Protein kinase C regulates the activity and stability of serotonin N‐acetyltransferase

Abstract

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/14-3-3f complex. PMA did not affect the basal level of cAMP and did not involve the potentiation of the cAMP production by forskolin, indicating that PKCdependent 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 a 1 -adrenergic specific agonist phenylephrine. Phenylephrine caused the phosphorylation of endogenous AANAT whereas GF109203X or prazosin, an a 1 -adrenergic-specific antagonist, markedly inhibited it. These results suggest that AANAT was phosphorylated at Thr29 by PKC activation through the a 1 -adrenergic receptor in rat pineal glands, and that its phosphorylation might contribute to the stability and the activity of AANAT.

Regulation of p53 by Activated Protein Kinase C-δ during Nitric Oxide-induced Dopaminergic Cell Death

“…Immunoblot Analysis-Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted as described previously (20). Bands were visualized with the ECL detection system.…”

Cellular Stability of Serotonin N-Acetyltransferase Conferred by Phosphonodifluoromethylene Alanine (Pfa) Substitution for Ser-205

“…DISCUSSION AANAT protein is thought to become phosphorylated at night at two putative protein kinase A sites, Thr-31 and Ser-205, in response to the elevation of cyclic AMP produced by ␤-and ␣ 1 -adrenergic receptor activation of pinealocytes (Fig. 1A); a contribution from calcium phospholipid-dependent protein kinase is also possible (4,5,9,24). Such post-translational modifications may serve as important functional switches to regulate the AANAT protein, which in turn controls melatonin production.…”

Dual Roles of P2 Purinergic Receptors in Insulin-stimulated Leptin Production and Lipolysis in Differentiated Rat White Adipocytes

“…3 H]cAMP competition assay as we described previously (27) with some modification. To determine the cAMP production induced by agonists, white adipose cells were stimulated with agonists for 20 min in the presence of the phosphodiesterase inhibitor Ro 20-1724 (5 M), and the reaction was quickly terminated by three repeated cycles of freezing and thawing.…”