The relationship between kinase pathways in pluripotency and cell cycle progression

Pluripotency is the ability of a stem cell to differentiate into any cell type, governed in part by intricate kinase-mediated phosphorylation networks. These regulatory pathways coordinate pluripotency, differentiation, and cell cycle progression. Mouse embryonic stem cells (mESCs) are pluripotent that can proliferate indefinitely as undifferentiated cells in vitro. While previous studies have identified phosphorylation of key pluripotency factors like NANOG, SOX2, and OCT4 by kinases such as Cyclin E-CDK2, the broader interactions between pluripotency-maintaining and differentiation-promoting kinase pathways remain unclear. Using mass spectrometry data from synchronized mESCs collected across distinct cell cycle phases and enriched for phosphorylated peptides using TiO₂, we predicted kinase-substrate relationships that regulate pluripotency and cell cycle progression. Our analysis identified RPS6KB1 as a key regulator of OCT4, suggesting a central role in pluripotency maintenance. We hypothesize that inhibiting key kinases important in pluripotency, such as RPS6KB1, will result in differentiation, while inhibiting key kinases important for differentiation will maintain pluripotency. To test these predictions, we will apply small-molecule inhibitors to mESCs and assess changes in NANOG, SOX2, and OCT4 expression via immunohistochemistry. Additionally, we identified kinases involved in cell cycle transitions, such as PRKAA2, which is predicted to regulate S-phase progression. We predict that inhibition of such kinases will lead to phase-specific cell cycle arrest, which will be validated using flow cytometry to measure DNA content. This research underscores the critical roles of kinase pathways in pluripotency and cell cycle regulation, providing insights into potential therapeutic targets for stem cell-based treatments and cancer therapy.