Glycolytic Pfkp acts as a Lin41 protein kinase to promote endodermal differentiation of embryonic stem cells

Unveiling the principles governing embryonic stem cell (ESC) differentiation into specific lineages is critical for understanding embryonic development and for stem cell applications in regenerative medicine. Here, we establish an intersection between LIF‐Stat3 signaling that is essential for maintaining murine (m) ESCs pluripotency, and the glycolytic enzyme, the platelet isoform of phosphofructokinase (Pfkp). In the pluripotent state, Stat3 transcriptionally suppresses Pfkp in mESCs while manipulating the cells to lift this repression results in differentiation towards the ectodermal lineage. Pfkp exhibits substrate specificity changes to act as a protein kinase, catalyzing serine phosphorylation of the developmental regulator Lin41. Such phosphorylation stabilizes Lin41 by impeding its autoubiquitination and proteasomal degradation, permitting Lin41‐mediated binding and destabilization of mRNAs encoding ectodermal specification markers to favor the expression of endodermal specification genes. This provides new insights into the wiring of pluripotency‐differentiation circuitry where Pfkp plays a role in germ layer specification during mESC differentiation. Synopsis Pfkp has a non‐glycolytic activity in mESC differentiation, where it catalyzes Lin41 phosphorylation, protecting Lin41 from proteasomal destruction. In turn, Lin41 promotes endodermal differentiation of mESCs by binding to and destabilizing mRNAs encoding ectodermal specification markers. Pfkp is transcriptionally repressed in mESCs through LIF‐Stat3 signaling. Lifting Stat3 repression promotes Pfkp expression and its phosphorylation of Lin41. Stabilized Lin41 disrupts expression of ectodermal mRNAs during differentiation. Graphical Abstract Pfkp has a non‐glycolytic activity in mESC differentiation, where it catalyzes Lin41 phosphorylation, protecting Lin41 from proteasomal destruction. In turn, Lin41 promotes endodermal differentiation of mESCs by binding to and destabilizing mRNAs encoding ectodermal specification markers.