Lyn regulates creatine uptake in an imatinib-resistant CML cell line.
Lyn regulates creatine uptake in an imatinib-resistant CML cell line.
复制标题
Lyn 调节伊马替尼耐药 CML 细胞系中肌酸的摄取。
DOI：
10.1016/j.bbagen.2019.129507
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Graves,LeeM
中科院分区：
文献类型：
--
作者：
Okumu,DenisO;Aponte-Collazo,LucasJ;Dewar,BrianJ;Cox,NathanJ;East,MichaelP;Tech,Katherine;McDonald,IanM;Tikunov,AndreyP;Holmuhamedov,Ekhson;Macdonald,JeffreyM;Graves,LeeM
研究背景甲磺酸伊马替尼（Imatinib mesylate，imatinib）因其显著的血液学和细胞遗传学反应而成为治疗初治慢性粒细胞白血病（chronic myeloid leukemia，CML）的一线药物。我们以前证明，伊马替尼耐药的CML细胞（Myl-R）包含升高的林恩活动和细胞内肌酸池相比，伊马替尼敏感的Mylcells. MethodsStable同位素代谢标记，媒体肌酸耗尽，Na +/K +-ATP酶抑制剂实验进行调查肌酸池在Myl-R细胞的起源。抑制和shRNA敲低进行调查的具体作用，林恩在调节Na +/K +-ATP酶和肌酸uptake.ResultsInhibition的Na +/K +-ATP酶泵（哇巴因，洋地黄毒苷），细胞外肌酸或林恩激酶（泊那替尼，达沙替尼）的抑制耗尽，证明增强肌酸积累在Myl-R细胞依赖于从生长介质中的摄取。肌酸摄取不依赖于Na +/肌酸同向转运体（SLC 6A 8）的表达或新生合成。蛋白质印迹分析表明，酪氨酸10（Y10），一个已知的调节磷酸化位点上的Na +/K +-ATP酶的磷酸化与林恩活性相关。在HEK293细胞中过表达林恩增加Na +/K +-ATP酶的Y10磷酸化（pY10），而林恩抑制或shRNA敲低降低Na +/K +-ATP酶pY10并减少Myl-R细胞中的肌酸积累。与Myl-R细胞中摄取增强一致，环肌酸（Ccr），一种细胞毒性肌酸类似物，结论林恩可通过磷酸化和调节Na +/K +-ATP酶泵活性来影响肌酸的摄取，这些研究证实了激酶对Na +/K +-ATP酶活性的调节作用。ATP酶是调节细胞肌酸摄取和能量代谢的关键。
BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.MethodsStable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake.ResultsInhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression orde novosynthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells.ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity.General significanceThese studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.