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

Abstract

Cytokines such as interferon-gamma (IFN-γ), 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-γ 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-γ 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-γ (IFN-γ(95–132)) containing the polybasic sequence¹²⁶RKRKRSR¹³² 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 sequence¹²⁶RKRKRSR¹³² is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-γ(95–132) but not the peptide IFN-γ(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-γ(95–132). This observation suggests that the NLS in IFN-γ may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-γ, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-γ(95–132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-γ 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-γ is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-γ.