Increased Site-Specific Phosphorylation of Tyrosine Hydroxylase Accompanies Stimulation of Enzymatic Activity Induced by Cessation of Dopamine Neuronal Activity

Abstract

Activation of striatal dopamine (DA) neurons by neuroleptic treatment or by electrical stimulation of the nigrostriatal pathway increases the activity of tyrosine hydroxylase (TH). The increase is mediated by phosphorylation of the enzyme. However, abolition of DA neuronal activity [by gamma-butyrolactone (GBL) treatment or transection of the nigrostriatal pathway] also increases TH activity. Quantitative blot immunolabeling experiments using site- and phosphorylation state-specific antibodies to TH demonstrated that GBL treatment (750 mg/kg, 35 min) significantly increased phosphorylation at Ser19 (+40%) and Ser40 (+217%) without altering Ser31 phosphorylation. Concomitantly, GBL treatment [along with the 3,4-dihydroxyphenylalanine (dopa) decarboxylase inhibitor NSD-1015, 100 mg/kg, 30 min] increased in vivo striatal dopa accumulation and in vitro TH activity 3-fold. Likewise, cerebral hemitransection of the nigrostriatal pathway significantly increased phosphorylation of TH at Ser19 (+89%) and Ser40 (+158%) but not at Ser31; dopa levels were increased accordingly (+191%). Kinetic analysis of TH activity established that GBL treatment and hemitransection primarily decreased the Km for the cofactor tetrahydrobiopterin (3-fold). The effects of GBL and hemitransection were abolished or attenuated by pretreatment with the DA agonist R-(-)-N-n-propylnorapomorphine (NPA; 30 microgram/kg, 40 min), presumably via stimulation of inhibitory presynaptic DA autoreceptors. NPA dose-response curves for reversal of GBL-induced dopa accumulation and Ser40 phosphorylation were identical; however, only the highest dose of NPA reversed the small and variable increase in Ser19 phosphorylation. Thus, TH activity seems to be regulated by phosphorylation in both hyper- and hypoactive striatal DA neurons; in the latter case, activation seems to be caused by selective phosphorylation of Ser40.

Neurochemical characterization of tyrosine hydroxylase-immunoreactive interneurons in the developing rat cerebral cortex

“…Because TH activity is regulated by phosphorylation, we examined whether the TH in cortical neurons was phosphorylated by using an antibody against TH phosphorylated at Ser19 (TH phosphoserine19), one of three serine residues, including Ser31 and Ser40, whose phosphorylation is regulated physiologically (Xu et al, 1998;Lew et al, 1999;Witkovsky et al, 2004;Bobrovskaya et al, 2004). Brains from P16 to P40 rats were examined: P16 (n=3 brains), P18 (n=2), P20 (n=1), P24 (n=2), P34 (n=3) and P40 (n=1).…”

“…Additionally, in the developing rat, labeling of the cortical TH-IR neurons was insensitive to 6-hydroxydopamine toxicity, and no endogenous catecholamines were detected in cortical somata (Berger et al, 1985). To address whether or not TH in cortical neurons is active, we examined the phosphorylation of TH at Ser19 because a variety of physiological stimuli induce TH phosphorylation at this residue and at Ser31 and/or Ser40 (Lew et al, 1999;Bobrovskaya et al, 2004;Witkovsky et al, 2004). The majority of cortical interneurons identified by the phosphorylation-independent TH antibody also contained TH phosphorylated at Ser19 in the postnatal and adult rat.…”

“…No immunoreactivity for active caspase 3 was observed in cortical TH cells. Because phosphorylation regulates TH enzyme activity (Xu et al, 1998;Lew et al, 1999;Witkovsky et al, 2004;Bobrovskaya et al, 2004), we assessed TH activity and the possibility of L-3,4-dihydroxyphenylalanine (L-DOPA) production in these cells by immunostaining for one of the phosphorylated forms of TH. The majority of cortical TH-IR cells were colabeled by an antibody against phosphorylated TH.…”

Genetic Deletion of Trace Amine 1 Receptors Reveals Their Role in Auto-Inhibiting the Actions of Ecstasy (MDMA)

“…The peptide sequences and procedures to generate the antibodies toward TH have been published previously (Haycock et al, 1998;Lew et al, 1999). The site-and phosphorylation state-specific antibodies show selective reactivity of the phospho versus nonphospho forms of TH (Haycock et al, 1998;Lew et al, 1999). Antibody binding was detected by enhanced chemiluminescence (GE Healthcare) and quantified by densitometry (NIH IMAGE 1.61 software).…”

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…Ser19, Ser31 and Ser40, by a variety of protein kinases, indicating that its regulation may involve multiple signalling pathways. Studies on TH from several species suggest that Ser40 is the main site involved in direct activation of TH (Sutherland et al 1993;Lew et al 1999;Salvatore et al 2000). This serine residue is phosphorylated in vitro by cAMP-dependent kinase (PKA), protein kinase C (PKC), mitogen-activated protein kinase-activated protein kinase 1 (MAPKAP-K1) and Ca 2+ /calmodulin-dependent protein kinase II (CaM-KII; Kappock and Caradonna 1996).…”