Sponder Gerhard, Abdulhanan Nasrin, Fröhlich Nadine, Mastrototaro Lucia, Aschenbach Jörg R, Röntgen Monika, Pilchova Ivana, Cibulka Michal, Racay Peter, Kolisek Martin
Institute of Veterinary-Physiology, Free University of Berlin, Berlin, Germany.
PerkinElmer Life and Analytical Sciences GmbH, Rodgau, Germany.
Oncotarget. 2017 Dec 22;9(4):5084-5104. doi: 10.18632/oncotarget.23598. eCollection 2018 Jan 12.
The Na/Mg exchanger SLC41A1 (A1), a key component of intracellular Mg homeostasis (IMH), is the major cellular Mg efflux system, and its overexpression decreases [Mg]. IMH plays an important role in the regulation of many cellular processes, including cellular signaling. However, whether the overexpression of A1 and the consequent drop of [Mg] impact on intracellular signaling is unknown. To examine the latter, we utilized dynamic mass redistribution (DMR) assay, PathScan RTK signaling antibody (PRSA) array, confirmatory Western blot (WB) analyses of phosphorylation of kinases selected by PRSA, and mag-fura 2-assisted fast filter spectrometry (FFS). We demonstrate here that the overexpression of A1 quantitatively and qualitatively changes the DMR signal evoked by the application of PAR-1-selective activating peptide and/or by changing [Mg] in HEK293 cells. PRSA profiling of the phosphorylation of important signaling nodes followed by confirmatory WB has revealed that, in HEK293 cells, A1 overexpression significantly attenuates the phosphorylation of Akt/PKB on Thr and/or Ser and of Erk1/2 on Thr/Tyr in the presence of 0 or 1 mM (physiological) Mg in the bath solution. The latter is also true for SH-SY5Y and HeLa cells. Overexpression of A1 in HEK293 cells significantly lowers [Mg] in the presence of [Mg] = 0 or 1 mM. This correlates with the observed attenuation of prosurvival Akt/PKB - Erk1/2 signaling in these cells. Thus, A1 expression status and [Mg] (and consequently also [Mg]) modulate the complex physiological fingerprint of the cell and influence the activity of kinases involved in anti-apoptotic and, hence, pro-survival events in cells.
钠/镁交换体SLC41A1（A1）是细胞内镁稳态（IMH）的关键组成部分，是主要的细胞镁外流系统，其过表达会降低[Mg]。IMH在包括细胞信号传导在内的许多细胞过程的调节中起重要作用。然而，A1的过表达以及随之而来的[Mg]下降是否会影响细胞内信号传导尚不清楚。为了研究后者，我们利用了动态质量重分布（DMR）分析、PathScan RTK信号抗体（PRSA）阵列、对PRSA选择的激酶磷酸化进行的验证性蛋白质印迹（WB）分析以及镁-呋喃2辅助快速过滤光谱法（FFS）。我们在此证明，A1的过表达在数量和质量上改变了在HEK293细胞中应用PAR-通道1选择性激活肽和/或改变[Mg]所诱发的DMR信号。对重要信号节点磷酸化的PRSA分析随后进行验证性WB，结果显示，在浴液中存在0或1 mM（生理浓度）镁的情况下，在HEK293细胞中，A1过表达显著减弱了苏氨酸和/或丝氨酸上Akt/PKB以及苏氨酸/酪氨酸上Erk1/2的磷酸化。在SH-SY5Y和HeLa细胞中也是如此。在[Mg] = 0或1 mM存在的情况下，HEK293细胞中A1的过表达显著降低了[Mg]。这与这些细胞中观察到的促生存Akt/PKB - Erk1/2信号传导的减弱相关。因此，A1的表达状态和[Mg]（以及因此的[Mg]）调节细胞的复杂生理特征，并影响参与细胞抗凋亡及因此促生存事件的激酶的活性。
