IDENTIFICATION OF THE SITE OF INTERACTION OF THE 14-3-3-PROTEIN WITH PHOSPHORYLATED TRYPTOPHAN-HYDROXYLASE

The 14-3-3 protein family plays a role in a wide variety of cell signa ling processes including monoamine synthesis, exocytosis, and cell cyc le regulation, but the structural requirements for the activity of thi s protein family are not known, We have previously shown that the 14-3 -3 protein binds with and activates phosphorylated tryptophan hydroxyl ase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmi tter serotonin) and proposed that this activity might be mediated thro ugh the COOH-terminal acidic region of the 14-3-3 molecules. In this r eport we demonstrate, using a series of truncation mutants of the 14-3 -3 eta isoform expressed in Escherichia coli, that the COOH-terminal r egion, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3- 3 box I, is one of the structural regions whose sequence is highly con served beyond species, allowing that the plant 14-3-3 isoform (GF14) c ould also activate rat brain TPH. The 14-3-3 box I is the first functi onal region whose activity has directly been defined in the 14-3-3 seq uence and may represent a common structural element whereby 14-3-3 int eracts with other target proteins such as Raf-1 kinase, The result is consistent with the recently published crystal structure of this prote in family, which suggests the importance of the negatively charged gro ove like structure in the ligand binding.