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

Abstract

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...

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

“…Notably, IFN-␥ treatment did not cause autophagy in K562 cells. Consistent with previous reports (25,26), inactivating the mammalian target of rapamycin increased STAT1 activation. In addition, the combination of IFN-␥ and CCI-779, a rapamycin analog that inhibits the mammalian target of rapamycin, increases anticancer activities through apoptosis (27).…”

Inhibition of Mammalian Target of Rapamycin Augments Lipopolysaccharide-Induced Lung Injury and Apoptosis

“…Moreover, we recently reported the suppression of STAT1-dependent genes (e.g., Fas, caspase-1, inducible NO synthase) by mTOR (16), and reasoned that inactivation of mTOR would enhance apoptosis in vivo. In the current study, administration of intratracheal LPS significantly increased levels of cleaved caspase-3 (8.4 versus 4.2 positive cells per high-power field; p , 0.05; Fig.…”

“…We recently iden-tified a physical and functional interaction between mTOR and STAT1 (15). In intact cells, inhibition of mTOR kinase activity lead to increased STAT1 nuclear content and transcription of IFNsensitive apoptosis genes (16). Based on these studies, we hypothesized that mTOR blockade with rapamycin would augment LPS-induced injury and apoptosis in the lung, as well as the STAT1 transcriptional response in vivo.…”

mTOR Signaling in Lymphangioleiomyomatosis

“…104 Interestingly, PKCd is a kinase for the transcription factor STAT1 kinase, and was detected in a rapamycin-sensitive macromolecular complex with mTOR and STAT1. [109][110][111] …”

“…[117][118][119] Conversely, inactivation of mTOR or its associated phosphatase complex, promoted its association with STAT1, enhanced STAT1 nuclear content, and amplified the transcription of STAT1-dependent anti-proliferative and proapoptotic genes. 109 The balance between STAT1 and STAT3 is dysregulated in LAM, and is thought to control the fate of neoplastic cells. 120 mTOR also activates NF-kB, a pro-proliferative transcription factor implicated in the pathogenesis of cancer.…”