24 research outputs found
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 â 720,000 ± 50,000, a radius of gyration of 64 ± 2 Ă
and a hydrodynamic radius of â 86 Ă
were obtained. Electron micrographs show a hollow cylinder-like particle with a diameter of 120 Ă
, n height of 170 Ă
and a diameter of 40 Ă
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 ValiditĂ€t der kontralateralen Suppression otoakustischer Emissionen bei auditiven Selektionsstörungen als Element der PhĂ€notypisierung fĂŒr genetische Untersuchungen
Ziele sind die Bestimmung der ValiditĂ€t der TEOAE-Suppressionsamplitude als diagnostisches Kriterium der Funktion des medialen olivocochleĂ€ren Systems (MOCS) zur Diagnostik auditiver Selektionsstörungen und die Suche nach Cutoff-Kriterien mit hinreichender SensitivitĂ€t und SpezifitĂ€t. Einschlusskriterien waren: regelgerechtes Tonaudiogramm, Sprachaudiogramm und Tympanogramm, IQ >=85 und Ausschluss von AD/-HS. Die PhĂ€notypisierung der bislang 91 Kinder erfolgte ĂŒber die Hörtests "Sprache im StörgerĂ€usch" 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 gegenĂŒber der Kontrollgruppe (n=29) bei vergleichbaren TEOAE-Ausgangsamplituden. Die Daten deuten eine gute SpezifitĂ€t der TEOAE-Suppression fĂŒr auditive Selektionsstörungen an. FĂŒr 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 EntitĂ€ten darstellen