The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

We have recently shown that the nuclear localization of IFN gamma is mediat ed by a polybasic nuclear localization sequence (NLS) in its C terminus. Th is NLS is required for the full expression of biological activity of IFN ga mma, both extracellularly and intracellularly. We now show that this NLS pl ays an integral intracellular role in the nuclear translocation of the tran scription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95-133) containing the NLS block ed the induction of STAT1 alpha nuclear translocation. The antibodies had n o effect on nuclear translocation of STAT1 alpha in IFN alpha treated cells . A deletion mutant of human IFN gamma, IFN gamma(1-123), which is devoid o f the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K-d si milar to that of the wild-type protein. Deletion of the NLS specifically ab olished the ability of IFN gamma(1-123) to initiate the nuclear translocati on of STAT1 alpha, which is required for the biological activities of IFN g amma following binding to the IFN gamma receptor complex. Thus, the NLS reg ion appears to contribute minimally to extracellular high-affinity receptor -ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal N LS domain of IFN gamma with a K-d approx. 3x10(-8) M-1 has been described b y previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-termin al NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked t he nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 a lpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95 -133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Delet ion of the NLS moth specifically abrogated the ability of this intracellula r peptide to cause STAT1 alpha nuclear translocation. In cells activated wi th IFN gamma, IFN gamma was found to as part of a complex that contained ST AT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha n uclear import. The tyrosine phosphorylation of STAT1 alpha, the formation o f the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclea r translocation of STAT1 alpha were all found to be dependent on the presen ce of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellular ly to directly regulate the activation and ultimate nuclear translocation S TAT1 alpha.