BACKGROUND Imatinib 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. METHODS Stable 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. RESULTS Inhibition 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 or de novo synthesis. 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. CONCLUSIONS These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. GENERAL SIGNIFICANCE These studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

中文翻译：

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Lyn调节对伊马替尼耐药的CML细胞系中的肌酸摄取。

背景技术甲磺酸伊马替尼（imatinib）由于其显着的血液学和细胞遗传学反应，是新诊断的慢性粒细胞白血病（CML）的一线治疗。我们先前证明，与对伊马替尼敏感的Myl细胞相比，对伊马替尼具有抵抗力的CML细胞（Myl-R）包含升高的Lyn活动和细胞内肌酸池。方法进行稳定的同位素代谢标记，中性肌酸耗竭和Na + / K + -ATPase抑制剂实验，以研究Myl-R细胞中肌酸池的来源。进行了抑制作用和shRNA敲低研究以研究Lyn在调节Na + / K + -ATPase和肌酸摄取中的特定作用。结果抑制Na + / K + -ATPase泵（哇巴因，洋地黄毒苷），清除细胞外肌酸或抑制Lyn激酶（ponatinib，dasatinib），证明了Myl-R细胞中肌酸积累的增加取决于生长培养基的摄取。肌酸的摄取与Na + /肌酸同向转运蛋白（SLC6A8）的表达或从头合成无关。蛋白质印迹分析表明，Tyr 10（Y10）（一个已知的调节磷酸化位点）上的Na + / K + -ATPase的磷酸化与Lyn活性相关。Lyn在HEK293细胞中的过表达增加了Na + / K + -ATPase的Y10磷酸化（pY10），而Lyn抑制或shRNA敲低则降低了Na + / K + -ATPase pY10并降低了Myl-R细胞中的肌酸蓄积。与Myl-R细胞摄取的增加相一致，与Myl细胞相比，一种具有细胞毒性的肌酸类似物环肌酸（Ccr）导致Myl-R的活力显着降低。结论这些数据表明Lyn可以通过Lyn依赖的磷酸化和Na + / K + -ATPase泵浦活性的调节来影响肌酸的摄取。一般意义这些研究发现，Na + / K + -ATPase的激酶调节在调节细胞中的肌酸摄取和能量代谢中起着关键作用。