The reversible tyrosine phosphorylation of proteins, modulated by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs), regulates the cellular response to a wide variety of stimuli. It is established that protein kinases possess discrete sets of substrates and that substrate recognition is often dictated by the presence of consensus phosphorylation sites. Here, we have extended this concept to the PTPs and demonstrated that (E/D)-pY-pY-(R/K) is a consensus substrate recognition motif for PTP1B. We have shown that JAK2 and TYK2 are substrates of PTP1B and that the substrate recognition site within theses kinases is similar to the site of dephosphorylation previously identified within the insulin receptor. A substrate-trapping mutant of PTP1B formed a stable interaction with JAK2 and TYK2 in response to interferon stimulation. Expression of wild type or substrate-trapping mutant PTP1B inhibited interferon-dependent transcriptional activation. Finally, mouse embryo fibroblasts deficient in PTP1B displayed subtle changes in tyrosine phosphorylation, including hyperphosphorylation of JAK2. The closely related JAK family member, JAK1, which does not match the consensus dephosphorylation site, was not recognized as a substrate. These data illustrate that PTP1B may be an important physiological regulator of cytokine signaling and that it may be possible to derive consensus substrate recognition motifs for other members of the PTP family, which may then be used to predict novel physiological substrates.

Interferons (IFNs) are the primary innate antiviral cytokines in mammalian cells and also regulate aspects of the adaptive immune response (3,5,27). Both IFN-␣/␤ and IFN-␥ can induce an antiviral state in cells as an end point of signal transduction through the JAK-STAT pathway. The general paradigm for this signaling pathway involves IFN-dependent receptor-mediated tyrosine phosphorylation of latent cytoplasmic STAT proteins to produce DNA-binding oligomers that are competent for nuclear translocation. IFN-␣/␤ responses are mediated primarily by an activated transcription complex, ISGF3, that consists of heterodimeric STAT1 and STAT2 in association with a DNA binding subunit, IRF9. ISGF3 binds to a DNA element, ISRE, in IFN-␣/␤ target gene promoters, inducing their transcription. IFN-␥ responses are also mediated by a STAT-containing transcription complex consisting of STAT1 homodimers. The STAT1 homodimer binds to a DNA element termed GAS to induce the transcription of a distinct but overlapping set of IFN-␥ target genes. In both cases, these IFN-stimulated gene products produce a general antiviral state in the cell that inhibits the replication of diverse virus species.The Paramyxoviridae family represents a wide variety of enveloped minus-strand RNA viruses including a number of established and emerging human and animal pathogens (12). Several paramyxoviruses, particularly those in the Rubulavirus genus, have been recently demonstrated to interfere with IFNinduced antiviral responses by targeting the STAT1 and/or STAT2 proteins for proteasomal degradation. For example, mumps virus and simian virus 5 (SV5) target STAT1 for degradation, while human parainfluenza virus type 2 (HPIV2) targets STAT2 (7,11,20).The exact mechanisms underlying STAT protein targeting by the rubulaviruses are not completely understood, but it is clear that the anti-STAT effects are mediated by a single viral protein, termed V (7). The V protein is not related to any cellular proteins and does not appear to be a protease itself; instead, it acts in concert with cellular factors to mediate STAT degradation. For SV5-induced STAT1 degradation and HPIV2-induced STAT2 degradation, the targeting complex requires at least V, STAT1, and STAT2 to be present in the host cell (21). The role for a nontarget STAT protein in a STAT-targeting complex is not apparent, but available evidence indicates that the requirement for the confederate STAT protein is completely independent of IFN-␣/␤ signal transduction or ISGF3 functions and can be supplied by a STAT protein N-terminal fragment (21). Moreover, the ability of SV5 to induce the degradation of STAT1 and to antagonize IFN signaling is species specific. IFN evasion by SV5 is very efficient in primate cells but restricted in murine cells (6). The cellular basis for the differential ability of murine and human cells to create an innate antiviral response to SV5 was recently uncovered (19). The failure of SV5 to antagonize IFN responses in the mouse system is not due to intrinsic defects in the SV5 pro...

