scholarly journals The Linker Domain of Stat1 Is Required for Gamma Interferon-Driven Transcription

1999 ◽  
Vol 19 (7) ◽  
pp. 5106-5112 ◽  
Author(s):  
Edward Yang ◽  
Zilong Wen ◽  
Richard L. Haspel ◽  
Jue J. Zhang ◽  
James E. Darnell
Keyword(s):  
Dna Binding ◽  
Nuclear Translocation ◽  
Contact Point ◽  
Sh2 Domain ◽  
Gamma Interferon ◽  
Transactivation Domain ◽  
Dimerization Domain ◽  
Transcriptional Responses ◽  
Ifn Γ ◽  
Linker Domain

ABSTRACT Upon binding of gamma interferon (IFN-γ) to its receptor, the latent transcription factor Stat1 becomes phosphorylated, dimerizes, and enters the nucleus to activate transcription. In response to IFN-α, Stat1 binds to Stat2 in a heterodimer that recruits p48, an IRF family member, to activate transcription. A number of functional domains of the STATs, including a C-terminal transactivation domain, a dimerization domain, and an SH2 domain, are known. However, the highly conserved residues between the DNA binding and SH2 domains (463 to 566), recently christened the linker domain on the basis of crystallographic studies, have remained without a known function. In the present study, we report that KE544-545AA point mutants in Stat1 abolish transcriptional responses to IFN-γ but not to IFN-α. We further show that this mutant Stat1 undergoes normal phosphorylation, nuclear translocation, and DNA binding. Taken together with recent structural evidence, these results suggest that the linker domain acts as a critical contact point during the construction of a Stat1-driven transcriptional complex.

scholarly journals Helicobacter pylori Disrupts STAT1-Mediated Gamma Interferon-Induced Signal Transduction in Epithelial Cells

2004 ◽  
Vol 72 (1) ◽  
pp. 537-545 ◽  
Author(s):  
David J. Mitchell ◽  
Hien Q. Huynh ◽  
Peter J. M. Ceponis ◽  
Nicola L. Jones ◽  
Philip M. Sherman
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Gamma Interferon ◽  
Cell Protein ◽  
Stat1 Signaling ◽  
Ifn Γ ◽  
H Pylori

ABSTRACT Infection with Helicobacter pylori is chronic despite a vigorous mucosal immune response characterized by gastric T-helper type 1 cell expansion and gamma interferon (IFN-γ) production. IFN-γ signals by activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1); however, the effect of H. pylori infection on IFN-γ-STAT1 signaling is unknown. We infected human gastric (MKN45 and AGS) and laryngeal (HEp-2) epithelial cell lines with type 1 and type 2 H. pylori strains and then stimulated them with IFN-γ. Western blotting of whole-cell protein extracts revealed that infection with live, but not heat-killed, H. pylori time-dependently decreased IFN-γ-induced STAT1 tyrosine phosphorylation. Electrophoretic mobility shift assay of nuclear protein extracts demonstrated that H. pylori infection reduced IFN-γ-induced STAT1 DNA binding. STAT1 was unable to translocate from the cytoplasm to the nucleus in H. pylori-infected HEp-2 cells examined by immunofluorescence, and reverse transcription-PCR showed that IFN-γ-induced interferon regulatory factor 1 expression was inhibited. These effects were independent of the cagA, cagE, and VacA status of the infecting H. pylori strain. Furthermore, neither H. pylori culture supernatants nor conditioned medium from H. pylori-infected MKN45 cells inhibited IFN-γ-induced STAT1 tyrosine phosphorylation, suggesting that inhibition is independent of a soluble epithelial or bacterial factor but is dependent on bacterial contact with epithelial cells. H. pylori disruption of IFN-γ-STAT1 signaling in epithelial cells may represent a mechanism by which the bacterium modifies mucosal immune responses to promote its survival in the human host.

scholarly journals Dimerization of the Papillomavirus E2 Protein Is Required for Efficient Mitotic Chromosome Association and Brd4 Binding

