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

Abstract

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. 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-␥.The interferons (IFNs) 1 are cytokines that use the well studied JAK/STAT pathway for signal transduction to the nucleus (1). This pathway is initiated by the binding of the ligand to the extracellular domain of the appropriate receptor complex followed by the activation of select members of the JAK family of tyrosine kinases at the intracellular cytoplasmic domain of the receptor subunits. These tyrosine kinases in turn phosphorylate appropriate members of the STAT family of transcription factors present in the cytoplasm, thereby targeting these factors, through unknown mechanisms, for translocation to the nucleus to activate transcription. Transcription factors the size of STATs must be taken into the cell nucleus by an active transport process through the nuclear pore complex.Active nuclear import of a large number of nuclear proteins occurs through the Ran/importin pathway (for review, see Ref.2). In this pathway, the nuclear protein initially binds to a heterodimeric nuclear transport protein called importin which contains a ␣ and ␤ subunit. Nuclear proteins interact with the ␣ subunit, importin ␣, of the transporter via a specific nuclear localization sequence (NLS). These NLSs generally consi...

Protein Interactions Involved in Nuclear Import of the Agrobacterium VirE2 Protein in Vivo and in Vitro

“…In these complexes, VIP1 likely functions as a molecular adapter between VirE2 and karyopherin ␣, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components. That VIP1 does act as such an adaptor rather than the import factor itself was supported by our observations that nuclear accumulation of VirE2 in the presence of VIP1 was inhibited by the SV40 NLS peptide known to specifically block the karyopherin ␣-dependent nuclear import pathway (39,40). Because VIP1 appears to be found only in plants but not in yeast or animal cells, VirE2 nuclear import also occurs only in plant cells.…”

“…The amounts of karyopherin ␣ in the in vitro import system can be effectively depleted by the addition of competing concentrations of SV40 NLS peptide, which is known to specifically bind karyopherin ␣ (37, 38), preventing it from promoting nuclear import of the labeled substrate (39,40). Indeed, addition of the SV40 NLS peptide conjugated to a BSA carrier virtually blocked accumulation of R-VirE2 in the nuclei of permeabilized HeLa cells even in the presence of VIP1 (Fig.…”

Selective delivery of IFN‐γ to renal interstitial myofibroblasts: a novel strategy for the treatment of renal fibrosis

“…Biologic effects of IFN-g take place via the nuclear signaling sequence (NLS), which is present in its C terminus region (63,64). This region is able to modulate IFN-g-responsive genes through activation of the JAK/STAT (Janus kinase/ signal transducers and activators of transcription signaling pathway) pathway (65,66).…”

Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

“…4b). Compared to other mammalian and piscine IFN-γ molecules, the C-terminal region of the Xenopus IFN-γ contains a well-conserved nuclear localization signal motif (KKRK) which is required for nuclear translocation and protein function (Subramaniam et al 1999). The Xenopus IFN-γ shares relatively high homology with the IFN-γs from higher vertebrates (34.0% and 36.5% identity with human and chicken IFN-γ) than piscine IFN-γs (17.7-23.7% identity).…”