Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases:: Ste20-related proline-alanine-rich kinase and WNK4
Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases:: Ste20-related proline-alanine-rich kinase and WNK4
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DOI：
10.1152/ajpcell.00037.2005
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发表时间：
2006-01-01
影响因子：
5.5
通讯作者：
Delpire, E
中科院分区：
文献类型：
--
作者：
Gagnon, KBE;England, R;Delpire, E
在本研究中，我们已经证明了Ste 20相关的富含脯氨酸-丙氨酸激酶（SPAK），WNK 4 [不含赖氨酸（K）]和广泛表达的Na+-K+-2Cl（-）协同转运蛋白1型（NKCC 1）之间的功能相互作用。NKCC 1的功能，我们在非洲爪蟾卵母细胞的等渗（基础）和高渗（刺激）条件下测量，SPAK和WNK 4单独表达时，不受影响。与此相反，这两种激酶与NKCC 1的表达导致协同转运蛋白活性的显着增加和对外部渗透压或细胞体积的不敏感性。NKCC 1的活化依赖于SPAK的催化活性，也可能依赖于WNK 4的催化活性，因为其催化结构域的突变导致缺乏协同转运蛋白刺激。我们的酵母双杂交实验的结果表明，WNK 4不直接与NKCC 1相互作用，但与SPAK相互作用。功能实验表明，SPAK与WNK 4的结合也是必需的，因为SPAK相互作用缺陷的WNK 4突变体（Phe 997 Ala）不增加NKCC 1活性。我们还表明，K+-Cl-协同转运蛋白2型（KCC 2），神经元特异性KCl协同转运蛋白的运输功能，在等渗和低渗条件下的两种激酶的表达减少。我们的数据与WNK 4与SPAK相互作用一致，SPAK反过来磷酸化并激活NKCC 1，磷酸化并灭活KCC 2。
In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine ( K)], and the widely expressed Na+-K+-2Cl(-) cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic ( basal) and hyperosmotic ( stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction- deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.