Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

Phosphoregulation of the intracellular termini of K+-Cl- cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

The Prerequisites for Central Tolerance Induction against Citrullinated Proteins in the Mouse.

OBJECTIVES: To assess the prerequisites for negative selection of peptidylcitrulline-specific T cells in the thymus. In detail, we here analyzed murine medullary thymic epithelial cells for the expression of peptidylarginine deiminases (PAD) and subsequent citrullination. METHODS: Medullary thymic epithelial cells were sorted, their mRNA was isolated and the expression of Pad genes was analyzed by quantitative PCR. Citrullination was detected by Western Blot in lysates of sorted medullary thymic epithelial cells and histologically by immunofluorescence of thymic thin sections. RESULTS: Pad2 and Pad4 are the main Pad isoforms expressed in mature medullary thymic epithelial cells of the mouse and their levels of expression are comparable to that of insulin (Ins2), another highly and promiscuously expressed protein in the thymus. Citrullination was detected in medullary thymic epithelial cells as shown by Western Blot and immunofluorescence. CONCLUSIONS: Even though we here show that the murine thymus harbors the prerequisites for central tolerance to PAD and citrullinated peptides, it remains an open question whether the emergence of peptidylcitrulline-specific T cells and of autoantibodies recognizing citrullinated epitopes is caused by a failure of central or peripheral tolerance mechanisms.