Phosphorylation and activation of brain tryptophan hydroxylase: identification of serine-58 as a substrate site for protein kinase A.
Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin-dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation-dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S-transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full-length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1-98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99-444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine-58 to arginine resulted in the expression of a full-length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine-58 within the regulatory domain of the enzyme.
Kuhn
D M
DM
Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan, U.S.A.
Arthur
R
R
States
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014365:Tryptophan Hydroxylase / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014365:Tryptophan Hydroxylase; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases