Winge Ingeborg, McKinney Jeffrey A, Ying Ming, D'Santos Clive S, Kleppe Rune, Knappskog Per M, Haavik Jan
Department of Biomedicine, University of Bergen, Jonas Liesv. 91,5009 Bergen, Norway.
Biochem J. 2008 Feb 15;410(1):195-204. doi: 10.1042/BJ20071033.
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.
色氨酸羟化酶（TPH）催化5-羟色胺合成中的限速步骤，它有两种同工型：TPH1，主要存在于外周组织和松果体中；TPH2，一种神经元形式。在本研究中，人TPH2在大肠杆菌和人胚肾（HEK）-293细胞中表达，并用几种不同的哺乳动物蛋白激酶进行磷酸化。TPH2被蛋白激酶A（PKA）迅速磷酸化，磷酸化化学计量比为每摩尔亚基2摩尔磷酸，但被Ca²⁺/钙调蛋白依赖性蛋白激酶II磷酸化的化学计量比仅为0.2。通过质谱、磷酸特异性抗体以及对几个可能的磷酸化位点（即Ser19、Ser99、Ser104和Ser306）进行定点诱变确定，这两种激酶都使Ser19磷酸化，但PKA还使Ser104磷酸化。平均而言，纯化的野生型（WT）TPH2在PKA磷酸化后活性提高了30%，对突变酶的研究表明，酶的激活主要是由于Ser19磷酸化。在表达TPH2的HEK-293细胞中，该位点的磷酸化化学计量比高达50%，用福司可林处理后，酶活性和磷酸化化学计量比进一步增加。纯化的PKA磷酸化TPH2与14-3-3蛋白γ、ε和BMH1高亲和力结合，导致酶稳定性和活性进一步增加。这表明14-3-3蛋白可能在巩固和加强磷酸化对TPH2的作用中发挥作用，并且它们可能对神经系统中5-羟色胺功能的调节很重要。
