Reciprocal Regulation of Akt and Oct4 Promotes the Self-Renewal and Survival of Embryonal Carcinoma Cells

College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Molecular cell (Impact Factor: 14.02). 10/2012; 17(4). DOI: 10.1016/j.molcel.2012.08.030
Source: PubMed


Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, posttranslational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization, and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.

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Available from: Ying Yang, Oct 28, 2015
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    • "Immunoblotting and immunoprecipitation was conducted as described previously [10] "
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    ABSTRACT: The octamer-binding transcription factor 4 (Oct4) is essential for maintaining the self-renewal and pluripotency of embryonic stem cells (ESCs). Post-translational modifications (PTMs) of Oct4 critically control its structure, function and intracellular localization. However, determination of Oct4 PTM profiles has largely been restricted by the quantity and purity of the Oct4 protein samples required for mass spectrometric analyses. In this study, by incubating the E. coli-derived His-tagged Oct4 proteins with the whole cell lysates of a variety of human cells followed by retrieving the reacted Oct4 proteins with the Ni-NTA beads, we developed a labor- and cost-effective in vitro PTM method that allowed for mass spectrometric determination of the phosphorylation profiles of Oct4 proteins exposed to various cell-free systems. A number of Oct4 phosphorylation sites that were commonly present in all the cell-free systems or specifically present in a particular cellular context were identified, indicating that Oct4 is controlled by both common and distinct PTM regulatory pathways. Our work provided a proof-of-concept that such a cell-free system-based in vitro PTM approach can be applied to systematically map out the physiologically-relevant PTM sites in Oct4 proteins, which opened up an avenue to fully decipher the Oct4 PTM barcodes in various cellular contexts.
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    • "Typically, expression of these markers is lost 24–36 h following removal of LIF. Reciprocal roles for PKB and Oct-3/4 in positively regulating each other in embryonal carcinoma cells (ECCs), the malignant equivalent of ES cells, has also emerged (Lin et al., 2012). PKB was shown to phosphorylate and positively regulate the protein stability, nuclear localization, and transcriptional activity of Oct-3/4. "
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    • "In addition, translation of OCT3/4 is mediated by micro-RNAs, specifically miR-145 [238] [239] and miR- 34a [240]. Also, post-translational modification of OCT3/4, i.e. phosphorylation by AKT has been shown to change its affinity/specificity for DNA binding sites, potentially changing the functional impact of this transcription factor [241]. Like OCT3/4 and in line with their pluripotent phenotype, the homeodomain NANOG is only detectable in the TGCC stem cell components SE and EC and in CIS and not in differentiated NS derivatives [18] [242] (Fig. 1). "
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