PXD049339-2

PXD049339 is an original dataset announced via ProteomeXchange.

Title	An Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of N-Acetylglucosamine Kinase
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:41:00.108.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Max Ruwolt
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2024-02-12 09:35:08	ID requested
1	2024-05-22 02:22:52	announced
⏵ 2	2024-10-22 06:41:00	announced	2024-10-22: Updated project metadata.

10.1021/JACS.4C03069;

submitter keyword: pulldown, affinity-purification MS, LFQ, phosphorylation,pyrophosphorylation

Dorothea Fiedler
contact affiliation	Leibniz-Forschungsinstitut fuer Molekulare Pharmakologie Humboldt-Universitaet zu Berlin
contact email	fiedler@fmp-berlin.de
lab head
Max Ruwolt
contact affiliation	Leibniz-Forschungsinstitut for Molecular Pharmacology
contact email	ruwolt@fmp-berlin.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD049339
     1. Label: PRIDE project
     2. Name: An Uncommon Phosphorylation Mode Regulates the Activity and Protein Interactions of N-Acetylglucosamine Kinase