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

This is the final version. Available from the publisher via the DOI in this record.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-sitesNational Natural Science Foundation of Chin

STRUCTURE AND RNA-CONTENT OF THE PROSOMES

AbstractDuck erythroblast, prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo-)electron microscopy. A molecular weight of ≈ 720,000 ± 50,000, a radius of gyration of 64 ± 2 Å and a hydrodynamic radius of ≈ 86 Å were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 Å, n height of 170 Å and a diameter of 40 Å for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence

Multizentrische Studie zur diagnostischen Validität der kontralateralen Suppression otoakustischer Emissionen bei auditiven Selektionsstörungen als Element der Phänotypisierung für genetische Untersuchungen

Ziele sind die Bestimmung der Validität der TEOAE-Suppressionsamplitude als diagnostisches Kriterium der Funktion des medialen olivocochleären Systems (MOCS) zur Diagnostik auditiver Selektionsstörungen und die Suche nach Cutoff-Kriterien mit hinreichender Sensitivität und Spezifität. Einschlusskriterien waren: regelgerechtes Tonaudiogramm, Sprachaudiogramm und Tympanogramm, IQ >=85 und Ausschluss von AD/-HS. Die Phänotypisierung der bislang 91 Kinder erfolgte über die Hörtests "Sprache im Störgeräusch" und "BILD-Test" sowie die Messung der TEOAE-Suppressionsamplitude (Cutoff-Kriterium <0,7 dB) in vier Inanspruchnahme-Gruppen (auditive Selektionsstörung, MOCB-Störung, beides, keine) und einer Kontrollgruppe. Kinder mit Selektionsstörung (n=16) zeigen beidseits eine signifikant niedrigere mittlere TEOAE-Suppression gegenüber der Kontrollgruppe (n=29) bei vergleichbaren TEOAE-Ausgangsamplituden. Die Daten deuten eine gute Spezifität der TEOAE-Suppression für auditive Selektionsstörungen an. Für statistisch gesicherte Analysen und die Bestimmung von Cutoff-Werten mittels ROC-Kurven erscheinen die Fallzahlen noch zu gering. Weiterhin wird mit Hilfe von genomweiten Assoziationsanalysen und Kandidatengenanalysen untersucht, ob die auditive Selektionsstörung/MOCS-Dysfunktion genetische Entitäten darstellen