Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation
Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation
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DOI：
10.1111/j.1742-4658.2009.07346.x
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发表时间：
2009-11-01
期刊：
影响因子：
5.4
通讯作者：
Becker, Walter
中科院分区：
文献类型：
--
作者：
Goeckler, Nora;Jofre, Guillermo;Becker, Walter
DYRK1A是一种双特异性蛋白激酶，在激活环中自磷酸化一个保守的酪氨酸残基，但只磷酸化丝氨酸或苏氨酸残基的外源底物。DYRKs的酪氨酸自磷酸化是在翻译过程中发生的一次性事件，并诱导激酶的激活。在这里，我们在体外和培养细胞中都将-碳碱生物碱毒碱定性为DYRK1A的有效和特异性抑制剂。对DYRK家族四种激酶的体外比较分析表明，鼠碱抑制DYRK1A对底物磷酸化的作用比对密切相关的DYRK1B激酶对底物磷酸化的作用更强[一半最大抑制浓度（IC50）分别为33 nm和166 nm]，以及对更远的家族成员DYRK2和DYRK4（分别为1.9 μ m和80 μ m）的抑制作用更强。在细菌体外翻译系统中，要抑制DYRK1A翻译中间体酪氨酸的自磷酸化需要更高浓度的毒草碱（IC50 = 1.9 μ m）。重要的是，在培养细胞中，鼠碱抑制DYRK1A对特定底物的磷酸化，其效力与在体外观察到的相似（IC50 = 48 nm），对细胞的活力没有负面影响。21号染色体上DYRK1A基因的过度表达与唐氏综合症中观察到的神经元发育改变有关。在这里，我们发现伤害碱干扰培养的海马神经元的神经细胞发生。综上所述，我们的数据表明，毒蜂碱抑制DYRK1A底物磷酸化比抑制酪氨酸自磷酸化更有效，并为DYRK1A在调节神经突形成中的作用提供了证据。
DYRK1A is a dual-specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one-off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta-carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC50) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 mu m and 80 mu m, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC50 = 1.9 mu m). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC50 = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.