蛋白激酶A抑制剂H-89通过调节核糖体蛋白S6激酶1的磷酸化阻断SP600125诱导的CMK细胞多倍体化  

Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1

作　　者：赵松[1] 杨金刚[1] 李长岭[1] 邢思宁[1] 于颖[1] 刘硕[1] 蒲菲菲[1] 马东初[1] ZHAO Song YANG Jingang LI Changling XING Sining YU Ying LIU Shuo PU Feifei MA Dongchu(Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China)

机构地区：[1]沈阳军区总医院医学实验科,辽宁沈阳110016

出　　处：《细胞与分子免疫学杂志》2016年第10期1321-1326,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(61302003;31571398)

摘　　要：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 investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1（ S6K1） on the polyploidization of megakaryocytes. Methods SP600125,a c-Jun N-terminal kinase（ JNK） inhibitor,and H-89,a c AMP-dependent protein kinase（ PKA） inhibitor,were used to treat CMK cel s separately or in combination. With propidium iodide（ PI） to dye DNA in the treated cells,the relative DNA content was detected by flow cytometry,and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1（ S6K1）,an important mammalian target of rapamycin（ m TOR） downstream target molecule, was analyzed by Western blotting.Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time,it increased the phosphorylation of S6K1 at Thr421 / Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization,but also decreased the phosphorylation of S6K1 at Thr421 / Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably,both H-89 and SP600125 inhibited the activity of PKA. Moreover,the two drugs further inhibited the activity of PKA when used together. Therefore,these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state,rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

关 键 词：核糖体蛋白S6激酶1(S6K1) 巨核细胞 多倍体化 

分 类 号：Q279[生物学—细胞生物学] R329-33[医药卫生—人体解剖和组织胚胎学]