study about melatonin
Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205
Surajit Ganguly * , Joan L. Weller * , Anthony Ho , Philippe Chemineau , Benoit Malpaux , and David C. Klein *, ¶
*Section on Neuroendocrinology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4480; Department of Physiology, University of Alberta, Edmonton, AB, Canada T6G 2H7; and Unité Mixte de Recherche Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université F. Rabelais, Haras Nationaux, 37380 Nouzilly, France
Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved December 10, 2004 (received for review September 20, 2004)
The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin N-acetylserotonin melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be 55% phosphorylated at night, when T31 is 40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3 in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3 through pT31 or pS205. Two-site binding lowers the Km for arylalkylamine substrate to 30 µM. In contrast, single-site pS205 binding increases the Km to 1,200 µM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by 40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1–2)-COOH, referred to here as mode III