DNA-dependent Protein Kinase Interacts with Antigen Receptor Response Element Binding Proteins NF90 and NF45

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...

Section: Fig 7 Nf90 Modulates Pkr Autophosphorylation Activitymentioning

confidence: 83%

“…Additionally, NF90 and NF45 were co-immunoprecipitated with antibodies directed against PKR (lanes 2 and 7). Since NF45 does not directly interact with PKR in vitro, 3 we conclude that PKR is found in complexes with NF90 and NF45 in both cytosolic and nuclear extracts.…”

Section: Nf90-pkr Interactionsmentioning

confidence: 98%

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

Parker

Fierro-Monti

Mathews

2001

“…Data bank analysis indicates that the NFARs may be targeted by a number of kinases in the cell, since consensus phosphorylation sites for both protein kinase C and casein kinase II are evident. Finally, a recent report also indicates that the partial NFAR clone NF-90 may also be a substrate for DNA-PK (70).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Two Evolutionarily Conserved, Alternatively Spliced Nuclear Phosphoproteins, NFAR-1 and -2, That Function in mRNA Processing and Interact with the Double-stranded RNA-dependent Protein Kinase, PKR

Saunders

Perkins

Balachandran

et al. 2001

114

148

We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with doublestranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/ SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.

“…Many studies have shown that Ku interacts with DNA substrates to form protein⅐DNA complexes in electrophoresis mobility shift assays. However, DNA-PKcs does not interact with DNA in this assay, and the interaction among DNA-PKcs, Ku, and DNA is weak in the absence of cross-linkers (30). Here, we used 0.1% glutaraldehyde to stabilize the complex.…”

Section: Ku Mediates the Interaction Between Wrn And Dna-pkcsmentioning

confidence: 99%

Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

Karmakar¹,

Piotrowski²,

Brosh³

et al. 2002

150

133

Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 335 exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNAdependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb. Werner syndrome (WS)1 is a human autosomal recessive disorder characterized by early onset of premature aging characteristics including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. In addition, WS patients exhibit an increased incidence of diabetes mellitus type 2, hypertension, and malignancies (1). The gene (WRN), defects in which are responsible for WS, encodes a 1,432-amino acid protein (WRN) (2) that has both 3Ј35Ј helicase and 3Ј35Ј exonuclease activities (3-7). Although WRN appears to play an important role in DNA metabolism, the precise cellular roles of both the helicase and exonuclease activities of WRN remain to be determined.Cells from patients with WS show premature replicative senesence compared with cells derived from normal individuals (8). The WS cellular phenotype suggests correlations among faulty DNA metabolism, genomic instability, and senescence. WS cells show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO) (9), topoisomerase inhibitors (10), and certain DNA cross-linking agents (11). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (12, 13). These studies suggest that WRN plays an important role in DNA metabolism possibly by participating in DNA repair, re...