14-3-3zeta contributes to tyrosine hydroxylase activity in MN9D cells: localization of dopamine regulatory proteins to mitochondria.
The 14-3-3 proteins stimulate the activation of tyrosine hydroxylase (TH), the rate-limiting catecholamine biosynthetic enzyme. To explore if particular endogenous 14-3-3 isoforms specifically affected TH activity and dopamine synthesis, we utilized rodent nigrostriatal tissues and midbrain-derived MN9D dopaminergic cells. Extracts from ventral midbrain and MN9D cells contained similar pools of 14-3-3 mRNAs, with 14-3-3zeta being relatively abundant in both. Protein levels of 14-3-3zeta were also abundant. [(32)P]Orthophosphate labeling of MN9D cells, followed by co-immunoprecipitation with pan-TH and pan-14-3-3 antibodies brought down similar amounts of phosphorylated TH in each, confirming that 14-3-3-bound phosphorylated TH in our cells. Co-immunoprecipitation of striatal tissues with a pan-TH antibody precipitated 14-3-3zeta but not another potential TH regulatory isoform, 14-3-3eta. In whole cell extracts from MN9D cells after 14-3-3 small interfering RNA treatments, we found that 14-3-3zeta knockdown significantly reduced TH activity and dopamine synthesis whereas knockdown of 14-3-3eta had no effect. 14-3-3zeta was found co-localized on mitochondria with TH, and its knockdown by small interfering RNA reduced TH phosphorylation and TH activity in the mitochondrial pool. Together the data support a role for 14-3-3zeta as an endogenous activator of TH in midbrain dopaminergic neurons and furthermore, identify mitochondria as a potential novel site for dopamine synthesis, with implications for Parkinson disease.
Wang
Jian
J
Departments of Neurology and Pharmacology & Chemical Biology.
Lou
Haiyan
H
Pedersen
Courtney J
CJ
Smith
Amanda D
AD
Perez
Ruth G
RG
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine