The carboxyl terminus of interferon-gamma contains a functional polybasic nuclear localization sequence

Cytokines such as interferon-gamma (IFN-gamma), which utilize the well stud ied JAK/STAT pathway for nuclear signal transduction, are themselves transl ocated to the nucleus. The exact mechanism for the nuclear import of IFN-ga mma or the functional role of the nuclear translocation of ligand in signal transduction is unknown, We show in this study that nuclear localization o f IFN-gamma is driven by a simple polybasic nuclear localization sequence ( NLS) in its COOH terminus, as verified by its ability to specify nuclear im port of a heterologous protein allophycocyanin (APC) in standard import ass ays in digitonin-permeabilized cells. Similar to other nuclear import signa ls, we show that a peptide representing amino acids 95-132 of IFN-gamma (IF N-gamma(95-132)) containing the polybasic sequence (RKRKRSR132)-R-126 was c apable 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, del etions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence (RKRKRSR132)-R-126 is necessary and sufficient for nuclear localization. FurthePmore, nuclear import was strongly blocked by competition with the cognate peptide IFN-gamma(95-132) but Plot the pept ide IFN-gamma(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A pepti de containing the prototypical polybasic NLS sequence of the SV40 large T-a ntigen was also able to inhibit the nuclear import mediated by IFN-gamma(95 -132), This observation suggests that the NLS in IFN-gamma may function thr ough the components of the Ran/importin pathway utilized by the SV40 T-NLS, Finally, we show that intact IFN-gamma, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the pe ptide IFN-gamma(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-gamma was also transported into the nucleus through the Ran/importin pa thway. Previous studies have suggested a direct intracellular role for IFN- gamma in the induction of its biological activities. Based on our data in t his study, we suggest that a key intracellular site of interaction of IFN-g amma is the one with the nuclear transport mechanism that occurs via the NL S in the COOH terminus of lFN-gamma.