Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines

Abstract

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.

Figure 1. Effect of SP600125 on proliferation, viability, polyploidy, expression of cyclin B1, cyclin D3, c-Myc, and survivin, and the translation-related proteins of Dami and CMK cells.

Dami and CMK cells were seeded at 2×10⁵/ml in RPMI 1640 medium containing 10% FCS and treated with SP600125 at different concentrations for different periods of time as indicated. Dami and CMK cells treated with DMSO were used as a control. (A, B) The cell number and viability, presented as the mean±SEM, were determined from 4 separate experiments. (C) Representative DNA histograms of SP600125-induced Dami and CMK cells analyzed by flow cytometry. (D) Morphology was analyzed by Wright-Giemsa staining of cytocentrifuged preparations of Dami and CMK cells induced by SP600125 or nocodazole (Original magnification, 1000×). The Dami and CMK cells treated with DMSO or with SP600125 were lysed, and equal amounts of protein were loaded for western blots to evaluate the protein levels of cyclin B1, cyclin D3, c-Myc, and survivin (E), and the phosphorylation and protein levels of S6K1, eIF4E and 4E-BP1(F). β-actin was used as an internal control.

Figure 2. H-89 blocked polyploidization of the SP600125 treated-Dami and CMK cells.

Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment without or with H-89 at 10 µM or 20 µM for 1 hour. Dami and CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy (A). The data are presented as the mean±SEM polyploidy and were obtained from 4 separate experiments (B). All bar graphs depict the means ± SEM; *p<0.05. The cells were lysed, and equal amounts of protein were analyzed by western blot for cyclin B1, cyclin D3, c-Myc, and survivin (C). The phosphorylation and protein levels of S6K1, eIF4E and 4E-BP1 (D). β-actin was used as an internal control.

Figure 3. H-89 inhibited the activity of S6K1 directly.

(A) Docking studies were performed to evaluate the binding of H-89 to S6K1 using AutoDock 4.2 software. H-89 is predicted to bind into the hydrophobic cleft between the N- and C-terminal domains of unphosphorylated S6K1 (PDB: 3A61). (B) The activity of S6K1 was assayed in vitro in the presence of H-89 at increasing concentrations as indicated. The data are presented as the mean±SEM of the percentage of kinase activity relative to the control measured in the presence of DMSO and were obtained from 3 separate experiments.

Figure 4. H-89 blocked the polyploidization of Dami and CMK cells independent of PKA.

Dami and CMK cells were treated with SP600125 at 32 µM for 72 hours after pretreatment with or without H-89 at increasing concentrations as indicated for 1 hour. Dami or CMK cells treated with DMSO were used as the vehicle-treated control, and Dami or CMK cells treated with H-89 alone were used as the pretreatment control. (A) The cells were lysed, and equal amounts of protein were analyzed by western blot for Phospho-PKA Substrate (RRXS*/T*), Phospho-(Ser/Thr) PKA Substrate, S6K1, phospho-S6K1 (Thr421/Ser424), and phospho-S6K1 (Thr389). (B) The cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy.

Figure 5. Partial inhibition of phosphorylation of S6K1 at Thr421/Ser424 is not sufficient to block polyploidization.

Dami and CMK cells were incubated with SP600125 at 32 µM for 72 hours after pretreatment with or without PD184352 (2 µM), U0126 (10 µM), or LY294002 (30 µM) for 1 hour. Dami and CMK cells treated with DMSO were used as the control. After incubation, the cells were fixed, stained with PI and analyzed with a flow cytometer for DNA ploidy. (A) A typical representative DNA histogram. (B) The data are presented as the mean±SEM level of polyploidy and were obtained from 4 separate experiments; *p<0.05. (C) The remaining cells were lysed, and equal amounts of protein were analyzed by western blot for p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), Akt, phospho-Akt (Ser473), S6K1, phospho-S6K1 (Thr421/Ser424), and phospho-S6K1 (Thr389). β-actin was used as an internal control.

Figure 6. Effects of S6K1 mutant plasmids on SP600125-induced Dami cells.

Dami cells were transfected with or without S6K1-WT, S6K1-T389E, S6K1-T389A and S6K1-D3E and cultured overnight. The cells were then induced with SP600125 for 72 hours after pretreatment with or without LY294002 for 1 hour. (A) Representative DNA histograms of Dami cells in each condition. (B) The data are presented as the mean±SEM of the level of polyploidy and were obtained from 4 separate experiments; *p<0.05.