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

Abstract

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...

Double-stranded RNA-dependent Protein Kinase Phosphorylation of the α-Subunit of Eukaryotic Translation Initiation Factor 2 Mediates Apoptosis

“…It has been proposed that PKR mediates apoptosis at the transcriptional level. For example, PKR signals through STAT1 and STAT3 (36,43,44), interferon regulatory factor 1 (IRF1) (16,45), IRF3 (46), p53 (17,47,48), and the IKK complex to regulate transcription during proinflammatory (16,17,40,45) and/or proapoptotic responses (12,40,45,49). PKR-dependent apoptosis was also associated with Fas-associated death domain (FADD)-mediated activation of caspase 8 (50,51) and up-regulation of Fas and Bax (18,52,53).…”

“…These results suggest that eIF2␣ phosphorylation may inhibit the translation of a short lived inhibitor of apoptosis, such as p53 (36) or inhibitors of caspase activation (IAPs). Most IAPs contain a C-terminal RING-Zinc finger domain that has ubiquitin ligase (E3) activity and is responsible for their rapid degradation mediated by the proteasome (37).…”

eIF2α Kinase PKR Modulates the Hypoxic Response by Stat3-Dependent Transcriptional Suppression of HIF-1α

“…T-cell protein tyrosine phosphatase −/− (TC-PTP −/− ) MEFs and their isogenic counterparts were cultured as described (18). HT1080 expressing GyrB.PKR were maintained as previously described (19 Protein extraction, immunoblot analysis, and immunoprecipitation Protein extraction, immunoblotting, and immunoprecipitation were performed as described (18).…”

“…T-cell protein tyrosine phosphatase −/− (TC-PTP −/− ) MEFs and their isogenic counterparts were cultured as described (18). HT1080 expressing GyrB.PKR were maintained as previously described (19 Protein extraction, immunoblot analysis, and immunoprecipitation Protein extraction, immunoblotting, and immunoprecipitation were performed as described (18). For immunoblotting and/or immunoprecipitation, the following antibodies were used: mouse monoclonal antibody for mouse HIF-1α (R&D Systems), rabbit HIF-2α (Novus Biologicals), anti-TC-PTP mouse monoclonal antibody (18), mouse monoclonal antibody for actin (Clone C4, ICN Biomedicals, Inc), rabbit anti-tubulin (Chemicon), mouse monoclonal against PKR (F9; ref.…”

STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells

“…27 Briefly, 100 g of nuclear protein, extracted from untreated or IL-6-treated CLL cells, was incubated, with or without 2.5 g TC-PTP (synthesized by S.S. and X.C. ), in buffer (250nM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, pH 7.2, 50mM NaCl, 5mM DTT, 2.5mM ethylenediaminetetraacetic acid) for 30 minutes at 30°C in a final volume of 50 L. After the enzymatic reaction was completed, Western blot analysis was performed to detect STAT3 phosphorylation, and electrophoretic mobility shift assay (EMSA) was used to determine STAT3-DNA binding, as described in "EMSA.…”