Amino-terminal analysis of tryptophan hydroxylase: Protein kinase phosphorylation occurs at serine-58

作者：Kumer SC; Mockus SM; Rucker PJ; Vrana KE

来源：Journal of Neurochemistry, 1997, 69(4): 1738-1745.

• tryptophan hydroxylase
• protein kinase A
• phosphorylation
• deletion mutagenesis

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., N Delta 50, N Delta 60, N Delta 90, N Delta 106, and N Delta 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 (N Delta 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 enzyme to serve as a substrate for PKA, yet the native and N Delta 50 enzymes were phosphorylated. 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.

• 出版日期1997-10