Protein kinase C regulates the activity and stability of serotonin N-acetyltransferase.
Effects of protein kinase C on protein stability and activity of rat AANAT were investigated in vitro and in vivo. When COS-7 cells transfected with AANAT cDNA were treated with phorbol 12-myristate 13-acetate (PMA), both the activity and protein level of AANAT were increased. These effects of PMA were blocked by GF109203X, a specific inhibitor of PKC. Moreover, PMA increased the phosphorylation of AANAT and induced the formation of AANAT/14-3-3zeta complex. PMA did not affect the basal level of cAMP and did not involve the potentiation of the cAMP production by forskolin, indicating that PKC-dependent activation of adenylyl cyclase was excluded in transfected COS-7 cells. To identify which amino acids were phosphorylated by PKC, several conserved Thr and Ser residues in AANAT were targeted for site-directed mutagenesis. Mutations of Thr29 and Ser203 prevented the increase of enzymatic activity and protein level mediated by PMA. To explore the nature of AANAT phosphorylation, purified rat AANAT was subjected to in vitro PKC kinase assay. PKC directly phosphorylated the rat recombinant AANAT. The phosphopeptides identified by mass spectrometric analysis, and western blotting indicated that Thr29 was one of target sites for PKC. To confirm the effects of the physiological activation of PKC, rat pineal glands were treated with alpha(1)-adrenergic specific agonist phenylephrine. Phenylephrine caused the phosphorylation of endogenous AANAT whereas GF109203X or prazosin, an alpha(1)-adrenergic-specific antagonist, markedly inhibited it. These results suggest that AANAT was phosphorylated at Thr29 by PKC activation through the alpha(1)-adrenergic receptor in rat pineal glands, and that its phosphorylation might contribute to the stability and the activity of AANAT.
Choi
Bo-Hwa
BH
Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Korea.
Chae
Hee-Don
HD
Park
Tae-Ju
TJ
Oh
Jisun
J
Lim
Jinkyu
J
Kang
Shin-Sung
SS
Ha
Hyunjung
H
Kim
Kyong-Tai
KT
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects / Q000502:physiology; D008297:Male; D016297:Mutagenesis, Site-Directed; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010870:Pineal Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D020536:Enzyme Activators; D010446:Peptide Fragments; D001191:Arylamine N-Acetyltransferase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate