Signal Transduction Mechanism of A Peptide Mimetic of Interferon-γ

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Abstract
The C-terminus of interferon-γ (IFNγ) contains a nuclear localization sequence (NLS) required for the activation and nuclear translocation of the transcription factor STAT1α and induction of IFNγ-activated genes. On the basis of this and other studies, we developed a peptide mimetic of IFNγ that possesses the IFNγ functions of antiviral activity and upregulation of MHC class II molecules. The mimetic also shares with IFNγ the ability to induce the activation and nuclear translocation of STAT1α and the IFNγ receptor (IFNGR)-1 subunit. The mimetic, IFNγ(95−132), is a peptide that consists of the C-terminal residues 95−132 of murine IFNγ and contains a required α-helical domain and the NLS of IFNγ. In this study, we determined the mechanism of the intracellular action of the mimetic at the level of signal transduction. We show that the mimetic mediates the nuclear transport of IFNGR-1 through its interaction with IFNGR-1 cytoplasmic region 253−287 via both the helical region and the NLS of IFNγ(95−132). Alanine substitutions of the NLS of the mimetic showed that the NLS was required for nuclear translocation and that the nuclear transport properties of the mimetic correlated with its ability to bind IFNGR-1. These data also show that the NLS of IFNγ(95−132) can interact simultaneously with IFNGR-1 and the nuclear import machinery. We found that in in vitro nuclear transport assays tyrosine-phosphorylated STAT1α failed to undergo nuclear translocation in the presence of nuclear import factors, but was transported to nucleus in the presence of IFNγ(95−132) and JAK2-phosphorylated IFNGR-1, to which STAT1α binds, as a complex of IFNγ(95−132)/IFNGR-1/STAT1α. Thus, the mimetic, which possesses IFNγ function, is directly involved as a chaperone in the nuclear transport of STAT1α and shares this mechanism of action with that previously described for IFNγ. The mimetic, like IFNγ, is able to upregulate the tumor suppressor p21WAF1/CIP1, a direct target of STAT1α, and this ability requires the NLS of the mimetic. However, unlike IFNγ, the mimetic is unable to downregulate c-myc and hence does not inhibit the cycling of cells. This suggests that IFNγ has additional functions that are not tied directly to the nuclear translocation of STAT1α.