Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1
Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1
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DOI：
10.1371/journal.pone.0232967
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发表时间：
2020-05
期刊：
影响因子：
3.7
通讯作者：
Jinwei Zhang;Antje Cordshagen;I. Medina;H. Nothwang;J. Wiśniewski;M. Winklhofer;Anna-Maria Hartmann
中科院分区：
文献类型：
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作者：
Jinwei Zhang;Antje Cordshagen;I. Medina;H. Nothwang;J. Wiśniewski;M. Winklhofer;Anna-Maria Hartmann
KCC 2和NKCC 1在快速抑制性神经传递的发展和维持中的关键作用及其在严重人类疾病中的意义引起了人们对转录后调节机制如（去）磷酸化的兴趣。调节激酶和磷酸酶活性的星形孢菌素（广泛激酶抑制剂）和N-乙基马来酰亚胺（NEM）可增强KCC 2活性并降低NKCC 1活性。在这里，我们研究了这种相互调节的监管机制，质谱和免疫印迹分析使用磷酸特异性抗体。我们的分析表明，应用星形孢菌素或NEM使KCC 2的Thr 1007和NKCC 1的Thr 203、Thr 207和Thr 212去磷酸化。KCC 2的Thr 1007和NKCC 1的Thr 207和Thr 212的去磷酸化先前被证明可以激活KCC 2并抑制NKCC 1。此外，这两种试剂的应用导致SPAK的T环和S环磷酸化位点Thr 233和Ser 373的去磷酸化，SPAK是介导KCC 2和NKCC 1磷酸化的WNK-SPAK/OSR 1信号传导模块中的关键激酶。总之，这些结果表明，KCC 2和NKCC 1通过星形孢菌素和NEM的相互调节是基于WNK-SPAK/OSR 1信号传导。KCC 2的关键调节磷酸化位点Ser 940在星形孢菌素和NEM处理后KCC 2的增强活化中并不关键参与，因为两种试剂对其磷酸化状态具有相反的作用。最后，NEM作用于Ser 940的组织特异性方式，如HEK 293细胞和未成熟培养的海马神经元中的比较分析所示。总之，我们的分析鉴定了对星形孢菌素或NEM应用有响应的磷酸位点。这为更好地理解不同磷酸化位点的协同作用提供了重要信息。
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.