Measles virus, a paramyxovirus of the Morbillivirus genus, is responsible for an acute childhood illness that infects over 40 million people and leads to the deaths of more than 1 million people annually (C. J. Murray and A. D. Lopez, Lancet 349:1269-1276, 1997). Measles virus infection is characterized by virus-induced immune suppression that creates susceptibility to opportunistic infections. Here we demonstrate that measles virus can inhibit cytokine responses by direct interference with host STAT protein-dependent signaling systems. Expression of the measles V protein prevents alpha, beta, and gamma interferon-induced transcriptional responses. Furthermore, it can interfere with signaling by interleukin-6 and the non-receptor tyrosine kinase, v-Src. Affinity purification demonstrates that the measles V protein associates with cellular STAT1, STAT2, STAT3, and IRF9, as well as several unidentified partners. Mechanistic studies indicate that while the measles V protein does not interfere with STAT1 or STAT2 tyrosine phosphorylation, it causes a defect in IFN-induced STAT nuclear accumulation. The defective STAT nuclear redistribution is also observed in measles virusinfected cells, where some of the STAT protein is detected in cytoplasmic bodies that contain viral nucleocapsid protein and nucleic acids. Interference with STAT-inducible transcription may provide a novel intracellular mechanism for measles virus-induced cytokine inhibition that links innate immune evasion to adaptive immune suppression.Cytokine signal transduction is essential for normal immune function and controls the quality of responses to a wide variety of microbial infections. Innate and adaptive host responses to virus infections are regulated by autocrine and paracrine cytokine signaling systems. For most cytokines, receptor binding triggers an intracellular signaling cascade involving one or more signal transducer and activator of transcription (STAT) proteins. Diverse cytokine and growth factor signaling pathways produce active STAT transcription factors that specify mRNA induction profiles (26). For example, the alpha and beta interferon (IFN-␣/␤) family is of primary importance for both innate and adaptive antiviral immunity (reviewed in references 1, 49, and 53). In the innate antiviral system, IFN-␣/␤ initiates a receptor-mediated signaling system that produces an activated STAT1-STAT2-IRF9 heterotrimeric transcription complex known as ISGF3 (27). The ISGF3 complex translocates to the nucleus, where it can bind to target gene promoter elements and induce the transcription of host antiviral genes. Similarly, IFN-␥, a cytokine that mediates both innate and adaptive immune responses critical for defense against microbial infections and cancer (21), activates a homodimeric STAT1 transcription factor, GAF (9). Interleukin 6 (IL-6), a cytokine required for acute-phase responses, liver regeneration, B-cell maturation, and macrophage differentiation, activates STAT3 homodimers, a response in common with growth factor pathways (69) and onco...

Type I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3 gamma/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery.

Mumps virus is a common infectious agent of humans, causing parotitis, meningitis, encephalitis, and orchitis. Like other paramyxoviruses in the genus Rubulavirus, mumps virus catalyzes the proteasomal degradation of cellular STAT1 protein, a means for escaping antiviral responses initiated by alpha/beta and gamma interferons. We demonstrate that mumps virus also eliminates cellular STAT3, a protein that mediates transcriptional responses to cytokines, growth factors, nonreceptor tyrosine kinases, and a variety of oncogenic stimuli. STAT1 and STAT3 are independently targeted by a single mumps virus protein, called V, that assembles STAT-directed ubiquitylation complexes from cellular components, including STAT1, STAT2, STAT3, DDB1, and Cullin4A. Consequently, mumps virus V protein prevents responses to interleukin-6 and v-Src signals and can induce apoptosis in STAT3-dependent multiple myeloma cells and transformed murine fibroblasts. These findings demonstrate a unique cytokine and oncogene evasion property of mumps virus that provides a molecular basis for its observed oncolytic properties.Gene expression and biological responses to cytokines and polypeptide growth factors are often mediated by signal transducer and activator of transcription (STAT) proteins (12). The activities of various STAT proteins control cell growth and differentiation, organogenesis, embryonic development, and host responses to cancer and infection. Inappropriate activation of STAT signaling is frequently observed in human diseases, including inflammation, asthma, autoimmunity, and cancer, suggesting that inhibitors of STAT factors might be of wide-ranging therapeutic value (reviewed in reference 55 and references therein).Of the seven mammalian STAT proteins, STAT1, STAT2, and STAT3 exhibit the broadest expression profiles and respond to activating stimuli in most cell types (70, 71). A transcription factor complex, ISGF3, containing activated STAT1 and STAT2 is essential for induction of alpha/beta interferon (IFN-␣/␤) target genes that establish an innate cellular antiviral state (54). Similarly, the transcriptional response to IFN-␥ is mediated by a STAT1 homodimer that is required for IFN-␥-dependent innate and adaptive immune responses (31). The third widely expressed STAT protein, STAT3, is activated by many cytokine systems, including interleukin 6 (IL-6), leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, and leptin (reviewed in reference 28). STAT3 is also activated by growth factor receptors with intrinsic protein tyrosine kinase activity (e.g., platelet-derived and epidermal growth factor receptors [53,61]) as well as cellular and viral cytoplasmic tyrosine kinases (e.g., c-Src and v-Src [6,68]).Targeted disruption of STAT3 in mice results in early embryonic lethality (57). Tissue-specific disruption has revealed diverse STAT3 functions in mammary gland, liver, keratinocytes, thymus, blood, and neurons that are involved in growth and differentiation, inflammation, liver regeneration, acutephase responses...