Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding.
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 Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). 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 Ser(19). 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 gamma, epsilon 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.
Winge
Ingeborg
I
Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway.
McKinney
Jeffrey A
JA
Ying
Ming
M
D'Santos
Clive S
CS
Kleppe
Rune
R
Knappskog
Per M
PM
Haavik
Jan
J
D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D053719:Tandem Mass Spectrometry; D014365:Tryptophan Hydroxylase / Q000737:chemistry / Q000378:metabolism*
D048948:14-3-3 Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D014365:Tryptophan Hydroxylase