DEK in the synovium of patients with juvenile idiopathic arthritis: characterization of DEK antibodies and posttranslational modification of the DEK autoantigen.

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.

The significance of protein maturation by plastidic type I signal peptidase 1 for thylakoid development in Arabidopsis chloroplasts.

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.

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK.

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.

Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1) during osmotic cell shrinkage.

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 (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P 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.