Journal of Cell Science, Vol 113, Issue 15 2771-2781, Copyright © 2000 by Company of Biologists

JOURNAL ARTICLES

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

PS Subramaniam, J Larkin, MG Mujtaba, MR Walter and HM Johnson
Dept Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA. prem@ufl.edu.

We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95-133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1-123), which is devoid of 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) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1-123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region 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 NLS domain of IFN gamma with a K(d) approx. 3 x 10(-8) M(-1) has been described by 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-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the 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 alpha 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. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. M. Ahmed, J. P. Martin, and H. M. Johnson IFN Mimetic as a Therapeutic for Lethal Vaccinia Virus Infection: Possible Effects on Innate and Adaptive Immune Responses J. Immunol., April 1, 2007; 178(7): 4576 - 4583. [Abstract] [Full Text] [PDF]

				M. G. Mujtaba, C. B. Patel, R. A. Patel, L. O. Flowers, M. A. Burkhart, L. W. Waiboci, J. Martin, M. I. Haider, C. M. Ahmed, and H. M. Johnson The Gamma Interferon (IFN-{gamma}) Mimetic Peptide IFN-{gamma}(95-133) Prevents Encephalomyocarditis Virus Infection both in Tissue Culture and in Mice. Clin. Vaccine Immunol., August 1, 2006; 13(8): 944 - 952. [Abstract] [Full Text] [PDF]

				C. M. I. Ahmed and H. M. Johnson IFN-{gamma} and Its Receptor Subunit IFNGR1 Are Recruited to the IFN-{gamma}-Activated Sequence Element at the Promoter Site of IFN-{gamma}-Activated Genes: Evidence of Transactivational Activity in IFNGR1 J. Immunol., July 1, 2006; 177(1): 315 - 321. [Abstract] [Full Text] [PDF]

				A. P. Costa-Pereira, H. M. Hermanns, H. Is'harc, T. M. Williams, D. Watling, V. Arulampalam, S. J. Newman, P. C. Heinrich, and I. M. Kerr Signaling through a Mutant IFN-{gamma} Receptor J. Immunol., November 1, 2005; 175(9): 5958 - 5965. [Abstract] [Full Text] [PDF]

				J. Zou, A. Carrington, B. Collet, J. M. Dijkstra, Y. Yoshiura, N. Bols, and C. Secombes Identification and Bioactivities of IFN-{gamma} in Rainbow Trout Oncorhynchus mykiss: The First Th1-Type Cytokine Characterized Functionally in Fish J. Immunol., August 15, 2005; 175(4): 2484 - 2494. [Abstract] [Full Text] [PDF]

				C. M. I. Ahmed, M. A. Burkhart, P. S. Subramaniam, M. G. Mujtaba, and H. M. Johnson Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein J. Virol., May 1, 2005; 79(9): 5632 - 5639. [Abstract] [Full Text] [PDF]

				M. Ito, M. Nakasato, T. Suzuki, S. Sakai, M. Nagata, and F. Aoki Localization of Janus Kinase 2 to the Nuclei of Mature Oocytes and Early Cleavage Stage Mouse Embryos Biol Reprod, July 1, 2004; 71(1): 89 - 96. [Abstract] [Full Text] [PDF]

				K. Schroder, P. J. Hertzog, T. Ravasi, and D. A. Hume Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol., February 1, 2004; 75(2): 163 - 189. [Abstract] [Full Text] [PDF]

				T. Meyer, A. Marg, P. Lemke, B. Wiesner, and U. Vinkemeier DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1 Genes & Dev., August 15, 2003; 17(16): 1992 - 2005. [Abstract] [Full Text] [PDF]

				C. M. I. Ahmed, M. A. Burkhart, M. G. Mujtaba, P. S. Subramaniam, and H. M. Johnson The role of IFN{gamma} nuclear localization sequence in intracellular function J. Cell Sci., August 1, 2003; 116(15): 3089 - 3098. [Abstract] [Full Text] [PDF]

				K. Melen, R. Fagerlund, J. Franke, M. Kohler, L. Kinnunen, and I. Julkunen Importin {alpha} Nuclear Localization Signal Binding Sites for STAT1, STAT2, and Influenza A Virus Nucleoprotein J. Biol. Chem., July 18, 2003; 278(30): 28193 - 28200. [Abstract] [Full Text] [PDF]

				S. Olsnes, O. Klingenberg, and A. Wiedlocha Transport of Exogenous Growth Factors and Cytokines to the Cytosol and to the Nucleus Physiol Rev, January 1, 2003; 83(1): 163 - 182. [Abstract] [Full Text] [PDF]

				B. A. Torres, G. Q. Perrin, M. G. Mujtaba, P. S. Subramaniam, A. K. Anderson, and H. M. Johnson Superantigen Enhancement of Specific Immunity: Antibody Production and Signaling Pathways J. Immunol., September 15, 2002; 169(6): 2907 - 2914. [Abstract] [Full Text] [PDF]

				P. S. Subramaniam and H. M. Johnson Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-{gamma}, Its Receptor Chain IFN-{gamma} Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1{alpha} J. Immunol., August 15, 2002; 169(4): 1959 - 1969. [Abstract] [Full Text] [PDF]

				K. Melen, L. Kinnunen, and I. Julkunen Arginine/Lysine-rich Structural Element Is Involved in Interferon-induced Nuclear Import of STATs J. Biol. Chem., May 4, 2001; 276(19): 16447 - 16455. [Abstract] [Full Text] [PDF]