Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology

Publication date: Available online 12 July 2019Source: Journal of Molecular BiologyAuthor(s): Meret Schmidhauser, Peter F. Renz, Panagiota Tsikrika, Remo Freimann, Anton Wutz, Jeffrey L. Wrana, Tobias A. BeyerAbstractModifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC) and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed. Here, we combined molecular and cellular biology with CRISPR/Cas9 mediated genome engineering to pinpoint the function of Serine 12 of OCT4 in ESCs. Using chemical inhibitors and an antibody specific to OCT4 phosphorylated on S12, we identified Cyclin Dependent Kinase (CDK) 7 as upstream kinase. Surprisingly, generation of isogenic mESCs that endogenously ablate S12 revealed no effects on pluripotency and self-renewal, potentially due to compensation by other phosphorylation events. Our approach reveals that modification of distinct amino acids by precise genome engineering can help to clarify the functions of post-translational modifications on proteins encoded by essential gene in an endogenous context.Graphical abstract
Source: Journal of Molecular Biology - Category: Molecular Biology Source Type: research