Nuclear Factor 90 Is a Substrate and Regulator of the Eukaryotic Initiation Factor 2 Kinase Double-stranded RNA-activated Protein Kinase

Abstract

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNAindependent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNAdependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed. Nuclear factor 90 (NF90)1 is a cellular protein initially purified in complex with nuclear factor 45 (NF45) through its ability to bind to the antigen response recognition element in the interleukin-2 promoter (1). Independently, our laboratory identified NF90 as a cellular protein that interacts with VA RNA II , a structured adenoviral RNA, and with double-stranded RNA (dsRNA) (2). NF90 is one of the most abundant dsRNAbinding proteins in human cells, interacting preferentially with highly structured RNAs, in the order dsRNA Ͼ VA RNA II Ͼ VA RNA I Ͼ single stranded RNA (ssRNA). NF90 and NF45 have also been purified from human placental extracts in association with the protein synthesis eukaryotic initiation factor 2 and the catalytic subunit of the DNA-activated protein kinase DNA-PK (3). NF90 stabilizes the interactions between the catalytic subunit of DNA-PK and its regulatory heterodimeric DNA-binding subunits, Ku, in vitro, and it serves as a substrate for the activated DNA-PK in vitro.NF90 contains no recognized DNA binding motifs; rather it contains two dsRNA-binding motifs (dsRBMs) which lie downstream of a bipartite nuclear localization signal.2 Several homologues of NF90 have since been identified, including Xenopus 4F.1 (4), Spnr (5), and ILF3 (6) in mouse, and p74 in rat (7). All of these proteins share homology at their N terminus to a mouse protein, Zfr, a protein of unknown function which has a conserved homologue in Drosophila (8) (Fig. 1A). Downstream of this Zfr homology domain is a region of homology which these proteins share with NF45, which we term the NF45 homology domain.Several NF90 homologues and variants have been identified in human cells, and recent work from Duchange and colleagues (9) has helped clarify the relationships of several of these proteins. Alternatively spliced variants of the protein exist...

NF90 Exerts Antiviral Activity through Regulation of PKR Phosphorylation and Stress Granules in Infected Cells

“…NF90 is one of the evolutionarily conserved members of the dsRNA-binding protein family and is expressed abundantly in various human cells. Previous studies (6,7,9,10,13,40) identified and confirmed PKR as the key interacting partner for NF90 in cells, in addition to its function in regulating IL-2 gene expression in T cells (4). PKR plays an important role in mediating innate immunity to viral infection and is required for NF90 antiviral activity, as demonstrated in a vesicular stomatitis virus infection experiment using PKR-knockout (PKR…”

“…It was suggested that binding of either dsRNA or PACT to PKR may interrupt intramolecular interaction between the kinase and dsRNA-binding domains, leading to an open conformation of the PKR molecule and allowing autophosphorylation to occur (49). NF90 is phosphorylated by PKR in its RBD (6). Previous studies (7,9,29) showed that NF90 interacts with PKR in the yeast two-hybrid and GST-pull down assays.…”

“…Previous reports (6,7,9) demonstrated that NF90 interacts with PKR and is one of the substrates of PKR. The phosphorylated form of NF90, MPP4, also was identified as a phosphoprotein detected during the M phase of the cell cycle (9,39).…”

“…NF90 was shown previously to interact with PKR using yeast twohybrid and GST pull-down assays (6,29). To verify their interaction in mammalian cells and map the interacting domains on both proteins, we first tested interactions between Flag-tagged full-length NF90 and V5-tagged full-length and C-terminal-and N-terminaltruncated versions of PKR (Fig.…”

“…However, this same study (29) found no evidence of NF90 activation of PKR in an in vitro assay. Conversely, it was found that NF90 inhibited PKR phosphorylation, presumably through competition for dsRNA binding, because the C-terminal of NF90 possesses RNAbinding ability (6).…”