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bioRxiv · 10.1101/2024.02.23.581760

An uncommon phosphorylation mode regulates the activity and protein-interactions of N-acetylglucosamine kinase

Celik, A.·Beyer, I.·Fiedler, D.
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Abstract

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 N-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 regent. A significant reduction in GlcNAc kinase activity was observed upon phosphorylation, and near-complete inactivation upon pyrophosphorylation. The formation of ppS76-NAGK proceeded via an ATP-dependent autocatalytic process, and once formed, ppS76-NAGK displayed notable stability towards dephosphorylation in mammalian cell lysates. Proteomic examination unveiled a distinct set of protein-protein interactions for ppS76-NAGK, suggesting an alternative function, independent of its kinase activity. Overall, a significant regulatory role of pyrophosphorylation on NAGK activity was uncovered, providing a strong incentive to investigate the influence of this unusual phosphorylation mode on other kinases.

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Celik, A., Beyer, I., Fiedler, D.. 2024-02-24. An uncommon phosphorylation mode regulates the activity and protein-interactions of N-acetylglucosamine kinase. https://doi.org/10.1101/2024.02.23.581760

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