O-GlcNAcylation regulates tyrosine hydroxylase serine 40 phosphorylation and l-DOPA levels

β-O-linked-N-acetylglucosamine (O-GlcNAcylation) is a post-translational modification (PTM) characterized by the covalent attachment of a single moiety of N-acetylglucosamine (GlcNAc) on serine/threonine residues in proteins. Tyrosine hydroxylase (TH), the rate-limiting step enzyme in the catecholamine synthesis pathway and responsible for the production of the dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), has its activity regulated by phosphorylation. Here, we show an inverse feedback mechanism between O-GlcNAcylation and phosphorylation of TH at serine 40 (TH pSer40). First, we showed that, during PC12 cells neuritogenesis, TH O-GlcNAcylation decreases concurrently with the increase of pSer40. In addition, an increase in O-GlcNAcylation induces a decrease in TH pSer40 only in undifferentiated PC12 cells, whereas the decrease in O-GlcNAcylation leads to an increase in TH pSer40 levels in both undifferentiated and differentiated PC12 cells. We further show that this feedback culminates on the regulation of l-DOPA intracellular levels. Interestingly, it is noteworthy that decreasing O-GlcNAcylation is much more effective on TH pSer40 regulation than increasing its levels. Finally, ex vivo analysis confirmed the upregulation of TH pSer40 when O-GlcNAcylation levels are reduced in dopaminergic neurons from C57Bl/6 mice. Taken together, these findings demonstrate a dynamic control of l-DOPA production by a molecular cross talk between O-GlcNAcylation and phosphorylation at Ser40 in TH.NEW & NOTEWORTHY This study shows how β-O-linked-N-acetylglucosamine (O-GlcNAcylation) modulates tyrosine hydroxylase (TH) activity, revealing a negative feedback loop with Ser40 phosphorylation both in vitro and ex vivo, which directly influences on l-3,4-dihydroxyphenylalanine (l-DOPA) production. These findings offer insights into neurotransmitter homeostasis regulation, with implications for understanding and potentially treating disorders linked to aberrant catecholamine signaling.