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

被引:85
作者
Itagaki, C
Isobe, T
Taoka, M
Natsume, T
Nomura, N
Horigome, T
Omata, S
Ichinose, H
Nagatsu, T
Greene, LA
Ichimura, T
机构
[1] Niigata Univ, Fac Sci, Dept Biochem, Niigata 9502181, Japan
[2] Tokyo Metropolitan Univ, Fac Sci, Dept Chem, Tokyo 1920397, Japan
[3] JST, ERATO, Calciosignal Net Project, Tokyo 1130021, Japan
[4] Fujita Hlth Univ, Inst Comprehens Med Sci, Aichi 4701192, Japan
[5] Columbia Univ, Coll Phys & Surg, Dept Pathol, New York, NY 10032 USA
[6] Columbia Univ, Coll Phys & Surg, Ctr Neurobiol & Behav, New York, NY 10032 USA
关键词
D O I
10.1021/bi9914255
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated 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 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 Ca2+ 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, Ca2+-dependent phosphorylation of TH.
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收藏
页码:15673 / 15680
页数:8
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