Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

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.

巨核细胞（Megakaryocytes, MKs）是少数会发生多倍体化的细胞类型之一，但目前这类细胞被定向诱导为多倍体的具体分子机制尚未完全阐明。本研究中，我们通过SP600125诱导Dami与CMK细胞，成功构建了两种相对同步的多倍体细胞模型。研究发现，SP600125可诱导Dami和CMK细胞发生多倍体化，同时伴随核糖体蛋白S6激酶1（ribosomal protein S6 kinase 1, S6K1）在Thr421/Ser424位点的磷酸化，以及Thr389位点的去磷酸化。环腺苷酸依赖性蛋白激酶（cAMP-dependent protein kinase, PKA）抑制剂H-89可通过直接结合S6K1，部分阻断该多倍体化过程，使S6K1在Thr421/Ser424位点发生去磷酸化、Thr389位点发生磷酸化，且该调控过程不依赖于PKA。过表达雷帕霉素抗性的S6K1突变体，可进一步增强LY294002对SP600125诱导的Dami与CMK细胞多倍体化的抑制作用。此外，SP600125还可诱导源自慢性髓性白血病患者的Meg-01细胞发生多倍体化，且该过程未伴随S6K1磷酸化水平的显著变化。同时，SP600125可诱导源自红白血病患者的HEL细胞发生多倍体化，且可检测到S6K1在Thr389位点的磷酸化。但SP600125处理后，Meg-01与HEL细胞的多倍体化程度均低于其诱导的Dami和CMK细胞；尽管H-89可使这两种细胞系中S6K1的Thr389位点磷酸化水平升高，但H-89并未阻断这两种细胞的多倍体化过程。鉴于Dami和CMK细胞系源自急性巨核细胞白血病（acute megakaryocytic leukemia, AMKL）患者且高表达血小板特异性抗原，我们的研究结果表明：SP600125诱导的多倍体化具有细胞类型特异性；这类细胞系的分化程度更高；S6K1的Thr421/Ser424位点磷酸化与Thr389位点去磷酸化，可能与其他信号通路协同作用，在SP600125诱导的这类细胞系多倍体化过程中发挥重要作用。