摘要
目的:探讨JNK抑制剂SP600125诱导巨核细胞白血病系多倍体化的调控机制。方法:用SP600125和3种抑制剂(PD184352、U0126和LY294002)处理Dami和CMK细胞,用点突变技术对核糖体蛋白S6激酶1(S6K1)C端自抑制假底物结构域和疏水基序突变构建的S6K1质粒转染细胞,流式细胞术分析细胞的DNA倍性,Western印迹检测S6K1、MAPK和Akt蛋白的表达及磷酸化修饰位点的变化。结果:当单独使用3种抑制剂时,对Dami和CMK细胞的倍性无影响。当SP600125与3种抑制剂联合时,尽管PD184352下调了p44/42 MAPK在Thr202/Tyr204位点的磷酸化,但其并不抑制SP600125诱导的Dami和CMK细胞的多倍体化;相反,U0126抑制SP600125诱导的Dami和CMK细胞的多倍体化,但并不下调p44/42 MAPK的磷酸化;而LY294002增加Akt的磷酸化,并阻断SP600125诱导的Dami和CMK细胞的多倍体化。这3种抑制剂均在SP600125诱导的多倍体Dami和CMK细胞中部分抑制S6K1的Thr421/Ser424磷酸化,但并不增加S6K1的Thr389磷酸化。转染S6K1突变质粒的Dami细胞,并没有对SP600125诱导的多倍体化产生影响,同时,无论是模拟磷酸化或去磷酸化的Thr389突变均对SP600125诱导的多倍体化无影响,且未显现出与LY294002的协同作用。C端自抑制假底物结构域突变质粒(S6K1-D3E)可以进一步阻断SP600125诱导的已经被LY294002部分阻断的Dami细胞多倍体化。结论:S6K1的C端自抑制假底物结构域磷酸化与Akt去磷酸化协同调节SP600125诱导巨核细胞白血病系的多倍体化。
Objective： To investigate the mechanism of SP600125-induced polyploidization of megakaryocytic leukemia cell lines.Methods： Dami and CMK cells were treated with SP600125 and three inhibitors PD184352, U0126 and LY294002. Meanwhile, we constructed plasmid encoding mutated ribosomal protein S6 kinase 1（S6K1） by point mutagenesis targeting the C-terminal autoinhibitory pseudosubstrate domain and the hydrophobic motif of S6K1 to transfecte cells. The DNA ploidy was analyzed by flow cytometry. The expression and phosphorylation of S6K1, MAPK and Akt were detected by Western blot.Results： When three inhibitors were used alone, there was no effect on the ploidy of Dami and CMK cells. When SP600125 was combined with three inhibitors, PD184352 did not inhibit SP600125-induced polyploidization of Dami and CMK cells, although it decreased the phosphorylation of p44/42 MAPK at Thr^202/Tyr^204. In contrast, U0126 inhibited SP600125-induced polyploidization of Dami and CMK cells, but did not decreased p44/42 MAPK phosphorylation. While LY294002 increased the phosphorylation of Akt and blocked SP600125-induced polyploidization of Dami and CMK cells. Notably, these three inhibitors, in SP600125-induced polyploidization of Dami and CMK cells, partially inhibited the phosphorylation of S6K1 at Thr^421/Ser^424, but did not increase the phosphorylation of S6K1 at Thr^389. Dami cells that transfected with S6K1 mutant plasmid did not affect SP600125-induced polyploidization. At the same time, either the phosphorylation or dephosphorylation mutations with Thr389 sites had no effect on SP600125-induced polyploidization, and it did not show synergistic effect with LY294002. However, C-terminal autoinhibitory pseudosubstrate domain mutant plasmid（S6K1-D3E） could further block polyploidization of SP600125-induced polyploidization of Dami cells that had been partially blocked by LY294002.Conclusion： SP600125-induced polyploidization of megakaryocytic leukemia cell lines by the regulation of S6K1 C-terminal autoinhibitory pse
出处
《生物技术通讯》
CAS
2017年第3期233-241,共9页
Letters in Biotechnology
基金
国家自然科学基金(61302003
31571398)
