[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].
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 cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells 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 (mTOR) 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.
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Song
S
Zhao
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Jingang
J
Yang
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Changling
C
Li
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Sining
S
Xing
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Ying
Y
Yu
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Shuo
S
Liu
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China.
Feifei
F
Pu
Department of Experimental Medicine, General Hospital, Shenyang Military Area Command, Shenyang 110016, China. *Corresponding author, E-mail: mdc580819@sina.com.
Dongchu
D
Ma
D000873:Anthracenes / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology*; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D062105:Molecular Docking Simulation; D010766:Phosphorylation; D011123:Polyploidy; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*
D000873:Anthracenes; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C432165:pyrazolanthrone; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C000620914:ribosomal protein S6 kinase 1, human; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide