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目的研究核糖体蛋白S6激酶1(S6K1)翻译后修饰在巨核细胞倍体化调控方面的作用。方法 c-Jun氨基末端激酶(JNK)抑制剂SP600125和蛋白激酶A(PKA)抑制剂H-89单独或联合处理CMK原始巨核细胞白血病细胞。碘化丙啶(PI)染色,结合流式细胞术检测DNA的相对量,从而进行DNA倍体分析;Western blot法检测哺乳动物雷帕霉素靶蛋白(m TOR)下游靶分子S6K1表达和磷酸化修饰(Thr389和Thr421/Ser424)的变化。使用分子对接和激酶活性检测分析H-89与S6K1结合的关系以及对激酶活性的影响。结果 SP600125以时间和剂量依赖的方式诱导CMK细胞多倍体化,同时,上调S6K1的Thr421/Ser424磷酸化和下调Thr389的磷酸化。H-89不但部分阻断SP600125诱导CMK细胞多倍体化,而且下调S6K1的Thr421/Ser424磷酸化和上调Thr389磷酸化。分子对接和激酶活性分析发现H-89通过占据ATP结合位点,抑制S6K1活性。值得注意的是,H-89和SP600125均抑制PKA的活性,而且两者联合进一步抑制了PKA的活性,表明H-89阻断SP600125诱导CMK多倍体化与其对PKA的作用无关,而与S6K1磷酸化修饰状态的改变有关。结论 H-89通过调节S6K1磷酸化阻断SP600125诱导CMK细胞多倍体化。
Objective To study the role of posttranslational modification of ribosomal protein S6 kinase 1 (S6K1) in the regulation of megakaryocyte ploidy. Methods CMK primary megakaryocyte leukemic cells were treated singly or in combination with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the protein kinase A (PKA) inhibitor H-89. Propidium iodide (PI) staining and flow cytometry were used to detect the relative amount of DNA, so DNA ploidy analysis was carried out. Western blot was used to detect the expression of S6K1, a downstream target of mammalian target of rapamycin (m TOR) Changes in the modifications (Thr389 and Thr421 / Ser424). The relationship between H-89 binding to S6K1 and its effect on kinase activity was analyzed using molecular docking and kinase activity assays. Results SP600125 induced CMK cell polyploidization in a time-and dose-dependent manner, up-regulated Thr421 / Ser424 phosphorylation of S6K1 and down-regulated Thr389 phosphorylation. H-89 partially blocked SP600125-induced polyploidization of CMK cells and down-regulated Thr421 / Ser424 phosphorylation of S6K1 and up-regulated Thr389 phosphorylation. Molecular docking and kinase activity assays revealed that H-89 inhibits S6K1 activity by occupying the ATP binding site. It is noteworthy that both H-89 and SP600125 inhibited the activity of PKA, and the combination of the two further inhibited the activity of PKA, indicating that H-89 block SP600125-induced CMK polyploidy has nothing to do with the effect of PKA, and S6K1 Phosphorylation modification of the state of the change. Conclusion H-89 can induce polyclonalization of CMK cells by blocking the phosphorylation of S6K1.