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Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation

Nature Structural & Molecular Biology volume 10, pages 1054–1057 (2003)Cite this article

Abstract

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.

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Figure 1: Design, semisynthesis and analysis of AANAT mutants to address the roles of Thr31 phosphorylation in AANAT stabilization in vivo.
Figure 2: Evaluation of semisynthetic AANATs in vitro and in vivo.

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Acknowledgements

We thank D.B. Murphy and C.J. Janetopoulos for advice on microinjection and immunofluorescence experiments. We are grateful for financial support from the US National Institutes of Health and the Ellison Medical Foundation. Mass spectrometry analysis was carried out in the AB Mass Spectrometry/Proteomics Facility at Johns Hopkins School of Medicine with support from a US National Center for Research Resources shared instrumentation grant, the Johns Hopkins Fund for Medical Discovery and the Institute for Cell Engineering.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, 21205, Maryland

    Weiping Zheng, Zhongsen Zhang & Philip A Cole

  2. Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 20892, Maryland

    Surajit Ganguly, Joan L Weller & David C Klein

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  1. Weiping Zheng
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Correspondence to Philip A Cole.

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Zheng, W., Zhang, Z., Ganguly, S. et al. Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation. Nat Struct Mol Biol 10, 1054–1057 (2003). https://doi.org/10.1038/nsb1005

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