cAMP Regulation of ArylalkylamineN-Acetyltransferase (AANAT, EC 2.3.1.87)

Abstract

Arylalkylamine N-acetyltransferase (serotonin Nacetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells ϳ8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steadystate levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

An Intramolecular Disulfide Bridge as a Catalytic Switch for Serotonin N-Acetyltransferase

“…For this purpose, we established COS7 cells (SYN-8 and SYM-12) in which either AA-NAT or mutated AA-NAT is constitutively expressed. The specific activity of AA-NAT is 2.59 Ϯ 0.04 nmol/min/mg protein (n ϭ 5), which is comparable to that reported elsewhere (32). H 2 O 2 at either 1 or 2 mM was added to the COS7 cells for a short time to exert oxidative stress without affecting cell viability according to previous reports (33,34).…”

Role of a pineal cAMP-operated arylalkylamine N- acetyltransferase/14-3-3-binding switch in melatonin synthesis

“…After 48 h, cells were treated with forskolin and incubated with 5-methoxytryptamine (100 M). Intracellular AANAT activity, i.e., activity in intact cells, was estimated from the amount of melatonin in the media (12). Forskolin treatment increased intracellular AANAT activity 10-fold in cells transfected with wild-type hAANAT but not in cells with the Thr 31 3 Ala mutant (Table 3).…”

“…Treatment of AANAT-transfected cells with an activator of adenylyl cyclase increases AANAT activity in intact cells, with relatively little increase in the amount of AANAT protein (12). To investigate the involvement of PK-A͞14-3-3 site in this activation of AANAT, COS7 cells were transfected either with wild-type hAANAT or with a form in which the PK-A͞14-3-3 site was mutated (Thr 31 3 Ala).…”

Arylalkylamine N-Acetyltransferase: “the Timezyme”

“…Ejection of CoASH from the binding pocket requires tyrosine (Tyr 168 )-dependent protonation of CoAS (32,40). A synthetic mimic of the ternary complex, CoA-S-N-acetyltryptamine, is a highly potent and specific AANAT inhibitor (27,31,32,38,41).…”