Faculty Bibliography

Interferons induce transcriptional activation through tyrosine phosphorylation of the latent, cytoplasmic transcription factor interferon-stimulated gene factor-3 (ISGF-3). Growth factors and cytokines were found to use a similar pathway: The 91-kilodalton subunit of ISGF-3 was activated and tyrosine phosphorylated in response to epidermal growth factor (EGF), platelet-derived growth factor, and colony stimulating factor-1. The tyrosine phosphorylated factor acquired DNA binding activity and accumulated in nuclei. Activation required the major sites for autophosphorylation on the EGF receptor that bind Src homology region 2 domain-containing proteins implicated in Ras activation. However, activation of this factor was independent of the normal functioning of Ras

We have previously reported three types of DNA-protein complexes, formed specifically with the interferon-stimulable response elements (ISRE) in the 5' flanking DNA of the interferon-inducible 6-16 and 9-27 genes, a type-I interferon-inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type-I and type-II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band-shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9-27 ISRE probe the M, G and C1/C2 complexes all contain the gamma subunit of ISGF3, which is present constitutively but is induced in response to IFN-alpha (to yield M) or IFN-gamma (to yield G). In contrast, a 6-16 ISRE probe forms band-shift complexes with IFN-alpha-inducible and IFN-gamma-inducible IRF1 and IRF2. With a 6-16 ISRE probe, therefore, M and G each correspond to two complexes which co-migrate in band-shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9-27 ISRE than the 6-16 ISRE, whereas the reverse is true for E (ISGF3) and its gamma subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band-shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6-16 and 9-27 probes are dominated by a dinucleotide sequence in the centre of the 14-nucleotide 'core' ISRE. In contrast, preferential binding of E (ISGF3) by the 39-nucleotide 9-27 ISRE-containing sequence, although ISRE dependent, appears to be mediated by sequences 3' of the 'core' ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5' flanking region and 'core' ISRE sequences from the 6-16 gene and sequences immediately 3' of the 'core' 9-27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo-ISRE sequence close to ISRE in the 9-27 gene. The precise roles of IRF1 and IRF2 in the induction of IFN-beta and the control of interferon-inducible gene expression remain to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha interferon (IFN-alpha) induces the transcription of a large set of genes through activation of multimeric transcription factor ISGF3. This factor can be dissociated into two protein components, termed ISGF3 gamma and ISGF3 alpha. ISGF3 gamma is a 48-kDa protein related at the amino terminus to members of the IFN-regulatory factor (IRF) and Myb families of DNA-binding proteins; ISGF3 alpha consists of three polypeptides of 84, 91, and 113 kDa that self-assemble to form an activated component in response to IFN-alpha. DNA-binding studies indicated that ISGF3 gamma binds DNA alone, recognizing the IFN-stimulated response element, while the ISGF3 alpha polypeptides alone display no specific interactions with DNA. A complex between ISGF3 gamma and activated ISGF3 alpha binds the IFN-stimulated response element with much greater affinity than does the 48-kDa ISGF3 gamma protein alone. The DNA-binding domain of ISGF3 gamma and regions responsible for protein-protein interaction with ISGF3 alpha were identified by using deleted forms of ISGF3 gamma expressed in vitro. The amino-terminal region of ISGF3 gamma homologous to the IRF and Myb proteins was sufficient for interaction with DNA and displayed the binding specificity of the intact protein; phosphorylation of this region was necessary for activity. A second region of 160 amino acids separated from the DNA-binding domain by over 100 amino acids contained a domain capable of associating with ISGF3 alpha and was sufficient to confer specific ISGF3 alpha interaction to a heterologous protein. Interaction of the ISGF3 alpha component with the protein interaction domain of ISGF3 gamma altered the DNA-binding specificity of the resulting complex, suggesting that one or more of the ISGF3 alpha polypeptides make base-specific contacts with DNA. This interaction defines a mechanism through which IRF-like proteins complexed with regulatory components can display novel DNA-binding specificities

