Nucleocytoplasmic shuttling of IPAS by its unique nuclear import and export signals unshared with other HIF-3α splice variants

Inhibitory Per/Arnt/Sim (PAS) domain protein (IPAS) is a splice variant of hypoxia-inducible factor (HIF)-3 alpha, and possesses two entirely different functions. One is as a transcriptional repressor against HIF-dependent hypoxic gene activation. The other is as a pro-apoptotic factor by direct binding to the pro-survival protein Bcl-x(L) and its related proteins on mitochondria. Presently, the regulatory mechanism that determines the intracellular distribution of IPAS to fulfill each of the two functions is unknown. As a first step towards elucidation of the mechanism, nucleocytoplasmic transport signals of IPAS were explored. A bipartite-like nuclear localization signal (NLS) was found in the N-terminal region by the deletion and mutation analysis of EGFP-IPAS. In addition, the helix-loop-helix domain showed weak nuclear import/retention activity. A leptomycin B-sensitive nuclear export signal (NES) was localized in the C-terminal region of the protein. A proline-rich region supported the NES activity. These NLS and NES are not carried by the other variants of HIF-3 alpha due to differential exon usage. These results strongly suggest that IPAS is a nucleocytoplasmic shuttling protein.