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

Abstract

O-GlcNAcylation is a post-translational modification characterized by the covalent attachment of a single moiety of GlcNAc on serine/threonine residues in proteins. Tyrosine hydroxylase (TH), the rate-limiting step enzyme in the catecholamine synthesis pathway and responsible for production of the dopamine precursor, 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, while 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 crosstalk between O-GlcNAcylation and phosphorylation at Ser40 in tyrosine hydroxylase.