biostudies-literatureCelik ADeutsche Forschungsgemeinschaft14807-14815https://www.ebi.ac.uk/biostudies/studies/S-EPMC11140747biostudies-literatureUnknown146(21)While the function of protein phosphorylation in eukaryotic cell signaling is well established, the role of a closely related modification, protein pyrophosphorylation, is just starting to surface. A recent study has identified several targets of endogenous protein pyrophosphorylation in mammalian cell lines, including <i>N</i>-acetylglucosamine kinase (NAGK). Here, a detailed functional analysis of NAGK phosphorylation and pyrophosphorylation on serine 76 (S76) has been conducted. This analysis was enabled by using amber codon suppression to obtain phosphorylated pS76-NAGK, which was subsequently converted to site-specifically pyrophosphorylated NAGK (ppS76-NAGK) with a phosphorimidazolide reagent. A significant reduction in GlcNAc kinase activity was observed upon phosphorylation and neaJournal of the American Chemical SocietyAn Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of &lt;i&gt;N&lt;/i&gt;-Acetylglucosamine Kinase.PMC11140747469186007Beyer ICelik AFiedler DfalseAn Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of &lt;i&gt;N&lt;/i&gt;-Acetylglucosamine Kinase.While the function of protein phosphorylation in eukaryotic cell signaling is well established, the role of a closely related modification, protein pyrophosphorylation, is just starting to surface. A recent study has identified several targets of endogenous protein pyrophosphorylation in mammalian cell lines, including <i>N</i>-acetylglucosamine kinase (NAGK). Here, a detailed functional analysis of NAGK phosphorylation and pyrophosphorylation on serine 76 (S76) has been conducted. This analysis was enabled by using amber codon suppression to obtain phosphorylated pS76-NAGK, which was subsequently converted to site-specifically pyrophosphorylated NAGK (ppS76-NAGK) with a phosphorimidazolide reagent. A significant reduction in GlcNAc kinase activity was observed upon phosphorylation and nea2024-01-01T00:00:00Z2024 May2026-06-03T06:49:10.979Z2026-04-25T03:21:51.862ZS-EPMC111407473873335310.1021/jacs.4c03069