Ubiquitination Participates in the Lysosomal Degradation of Na,K-ATPase in Steady-State Conditions
Ubiquitination Participates in the Lysosomal Degradation of Na,K-ATPase in Steady-State Conditions
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DOI：
10.1165/rcmb.2008-0365oc
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发表时间：
2009-12-01
影响因子：
6.4
通讯作者：
Sznajder, Jacob I.
中科院分区：
文献类型：
--
作者：
Lecuona, Emilia;Sun, Haiying;Sznajder, Jacob I.
肺泡上皮细胞（AEC）Na，K-ATP酶有助于Na+的载体转运，并在保持肺无水肿中起重要作用。我们确定，细胞表面标记生物素和免疫荧光，约30%的总Na，K-ATP酶是在稳态条件下的AEC的质膜。质膜Na，K-ATP酶的半衰期约为4小时，新的Na，K-ATP酶掺入质膜对Brefeldin A敏感。用双吲哚马来酰亚胺（10 μ M）抑制蛋白激酶C（PKC）和用表达显性阴性PKC ζ的腺病毒感染都能防止Na，K-ATP酶降解。在表达缺乏PKC磷酸化位点的Na，K-ATP酶α 1亚基的细胞中，质膜Na，K-ATP酶的半衰期中度增加。我们还发现，在稳态条件下，Na，K-ATP酶是泛素化的，蛋白酶体抑制剂阻止其降解。有趣的是，突变的四个赖氨酸被描述为必要的泛素化和内吞的Na，K-ATP酶在有害的条件下没有影响其在稳态条件下的半衰期。溶酶体抑制剂可抑制Na，K-ATP酶的降解，在标记和追踪质膜Na，K-ATP酶4 h后，发现Na，K-ATP酶与溶酶体共定位。因此，我们提供的证据表明，磷酸化和泛素化是必要的质膜Na，K-ATP酶的溶酶体在肺泡上皮细胞的稳态降解。
The alveolar epithelial cell (AEC) Na,K-ATPase contributes to vectorial Na+ transport and plays an important role in keeping the lungs free of edema. We determined, by cell surface labeling with biotin and immunofluorescence, that approximately 30% of total Na,K-ATPase is at the plasma membrane of AEC in steady-state conditions. The half-life of the plasma membrane Na,K-ATPase was about 4 hours, and the incorporation of new Na,K-ATPase to the plasma membrane was Brefeldin A sensitive. Both protein kinase C (PKC) inhibition with bisindolylmaleimide (10 mu M) and infection with an adenovirus expressing dominant-negative PKC zeta prevented Na,K-ATPase degradation. In cells expressing the Na,K-ATPase alpha 1-subunit lacking the PKC phosphorylation sites, the plasma membrane Na,K-ATPase had a moderate increase in half-life. We also found that the Na,K-ATPase was ubiquitinated in steady-state conditions and that proteasomal inhibitors prevented its degradation. Interestingly, mutation of the four lysines described to be necessary for ubiquitination and endocytosis of the Na,K-ATPase in injurious conditions did not have an effect on its half-life in steady-state conditions. Lysosomal inhibitors prevented Na,K-ATPase degradation, and co-localization of Na,K-ATPase and lysosomes was found after labeling and chasing the plasma membrane Na,K-ATPase for 4 hours. Accordingly, we provide evidence suggesting that phosphorylation and ubiquitination are necessary for the steady-state degradation of the plasma membrane Na,K-ATPase in the lysosomes in alveolar epithelial cells.