Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.
Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at 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 same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.
Itagaki
C
C
Department of Biochemistry, Faculty of Science, Niigata University, Japan.
Isobe
T
T
Taoka
M
M
Natsume
T
T
Nomura
N
N
Horigome
T
T
Omata
S
S
Ichinose
H
H
Nagatsu
T
T
Greene
L A
LA
Ichimura
T
T
D048948:14-3-3 Proteins; D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D016716:PC12 Cells; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D020033:Protein Isoforms; D011506:Proteins; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases