Zhou Guofei, Dada Laura A, Chandel Navdeep S, Iwai Kazuhiro, Lecuona Emilia, Ciechanover Aaron, Sznajder Jacob I
Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, 240 E. Huron, Chicago, IL 60611, USA.
FASEB J. 2008 May;22(5):1335-42. doi: 10.1096/fj.07-8369com. Epub 2007 Dec 11.
Hypoxia inhibits Na-K-ATPase activity and leads to its degradation in mammalian cells. Von Hippel Lindau protein (pVHL) and hypoxia inducible factor (HIF) are key mediators in cellular adaptation to hypoxia; thus, we set out to investigate whether pVHL and HIF participate in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase. We found that in the presence of pVHL hypoxia decreased Na-K-ATPase activity and promoted the degradation of plasma membrane Na-K-ATPase. In pVHL-deficient cells, hypoxia did not decrease the Na-K-ATPase activity and the degradation of plasma membrane Na-K-ATPase was prevented. pVHL-mediated degradation of Na-K-ATPase required the functional pVHL E3 ligase and Ubc5 since pVHL mutants and dominant-negative Ubc5 prevented Na-K-ATPase from degradation. The generation of reactive oxygen species was necessary for pVHL-mediated Na-K-ATPase degradation during hypoxia. Desferrioxamine, which stabilizes HIF1/2alpha, did not affect the half-life of plasma membrane Na-K-ATPase. In addition, stabilizing HIF1/2alpha by infecting mammalian cells with adenoviruses containing the oxygen-dependent degradation domain of HIF1alpha did not affect the plasma membrane Na-K-ATPase degradation. In cells with suppression of pVHL by short hairpin RNA, the Na-K-ATPase was not degraded during hypoxia, whereas cells with knockdown of HIF1/2alpha retained the ability to degrade plasma membrane Na-K-ATPase. These findings suggest that pVHL participates in the hypoxia-mediated degradation of plasma membrane Na-K-ATPase in a HIF-independent manner.
缺氧会抑制哺乳动物细胞中钠钾ATP酶的活性并导致其降解。冯·希佩尔-林道蛋白（pVHL）和缺氧诱导因子（HIF）是细胞适应缺氧的关键介质；因此，我们着手研究pVHL和HIF是否参与缺氧介导的质膜钠钾ATP酶的降解。我们发现，在有pVHL存在的情况下，缺氧会降低钠钾ATP酶的活性并促进质膜钠钾ATP酶的降解。在缺乏pVHL的细胞中，缺氧不会降低钠钾ATP酶的活性，并且质膜钠钾ATP酶的降解受到抑制。pVHL介导的钠钾ATP酶降解需要功能性的pVHL E3连接酶和Ubc5，因为pVHL突变体和显性负性Ubc5可阻止钠钾ATP酶的降解。缺氧期间，活性氧的产生是pVHL介导的钠钾ATP酶降解所必需的。去铁胺可稳定HIF1/2α，但不影响质膜钠钾ATP酶的半衰期。此外，用含有HIF1α氧依赖性降解结构域的腺病毒感染哺乳动物细胞来稳定HIF1/2α，并不影响质膜钠钾ATP酶的降解。在通过短发夹RNA抑制pVHL的细胞中，缺氧期间钠钾ATP酶不会降解，而敲低HIF1/2α的细胞仍保留降解质膜钠钾ATP酶的能力。这些发现表明，pVHL以不依赖HIF的方式参与缺氧介导的质膜钠钾ATP酶的降解。
