ABSTRACT
Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins , Repressor Proteins , Transcription Factors , Transcriptional Activation , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , DNA/genetics , DNA-Binding Proteins/genetics , Genes, MHC Class II/genetics , Genes, Reporter , Humans , Interferon Regulatory Factor-2 , Leydig Cells/metabolism , Male , Mice , Molecular Sequence Data , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Secondary , RNA, Messenger/analysis , RNA, Messenger/genetics , Sequence Alignment , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolism , TransfectionABSTRACT
Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Nuclear Proteins , Proteins/chemistry , Proteins/metabolism , Receptors, Thyroid Hormone , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Transcription Factors , 3T3 Cells , Alanine/chemistry , Animals , B-Lymphocytes/metabolism , Blotting, Northern , COP9 Signalosome Complex , COS Cells , DNA/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Gene Library , Glutathione Transferase/metabolism , HL-60 Cells , HeLa Cells , Humans , Interferon Regulatory Factors , Mice , Models, Biological , Multiprotein Complexes , Mutagenesis, Site-Directed , Peptide Hydrolases , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , Repressor Proteins/genetics , Serine/chemistry , Signal Transduction , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
IFN regulatory factors (IRFs) constitute a family of transcription factors that are involved in IFN signaling and the development and differentiation of the immune system. Targeted gene disruption studies in mice assigned their primary role to the immune system. Two lymphoid-specific IRF members, IFN consensus sequence binding protein (ICSBP) and IRF-4, bind target DNA with greater efficiency following interaction with two transcription factors, PU.1 and E47, leading to transcriptional synergy. PU.1 and E47 are essential for proper differentiation and maturation of lymphoid cells. In addition, ICSBP interacts with two IRF members, IRF-1 and IRF-2, which also have central roles in the regulation of cell-mediated immunity. Previously, we identified a region in ICSBP, termed the IRF association domain (IAD), that is conserved in all IRFs (excluding IRF-1 and IRF-2) and is essential for its interactions with other IRF proteins. Here we show that the IAD is an independent module used by ICSBP and IRF-4 for protein-protein interactions. In addition, an IAD of IRF-2 (IAD2), necessary for interaction with ICSBP, was identified and found to be conserved in IRF-1. The IAD2 shares similar characteristics with the PEST domain that is essential for the interaction of PU.1 with IRF-4. We also show that the ICSBP DNA binding domain is indispensable for the formation of DNA binding heterocomplexes and transcriptional activity. Therefore, our results shed light on the molecular mechanisms that affect IRF activities in the immune system via discrete functional domains.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Interferons/metabolism , Transcription Factors/metabolism , 3T3 Cells , Amino Acid Motifs/immunology , Amino Acid Sequence , Animals , Consensus Sequence/immunology , DNA/physiology , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/physiology , Interferon Regulatory Factor-2 , Interferon Regulatory Factors , Interferons/physiology , Mice , Molecular Sequence Data , Protein Structure, Tertiary/physiology , Proto-Oncogene Proteins/metabolism , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , TCF Transcription Factors , Trans-Activators/metabolism , Transcription Factor 7-Like 1 Protein , Transcription, Genetic/immunologyABSTRACT
Two families of transcription factors mediate interferon (IFN) signaling. The first family, signal transducers and activators of transcription (STATs), is activated within minutes of IFN treatment. Specific phosphorylation events lead to their translocation to the nucleus, formation of transcriptional complexes, and the induction of the second family of transcription factors termed interferon regulatory factors (IRFs). Interferon consensus sequence binding protein (ICSBP) is a member of IRF family that is expressed only in cells of the immune system and acts as a transcriptional repressor. ICSBP binds DNA through the association with other transcription factors such as IRF-1 or IRF-2. In this communication, the domain that is involved in protein-protein interactions was mapped to the carboxyl terminus of ICSBP. This domain is also important for mediating ICSBP-repressing activity. In vitro studies demonstrated that direct binding of ICSBP to DNA is prevented by tyrosine (Tyr) phosphorylation. Yet, Tyr-phosphorylated ICSBP can bind target DNA only through the association with IRF-2 and IRF-1. This type of phosphorylation is essential for the formation of heterocomplexes. Tyr-phosphorylated ICSBP and IRF-2 are detected in expressing cells constitutively, and Tyr-phosphorylated IRF-1 is induced by IFN-gamma. These results strongly suggest that like the STATs, the IRFs are also modulated by Tyr phosphorylation that affects their biological activities.
