An Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of N-Acetylglucosamine Kinase

Reversible protein phosphorylation serves as a pivotal signaling mechanism in eukaryotic cells, contrasting with the poorly understood protein pyrophosphorylation, a posttranslational modification (PTM) whose functions in living organisms remain elusive. Unlike kinase-mediated protein phosphorylation, pyrophosphorylation is thought to be installed non-enzymatically, when a highly phosphorylated inositol pyrophosphate (PP-InsPs) transfers its high energy β-phosphoryl group onto the pre-existing phosphoryl group of a protein substrate. Our group recently presented the initial evidence of endogenous protein pyrophosphorylation in mammalian cell lines, including N-acetylglucosamine kinase (NAGK). Here we report, the first site-selective pyrophosphorylation of NAGK, strategically modified at its endogenous site to evaluate the modification's impact on protein activity. Biochemical evaluation revealed a significant reduction in GlcNAc kinase activity upon phosphorylation and near-complete inactivation upon pyrophosphorylation at serine 76 (S76), highlighting pyrophosphorylation’s unique regulatory function facilitated by an ATP-mediated autocatalytic process. ppS76-NAGK demonstrated notable stability in mammalian cell lysates, hinting at alternative functional outcomes. Proteomic examination unveiled distinct protein-protein interactions for ppS76-NAGK, suggesting involvement in diverse cellular pathways. In summary, this study enhances our comprehension of protein pyrophosphorylation and its significance for kinase regulation and protein-protein interactions, paving the way for further exploration of pyrophosphorylated kinases.