Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes
Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes
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DOI：
10.1074/jbc.m109.072553
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Chiba, Kazuyoshi
中科院分区：
文献类型：
--
作者：
Harada, Kaori;Fukuda, Eriko;Chiba, Kazuyoshi
海星卵母细胞在卵巢中的第一次减数分裂中期停滞（MI停滞），并在产卵到海水中后恢复减数分裂。MI停滞通过较低的细胞内pH（pH（i））维持，并通过细胞碱化从停滞中释放。为了阐明卵母细胞中pH（i）的调控，我们克隆了海星（Asterina pectinifera）Na+/H+交换器3（ApNHE 3）在卵母细胞质膜中的表达。ApNHE 3的胞质结构域含有p90核糖体S6激酶（p90 Rsk）磷酸化位点，并且向未成熟卵母细胞注射组成型活性p90 Rsk和上游调节剂Mos，刺激pH升高（i）。这种增加被NHE抑制剂5-（N-乙基-N-异丙基）-阿米洛利和特异性Rsk抑制剂SL 0101阻断。MAPK激酶（MEK）抑制剂U 0126阻断了Mos诱导的pH（i）升高，但不能阻断p90 Rsk诱导的pH（i）升高，表明Mos-MEK-MAPK-p90 Rsk途径促进ApNHE 3活化。在无细胞提取物中，Mos-MEK-MAPK-p90 Rsk途径使ApNHE 3在Ser-590、-606和-673处磷酸化。当p90 Rsk依赖的ApNHE 3磷酸化被显性负性C末端片段或中和抗体阻断时，p90 Rsk诱导的未成熟卵母细胞pH（i）升高被抑制。然而，在MAPK激活之前，ApNHE 3通过上游磷脂酰肌醇3-激酶途径上调，并且活性状态维持至产卵，这表明p90 Rsk依赖的ApNHE 3磷酸化不太可能是参与MI停滞退出的主要调节机制。减数分裂完成后，未受精卵保持其升高的pH（i）（类似于7.4），直到细胞凋亡开始。我们认为p90 Rsk/ApNHE 3依赖性pH升高通过延迟细胞凋亡的启动来增加受精成功率。
Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na+/H+ exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH(i). This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH(i) increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH(i) increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH(i) (similar to 7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH(i) increases fertilization success by delaying apoptosis initiation.