2008 ◽  
Vol 82 (15) ◽  
pp. 7298-7305 ◽  
Author(s):  
Juan Cardenas-Mora ◽  
Jonathan E. Spindler ◽  
Moon Kyoo Jang ◽  
Alison A. McBride
Keyword(s):  
Dna Binding ◽  
Fluorescent Protein ◽  
Binding Domain ◽  
Bovine Papillomavirus ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Mitotic Chromosomes ◽  
Dimerization Domain ◽  
E2 Protein ◽  
Binding Function

ABSTRACT The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.

scholarly journals Vaccinia Virus Blocks Gamma Interferon Signal Transduction: Viral VH1 Phosphatase Reverses Stat1 Activation

2001 ◽  
Vol 75 (7) ◽  
pp. 3185-3196 ◽  
Author(s):  
Pilar Najarro ◽  
Paula Traktman ◽  
John A. Lewis
Keyword(s):  
Signal Transduction ◽  
Vaccinia Virus ◽  
Okadaic Acid ◽  
Nuclear Translocation ◽  
Gamma Interferon ◽  
Viral Protein Synthesis ◽  
Host Immune Responses ◽  
Infected Cells ◽  
Ifn Γ

ABSTRACT We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-γ) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-γ inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-γ. While phosphorylation of other proteins in the IFN-γ pathway was not affected, activation of Stat1 by other ligand-receptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na3VO4 but not to okadaic acid. Addition of Na3VO4 but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-γ signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-γ signal cascade through the dephosphorylating activity of the viral phosphatase VH1.

scholarly journals Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

2015 ◽  
Vol 112 (48) ◽  
pp. 14811-14816 ◽  
Author(s):  
Claudia Mertens ◽  
Bhagwattie Haripal ◽  
Sebastian Klinge ◽  
James E. Darnell
Keyword(s):  
Dna Binding ◽  
Tyrosine Phosphorylation ◽  
Transcriptional Activation ◽  
Sh2 Domain ◽  
Binding Domain ◽  
Structural Basis ◽  
Dna Binding Domain ◽  
Functional Interaction ◽  
Drug Candidates ◽  
Linker Domain

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

scholarly journals Phosphorylation-Induced Dimerization of Interferon Regulatory Factor 7 Unmasks DNA Binding and a Bipartite Transactivation Domain

2000 ◽  
Vol 20 (23) ◽  
pp. 8803-8814 ◽  
Author(s):  
Isabelle Marié ◽  
Eric Smith ◽  
Arun Prakash ◽  
David E. Levy
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Active Form ◽  
Interferon Regulatory Factor ◽  
Transactivation Domain ◽  
Regulatory Factor ◽  
Dimerization Domain ◽  
Nuclear Retention ◽  
Interferon Regulatory Factor 7

ABSTRACT Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-α genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding.

scholarly journals The E2A-HLF Oncoprotein ActivatesGroucho-Related Genes and SuppressesRunx1

2001 ◽  
Vol 21 (17) ◽  
pp. 5935-5945 ◽  
Author(s):  
Jinjun Dang ◽  
Takeshi Inukai ◽  
Hidemitsu Kurosawa ◽  
Kumiko Goi ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Fusion Gene ◽  
Chromosomal Translocation ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Representational Difference Analysis ◽  
Transactivation Domain ◽  
Dimerization Domain ◽  
Dna Binding Activity

ABSTRACT The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells, encodes a chimeric transcription factor consisting of the transactivation domain of E2A linked to the bZIP DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF). This oncoprotein blocks apoptosis induced by growth factor deprivation or irradiation, but the mechanism for this effect remains unclear. We therefore performed representational difference analysis (RDA) to identify downstream genetic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were differentially upregulated in E2A-HLF-positive cells after zinc induction. The corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptional corepressor that lacks DNA-binding activity on its own but can act in concert with other proteins to regulate embryologic development of the fly. Expression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected increased amounts of two additional Groucho-related proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell survival and activated Groucho-related genes. Among the transcription factors known to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis.

scholarly journals Akt Suppresses Apoptosis by Stimulating the Transactivation Potential of the RelA/p65 Subunit of NF-κB

