Canonical Bcl-2 Motifs of the Na+/K+ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?
Canonical Bcl-2 Motifs of the Na+/K+ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?
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DOI：
10.1159/000343366
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发表时间：
2013-02
影响因子：
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通讯作者：
P. Lauf;J. Heiny;J. Meller;Michael Lepera;L. Koikov;G. Alter;T. Brown;N. Adragna
中科院分区：
文献类型：
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作者：
P. Lauf;J. Heiny;J. Meller;Michael Lepera;L. Koikov;G. Alter;T. Brown;N. Adragna
背景/目的：白屈菜红碱[CET]是一种蛋白激酶C[PKC]抑制剂，是一种与BH3类似的BH3蛋白与BH1样蛋白结合的蛋白。观察CET对人晶状体上皮细胞(LECs)依赖的膜转运蛋白(Na+/K+泵/ATPase[NKP，NKA]、Na+-K+-2Cl+[NKCC]和K+-Cl-[KCC]共转运体，以及通道支持的K+丢失)的作用。方法：用NKP和NKCC抑制剂，以Rb+为同系物，用原子吸收/发射光谱法测定K+流失和K+摄取，用NO3-ˉ取代氯离子测定KCC。用~3H-哇巴因结合猪肾NKA，观察了CET存在和不存在的情况。使用ProSite结合BLAST比对以及基于二级结构和晶体结构预测的保守性和结构相似性分析，对BCL-2蛋白和NKA序列进行比对和基序鉴定和图谱绘制。结果：CET可抑制NKP和NKCC活性的90%(IC50值分别为∼35和∼15µM)，但对KCC活性无明显影响；在10-30µM时，∼可刺激K+丢失35%，对NKAα1亚基的磷酸化水平无影响。当CET浓度比配体高100倍时，~3H-哇巴因从猪肾NKA中被取代。NKA与含有促生存蛋白的BH1和BH3类基序的序列比对显示，NKA中有多个BH1类基序与CET或BH3基序相互作用。在所有的P型ATPase中也发现了一个NKA BH1样基序(ARAAEILARDGPN)。此外，NKA还具有第二个基序，类似于BH3区附近的Bcl-2。结论：研究结果支持CET通过与BH1样基序结合并通过构象变化破坏α1亚单位的催化活性来抑制NKP的假说。NKP蛋白通过其互补的BH1或BH3类基序与Bcl2蛋白相互作用，可能是正常和病理细胞功能的感受器，在细胞凋亡的初始阶段成为重要但尚未被识别的信号转导。CET对NKCC1和K+通道的作用可能涉及PKC调节机制；然而，BH1类基序的有限序列同源性不能排除直接作用。
Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by >90% (IC50 values ∼35 and ∼15 µM, respectively) without significant KCC activity change, and stimulated K+ loss by ∼35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. 3H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Conclusion: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α1 subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K+ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.