Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin
Hypertonicity activates Na+/H+ exchange through Janus kinase 2 and calmodulin
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DOI：
10.1074/jbc.m209883200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Raymond, JR
中科院分区：
文献类型：
--
作者：
Garnovskaya, MN;Mukhin, YV;Raymond, JR
1型钠氢交换剂（NHE-1）是一种普遍存在的电中性膜转运蛋白，在许多细胞中被高渗激活。NHE-1可能是容量恢复过程中Na+进入的重要途径，但NHE-1渗透调节的分子机制知之甚少。在本研究中，我们进行了一个重要的信号分子，可能参与高渗诱导的激活CHO-K1细胞中的NHE-1的筛选。高渗以浓度依赖性方式快速激活NHE-1，这通过质子微生理测量法和FLIPR（TM）（荧光成像板读数器）上的细胞内pH测量来评估。Ca 2 +/钙调蛋白（CaM）和Janus激酶2（Jak 2）的抑制剂减弱了这种激活，而钙螯合和蛋白激酶C，Ras-ERK 1/2途径，Src激酶和Ca 2 +/钙调蛋白依赖性酶的抑制剂都没有显着的效果。高渗还导致Jak 2和STAT 3（Jak 2的主要底物）和CaM的快速酪氨酸磷酸化。JAK 2和CaM的磷酸化被JAK 2的抑制剂AG 490阻断。免疫沉淀研究表明，高渗刺激包括CaM，Jak 2和NHE-1的信号复合物的组装。AG 490可阻断复合物的形成。因此，我们认为高渗在很大程度上通过以下途径诱导CHO-K1细胞中NHE-1的活化：高渗3 Jak 2磷酸化和CaM的活化3酪氨酸磷酸化3 CaM与NHE-1的结合--> NHE-1活化。
The type 1 sodium-hydrogen exchanger (NHE-1) is a ubiquitous electroneutral membrane transporter that is activated by hypertonicity in many cells. NHE-1 may be an important pathway for Na+ entry during volume restoration, yet the molecular mechanisms underlying the osmotic regulation of NHE-1 are poorly understood. In the present study we conducted a screen for important signaling molecules that could be involved in hypertonicity-induced activation of NHE-1 in CHO-K1 cells. Hypertonicity rapidly activated NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry and by measurements of intracellular pH on a FLIPR(TM) (fluorometric imaging plate reader). Inhibitors of Ca2+/calmodulin (CaM) and Janus kinase 2 (Jak2) attenuated this activation, whereas neither calcium chelation nor inhibitors of protein kinase C, the Ras-ERK1/2 pathway, Src kinase, and Ca2+/calmodulin-dependent enzymes had significant effects. Hypertonicity also resulted in the rapid tyrosine phosphorylation of Jak2 and STAT3 (the major substrate of Jak2) and CaM. Phosphorylation of Jak2 and CaM were blocked by AG490, an inhibitor of Jak2. Immunoprecipitation studies showed that hypertonicity stimulates the assembly of a signaling complex that includes CaM, Jak2, and NHE-1. Formation of the complex could be blocked by AG490. Thus, we propose that hypertonicity induces activation of NHE-1 in CHO-K1 cells in large part through the following pathway: hypertonicity 3 Jak2 phosphorylation and activation 3 tyrosine phosphorylation of CaM 3 association of CaM with NHE-1 --> NHE-1 activation.