Interferon regulatory factor subcellular localization is determined by a bipartite nuclear localization signal in the DNA-binding domain and interaction with cytoplasmic retention factors.

The transduction of type I interferon signals to the nucleus relies on activation of a protein complex, ISGF3, involving two signal transducers and activators of transcription (STAT) proteins, STAT1 and STAT2, and the interferon (IFN) regulatory factor (IRF) protein, p48/ISGF3gamma. The STAT subunits are cytoplasmically localized in unstimulated cells and rapidly translocate to the nucleus of IFN-stimulated cells, but the p48/ISGF3gamma protein is found in both the nucleus and the cytoplasm, regardless of IFN stimulation. Here, we demonstrate that p48 is efficiently and constitutively targeted to the nucleus. Analysis of the subcellular distribution of green fluorescent protein-p48 fragments indicates that p48 contains a bipartite nuclear retention signal within its amino-terminal DNA-binding domain. This signal is preserved in two other IRF proteins involved in immune responses, ICSBP and IRF4. Mutations to clustered basic residues within amino acids 50-100 of p48 or IRF4 disrupt their nuclear accumulation, and DNA-binding ability is not required for nuclear targeting. This is the only example of a nuclear localization signal for any ISGF3 component and assigns a second function to the IRF DNA-binding domain. We also demonstrate that the nuclear distribution of p48 is dramatically altered by coexpression of the STAT2 protein, indicating that STAT2 forms a cytoplasmic complex with p48, overriding the intrinsic p48 nuclear targeting. Retention by STAT2 may serve to regulate the activity of free p48 and/or guarantee that cytoplasmic pools of preassociated STAT2:p48 are available for rapid activation of the IFN response. These findings suggest that analogous mechanisms may exist for regulating the distribution of other IRF proteins.

[1]  J. Hiscott,et al.  Posttranslational regulation of IRF-4 activity by the immunophilin FKBP52. , 2000, Immunity.

[2]  M. Atchison,et al.  Differential expression and distinct functions of IFN regulatory factor 4 and IFN consensus sequence binding protein in macrophages. , 1999, Journal of immunology.

[3]  Wenmei Li,et al.  Interferon Consensus Sequence-binding Protein Is Constitutively Expressed and Differentially Regulated in the Ocular Lens* , 1999, The Journal of Biological Chemistry.

[4]  S. Majumder,et al.  p48/STAT-1α-Containing Complexes Play a Predominant Role in Induction of IFN-γ-Inducible Protein, 10 kDa (IP-10) by IFN-γ Alone or in Synergy with TNF-α , 1998, The Journal of Immunology.

[5]  F. Schaper,et al.  Functional domains of interferon regulatory factor I (IRF-1). , 1998, The Biochemical journal.

[6]  G. Blobel,et al.  Crystallographic Analysis of the Recognition of a Nuclear Localization Signal by the Nuclear Import Factor Karyopherin α , 1998, Cell.

[7]  T. Maniatis,et al.  Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. , 1998, Molecular cell.

[8]  N. Reich,et al.  Interferon Regulatory Factor 3 and CREB-Binding Protein/p300 Are Subunits of Double-Stranded RNA-Activated Transcription Factor DRAF1 , 1998, Molecular and Cellular Biology.

[9]  E. Nishida,et al.  Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF‐3 and CBP/p300 , 1998, The EMBO journal.

[10]  A. Aggarwal,et al.  Structure of IRF-1 with bound DNA reveals determinants of interferon regulation , 1998, Nature.

[11]  N. Reich,et al.  Distinct STAT Structure Promotes Interaction of STAT2 with the p48 Subunit of the Interferon-α-stimulated Transcription Factor ISGF3* , 1997, The Journal of Biological Chemistry.

[12]  G. Stark,et al.  Functional subdomains of STAT2 required for preassociation with the alpha interferon receptor and for signaling , 1997, Molecular and cellular biology.

[13]  J. Darnell,et al.  Interactions between STAT and non-STAT proteins in the interferon-stimulated gene factor 3 transcription complex , 1996, Molecular and cellular biology.

[14]  T. Taniguchi,et al.  Essential and non‐redundant roles of p48 (ISGF3γ) and IRF‐1 in both type I and type II interferon responses, as revealed by gene targeting studies , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[15]  D. Levy,et al.  Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[16]  U. Storb,et al.  Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. , 1995, Genes & development.

[17]  J. Darnell,et al.  A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain. , 1995, Genes & development.

[18]  J. Darnell,et al.  Contribution of STAT SH2 groups to specific interferon signaling by the Jak-STAT pathway , 1995, Science.

[19]  J. Darnell,et al.  Role of STAT2 in the alpha interferon signaling pathway , 1995, Molecular and cellular biology.

[20]  D. Levy,et al.  Two domains of ISGF3 gamma that mediate protein-DNA and protein-protein interactions during transcription factor assembly contribute to DNA-binding specificity , 1993, Molecular and cellular biology.

[21]  R. Aebersold,et al.  Subunit of an alpha-interferon-responsive transcription factor is related to interferon regulatory factor and Myb families of DNA-binding proteins , 1992, Molecular and cellular biology.

[22]  G. Stark,et al.  High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Stark,et al.  Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons , 1991, Molecular and cellular biology.

[24]  R. Laskey,et al.  Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: Identification of a class of bipartite nuclear targeting sequence , 1991, Cell.

[25]  D. Levy,et al.  Interferon-alpha regulates nuclear translocation and DNA-binding affinity of ISGF3, a multimeric transcriptional activator. , 1990, Genes & development.

[26]  J. Darnell,et al.  Synergistic interaction between interferon‐alpha and interferon‐gamma through induced synthesis of one subunit of the transcription factor ISGF3. , 1990, The EMBO journal.

[27]  J. Darnell,et al.  Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro. , 1989, Genes & development.

[28]  D. Baltimore,et al.  Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor , 1988, Cell.

[29]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.