title: Interaction of phosphorylated tryptophan hydroxylase with 14-3-3 proteins.
abstract: 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.
authors.affiliation: Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: U
authors.initials: U
authors.lastname: Banik
authors.forename: G A
authors.initials: GA
authors.lastname: Wang
authors.forename: P D
authors.initials: PD
authors.lastname: Wagner
authors.forename: S
authors.initials: S
authors.lastname: Kaufman
mesh_terms: D048948:14-3-3 Proteins; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D014365:Tryptophan Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D011506:Proteins; D006639:Histidine; D014446:Tyrosine 3-Monooxygenase; D014365:Tryptophan Hydroxylase