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

Abstract

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 3 N-acetylserotonin 3 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 Ϸ55% phosphorylated at night, when T31 is Ϸ40% 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 K m for arylalkylamine substrate to Ϸ30 M. In contrast, single-site pS205 binding increases the K m to Ϸ1,200 M. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the K m for substrates by Ϸ40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS͞pT (X 1-2)-COOH, referred to here as mode III.pineal ͉ circadian ͉ cAMP ͉ kinase T he daily rhythm in circulating melatonin is a highly conserved feature of vertebrate physiology; in all cases, high levels occur at night. Day͞night differences in circulating melatonin levels provide a hormonal analog signal of environmental lighting, which is used in a variety of ways to optimize circadian and circannual rhythms in physiology (1). The melatonin rhythm is controlled by large changes in the rate of melatonin production in the pineal gland; these changes reflect changes in the penultimate enzyme in melatonin synthesis, arylalkylamine Nacetyltransferase (AANAT) (2, 3). Changes in the activity of this enzyme are strongly influenced by cAMP-dependent binding to the bowl-shaped, dimeric 14-3-3 protein (4, 5). Binding is thought to protect the enzyme against proteasomal proteolysis (6, 7); moreover, binding to 14-3-3 increases the affinity of AANAT by Ϸ10-fold for arylalkylamine substrates, e.g., serotonin and tryptamine (4, 5).Pinealocyte cAMP is controlled in mammals through a photoneural system, which includes the eyes and the suprachiasmatic nucleus, the site of the master circadian clock (1). In lower vertebrates, cAMP is regulated by light acting directly on pinealocytes (8). It is likely that cAMP-dependent binding to 14-3-3 occurs in all vertebrates, based on the ubiquitous presence of 14-3-3 proteins and the presence of two putative cyclic AMP-dependent protein kinase (PKA) sit...

Photic Regulation of ArylalkylamineN-Acetyltransferase Binding to 14-3-3 Proteins in Retinal Photoreceptor Cells

“…Previously, the available evidence indicated only that light-evoked physiological changes in AANAT activity in retina and pineal were attributable to protein degradation (Gastel et al, 1998;Schomerus et al, 2000;Zatz et al, 2000;Falcon et al, 2001;Iuvone et al, 2002). A physiological effect of 14-3-3 binding on the K m of AANAT for amine substrates was inferred based on studies using expressed protiens Ganguly et al, 2001Ganguly et al, , 2005. The current results provide new insight on a mechanism whereby light can rapidly downregulate melatonin synthesis.…”

“…These two sequences correspond to those in ovine AANAT that mediate binding to 14-3-3 (Ganguly et al, , 2005Zheng et al, 2003Zheng et al, , 2005.…”

“…Phosphorylated AANAT was detected with a phosphospecific rabbit polyclonal antibody 3352 directed against rat pT 31 -AANAT 22-37 (Ganguly et al, , 2005. 14-3-3 was detected with a mouse monoclonal antibody 7A3, which detects most isoforms of 14-3-3 (R. Subramanian, H. Fu, and A. Levey, unpublished observation).…”

“…Roseboom et al (1994) showed that 14-3-3 copurified with AANAT from sheep pineal glands. Subsequent studies demonstrated that phosphorylation-dependent binding to 14-3-3 proteins activates AANAT and protects it from proteolytic degradation (Ganguly et al, , 2005Zheng et al, 2003Zheng et al, , 2005. In this study, we investigated the role of 14-3-3 complex formation in the photic regulation of AANAT in chicken photoreceptor cells.…”

Structure of the 14-3-3ζ–LKB1 fusion protein provides insight into a novel ligand-binding mode of 14-3-3

“…A proline residue at the pS/pT +2 position in both the mode I and II motifs causes a turn of the peptide away from the binding groove of 14-3-3, presumably to prevent steric hindrance. In addition to the two canonical phosphorylated ligand-binding modes, protein C-termini binding by 14-3-3 proteins (mode III) has been identified and the consensus sequence is pS/pTX 1-2 -COOH (Coblitz et al, 2005(Coblitz et al, , 2006Wü rtele et al, 2003;Ganguly et al, 2005). 14-3-3 proteins can also bind unphosphorylated peptides such as peptide R18 and a peptide from exoenzyme S (Petosa et al, 1998;Ottmann, Yasmin et al, 2007;Yang et al, 2006).…”