Interactions of β and γENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation
Interactions of β and γENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation
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DOI：
10.1074/jbc.m111717200
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发表时间：
2002-04-19
影响因子：
4.8
通讯作者：
Garty, H
中科院分区：
文献类型：
--
作者：
Shi, HK;Asher, C;Garty, H
上皮 Na+ 通道 (ENaC) 的磷酸化被认为在其调节中发挥作用。在这里，我们证明磷酸化 β 和 γ 亚基的羧基末端有利于它们与泛素连接酶 Nedd4 的相互作用并抑制通道活性。体外测定已鉴定出三种磷酸化 β 和 γENaC 羧基末端的蛋白激酶。其中一种磷酸化 betaThr-613 和 gammaThr-623，它们是紧邻 PY 基序的高度保守的 C 尾苏氨酸。 gammaThr-623 的磷酸化也已在爪蟾卵母细胞表达的通道中体内得到证实，将 betaThr-613 和 gammaThr-623 突变为丙氨酸可使通道活性增加 3.5 倍。已使用表面等离子共振研究了上述磷酸化对 ENaC 和 Nedd4 之间相互作用的影响。由于较高的结合速率常数，在 beta613 或 gamma623 位点具有磷酸苏氨酸的肽与 Nedd4 的 WW 结构域的结合比非磷酸化类似物好两到三倍。使用多种不同的方法证明作用于 betaThr-613 和 gammaThr-623 的蛋白激酶是细胞外调节激酶 (ERK)。表明 ERK 介导的 betaThr-613 和 gammaThr-623 磷酸化通过促进其与 Nedd4 的相互作用来下调该通道。
Phosphorylation of the epithelial Na+ channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.