Novel AKT phosphorylation sites identified in the pluripotency factors OCT4, SOX2 and KLF4

Abstract

The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, selfrenewal and tumorigenesis in stem cells. However, although their transcriptional targets have been extensively studied, little is known about how these factors are regulated at the posttranslational level. In this study, we established an in vitro system to identify phosphorylation patterns of the OSKM factors by AKT kinase. OCT4, SOX2, KLF4 and c-MYC were expressed in Sf9 insect cells employing the baculoviral expression system. OCT4, SOX2 and KLF4 were localized in the nucleus of insect cells, allowing their easy purification to near homogeneity upon nuclear fractionation. All transcription factors were isolated as biologically active DNA-binding proteins. Using in vitro phosphorylation and mass spectrometry-based phosphoproteome analyses several novel and known AKT phosphorylation sites could be identified in OCT4, SOX2 and KLF4.

Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop

“…AKT has many well-known, crucial roles in proliferation, anti-apoptosis, protein synthesis, and metabolism. Recently, AKT has also been suggested to have regulatory signi cance in cancer stemness 54 because the AKT-dependent phosphorylation pattern has been identi ed within the key stemnessinducing transcription factors, OCT-4, SOX-2, and KLF4 55,56 . Moreover, additional modi cation patterns, including phosphorylation-dependent inhibition of ubiquitination and SUMOylation, have been identi ed for these stemness factors, implicating that overall control on stemness-related transcription factors by AKT-dependent regulatory mechanisms determines the cellular fate of stem cells.…”

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

“…Thus, mRNA coding phosphosite‐modified OLIG2 may be used in efficient mRNA‐driven motor neuron production from iPSCs. Phosphosites in the Yamanaka factors (e.g., SOX2) have also been reported to regulate their protein stability, complex formation, and ability in maintaining pluripotency . This knowledge will likely allow us to optimize current mRNA‐driven cell reprogramming system.…”

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells

“…Five days post-infection cells were harvested by centrifugation at 4°C for 10 min at 2000 g and washed twice with 50 mL PBS. As recombinant OCT4 localizes in the nucleus of Sf9 cells, nuclear extraction was conducted as described [50]. Briefly, cell pellets were suspended in hypotonic buffer (10 mM Na-HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM PMSF) and incubated for 15 min on ice.…”