Abstract
Interferons (IFNs) play critical roles in host defense by modulating gene expression via activation of signal transducer and activator of transcription (STAT) factors. IFN-alpha/beta also activates another transcription factor, nuclear factor kappaB (NF-kappaB), which protects cells against apoptotic stimuli. NF-kappaB activation requires the IFN-dependent association of STAT3 with the IFNAR1 chain of the IFN receptor. IFN-dependent NF-kappaB activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol 3-kinase (PI-3K) and Akt. Whereas constitutively active PI-3K and Akt induce NF-kappaB activation, Ly294002 (a PI-3K inhibitor), dominant-negative PI-3K, and kinase-dead Akt block IFN-dependent NF-kappaB activation. Moreover, dominant-negative PI-3K blocks IFN-promoted degradation of kappaBox alpha. Ly294002, a dominant-negative PI-3K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT3, PI-3K, and Akt are components of an IFN signaling pathway that promotes cell survival through NF-kappaB activation.
Highlights
Introduction of Phosphopeptides into Permeabilized CellsDaudi cells were permeabilized with streptolysin O as described previously [26]
To determine whether intracellular tyrosine residues of IFNAR1 are involved in IFN-dependent nuclear factor B (NF-B) activation, peptides corresponding to the amino acids surrounding the four intracellular tyrosine residues in the IFNAR1 chain were introduced into streptolysin O-permeabilized Daudi cells
These results indicate that NF-B activation is directed through the tyrosine phosphorylation of the conserved YSSQ motif in the IFNAR1 chain
Summary
Daudi cells were permeabilized with streptolysin O as described previously [26]. Phosphopeptides (5 M) corresponding to the amino acids surrounding intracellular tyrosine residues of IFNAR1 (Fig. 1A: PY466, INY[PO4]VFFPSL; PY481, IDEY[PO4]FSEQPL; PY527, HKKY[PO4]SSQTSQ; PY538, SGNY[PO4]SNEDES) or nonphosphorylated peptide (NPY527) were introduced into permeabilized cells. IFN-␣-treated (5,000 IU/ml; 15 min) cells were subjected to EMSA. IB␣ Degradation—At various times after IFN-␣ treatment, 1 ϫ 108 cells were lysed directly in Laemmli buffer, and equivalent amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, immunoblotted with specific affinity-purified rabbit anti-IB␣, and visualized by chemiluminescence with the ECL reagent (Amersham Pharmacia Biotech)
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