Interferon-alpha (IFN-alpha) and IFN-gamma regulate gene expression by tyrosine phosphorylation of several transcription factors that have the 91-kilodalton (p91) protein of interferon-stimulated gene factor-3 (ISGF-3) as a common component. Interferon-activated protein complexes bind enhancers present in the promoters of early response genes such as the high-affinity Fc gamma receptor gene (Fc gamma RI). Treatment of human peripheral blood monocytes or basophils with interleukin-3 (IL-3), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-gamma response region (GRR) located in the promoter of the Fc gamma RI gene. Although tyrosine phosphorylation was required for the assembly of each of these GRR binding complexes, only those formed as a result of treatment with IFN-gamma or IL-10 contained p91. Instead, complexes activated by IL-3 or GM-CSF contained a tyrosine-phosphorylated protein of 80 kilodaltons. Induction of Fc gamma RI RNA occurred only with IFN-gamma and IL-10, whereas pretreatment of cells with GM-CSF or IL-3 inhibited IFN-gamma induction of Fc gamma RI RNA. Thus, several cytokines other than interferons can activate putative transcription factors by tyrosine phosphorylation.

Interferon gamma (IFN gamma) induces the expression of early response genes by tyrosine phosphorylation of Jak kinases and transcription factors referred to as STAT proteins. The topology of the IFN gamma receptor is partially understood and the relationship between the alpha chain that binds the ligand and the beta chain that is required for signal transduction is undefined. In a cell line which expresses only the human alpha chain, we show that these cells did not activate Jak kinases or STAT proteins with human IFN gamma, even though Jak1 co-immunoprecipitated with the alpha chain. In cells unexposed to IFN gamma, Jak1 preferentially associated with the alpha chain, while Jak2 associated with the beta chain. There was evidence for Jak1 kinase activity in untreated cells. For Jak2, kinase activity was IFN gamma-dependent. Although the alpha chain was tyrosine-phosphorylated in response to ligand, we found no evidence for tyrosine phosphorylation of the beta chain. These data are consistent with a model of the IFN gamma receptor in which Jak1 associates with the alpha chain, whereas Jak2 associates with the beta chain. IFN gamma clusters at least two receptor units which results in the tyrosine phosphorylation of Jak1 and Jak2, the activation of Jak2 kinase activity, and the recruitment of STAT1 alpha resulting in its activation by tyrosine phosphorylation.

Gamma interferon (IFN-y) activates the formation of a DNA-binding protein complex (FcRF'y) that recognizes the gamma response region (GRR) of the promoter for the human high-affinity Fcey receptor. In a membrane-enriched fraction prepared from human peripheral blood monocytes, IFN-y activation of FcRF-y occurred within 1 min and was ATP dependent. Activation of FcRF'y required a tyrosine kinase activity, and recognition of the GRR sequence by FcRF'y could be abrogated by treatment with a tyrosine-specific protein phosphatase. Treatment of cells with vanadate alone resulted in the formation of FcRF'y without the need for EFN--y. UV cross-linking and antibody competition experiments demonstrated that the FcRF'y complex was composed of at least two components: the 91-kDa protein of the IFN-oa-induced transcription complex ISGF3 and a 43-kDa component that bound directly to the GRR. Therefore, specificity for IFN-induced transcriptional activation of early response genes requires at least two events: (i) ligand-induced activation of membrane-associated protein by tyrosine phosphorylation and (ii) formation of a complex composed of an activated membrane protein(s) and a sequence-specific DNA-binding component.

Although it has been well documented that the biological activities of gamma interferon (IFN-'y) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-y signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-'y activates the formation of the IFN--y activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-y. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-y. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-y was added. The addition of either agent 5 min after IFN-y had no effect. These results provide the first evidence for an IFNq-yregulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH-and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.