Enhanced Antiviral and Antiproliferative Properties of a STAT1 Mutant Unable to Interact with the Protein Kinase PKR

Wong, Andrew; Durbin, Joan E.; Li, Suiyang; Dever, Thomas E.; Decker, Thomas; Koromilas, Antonis E.

doi:10.1074/jbc.m011240200

Cited by 28 publications

(24 citation statements)

References 44 publications

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“…The shRNA constructs were transfected in phiNX retroviral packaging cells, which were selected with puromycin (2.5 g/ml; Sigma) and hygromycin (200 g/ml). Then, the resultant retroviruses were used to infect HT1080 cells expressing GyrB-wtPKR in the presence of Polybrene (6 g/ml; Sigma) as described (19). Targeted cells were selected with 2.5 g/ml puromycin for 2 weeks, and polyclonal populations were pooled, expanded.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation and Immunoblotting-Protein extraction and immunoprecipitation of Stat proteins were performed as described (18,19). Immunoblotting was performed as described (28).…”

Section: Methodsmentioning

confidence: 99%

“…Viral Infection-PKR ϩ/ϩ and PKR Ϫ/Ϫ MEFs (4 ϫ 10 5 ) were seeded in 6-cm plates and infected with vesicular stomatitis virus (VSV; Indiana strain) as described (19).…”

Section: Methodsmentioning

confidence: 99%

“…We previously reported that PKR physically interacts with Stat1, an interaction that is diminished in cells treated with IFN or double-stranded RNA (18,19).We showed that PKR impairs both the nuclear function of Stat1 and the induction of Stat1-dependent gene transcription in response to IFNs (18). Despite the clear inhibitory role of PKR in Stat1 function (18,19), the precise molecular events underlying this regulation have remained elusive. Herein, using mouse embryonic fibroblasts (MEFs) from a catalytic knock-out of PKR (20) and a conditionally active form of PKR (21,22), we demonstrate that PKR negatively regulates Stat1 and Stat3.…”

mentioning

confidence: 99%

“…Through its capacity to regulate protein synthesis, PKR has been considered as a major mediator of the antiviral and anti-proliferative effects of IFN (17). We previously reported that PKR physically interacts with Stat1, an interaction that is diminished in cells treated with IFN or double-stranded RNA (18,19).We showed that PKR impairs both the nuclear function of Stat1 and the induction of Stat1-dependent gene transcription in response to IFNs (18). Despite the clear inhibitory role of PKR in Stat1 function (18,19), the precise molecular events underlying this regulation have remained elusive.…”

mentioning

confidence: 99%

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The Catalytic Activity of the Eukaryotic Initiation Factor-2α Kinase PKR Is Required to Negatively Regulate Stat1 and Stat3 via Activation of the T-cell Protein-tyrosine Phosphatase

Wang

Raven

Baltzis

et al. 2006

Journal of Biological Chemistry

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The Stat 5 family of proteins plays a role in many cellular processes, including development, cell growth and proliferation, and apoptotic cell death (1, 2). These transcription factors remain latent in the cytoplasm until activated by extracellular signaling proteins, primarily cytokines and growth factors (1, 2). Stat1 plays an important role in the cellular response to interferon (IFN) and viral infection and in the regulation of proliferation and apoptosis (1). Stat1 knock-out mice are extremely susceptible to infection with viruses and other pathogens, demonstrating the essential role of this transcription factor in innate immunity (3, 4). At the molecular level, IFN treatment leads to Stat1 activation by phosphorylation at Tyr 701 mediated by activated Jaks, a family of tyrosine kinases that are associated with the cytoplasmic portions of IFN receptors (1). Tyrosine phosphorylation is essential for Stat1 dimerization, nuclear translocation, DNA binding, and gene transcription (1). Phosphorylation at Ser 727 in the C-terminal domain of Stat1 enhances its transactivation capacity (5). Stat3, another member of the Stat family, was discovered as a result of its response to interleukin-6 (IL-6) in hepatocytes (6). Stat3 is a major signal transducer downstream of gp130-like receptors, and its activation induces many responses, including proliferation of B-lymphocytes, activation of terminal differentiation and growth arrest in monocytes, and maintenance of the pluripotency of embryonic stem cells (1, 6). As with Stat1, Stat3 is tyrosine-phosphorylated at a site close to the C terminus and serine-phosphorylated within the transactivation domain (1, 7). Stat3 knock-out mice exhibit early embryonic lethality, and loss of Stat3 is even lethal in embryonic stem cells; therefore, Stat3 is also required for embryogenesis (6). Although both Stat1 and Stat3 are hyperphosphorylated in numerous cancers (8), Stat3 is categorized as a proto-oncogene, whereas Stat1 is generally believed to act as a tumor suppressor (9).The cell has developed numerous mechanisms to regulate both the duration and magnitude of Stat activation so that it can formulate appropriate responses to cytokine stimulation. Phosphorylation of Stats is inhibited by proteins known as SOCS (suppressors of cytokine signaling), which either interfere with activation of Jaks or compete with Stats for binding to cytokine receptors (10, 11). Stat activation is also limited by dephosphorylation of upstream signaling components (i.e. receptors and Jaks) by the SH2 domain-containing phosphatases (2, 10). In the nucleus, the transcriptional activities of Stats are regulated by PIAS (protein inhibitors of activated Stats), which prevent DNA binding (2), or by specific phosphatases that remove phosphate groups from the tyrosine residues of active Stat molecules (2, 10). In particular, the nuclear T-cell protein-tyrosine phosphatase (TC-PTP) (12) decreases both the cytokine-induced phosphorylation and transcriptional activities of Stat1 and Stat3, thus inhibiting the sig...

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Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation and Immunoblotting-Protein extraction and immunoprecipitation of Stat proteins were performed as described (18,19). Immunoblotting was performed as described (28).…”

Section: Methodsmentioning

confidence: 99%

“…Viral Infection-PKR ϩ/ϩ and PKR Ϫ/Ϫ MEFs (4 ϫ 10 5 ) were seeded in 6-cm plates and infected with vesicular stomatitis virus (VSV; Indiana strain) as described (19).…”

Section: Methodsmentioning

confidence: 99%