Maruyama Junichi, Kobayashi Yumie, Umeda Tsuyoshi, Vandewalle Alain, Takeda Kohsuke, Ichijo Hidenori, Naguro Isao
Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Sci Rep. 2016 Jan 6;6:18710. doi: 10.1038/srep18710.
The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway has been reported to be a crucial signaling pathway for triggering pseudohypoaldosteronism type II (PHAII), an autosomal dominant hereditary disease that is characterized by hypertension. However, the molecular mechanism(s) by which the WNK-SPAK/OSR1 pathway is regulated remain unclear. In this report, we identified WNK4 as an interacting partner of a recently identified MAP3K, apoptosis signal-regulating kinase 3 (ASK3). We found that WNK4 is phosphorylated in an ASK3 kinase activity-dependent manner. By exploring the ASK3-dependent phosphorylation sites, we identified Ser575 as a novel phosphorylation site in WNK4 by LC-MS/MS analysis. ASK3-dependent WNK4 Ser575 phosphorylation was mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway. Osmotic stress, as well as hypotonic low-chloride stimulation, increased WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway. ASK3 was required for the p38MAPK activation induced by hypotonic stimulation but was not required for that induced by hypertonic stimulation or hypotonic low-chloride stimulation. Our results suggest that the p38MAPK-MK pathway might regulate WNK4 in an osmotic stress-dependent manner but its upstream regulators might be divergent depending on the types of osmotic stimuli.
无赖氨酸K-与Ste20相关的富含脯氨酸/丙氨酸的激酶(SPAK)/氧化应激反应激酶1(OSR1)信号通路据报道是引发II型假性醛固酮增多症(PHAII)的关键信号通路，PHAII是一种以高血压为特征的常染色体显性遗传病。然而，WNK-SPAK/OSR1信号通路的调控分子机制仍不清楚。在本报告中，我们鉴定出WNK4是最近鉴定出的丝裂原活化蛋白激酶3(MAP3K)——凋亡信号调节激酶3(ASK3)的相互作用伴侣。我们发现WNK4以ASK3激酶活性依赖的方式被磷酸化。通过探索ASK3依赖的磷酸化位点，我们通过液相色谱-串联质谱(LC-MS/MS)分析鉴定出Ser575是WNK4中的一个新的磷酸化位点。ASK3依赖的WNK4 Ser575磷酸化由p38丝裂原活化蛋白激酶-丝裂原活化蛋白激酶激活的蛋白激酶(MK)信号通路介导。渗透应激以及低渗低氯刺激通过p38丝裂原活化蛋白激酶-MK信号通路增加WNK4 Ser575磷酸化。ASK3是低渗刺激诱导的p38丝裂原活化蛋白激酶激活所必需的，但不是高渗刺激或低渗低氯刺激诱导的p38丝裂原活化蛋白激酶激活所必需的。我们的结果表明，p38丝裂原活化蛋白激酶-MK信号通路可能以渗透应激依赖的方式调节WNK4，但其上游调节因子可能因渗透刺激的类型而异。
