SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

Abstract

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts

“…Interestingly, we found a SUMOylationindependent repression of Nanog promoter (Fig. 1H), evidencing again a different response in this scenario and recalling the observations in MEFs [26].…”

“…1B, dotted bars). Altogether, these results in iPSCs, along with the previously obtained in ESCs [26], demonstrate the SUMOylation-dependence of AKT1 for the induction of the Nanog promoter in different pluripotent contexts.…”

“…We have previously reported that AKT1 induces Nanog expression in a SUMOylation-dependent manner in mouse ESCs [26]. Interestingly, we found a SUMOylation-independent repression of Nanog promoter by AKT in the terminally differentiated cell line NIH/3T3 of mouse embryonic fibroblasts (MEFs), suggesting that AKT induction of Nanog promoter activity is not a universal effect [26]. These results led us to deepen into the cellular context dependence of this effect, looking for a hint about specific players or the underlying molecular mechanism.…”

“…This PTM regulates the function of a plethora of proteins, impacts on protein-protein interactions [21], and can even affect their subcellular localization [22][23][24][25]. We have previously reported that SUMOylation of AKT1 is crucial to induce the expression of the pluripotency TF Nanog in mouse ESCs [26]. Additionally, we have recently found that this PTM influences AKT1 subcellular compartmentalization and distribution, and also impacts on NANOGchromatin binding dynamics in ESCs [27].…”

The transcription factor OCT6 promotes the dissolution of the naïve pluripotent state by repressing Nanog and activating a formative state gene regulatory network

“…Transfected cells were either maintained in this state or differentiated for 24 h, either with or without Dox. Luciferase activity was analyzed using as previously reported 50 .…”