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J Biol Chem, Vol. 274, Issue 1, 403-407, January 1, 1999

The Carboxyl Terminus of Interferon- Contains a Functional Polybasic Nuclear Localization Sequence

From the Department of Microbiology and Cell Science and the ^§ Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32611

Cytokines such as interferon-gamma (IFN-), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN- or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN- is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95-132 of IFN- (IFN-(95-132)) containing the polybasic sequence ¹²⁶RKRKRSR¹³² was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH₂-terminal of this sequence did not affect the nuclear import. Thus, the sequence ¹²⁶RKRKRSR¹³² is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-(95-132) but not the peptide IFN-(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-(95-132). This observation suggests that the NLS in IFN- may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN- was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN- in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN- is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-.

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