Alpha interferon stimulates transcription by converting the positive transcriptional regulator ISGF3 from a latent to an active form. This receptor-mediated event occurs in the cytoplasm, with subsequent translocation of the activated factor to the nucleus. ISGF3 has two components, termed ISGF3 alpha and ISGF3 gamma. ISGF3 gamma serves as the DNA recognition subunit, while ISGF3 alpha, which appears to consist of three polypeptides, is a target for alpha interferon signaling and serves as a regulatory component whose activation is required to form ISGF3. ISGF3 gamma DNA-binding activity was identified as a 48-kDa polypeptide, and partial amino acid sequence has allowed isolation of cDNA clones. ISGF3 gamma translated in vitro from recombinant clones bound DNA with a specificity indistinguishable from that of ISGF3 gamma purified from HeLa cells. Sequencing of ISGF3 gamma cDNA clones revealed significant similarity to the interferon regulatory factor (IRF) family of DNA binding proteins in the amino-terminal 117 residues of ISGF3 gamma. The other IRF family proteins bind DNA with a specificity related to but distinct from that of ISGF3 gamma. We note sequence similarities between the related regions of IRF family proteins and the imperfect tryptophan repeats which constitute the DNA-binding domain of the c-myb oncoprotein. These sequence similarities suggest that ISGF3 gamma and IRF proteins and the c-myb oncoprotein use a common structural motif for DNA recognition. Recombinant ISGF3 gamma, like the natural protein, interacted with HeLa cell ISGF3 alpha to form the mature ISGF3 DNA-binding complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-alpha (IFN alpha) induces an immediate transcriptional response of a restricted set of genes in target cells. Specific transcription is mediated by the cytoplasmic activation of a transcription factor complex termed ISGF3. ISGF3 is a multimeric protein complex composed of a regulatory component (ISGF3 alpha), which is activated following IFN alpha treatment, and a DNA-binding component (ISGF3 gamma), which recognizes the IFN alpha-stimulated response element (ISRE). Following activation, ISGF3 alpha translocates to the nucleus where ISGF3 assembles as a high affinity complex on the ISRE. The biochemical basis for receptor-mediated activation of ISGF3 is unknown. We report that two potent protein kinase inhibitors, staurosporine and K-252a, ablated the transcriptional response to IFN alpha treatment. These inhibitors prevented the activation of the ISGF3 alpha component without affecting the ISGF3 gamma component, resulting in no accumulation of mature ISGF3 in nuclei of treated cells. Although these agents are potent inhibitors of protein kinase C (PKC), PKC does not mediate ISGF3 alpha activation. Down-regulation of PKC by chronic exposure of cells to 12-O-tetradecanoylphorbol-13-acetate, which led to complete loss of PKC-immunoreactive material, failed to ablate the transcriptional response to IFN alpha or the activation of ISGF3 alpha. The PKC-specific inhibitor calphostin C did not perturb activation or nuclear accumulation of ISGF3. We conclude that a novel, staurosporine/K-252a-sensitive kinase is required for ISGF3 activity and may participate in receptor-mediated signal transduction

Interferon-stimulated gene factor 3 (ISGF3) is the ligand-dependent transcriptional activator that, in response to interferon treatment, is assembled in the cell cytoplasm, is translocated to the nucleus, and binds the consensus DNA site, the interferon-stimulated response element. We have purified ISGF3 and identified its constituent proteins: a DNA-binding protein of 48 kDa and three larger polypeptides (84, 91, and 113 kDa), which themselves do not have DNA-binding activity. The multisubunit structure of ISGF3 most likely reflects its participation in receiving a ligand-dependent signal, translocating to the nucleus, and binding to DNA to activate transcription

