AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2
AMP-activated kinase inhibits the epithelial Na+ channel through functional regulation of the ubiquitin ligase Nedd4-2
复制标题
DOI：
10.1074/jbc.m606045200
复制
发表时间：
2006-09-08
影响因子：
4.8
通讯作者：
Hallows, Kenneth R.
中科院分区：
文献类型：
--
作者：
Bhalla, Vivek;Oyster, Nicholas M.;Hallows, Kenneth R.
我们最近发现，代谢感受器AMP激活的激酶(AMPK)通过减少质膜ENaC的表达来抑制上皮Na+通道(ENaC)，这种作用需要在泛素连接酶Nedd4-2的β-ENaC亚基的细胞质尾部存在结合基序。为了进一步研究Nedd4-2在AMPK调节ENaC中的作用，我们研究了AMPK激活对共表达ENaC和野生型(WT)或突变型Nedd4-2的非洲爪哇卵母细胞ENaC电流的影响。AMPK对ENAC的抑制在表达WT Nedd4-2的卵母细胞中被保留，但在表达显性-阴性(DN)或结构性活性(CA)Nedd4-2突变体的卵母细胞中被阻断，这表明AMPK依赖于Nedd4-2功能的调节。利用WT或突变形式的血清和糖皮质激素调节的激酶(SGK1)、蛋白激酶A的调节剂(PKA)或细胞外调节的激酶(ERK)的类似实验并未影响AMPK对ENaC的抑制，表明这些已知的调节Nedd4-2-ENaC相互作用的途径不起作用。在HEK-293细胞中表达的Nedd4-2在体外和体内都发生了AMPK依赖性的磷酸化，提示了Nedd4-2调节从而调节细胞ENaC活性的潜在机制。此外，在HEK-293细胞中，通过免疫共沉淀实验，细胞内AMPK的激活显著增强了β-ENaC亚单位与Nedd4-2的相互作用。综上所述，这些结果提示了一种新的ENaC调节机制，其中AMPK促进ENaC-Nedd4-2相互作用，从而通过增加依赖Nedd4-2的ENaC从质膜上回收来抑制ENaC。当AMPK激活时，依赖于AMPK的ENaC抑制可能限制代谢应激条件下细胞的Na+负荷。
We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the beta-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A(PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the beta-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.