide. Therefore, these substances were then applied to examine nuclear import mechanisms of IPMK. Thereby, we demonstrated that nuclear accumulation of IPMK is significantly lessened, but not abrogated by inhibition of conventional protein import. This indicates that IPMK is imported into the nucleus by both conventional and non-conventional pathways. Furthermore, intracellular distribution of an IPMK mutant with inactivated NLS is unaffected by inhibition of conventional protein import. Obviously, the conventional import of IPMK is entirely mediated by interaction of the Importin α/β heterodimer with IPMK's sole NLS motif (R(320)HRKIYTKKHH). Future research should focus on the hitherto unknown non-conventional import of IPMK and the potential impact of its dysregulation on IPMK signaling pathways regulating cellular growth and proliferation.