TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca2+/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser19, but PKA also phosphorylated Ser104, as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser19, Ser99, Ser104 and Ser306. On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser19. This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins γ, ϵ and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.
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Research Article|
January 29 2008
Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding
Ingeborg Winge;
Ingeborg Winge
*Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Jeffrey A. Mckinney;
Jeffrey A. Mckinney
*Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Ming Ying;
Ming Ying
*Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Clive S. D'Santos;
Clive S. D'Santos
†PROBE Proteomic Unit, Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Rune Kleppe;
Rune Kleppe
*Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Per M. Knappskog;
Per M. Knappskog
‡Center of Medical Genetics and Molecular Medicine, Haukeland University Hospital, 5021 Bergen, Norway
§Department of Clinical Medicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
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Jan Haavik
Jan Haavik
1
*Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway
∥Department of Psychiatry, Haukeland University Hospital, 5021 Bergen, Norway
1To whom correspondence should be addressed (email jan.haavik@biomed.uib.no).
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Publisher: Portland Press Ltd
Received:
July 30 2007
Revision Received:
October 22 2007
Accepted:
October 31 2007
Accepted Manuscript online:
October 31 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 410 (1): 195–204.
Article history
Received:
July 30 2007
Revision Received:
October 22 2007
Accepted:
October 31 2007
Accepted Manuscript online:
October 31 2007
Citation
Ingeborg Winge, Jeffrey A. Mckinney, Ming Ying, Clive S. D'Santos, Rune Kleppe, Per M. Knappskog, Jan Haavik; Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding. Biochem J 15 February 2008; 410 (1): 195–204. doi: https://doi.org/10.1042/BJ20071033
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