Role of NT5DC2 in tyrosine hydroxylase phosphorylation based on the analysis of NT5DC2-binding proteins

Abstract

The gene encoding 5'-nucleotidase domain-containing protein 2 (NT5DC2) has been associated with neuropsychiatric disorders related to the abnormality of dopamine activity in the brain. However, its physiological functions remain unclear. In this study, we analyzed the features of NT5DC2 that influence its binding with tyrosine hydroxylase (TH) and its effects on dihydroxyphenylalanine (DOPA) synthesis, using NT5DC2 overexpressed in PC12D cells by the pCMV vector. Western blot analysis revealed that the purified NT5DC2-DYKDDDDK-tag (NT5DC2-tag) protein can bind with the phosphorylated form of recombinant human TH type 1 (rhTH1), apart from the endogenous TH in PC12D cells. Proteomic analysis by mass spectrometry revealed that the purified NT5DC2-tag protein has the potential to bind to 41 proteins with multiple phosphorylation sites in PC12D cells (NT5DC2 binding proteins: positive, 391 sites/41 proteins; and negative, 85 sites/27 proteins). Overexpression of NT5DC2 in PC12D cells decreased DOPA levels in the medium. When the lysate of PC12D cells overexpressing NT5DC2 was incubated at 37 °C, the phosphorylated form of endogenous TH in PC12D cells decreased. This decrease was also detected when phosphorylated rhTH1 was incubated with purified NT5DC2-tag. Overall, our results suggest that NT5DC2 regulates DOPA synthesis by promoting the dephosphorylation of TH, similar to a phosphatase. Therefore, our study provides useful information for understanding various disorders associated with abnormalities in dopamine levels in the brain.

Keywords: DOPA; NT5DC2; Neuropsychiatric disorders; Tyrosine hydroxylase.