Amino‐Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine‐58

Abstract

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Nz~50, Nz~60, N~90,Nz~106, and NL~~116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Nz~116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzymeto serve as a substrate for PKA, yet the native and N~50enzymes were phosphotylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.

Tryptophan hydroxylase 2 (TPH2) gene variants associated with ADHD

“…Deletion of the first 106 aa in TPH1 had no effect on enzyme activity, but deletion of the first 116 aa (including those between 105 and 109) was found to abolish the enzyme activity demonstrating the importance of the region between residues 106 and 116 for TPH1 function. 25 It is plausible that DNA modification at the corresponding region in TPH2 (including aa 151-156) may influence the activity of the TPH2 enzyme consequently affecting the level of serotonin synthesis, a low level of which is thought to be correlated with ADHD. DNA sequence analysis shows that this motif maps to exon 4 of the TPH2 gene.…”

Expression and Characterization of the Catalytic Core of Tryptophan Hydroxylase

“…Natural sources such as brain have proved to contain too little enzyme for useful purification, resulting in very low amounts of protein with low specific activities (1, 2, 4). A number of groups have described preparations of TRH from recombinant sources, but these typically involved enzyme of low or indeterminate specific activity (16,37,38). The limited amounts of material available have severely restricted analyses of the mechanism of this important enzyme.…”

A Regulatory Domain in the N Terminus of Tryptophan Hydroxylase 2 Controls Enzyme Expression

“…5, C and D), suggesting that phosphorylation at Ser 19 is sufficient to produce the increased expression seen with forskolin-mediated PKA activation. The residual response to forskolin treatment seen with the S19A mutant may be attributable to phosphorylation of Ser 104 , a site homologous to Ser 58 in TPH1, which has been previously identified as a PKA target (24).…”

“…1). The regulatory domains of other members of the family, such as TPH1 and TH, have been shown to modulate enzyme activity and thermostability through various mechanisms, including phosphorylation by protein kinases (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In line with this, the extended N terminus of TPH2 contains a serine at position 19, which is a candidate for PKA phosphorylation (32) and has been recently shown to interact with 14-3-3 proteins in a phosphorylationdependent manner to increase protein stability (33).…”