We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein.Phosphorylation fluctuates during the cell cycle with a moderate peak during G 1 phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un-or underphosphorylated form of DEK.The DEK protein was initially identified as a fusion protein with CAN nucleoporin in a subtype of acute myeloid leukemia involving the t(6;9) chromosomal translocation (34). Subsequently, DEK was found to be the target of autoantibodies in several diseases, including systemic lupus erythematosus (9, 10, 38), juvenile rheumatoid arthritis (10, 32), and sarcoidosis (9, 10). Interestingly, DEK has also been linked to ataxia-telangiectasia, because a fragment of DEK cDNA reverses the mutagen-sensitive phenotype of cells from ATM patients (28). Despite the number of clinical observations, the biological function of DEK remains unclear (33).Several reports suggest an involvement of DEK in transcriptional regulation. By using cofractionation and coimmunoprecipitation, it was demonstrated that the transcriptional corepressor hDaxx associates with DEK (21). However, the exact function of DEK in hDaxx-mediated repression is not clear. DEK has also been found to be associated with the latencyassociated nuclear antigen, which is constitutively expressed in Kaposi's sarcoma-associated herpesvirus latent infection (25). The data indicate that the latency-associated nuclear antigen is tethered to chromatin through its interaction with the DEK protein and the methyl CpG binding protein MeCP2. In addition, DEK interacts with cell type-specific transcription factor AP-2␣ in vitro and stimulates transactivation activity of AP-2␣ over the APOE promoter (8). Thus, DEK could be involved in linking several different proteins to chromatin. It has been reported that DEK binds to DNA and specifically recognizes the peri-ets sites in the human immunodeficiency virus type 2 enhancer (11, 14, 15) and to class II major histocompatibility complex Y-box sequences (1). However, our experiments had shown that DEK recognizes DNA structures rather than DNA sequences and preferentially binds supercoiled and four-way junction DNAs (35). These data suggest that DEK functions as an architectural protein in chromatin. Indeed, DEK is a constituent of oligonucleosomes, generated by micrococcal nuclease digestion of chromatin in isolated nuclei (22) and associates with metaphase chromosomes (13). Purified DEK changes the topology of DNA in viral minichromosomes and reduces the accessibility of chromatin to DNA binding factors including comp...
Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/ arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK cosedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.DNA in the nucleus is organized into a hierarchy of structures with the nucleosome as the basic building block. It has become widely accepted that modification of nucleosome structure is an important mechanism that regulates the accessibility of chromatin to DNA binding factors (1, 2).In the search for factors that change the structure of chromatin and the replicational activity of chromatin templates, we recently identified the proto-oncogene protein DEK as a candidate protein that changes the topology of DNA in chromatin in vitro (3). DEK is a 43-kDa phosphoprotein that was first isolated as part of a fusion protein expressed in a subtype of acute myeloid leukemias with (t6;9) chromosomal translocations (4). DEK was later identified as an autoimmune antigen in patients with pauciarticular onset juvenile rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases (5-7). In addition, DEK has been reported to be a site-specific DNA binding factor, which recognizes a specific DNA element in the human immunodeficiency virus enhancer (8).In a recent study, it was demonstrated that DEK associates with splicing complexes through interactions promoted by SR 1 proteins. It was shown that DEK associates with mRNA in a splicing-dependent manner, indicating that it could function to coordinate splicing with subsequent steps in gene expression (9). In addition DEK was found in a ϳ335-kDa five-component complex at a conserved position 20 -24 nucleotides upstream of exon-exon junctions (10).Our recent experiments have identified DEK as a protein that induces alterations in the superhelical density of DNA in chromatin (3). The change in topology was only observed with chromatin but not with naked DNA and depends on...
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