The interaction of interferon-alpha (IFN-alpha) with a specific cell-surface receptor elicits physiological changes that rely on rapid transcriptional activation of a group of IFN-alpha-stimulated genes (ISGs). The IFN-stimulated response element (ISRE), a conserved regulatory element of all ISGs, is the target for transcriptional activation by the positive regulator IFN-stimulated gene factor-3 (ISGF3). We reported previously that post-translational activation of ISGF3 in the cytoplasm of IFN-alpha-treated cells requires two cytoplasmic activities (ISGF3 alpha and ISGF3 gamma) to produce an ISRE-binding complex that accumulates in the nucleus. In this study, we show that these activities are actually distinct subunits of the ISGF3 complex, which associate through noncovalent interaction. Sedimentation analysis, protein renaturation, and photoaffinity cross-linking of enriched preparations of cytoplasmic ISGF3 alpha and ISGF3 gamma and of nuclear ISGF3 demonstrated that ISGF3 gamma was a 48-kD polypeptide with intrinsic, low-affinity DNA-binding activity. Four polypeptides of 48, 84, 91, and 113 kD bound to the ISRE in vitro; the larger three polypeptides most likely compose the ISGF3 alpha component. These ISGF3 alpha polypeptides were unable to bind DNA alone but formed a DNA-binding complex in conjunction with ISGF3 gamma. The resulting heteromeric complex had the same ISRE-binding specificity as the individual ISGF3 gamma polypeptide but approximately 25-fold higher affinity. Whereas ISGF3 gamma partitioned between the cytoplasm and nucleus in unstimulated cells, ISGF3 alpha was stimulated to translocate to the nucleus only following IFN-alpha treatment, resulting in preferential nuclear accumulation of both ISGF3 alpha and ISGF3 gamma as a stable ISGF3-ISRE complex. This regulated nuclear translocation of an activated transcription factor subunit maintained the specificity and rapidity of the IFN-alpha signaling pathway

Interferon-stimulated gene factor 2 (ISGF2) was purified from HeLa cells treated with alpha interferon. The factor, a single polypeptide of 56 kilodaltons (kDa), bound both to the central 9 base pairs of the 15-base-pair interferon-stimulated response element (ISRE) that is required for transcriptional activation of interferon-stimulated genes and to the PRD-I regulatory element of the beta interferon gene. ISGF2 was a phosphoprotein, and dephosphorylation in vitro reduced its DNA-binding activity. However, conditions that changed the amount of ISGF2 did not change the phosphorylated isoforms in vivo. ISGF2 in unstimulated cells existed in trace amounts and was induced by both alpha interferon and gamma interferon as well as by virus infection. Plasmid-bearing Escherichia coli clones encoding ISGF2 were selected with antibody against purified ISGF2. Sequence analysis revealed that the ISGF2 protein was the same as that encoded by the cDNA clone IRF-1, which has been claimed to activate transcription of interferon genes. We show that transcription of the ISGF2 gene was induced by alpha interferon, gamma interferon, and double-stranded RNA. However, ISGF2 was neither necessary nor sufficient for induced transcription of the beta interferon gene, while the factor NF kappa B was clearly involved

Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus inducible elements (IE) in promoters of mouse alpha-interferon and human beta 1-interferon genes contain multiple copies of the hexanucleotide sequence AGT-GAA or its variants which are also found in the interferon-stimulated response element of genes transcriptionally induced by interferon. We have examined the similarities between virus and interferon induction of gene expression and the role of AGTGAA and AAT-GAA hexamers in these responses. Hybrid plasmids were constructed by inserting the IE region, the alpha 4 promoter, or the multiple copies of AGTGAA or AAT-GAA 5' to the inactive-45 human immunodeficiency-chloramphenicol acetyltransferase hybrid gene, and their inducible expression was studied in a transient expression assay. In L-cells, multiple hexamers were efficiently induced both by infection with Newcastle disease virus and by interferon treatment; while the alpha 4 promoter and the IE inducible region were induced predominantly by virus rather than by interferon. In order to dissociate the effect of virus and endogenous interferon on the induction process, we examined the gene expression in Vero cells, which have undergone homozygous deletion of type 1 interferon genes, and in VNPT-159 cells, which were derived from Vero cells by insertion of an inducible human interferon beta 1 gene. The results show that while the alpha 4 promoter was efficiently induced only by virus in both cell types, the constructs containing shorter segments of the IE were induced by both virus and interferon in Vero cells. However, the inducibility by interferon was not detected in VNPT-159 cells, suggesting that the presence of endogenous interferon suppresses interferon-induced expression of hexanucleotide repeats and the short inducible region. In contrast, virus inducibility of endogenous interferon-stimulated genes, ISG-15 and ISG-54, was about 100-fold more efficient in VNPT-159 cells than in Vero cells, suggesting that this induction is largely mediated through synthesis of endogenous interferon. Hence, endogenous interferon may play a role in the autoregulation of both interferon genes and interferon-stimulated genes