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

The four OSKM factors OCT4, SOX2, KLF4 and c-MYC are key transcription factors modulating pluripotency, self-renewal 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.