NDLI: Analysis of Serotonin N-Acetyltransferase Regulation in Vitro and in Live Cells Using Protein Semisynthesis†

Content Provider	PubMed Central
Author	Szewczuk, Lawrence M. Tarrant, Mary K. Sample, Vedangi Drury, William J. Zhang, Jin Cole, Philip A.
Abstract	Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3ζ recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3ζ. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with14-3-3ζ that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3ζ heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3ζ– AANAT interaction using 14-3-3ζ fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3ζ interaction.
Related Links	http://dx.doi.org/10.1021/bi801189d
Ending Page	10419
Page Count	13
Starting Page	10407
File Format	PDF
ISSN	00062960
e-ISSN	15204995
Journal	Biochemistry
Issue Number	39
Volume Number	47
Language	English
Publisher Date	2008-09-30
Access Restriction	Open
Subject Keyword	Biochemistry Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biochemistry

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