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

Area of Science:

• Stem cell biology
• Molecular biology
• Biochemistry

Background:

• The OSKM transcription factors (OCT4, SOX2, KLF4, c-MYC) are crucial for maintaining pluripotency, self-renewal, and are implicated in tumorigenesis.
• While their transcriptional roles are well-documented, their posttranslational regulation, particularly phosphorylation, remains largely unexplored.

Purpose of the Study:

• To investigate the posttranslational regulation of the OSKM factors by identifying their phosphorylation patterns mediated by AKT kinase.
• To establish an in vitro system for studying OSKM factor phosphorylation.

Main Methods:

• Expression of OCT4, SOX2, KLF4, and c-MYC in Sf9 insect cells using a baculoviral system.
• Nuclear fractionation and purification of OCT4, SOX2, and KLF4.
• In vitro kinase assays with AKT kinase.
• Mass spectrometry-based phosphoproteome analysis to identify phosphorylation sites.

Main Results:

• Successfully expressed and purified biologically active, DNA-binding OCT4, SOX2, and KLF4 from insect cell nuclear fractions.
• Identified several novel and known AKT kinase phosphorylation sites on OCT4, SOX2, and KLF4 through in vitro phosphorylation and phosphoproteome analysis.

Conclusions:

• This study provides novel insights into the posttranslational modification of key pluripotency factors by AKT kinase.
• The identified phosphorylation sites on OCT4, SOX2, and KLF4 may influence their function and regulation in stem cells.