Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. (1/524)

Shp-2 is an SH2 domain-containing protein tyrosine phosphatase. Although the mechanism remains to be defined, substantial experimental data suggest that Shp-2 is primarily a positive regulator in cell growth and development. We present evidence here that Shp-2, while acting to promote mitogenic signals, also functions as a negative effector in interferon (IFN)-induced growth-inhibitory and apoptotic pathways. Treatment of mouse fibroblast cells lacking a functional Shp-2 with IFN-alpha or IFN-gamma resulted in an augmented suppression of cell viability compared to that of wild-type cells. To dissect the molecular mechanism, we examined IFN-induced activation of signal transducers and activators of transcription (STATs) by electrophoretic mobility shift assay, using a specific DNA probe (hSIE). The amounts of STAT proteins bound to hSIE upon IFN-alpha or IFN-gamma stimulation were significantly increased in Shp-2(-/-) cells. Consistently, tyrosine phosphorylation levels of Stat1 upon IFN-gamma treatment and, to a lesser extent, upon IFN-alpha stimulation were markedly elevated in mutant cells. Furthermore, IFN-gamma induced a higher level of caspase 1 expression in Shp-2(-/-) cells than in wild-type cells. Reintroduction of wild-type Shp-2 protein reversed the hypersensitivity of Shp-2(-/-) fibroblasts to the cytotoxic effect of IFN-alpha and IFN-gamma. Excessive activation of STATs by IFNs was also diminished in mutant cells in which Shp-2 had been reintroduced. Together, these results establish that Shp-2 functions as a negative regulator of the Jak/STAT pathway. We propose that Shp-2 acts to promote cell growth and survival through two mechanisms, i.e., the stimulation of growth factor-initiated mitogenic pathways and the suppression of cytotoxic effect elicited by cytokines, such as IFNs.  (+info)

Dissociation between IFN-alpha-induced anti-viral and growth signaling pathways. (2/524)

The ability of IFN-alpha to induce an anti-viral state in a wide variety of cell types as well as to inhibit cellular growth has long been appreciated. It is less clear, however, whether both these effects lie downstream of a common signaling pathway. In this study we have taken advantage of an atypical human myeloma cell line (KAS-6/1) displaying a dramatic proliferative response to IFN-alpha in an effort to resolve the signaling requirements for IFN-alpha-induced anti-viral and growth regulatory effects. Thus, we have analyzed the ability of IFN-alpha to induce a number of known receptor-initiated events in this cell line and have compared these responses with those exhibited by a cell lineage- and maturation stage-matched myeloma cell line (ANBL-6) that displays typical IFN-alpha responsiveness. Despite the widely contrasting effects of IFN-alpha on cellular proliferation, IFN-alpha was shown to be comparable in its ability to induce the expression of early response genes as well as induce resistance to viral infection in both cell lines. By contrast, the effects of IFN-alpha on the activation of mitogen-activated protein kinase (MAPK) were strikingly distinct. Finally, although inhibition of MEK and MAPK activation had no effect on the induction of the anti-viral response, it completely blocked IFN-alpha-stimulated proliferation of the KAS-6/1 cells. In summary, our analysis of the role of the MAPK and anti-viral signaling pathways using these two cell lines suggests that the anti-viral and growth regulatory effects of IFN-alpha display a differential requirement for activation of the MAPK pathway.  (+info)

The intracellular domain of interferon-alpha receptor 2c (IFN-alphaR2c) chain is responsible for Stat activation. (3/524)

Type I IFNs activate the Jak-Stat signal transduction pathway. The IFN-alpha receptor 1 (IFN-alphaR1) subunit and two splice variants of the IFN-alphaR2 subunit, IFN-alphaR2c and IFN-alphaR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-gammaR2 or IFN-gammaR1 fused to the intracellular domains of IFN-alphaR1, IFN-alphaR2b, and IFN-alphaR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-gamma exhibits no activity on hamster cells, the use of the human IFN-gamma receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-alphaR2c intracellular domain. When chimeric receptors with the human IFN-gammaR1 extracellular domain and various human IFN-alpha receptor intracellular domains were expressed in hamster cells carrying the human IFN-gammaR2 subunit, only the IFN-alphaR2c subunit was capable of supporting IFN-gamma signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-alphaR2b nor the IFN-alphaR1 intracellular domain was able to recruit Stats or support IFN-gamma-induced biological activities. Thus, the IFN-alphaR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins.  (+info)

Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells. (4/524)

Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly up-regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors.  (+info)

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2. (5/524)

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.  (+info)

Human MxA protein protects mice lacking a functional alpha/beta interferon system against La crosse virus and other lethal viral infections. (6/524)

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.  (+info)

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies. (7/524)

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.  (+info)

Characterization of a soluble ternary complex formed between human interferon-beta-1a and its receptor chains. (8/524)

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-beta-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.  (+info)