The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the fulllength version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.

The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses to oxidant stress and in several signal transduction pathways. The nuclear factor I (NFI) family of transcription factors is sensitive to oxidative inactivation due to the presence of a conserved, oxidation-sensitive cysteine residue within the NFI DNA-binding domain. Here we show that restoration of the DNA-binding activity of oxidized NFI-C can be catalyzed in vitro by the cellular enzyme thioltransferase (glutaredoxin) coupled to GSH and GSSG reductase. To test whether GSHdependent pathways play a role in the maintenance of NFI activity in vivo, we used buthionine sulfoximine, an agent that inhibits GSH synthesis, and N-acetylcysteine, an agent that can replenish intracellular GSH. Pretreatment of HeLa cells with buthionine sulfoximine greatly potentiated the inactivation of NFI by the oxidizing agent diamide. Inclusion of N-acetylcysteine in the culture medium during the recovery period following diamide treatment increased the extent of restoration of NFI activity. These results suggest that maintenance of the DNA-binding activity of NFI proteins during oxidant stress in vivo requires a GSH-dependent pathway, likely involving thioltransferase-catalyzed reduction of the oxidation-sensitive cysteine residue on NFI.Modulation of DNA-binding activity by oxidation and reduction reactions (redox regulation) has been demonstrated for a number of transcription factors, including NF-B/Rel proteins, Fos and Jun proteins, and others (1-6). The mechanism of oxidative inactivation of DNA binding is through the oxidation of specific cysteine residues close to or within the DNA-binding domains of these proteins. This modulation of the DNA-binding activity of transcription factors by redox state has been proposed to be important both in cellular response to oxidant stress and in a number of signal transduction pathways including those mediated by tumor necrosis factor ␣ (7, 8). While the process of oxidative inactivation of DNA binding has been studied extensively in vitro, the biochemical pathways that mediate the reversible oxidation of oxidation-sensitive cysteine residues are only poorly understood. We are using the NFI 1 family of transcription factors as a model system to characterize the pathways for reversible oxidative inactivation of DNAbinding proteins in vitro and in vivo.The NFI family of site-specific DNA-binding proteins is required both for adenovirus DNA replication and for the expression of a large number of cellular and viral genes (9 -11). In vertebrates, this family of proteins is encoded by four distinct genes (NFI-A, NFI-B, NFI-C, and NFI-X), and the expression patterns of the four genes are regulated during cell differentiation and development (12, 13). We had shown previously that the DNA-binding activity of the NFI family of transcription factors was inactivated by oxidation in vitro (14). This sensitivity to oxidation requires the presence of a single oxidationsensitive cysteine ...

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2-5-oligoadenylate (2-5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C.

Background: NF-B post-translational modifications contribute to gene-specific transcriptional activation. Results: Symmetrical dimethylation of NF-B p65 by PRMT5 enhances CXCL10 induction in response to TNF-␣. Conclusion: Arginine methylation of p65 is a novel mechanism regulating inflammatory chemokine induction. Significance: Methylation of specific NF-B arginine residues contributes to promoter targeting and transcriptional activation.