Isolation and Characterization of a Human STAT1Gene Regulatory Element

Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth, primarily via regulation of protein synthesis. In Saccharomyces cerevisiae, TOR can also suppress the transcription of stress response genes by a mechanism involving Tap42, a serine/threonine phosphatase subunit, and the transcription factor Msn2. A physical association between mammalian TOR (mTOR) and the transcription factor signal transducer and activator of transcription-1 (STAT1) was recently identified in human cells, suggesting a similar role for mTOR in the transcription of interferon-␥-stimulated genes. In the current study, we identified a macromolecular protein complex composed of mTOR, STAT1, the Tap42 homologue ␣4, and the protein phosphatase 2A catalytic subunit (PP2Ac). Inactivation of mTOR enhanced its association with STAT1 and increased STAT1 nuclear content in PP2Ac-dependent fashion. Depletion of ␣4, PP2A, or mTOR enhanced the induction of early (i.e. IRF-1) and late (i.e. caspase-1, hiNOS, and Fas) STAT1-dependent genes. The regulation of IRF-1 or caspase-1 by mTOR was independent of other known mTOR effectors p70 S6 kinase and Akt. These results describe a new role for mTOR and ␣4/PP2A in the control of STAT1 nuclear content, and the expression of interferon-␥-sensitive genes involved in immunity and apoptosis.The macrocyclic lactone rapamycin (Sirolimus, Rapamune TM ), as well as its analogues temsirolimus (CCI-779, Torisel TM ) and everolimus (RAD-001, Certican TM ), are approved for immunosuppression after organ transplantation, treatment of renal cell carcinoma, and the prevention of coronary artery in-stent restenosis (1). Their only known target is mammalian target of rapamycin (mTOR), 3 a highly conserved protein that controls cell growth in response to mitogens and changes in cellular metabolism. The effects of mTOR on cell growth involve the phosphorylation of p70 S6 kinase (S6K) and the translation inhibitor 4E-BP1, key regulators of ribosomal biogenesis and the initiation of protein synthesis (2). In contrast to its role in the initiation of translation, the current study focuses on mTOR as a regulator of mammalian gene transcription. Studies in Saccharomyces cerevisiae have revealed possible mechanisms by which mTOR might control mammalian transcription factors. TOR-regulated transcriptional control pathways include ribosomal biogenesis, the nutrient deprivation response, and the stress response (3). TOR stimulates 35 S ribosomal RNA expression and ribosomal biogenesis in nutrientdependent fashion. Inhibition of TOR (e.g. rapamycin or amino acid depletion) reproduces a catabolic response in part by inhibiting rRNA synthesis. In the nutrient deprivation and stress responses, TOR controls the nuclear localization of key transcription factors by mechanisms that require its associated serine/threonine phosphatases (i.e. Pph21, Pph22, or Sit4) and their adaptor, Tap42 (3). Rapamycin blocks phosphorylation of the transcription factor Gln3, as well as its cytosolic scaffolding protein Ure...

Section: Discussionmentioning

confidence: 99%

Section: Inactivation Of Mtor Enhances the Induction Of Early Ifn-␥-smentioning

confidence: 99%

See 1 more Smart Citation

Inactivation of Mammalian Target of Rapamycin Increases STAT1 Nuclear Content and Transcriptional Activity in α4- and Protein Phosphatase 2A-dependent Fashion

Fielhaber¹,

Han²,

Tan

et al. 2009

“…8B), a transcription factor with tumor suppressor properties that has been shown to revert the transformed phenotype of Ras-transformed cells in vitro and in vivo (73) and plays a major role in transcriptional activation of STAT1 (52). Mouse embryonal fibroblasts homozygous for IRF-1 deficiency undergo transformation upon expression of an activated form of Ha-Ras and no longer require a cooperating oncogene, such as c-myc (74).…”

Section: Fig 3 Concentration-dependent Induction Of Stat2 By Ifn␥ Imentioning

confidence: 99%

Oncogenic Ki-Ras Inhibits the Expression of Interferon-responsive Genes through Inhibition of STAT1 and STAT2 Expression

Klampfer

Huang

Corner

et al. 2003

“…A number of studies have shown that various cell types, including primary chronic lymphocytic leukemia, human trophoblast, melanoma, and acute promyelocytic leukemia, are resistant to the actions of IFN because the induction of p48 protein is defective (9 -12). Additionally, it has been argued that IFN-␥ priming up-regulates p48-enhanced antiviral and antitumor effects of class I IFNs on IFN-resistant cells (13,14). Furthermore, recent studies have shown that p48 protein also binds to virus-inducible elements within the IFN-␣/␤ promoters.…”

mentioning

confidence: 99%

CCAAT/Enhancer-binding Protein β Mediates Interferon-γ-induced p48 (ISGF3-γ) Gene Transcription in Human Monocytic Cells

Xiao

Wang

Yang

et al. 2001

Previous studies have identified a novel interferonstimulated response element-like element, termed ␥-interferon-activating transcription element, within the interferon-stimulating gene factor-3␥ ( Interferon-stimulated gene factor-3 (ISGF3) 1 plays a crucial role in mediating the antiviral, antitumor, and immune responses induced by IFNs. The ISGF3 complex consists of a 48-kDa DNA-binding protein (p48 or ISGF3-␥) and two signal transducer and activators of transcription (STAT) proteins, STAT1 and STAT2. The p48 protein in the ISGF3 complex serves as the adaptor to redirect STAT multimers from their intrinsic palindrome sequence specificity and allows binding to the IFN-stimulated response element (ISRE) that occurs within the promoters of various IFN-stimulated genes (ISGs) (2). In addition to the ISRE element, a distinct cis-element termed IFN-␥-activated site (GAS) is also found within the promoters of many IFN-inducible genes and is bound by the STAT1 homodimer (3). The p48 protein belongs to the interferon regulatory factor (IRF)-myb family of transacting factors. This family of transcription factors can activate (IRF-1 and p48), repress (IRF-2 and interferon consensus sequence-binding protein), both activate and repress (IRF-4), and may activate or repress (IRF-3) transcription of target genes (4). Studies of p48 knockout mice have shown that p48 plays an essential role in both type I and type II IFN responses for activation of IFN-inducible genes and establishment of the antiviral state (5). A recent study showed that an ISRE-like sequence in the hepatitis B viral enhancer-1 could interact with the ISGF3 complex containing p48 and mediate the IFN-␣-induced suppression of enhancer activity (6). Other independent studies also demonstrated that overexpression of p48 in NIH-3T3 cells restored IFN responses of adenovirus E1A-expressing cells in which the IFN signaling was blocked by E1A (7,8). Although IFN and other cytokines induced by the Janus tyrosine kinase-STAT pathway have been well described, the mechanisms regulating expression of the p48 component of ISGF3 remain largely unknown. A number of studies have shown that various cell types, including primary chronic lymphocytic leukemia, human trophoblast, melanoma, and acute promyelocytic leukemia, are resistant to the actions of IFN because the induction of p48 protein is defective (9 -12). Additionally, it has been argued that IFN-␥ priming up-regulates p48-enhanced antiviral and antitumor effects of class I IFNs on IFN-resistant cells (13,14). Furthermore, recent studies have shown that p48 protein also binds to virus-inducible elements within the IFN-␣/␤ promoters. This evidence suggests a potentially new role for ISGF3 in that it may participate directly in the activation of IFN-␣/␤ promoters (15, 16). Therefore, understanding the transcriptional regulation of p48 is very important for elucidating the global functions of IFNs and signaling pathway networks among cytokines.Previously, we demonstrated that a novel IFN-␥-activated transcription element ...