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

• Published in: The Journal of biological chemistry 1997-10, Vol.272 (42), p.26219-26225
• Main Authors: Banik, Utpal, Wang, Guo-An, Wagner, Paul D., Kaufman, Seymour
• Format: Article
• Language: English
• Subjects: 14-3-3 Proteins; Animals; Brain - enzymology; Cattle; Enzyme Activation; Histidine - chemistry; Histidine - metabolism; Kinetics; Phosphorylation; Protein Binding; Proteins - metabolism; Rabbits; Tryptophan Hydroxylase - chemistry; Tryptophan Hydroxylase - genetics; Tryptophan Hydroxylase - metabolism; Tyrosine 3-Monooxygenase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.42.26219

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 ∼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 × 107m−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.