Role of the intracellular domain of the human type I interferon receptor 2 chain (IFNAR2c) in interferon signaling. Expression of IFNAR2c truncation mutants in U5A cells

A human cell line (U5A) lacking the type I interferon (IFN) receptor chain 2 (IFNAR2c) was used to determine the role of the IFNAR2c cytoplasmic domain in regulating IFN-dependent STAT activation, interferon-stimulated gene factor 3 (ISGF3) and c-sis-inducible factor (SIF) complex formation, gene expression, and antiproliferative effects. A panel of U5A cells expressing truncation mutants of IFNAR2c on their cell surface were generated for study. Janus kinase (JAK) activation was detected in all mutant cell lines; however, STAT1 and STAT2 activation was observed only in U5A cells expressing full-length IFNAR2c and IFNAR2c truncated at residue 462 (R2.462). IFNAR2c mutants truncated at residues 417 (R2. 417) and 346 (R2.346) or IFNAR2c mutant lacking tyrosine residues in its cytoplasmic domain (R2.Y-F) render the receptor inactive. A similar pattern was observed for IFN-inducible STAT activation, STAT complex formation, and STAT-DNA binding. Consistent with these data, IFN-inducible gene expression was ablated in U5A, R2.Y-F, R2.417, and R2.346 cell lines. The implications are that tyrosine phosphorylation and the 462-417 region of IFNAR2c are independently obligatory for receptor activation. In addition, the distal 53 amino acids of the intracellular domain of IFNAR2c are not required for IFN-receptor mediated STAT activation, ISFG3 or SIF complex formation, induction of gene expression, and inhibition of thymidine incorporation. These data demonstrate for the first time that both tyrosine phosphorylation and a specific domain of IFNAR2c are required in human cells for IFN-dependent coupling of JAK activation to STAT phosphorylation, gene induction, and antiproliferative effects. In addition, human and murine cells appear to require different regions of the cytoplasmic domain of IFNAR2c for regulation of IFN responses.     

Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1: similarities with type I interferon signaling

Interferon (IFN)-lambda 1, -lambda 2, and -lambda 3 are the latest members of the class II cytokine family and were shown to have antiviral activity. Their receptor is composed of two chains, interleukin-28R/likely interleukin or cytokine or receptor 2 (IL-28R/LICR2) and IL-10R beta, and mediates the tyrosine phosphorylation of STAT1, STAT2, STAT3, and STAT5. Here, we show that activation of this receptor by IFN-lambda 1 can also inhibit cell proliferation and induce STAT4 phosphorylation, further extending functional similarities with type I IFNs. We used IL-28R/LICR2-mutated receptors to identify the tyrosines required for STAT activation, as well as antiproliferative and antiviral activities. We found that IFN-lambda 1-induced STAT2 tyrosine phosphorylation is mediated through tyrosines 343 and 517 of the receptor, which showed some similarities with tyrosines from type I IFN receptors involved in STAT2 activation. These two tyrosines were also responsible for antiviral and antiproliferative activities of IFN-lambda 1. By contrast, STAT4 phosphorylation (and to some extent STAT3 activation) was independent from IL-28R/LICR2 tyrosine residues. Taken together, these observations extend the functional similarities between IFN-lambdas and type I IFNs and shed some new light on the mechanisms of activation of STAT2 and STAT4 by these cytokines.     

Involvement of the Gab2 scaffolding adapter in type I interferon signalling

Interferons (IFNs) are pleiotropic cytokines involved in the regulation of physiological and pathological processes. Upon interaction with their specific receptors, IFNs activate the Jak/STAT signalling pathway. Numerous studies suggest, however, that the classical Jak/STAT pathway cannot alone account for the wide range of IFN's biological effects. To better understand the role of alternative signalling pathways in the type I IFNs response, we analyzed novel tyrosine-phosphorylated proteins following IFN-alpha2 stimulation. We showed for the first time that the Grb2-associated binder 2 (Gab2) protein is differentially phosphorylated upon the IFN subtype employed and the cells stimulated. We demonstrated that IFNAR1 physically interacts with Gab2. Moreover, the cellular content of Gab2 varies as a function of IFN receptor chain expression levels, and in particular of the ratio of IFNAR1 to IFNAR2, suggesting that Gab2 and IFNAR2 compete for interaction with IFNAR1. Analysis of Gab2 deletion mutants indicates that IFNAR1 might interact with a Gab2 region containing p85-PI3'kinase binding sites. Our results shed new light on recent data involving both Gab2 and type I IFNs in osteoclastogenesis and oncogenesis.     

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.     

Formation of a uniquely stable type I interferon receptor complex by interferon beta is dependent upon particular interactions between interferon beta and its receptor and independent of tyrosine phosphorylation

Human type I interferons (IFN) require two receptor chains, IFNAR1 and IFNAR2c for high affinity (pM) binding and biological activity. Our previous studies have shown that the ligand dependent assembly of the type I IFN receptor chains is not identical for all type I IFNs. IFNbeta appears unique in its ability to assemble a stable complex of receptor chains, as demonstrated by the observation that IFNAR2c co-immunoprecipitates with IFNAR1 when cells are stimulated with IFNbeta but not with IFNalpha. The characteristics of such a receptor complex are not well defined nor is it understood if differential signaling events can be mediated by variations in receptor assembly. To further characterize the factors required for formation of such a stable receptor complex we demonstrate using IFN stimulated Daudi cells that (1) IFNAR2c co-immunoprecipitates with IFNAR1 even when tyrosine phosphorylation of receptor chains is blocked with staurosporine, and (2) IFNbeta1b but not IFNalpha2, is present in the immunoprecipitated receptor complex. These results demonstrate that the unique IFNbeta induced assembly of type I IFN receptor chains is independent of receptor tyrosine phosphorylation and the recruitment of additional proteins to the receptor by such events. Furthermore, the presence of IFNbeta1b in the immunoprecipitated IFN receptor complex suggests that IFNbeta interacts and binds differently to the receptor than IFNalpha2. These results suggest that the specific assembly of type I IFN receptor chains is ligand dependent and may represent an early event which leads to the differential biological responses observed among type I IFNs.     

Essential Role for the Mnk Pathway in the Inhibitory Effects of Type I Interferons on Myeloproliferative Neoplasm (MPN) Precursors

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.