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Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N -acetyltransferase mediated by phosphoserine-205

2005; National Academy of Sciences; Volume: 102; Issue: 4 Linguagem: Inglês

10.1073/pnas.0406871102

1091-6490

Surajit Ganguly, Joan L. Weller, Anthony D. Ho, Philippe Chemineau, Benoît Malpaux, David C. Klein,

Plant Molecular Biology Research

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 14-3-3ζ 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 14-3-3ζ through pT31 or pS205. Two-site binding lowers the K m for arylalkylamine substrate to ≈30 μM. In contrast, single-site pS205 binding increases the K m to ≈1,200 μM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the K m for substrates by ≈40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X 1–2 )-COOH, referred to here as mode III.