Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activat ed in situ in response to a variety of stimuli that increase intracellular Ca2+. We report here, using baculovirus-expressed TH, that the 14-3-3 prote in binds and activates the expressed TH when the enzyme is phosphorylated a t Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the s ame level as the non-mutated enzyme, but was a poor substrate for CaM kinas e II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopept ide (FRRAVpSELDA) corresponding to the part of the TH sequence, including p hosphoSer-19: inhibited the interaction between the expressed TH and 14-3-3 , while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-d ependent phosphorylation (Ser-40) had little effect on complex formation. T he complex was very stable with a dissociation constant of 3 nM. Furthermor e, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed tha t 14-3-3 formed a complex with endogenous TH when the cultured cells were e xposed to a high K+ concentration that increases intracellular Ca2+ and pho sphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protei n participates in the stimulus-coupled regulation of catecholamine synthesi s that occurs in response to depolarization-evoked, Ca2+-dependent phosphor ylation of TH.