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

Abstract

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 Thr 1007 of KCC2, and Thr 203 , Thr 207 and Thr 212 of NKCC1. Dephosphorylation of Thr 1007 of KCC2, and Thr 207 and Thr 212 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 Thr 233 and Ser 373 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 Ser 940 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 Ser 940 , 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.

Oxytocin administration in neonates shapes the hippocampal circuitry and restores social behavior in a mouse model of autism

“…We then asked if the altered DFGABA values could be due to alteration in the expression of the The quantitative western blot analysis of the total KCC2 protein expression in hippocampi of P7 mice did not reveal statistically significant difference of the amount of KCC2 between WT and Magel2 KO animals ( Figure 9 A-B). The ion-transport activity of KCC2 and its stability at the cellular plasma membrane also strongly depend on posttranslational modifications of multiple phosphorylation sites (50). We therefore applied in a next step phospho-site-specific antibodies, as they were previously shown to quantitatively monitor changes in KCC2…”

Role of the Cation-chloride-cotransporters in Cardiovascular Disease

“…Lee and team reported that phosphorylation of KCC2 specific residue, Ser940, by protein kinase C (PKC) have been shown to activate KCC2, an isoform exclusively expressed in the nervous system. However, the phosphorylation of highly conserved threonine residues in all KCCs are all associated with inactivation [26,[30][31][32]. Thus the phosphorylation-mediated-activation of KCC2 may be an adaptive mechanism to assist with KCC2 functions in the nervous system [31,33,34].…”

Role of the Cation-Chloride-Cotransporters in Cardiovascular Disease

“…Lee and team reported that phosphorylation of KCC2 specific residue, Ser940, by protein kinase C (PKC) has been shown to activate KCC2, an isoform exclusively expressed in the nervous system. However, the phosphorylation of highly conserved threonine residues in all KCCs is ubiquitously associated with inactivation [ 33 , 37 , 38 , 39 ]. Thus, the phosphorylation-mediated-activation of KCC2 may be an adaptive mechanism to assist with KCC2 functions in the nervous system [ 38 , 40 , 41 ].…”