Abstract
Interferon-alpha (IFN alpha) and platelet-derived growth factor (PDGF) each rapidly stimulate binding of nuclear factors from Balb/c 3T3 fibroblasts, to a 29-base pair regulatory sequence derived from the 5' upstream region of the murine 2-5A synthetase gene. This regulatory sequence contains a functional interferon-stimulated response element (ISRE) and also functions as a PDGF-responsive sequence. We show that IFN alpha induces binding of a protein of molecular mass 65 kDa to the ISRE. Constitutively expressed ISRE-binding proteins of 98 and 150 kDa are also demonstrated. Binding of inducible factors to the ISRE increases significantly within 15 min of IFN alpha or PDGF treatment. PDGF-induced binding is not mediated by IFN beta. The protein kinase inhibitors, staurosporine and K252a, block PDGF-induced ISRE binding and 2-5A synthetase gene expression. IFN alpha-induced ISRE binding and gene activation are not blocked by these inhibitors. Treatment of cells with 12-O-tetradecanoyl-13-acetate or dibutyryl cyclic AMP does not activate ISRE binding factors or 2-5A synthetase gene expression. PDGF responsiveness of the ISRE in vivo is also sensitive to staurosporine, indicating that inhibition of a protein kinase activity blocks the PDGF-specific transcriptional signal. Our data indicate the signal transduction pathway for IFN alpha-induced, ISRE-dependent transcription is distinct from the PDGF-induced ISRE response and is likely independent of cyclic AMP-dependent protein kinase and protein kinase C activities.
Highlights
From the Research Institute, Hospitalfor Sick Children and Department of Molecular and Medical Genetics, University of Toronto, Toronto M5G 1x8,Canada
Oligonucleotides were endsignal transduction by a wide variety of growth factors, we show that induction of IFN-stimulated response element (ISRE) binding and 2-5A synthetase gene expression by IFN-a and PDGF are differentially sensitive to potent inhibitorsof these kinases
We have previously shown that a plasmid carrying the ways mediating ISRE-dependent gene activation by IFNa Escherichia coli chloramphenicol acetyltransferase gene under and PDGF,we have used the protein kinase inhibitors, stau- the control of a single ISRE,is inducible by rosporine and K252a [28, 29]. 2-5A synthetasemRNAis IFNa and PDGFin A31 cells [7]
Summary
Cell pellets (2-4 X 10' cells) were extracted with buffer A [25] for 5 marked A and H are IFN-induced, and C represents a constitutive min on ice. Cells were pelleted for 10 s a t 10,000 X g, and the binding complex (see Fig. 2). We isolated each of these complexes from native band mobil- ent protein kinase C by treatment of cells with TPA did not ity shift gels, for cross-linking analysis. ConfluAe3n1t cells were treated for and 15 min with PDGF or IFNa, and crude nuclear extracts or WCE were analyzed for ISRE binding, using the. Induced ISRE bindingwas clearlyevident (Fig. 3a).T o ensure that equal amounts of active extract were assayed in each reaction, we measured binding to a labeled oligonucleotide representing regulatorysequences inthepromoter of the human IFNP gene, and termed IRE anterferongene _Regulatory Element, Ref. 30). " ential kinetic displayed in the inductionof binding by IFNa inhibitor: 0 0 K25S2lauro
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