Abstract
Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.
Highlights
Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine into L-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons
We investigated the functional role of Ser-31 phosphorylation, and our results indicate the association of THSer(P)-31 with the Golgi complex (GC) and the vesicular transport pathway, through vesicular monoamine transporter 2 (VMAT2) and ␣-synuclein (␣-syn). ␣-Syn is a synaptic vesicle associated-protein and a culprit in the development of Parkinson’s disease (PD) [30]
It is well established that PC12 can give rise to specific phenotypes as described previously in depth (39 – 41) and illustrated here when profiling different proteins of the dopamine pathway in the presence or absence of NGF stimulation by Western blotting (Fig. 1I), where we observe that THSer(P)-31 content increases in PC12Adh by 10% after NGF treatment, whereas it increases by 2-fold in PC12*, in agreement with previous studies [24]
Summary
The cellular distribution of the different phosphorylated forms of TH at Ser-19, Ser-31, and Ser-40 was investigated by immunofluorescence using specific antibodies. To rule out possible artifacts, we treated V5-TH1-S31E– transfected cells with the kinase inhibitors, and we compared the amounts of V5-TH1-S31E in the microsomal fractions, without finding significant differences (Fig. 3D) These results reinforce the specificity of the Ser-31 phosphorylation for TH vesicle association. R/S and nocodazole treatment (0 min of washout) showed a disrupted microtubule pattern compared with the controls, as well as a significantly decreased THSer(P)-31 signal both for the somas (34.99 Ϯ 11.7 versus 22.81 Ϯ 7.1; p Ͻ 0.001; n ϭ 50) and for the neurites (18.85 Ϯ 4.57 versus 8.22 Ϯ 2.9; p Ͻ 0.001; n ϭ 50) (Fig. 5, D and E). Our data demonstrate that phosphorylation at Ser-31 regulates TH association with vesicles and its transport to neurite terminals through interaction with VMAT2 and ␣-syn
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