The Interplay between O-GlcNAc And Phosphorylation on Tyrosine Hydroxylase Activity And Cathecolamine Synthesis in PC12 Cells

About 2% to 5% of all glucose that enters the cell is directed to the Hexosamine Biosynthetic Pathway (HBP), which has glutamine fructose‐6‐phosphate aminotransferase (GFAT) as the rate‐limiting step enzyme. HBP has UDP‐N‐acetylglucosamine (UDP‐GlcNAc) as a final product, which can be used as substrate for intracellular O‐GlcNAc; a post‐translational modification (PTM) resulting from the covalent attachment of a GlcNAc to the hydroxyl groups on serine and threonine residues in proteins. This reaction is catalyzed by O‐GlcNAc transferase (OGT), and the removal reaction of this monosaccharide is made by O‐GlcNAcase (OGA). The balance of the activity of such enzymes and UDP‐GlcNAc availability will regulate the levels of O‐GlcNAcylated proteins, with an O‐GlcNAcylation, similarly to phosphorylation, highly inducible, dynamic and active in many cellular processes. Tyrosine hydroxylase (TH) is the rate‐limiting step enzyme in catecholamine synthesis, responsible for hydroxylate L‐tyrosine at meta‐position to obtain L‐DOPA, the precursor of dopamine, which has a physiological role as a neurotransmitter. There are several supports on the literature that phosphorylation on serine 40 of TH increases its activity on a dynamical way, however, the mechanism of how O‐GlcNAc plays a role on this modulation remains unknown. We showed in PC12 cells treated with the pharmacological inhibitor of OGA Thiamet G (TMG); and nerve growth factor (NGF), a compound that induces these cells in a process of neuritogenesis, that O‐GlcNAcylation acts on the control of the phosphorylation levels of serine 40 in TH, where stimulation by 28% on the increase in phosphorylation at serine 40 decreases the levels of O‐GlcNAc in 26% compared to control; while the increase of intracellular O‐GlcNAc in 19% reduces the phosphorylation at serine 40 by 16%. In addition, HPLC analysis shows that the increase of intracellular O‐GlcNAc reduces the levels of L‐DOPA and dopamine by 80%. Finally, TH’s immunoprecipitation analysis reveals that it is O‐GlcNacylated, and this PTM regulates its activity. These data suggest a cellular mechanism that integrates carbohydrate metabolism (by HBP) with the catecholamine biosynthetic pathway in a neuritogenesis process; where competition between O‐GlcNAc and phosphate at serine 40 tyrosine hydroxylase site modulates its activity, controlling the synthesis of dopamine levels in PC12 cells. Support or Funding Information Funding: CNPq, CAPES, FAPERJ