Phosphorylation Of Tryptophan Hydroxylase Research Articles

Levetiracetam-mediated emotional behavior in heterozygous rolling Nagoya Ca V2.1 channel mutant mice

Ca V2.1, which is highly expressed in the nervous system, plays an essential role in presynaptic neurotransmitter release. Although recent data suggest that the antiepileptic drug levetiracetam (LEV) inhibits presynaptic Ca V2.1 activity, the precise physiological role of Ca V2.1/LEV-regulated emotional performance has not been elucidated. We examined whether Ca V2.1/LEV mediates emotional behavior using a combined pharmacologic and genetic approach. Heterozygous rolling Nagoya ( rol/+) mice carrying the Ca V2.1α 1 mutation demonstrated normal emotional behavior. Exposure to 75 mg/kg LEV, which had no effect in wild-type controls, reduced anxiety in elevated plus maze and light–dark exploration tests and reduced depression in forced swimming and tail suspension behavioral tests in rol/+ mice. Similar behavioral patterns in motor activity were noted in wild-type and rol/+ mice injected with 0–150 mg/kg LEV. The phosphorylation of tryptophan hydroxylase at serine-58 and serotonin concentration were increased in the brainstems of rol/+ mice injected with 75 mg/kg LEV but not in those of wild-type controls. These results indicate that Ca V2.1/LEV mediates serotonin signaling leading to alterations in emotion. Our results also indicate that a combination of subthreshold pharmacologic and genetic approaches can be used to study functional signaling pathways in neuronal circuits.

Emotional behavior in heterozygous rolling mouse Nagoya Cav2.1 channel mutant mice

Although rolling mouse Nagoya, a Ca(v)2.1α(1) mutant, exhibits ataxia and elevated serotonin concentrations, heterozygous mice have not been examined in detail. Patients with heterozygous mutations in this orthologous gene exhibit neurological disorders. To examine the emotional behavior of heterozygous mice, we used behavioral tasks and examined Ca(v)2.1α(1) message levels, tryptophan hydroxylase expression patterns, and monoamine concentrations in 2- and 22-month-old mice. Reduced anxiety in the elevated plus maze, light-dark exploration, and marble-burying behavioral tests and reduced depression in the forced swimming and tail suspension tests were observed in 22-month-old heterozygous mice compared to aged-matched wild-type mice. The levels of mutant-type Ca(v)2.1α(1) message, phosphorylation of tryptophan hydroxylase, and serotonin increased in the brainstems of 22-month-old heterozygous mice. No difference was observed between 2-month-old heterozygous and wild-type mice in these analyses. These findings suggest that heterozygous mice show age-related emotional changes due to alterations in the serotonin system associated with mutant-type Ca(v)2.1α(1), and that heterozygous mice may represent a novel model to delineate the interaction between Ca(v)2.1 function and synaptic transmission.

P.1.a.008 Catechol-O-methyltrasferase gene polymorphism (Val/Met) is not associated with bipolar disorder

Potential determinants of existing tryptophan hydroxylase (TPH) activity in vivo are (1) a supply of tryptophan via the tryptophan transporter, (2) de novo synthesis of tetrahydrobiopterin and efficient regeneration of this coenzyme, and (3) iron metabolism. TPH is an iron protein. Iron binding is relatively strong in TPH2, while it is very weak in TPH1. Owing to the poorly liganded iron of TPH, various complexities arise. Irreversible inactivation of TPH and neural deterioration seem to be caused by an H2O2 reaction with non-liganded or poorly liganded Fe2+. Within the cell, the amount of ferrous iron available to TPH is below saturation. The metabolism of iron may control serotonin production by attenuating actual TPH activity in the cell interior. Phosphorylation of TPH was studied in terms of its functional aspects with the use of brain TPH, while the identification of phosphorylation sites was mainly conducted on the basis of the amino acid sequence of TPH1. Protein turnover is cell-type specific in terms of its machinery, and its regulation is one of the major post-translational modifications of a protein. TPH degradation is likely linked to protein phosphorylation. Functionally, TPH, the rate-limiting enzyme in serotonin biosynthesis, is involved in neural development, which likely affects animal behaviors in the adult stages. Future studies will define the sensitive period during development when TPH activity is required for the fine-tuning of neuronal organization that is responsible for each behavior in the adult.

Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line.

We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121-127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation.

Interaction of Phosphorylated Tryptophan Hydroxylase with 14-3-3 Proteins

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25-30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by approximately 45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (Ka) of 4.5 x 10(7) M-1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation.