Amino-terminal analysis of tryptophan hydroxylase: protein kinase phosphorylation occurs at serine-58.
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., Ndelta50, Ndelta60, Ndelta90, Ndelta106, and Ndelta116 (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 (Ndelta116) 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 Ndelta50 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.
Kumer
S C
SC
Department of Physiology and Pharmacology and Center for the Neurobiological Investigation of Drug Abuse, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1083, U.S.A.
Mockus
S M
SM
Rucker
P J
PJ
Vrana
K E
KE
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017353:Gene Deletion; D010766:Phosphorylation; D017354:Point Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D014365:Tryptophan Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases