Department of Physiology, Pharmacology, and Medicine, University of Toledo College of Medicine, Toledo, Ohio 43614, USA.
J Biol Chem. 2013 May 10;288(19):13295-304. doi: 10.1074/jbc.M113.467381. Epub 2013 Mar 26.
It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells.
Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase.
A420P mutant has normal pumping but not signaling function.
Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.
在活细胞中，无法独立研究钠/钾-ATP 酶的泵送和信号功能。
无细胞和基于细胞的测定均表明，A420P 突变使钠/钾-ATP 酶失去Src 调节功能。
A420P 突变体具有正常的泵送功能，但没有信号功能。
鉴定Src 调节缺失突变体对于解决钠/钾-ATP 酶的生理作用至关重要。α1 钠/钾-ATP 酶具有泵送和信号功能。然而，在活细胞中，无法独立研究这些功能。我们从α1 钠/钾-ATP 酶的核苷酸结合域中鉴定出一个 20 个氨基酸的肽（Ser-415 至 Gln-434）（NaKtide），该肽在体外与Src 结合并抑制 Src。NaKtide 的 N 端适应了一个螺旋结构。体外激酶测定表明，用丙氨酸替换螺旋结构中含有大侧链的残基，可消除肽对 Src 的抑制作用。同样，通过脯氨酸替换破坏螺旋结构，无论是单个还是组合，都会降低 NaKtide 对 Src 的抑制效力。为了鉴定保留正常泵送功能但在 Src 调节中存在缺陷的突变α1，我们用携带 NaKtide 螺旋结构区域中 Ala 至 Pro 突变的野生型或突变型α1 的表达载体转染 Na/K-ATPaseα1 敲低的 PY-17 细胞，并生成了几个稳定的细胞系。我们发现，A416P、A420P 或 A425P 突变体的表达完全恢复了细胞中α1 的含量和泵浦能力。然而，与 A416P 不同的是，A420P 或 A425P 突变体均不能与细胞中的 Src 相互作用和调节。因此，这些两种突变体的表达导致哇巴因激活的信号转导和细胞生长受到显著抑制。因此，我们鉴定出一种具有正常泵送功能但信号转导功能缺失的α1 突变体。
