Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells

Biochem Biophys Res Commun. 2016 Apr 15;472(4):598-602. doi: 10.1016/j.bbrc.2016.03.022. Epub 2016 Mar 8.


Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway.

Keywords: 14-3-3 protein; Parkinson's disease; Phosphorylation; Proteasome; Tyrosine hydroxylase; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • PC12 Cells
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteolysis
  • Rats
  • Signal Transduction
  • Tyrosine 3-Monooxygenase / metabolism*
  • Ubiquitin Thiolesterase / antagonists & inhibitors
  • Ubiquitin Thiolesterase / metabolism*
  • Ubiquitination


  • USP14 protein, rat
  • Tyrosine 3-Monooxygenase
  • Ubiquitin Thiolesterase
  • Proteasome Endopeptidase Complex