Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205.
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 approximately 55% phosphorylated at night, when T31 is approximately 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 14-3-3zeta 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 14-3-3zeta through pT31 or pS205. Two-site binding lowers the Km for arylalkylamine substrate to approximately 30 microM. In contrast, single-site pS205 binding increases the Km to approximately 1,200 microM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by approximately 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.
Ganguly
Surajit
S
Section on Neuroendocrinology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4480, USA.
Weller
Joan L
JL
Ho
Anthony
A
Chemineau
Philippe
P
Malpaux
Benoit
B
Klein
David C
DC
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002940:Circadian Rhythm; D004789:Enzyme Activation; D007700:Kinetics; D008550:Melatonin / Q000096:biosynthesis*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D012756:Sheep
D048948:14-3-3 Proteins; D010768:Phosphoserine; D047068:Arylalkylamine N-Acetyltransferase; D008550:Melatonin