S6K1 is involved in polyploidization through its phosphorylation at Thr421/Ser424

Abstract

Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole-induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole-induced Dami cell model, we found that 4E-BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M-phase in cytoplasm and oscillated in nocodazole-induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E-BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12-myristate 13-acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 --> Gln in PMA-induced Dami cells increased ploidy whereas overexpression of rapamycin-resistant form of S6K1 containing the mutations Thr421 --> Glu and Ser424 --> Asp significantly dephosphorylated 4E-BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA-induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin-resistant form of S6K1 significantly reversed polyploidization of nocodazole-induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole-induced Dami cells. Taken together, these data suggested that S6K1/4E-BP1 pathway may play an important role in polyploidization of megakaryocytes.

Inhibition of mTOR Pathway Sensitizes Acute Myeloid Leukemia Cells to Aurora Inhibitors by Suppression of Glycolytic Metabolism

“…mTORC2 modulates cells metabolism through phosphorylating Akt/PKB and activating the downstream such as FoxO3a (19,39). Furthermore, knockout of rictor (one of the mTORC2 components) in mice liver (40), adipose (19), or muscle (20) cells impaired glycolysis and lipogenesis. These studies suggested that mTOR pathway played an important role in activating cellular bio-energetic processes contributed to cancer.…”

“…Furthermore, mTOR was associated with the development of polyploidization. Ma and colleagues showed that S6K1, a downstream of mTOR, was involved in polyploidization through its phosphorylation at Thr421/ Ser424 (20). In addition, activation of mTOR pathway was required for TSC2 þ/EK [EKer rat model carrying a germline insertional deletion in one copy of the Tsc2 (tuberous sclerosis 2) gene] smooth muscle cells of pulmonary lymphangioleiomyomatosis to undergo tetraploidization (21).…”

Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants With the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals

“…5364P). The polyclonal antibodies against EIF4EBP1, phosphoThr69-EIF4EBP1, and phospho-Ser64-EIF4EBP1 have been used extensively, and their specificity established in earlier studies (Gingras et al 2001a;Wang et al 2003;Ohne et al 2008;Ma et al 2009;Fonseca et al 2011;Fuchs et al 2011) including one report (Ellederova et al 2006) in which SDS-PAGE/Western blotting of in vitro matured pig oocytes showed specific reactions with bands of the appropriate sizes. After overnight incubation with primary antibody at 4°, the oocytes were washed three times in blocking buffer for 10 min each wash.…”

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

“…Moreover, the role of S6K1 and 4E-BP1 in the polyploidization of MKs is not clearly understood. Previously, we demonstrated that S6K1 was involved in polyploidization through its phosphorylation at Thr421/Ser424 during M phase in nocodazole-treated Dami cells [7] . However, in nocodazole-induced polyploidization, the cells are multinucleated (karyokinesis is not affected), while in MKs, the nuclei are polylobulated.…”