2000 ◽  
Vol 20 (5) ◽  
pp. 1626-1638 ◽  
Author(s):  
Lee V. Madrid ◽  
Cun-Yu Wang ◽  
Denis C. Guttridge ◽  
Arndt J. G. Schottelius ◽  
Albert S. Baldwin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Dominant Negative ◽  
Transactivation Domain ◽  
Iκb Kinase ◽  
Transactivation Potential ◽  
Dependent Signal ◽  
Phosphoinositide 3 Kinase ◽  
Induced Apoptosis

ABSTRACT It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65.

scholarly journals Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains

1999 ◽  
Vol 19 (4) ◽  
pp. 2465-2474 ◽  
Author(s):  
Rongtuan Lin ◽  
Yael Mamane ◽  
John Hiscott
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Virus Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Interferon Regulatory Factor ◽  
Phosphorylation Sites ◽  
Transactivation Domain ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3

ABSTRACT The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.

scholarly journals Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

2005 ◽  
Vol 79 (9) ◽  
pp. 5632-5639 ◽  
Author(s):  
Chulbul M. I. Ahmed ◽  
Marjorie A. Burkhart ◽  
Prem S. Subramaniam ◽  
Mustafa G. Mujtaba ◽  
Howard M. Johnson
Keyword(s):  
Antiviral Activity ◽  
Vaccinia Virus ◽  
Virulence Factor ◽  
Nuclear Translocation ◽  
Extracellular Domain ◽  
Gamma Interferon ◽  
Peptide Mimetics ◽  
Direct Role ◽  
African Green Monkey Kidney ◽  
Ifn Γ

ABSTRACT We have developed peptide mimetics of gamma interferon (IFN-γ) that play a direct role in the activation and nuclear translocation of STAT1α transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-γ receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-γ and prevents its interaction with the receptor. Human and murine IFN-γ mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-γ mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-γ, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-γ receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-γ was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-γ mimetics was not affected by B8R protein, while IFN-γ-induced upregulation was blocked. Finally, the mimetics, but not IFN-γ, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-γ can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.

scholarly journals Leishmania donovani Amastigotes Impair Gamma Interferon-Induced STAT1α Nuclear Translocation by Blocking the Interaction between STAT1α and Importin-α5

2010 ◽  
Vol 78 (9) ◽  
pp. 3736-3743 ◽  
Author(s):  
Christine Matte ◽  
Albert Descoteaux
Keyword(s):  
Leishmania Donovani ◽  
Nuclear Translocation ◽  
Protozoan Parasite ◽  
Gamma Interferon ◽  
Janus Kinase ◽  
Mouse Bone Marrow ◽  
Protein Levels ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
The Impact

ABSTRACT The protozoan parasite Leishmania donovani, the etiological agent of visceral leishmaniasis, is renowned for its capacity to sabotage macrophage functions and signaling pathways stimulated by activators such as gamma interferon (IFN-γ). Our knowledge of the strategies utilized by L. donovani to impair macrophage responsiveness to IFN-γ remains fragmentary. In the present study, we investigated the impact of an infection by the amastigote stage of L. donovani on IFN-γ responses and signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in mouse bone marrow-derived macrophages. The levels of IFN-γ-induced expression of major histocompatibility complex class II and inducible nitric oxide synthase (iNOS) were strongly reduced in L. donovani amastigote-infected macrophages. As the expression of those genes is mediated by the transcription factors STAT1α and IFN regulatory factor 1 (IRF-1), we investigated their activation in amastigote-infected macrophages treated with IFN-γ. We found that whereas STAT1α protein levels and the levels of phosphorylation on Tyr701 and Ser727 were normal, IRF-1 expression was inhibited in infected macrophages. This inhibition of IRF-1 expression correlated with a defective nuclear translocation of STAT1α, and further analyses revealed that the IFN-γ-induced STAT1α association with the nuclear transport adaptor importin-α5 was compromised in L. donovani amastigote-infected macrophages. Taken together, our results provide evidence for a novel mechanism used by L. donovani amastigotes to interfere with IFN-γ-activated macrophage functions and provide a better understanding of the strategies deployed by this parasite to ensure its intracellular survival.

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