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.
Banik
U
U
Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
G A
GA
Wagner
P D
PD
Kaufman
S
S
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
D048948:14-3-3 Proteins; D011506:Proteins; D006639:Histidine; D014446:Tyrosine 3-Monooxygenase; D014365:Tryptophan Hydroxylase