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...

Objective. DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes.Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA.Methods. DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS).Results. DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS.Conclusion. These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.Juvenile idiopathic arthritis (JIA), a polymorphic chronic inflammatory disease of unknown etiology, is

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids.

The Na+/H + Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.