Mechanism of dual specificity kinase activity of DYRK1A
Mechanism of dual specificity kinase activity of DYRK1A
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DOI：
10.1111/febs.12411
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发表时间：
2013-09-01
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：
Walte, Agnes;Rueben, Katharina;Becker, Walter
许多蛋白激酶的功能是由激活环中一个关键酪氨酸残基的磷酸化控制的。双特异性酪氨酸磷酸化调节激酶（DYRKs）在酪氨酸残基上自磷酸化，但在脂肪氨基酸上磷酸化底物。本研究探讨了DYRK1A及相关激酶双特异性激酶活性的机制。DYRK1A的酪氨酸自磷酸化在体外翻译过程中迅速发生，不依赖于非催化结构域或其他蛋白质。在细菌和哺乳动物细胞中的表达表明，DYRK1A的酪氨酸激酶活性并不局限于激活环中的共翻译自磷酸化。此外，成熟的DYRK1A仍然能够酪氨酸自磷酸化。缺乏激活环酪氨酸的DYRK1A和DYRK2点突变体显示酪氨酸激酶活性增强。一系列结构多样的DYRK1A抑制剂被用来从药理学上区分催化结构域的不同构象状态，这些构象状态被假设为解释双特异性激酶活性。所有被测试的化合物抑制底物磷酸化的效力都高于自磷酸化，但没有一种被测试的抑制剂对苏氨酸和酪氨酸激酶活性有不同的抑制作用。最后，缺乏激活环酪氨酸的相关周期蛋白依赖性激酶样激酶（clk）在体外和活细胞中都在酪氨酸上发生自磷酸化。我们提出了一个DYRK自激活模型，其中激活环中的酪氨酸自磷酸化稳定了催化结构域的构象，增强了丝氨酸/苏氨酸激酶活性，而不会使酪氨酸磷酸化失活。双特异性激酶活性的机制可能适用于依赖酪氨酸自磷酸化成熟的相关丝氨酸/苏氨酸激酶。
The function of many protein kinases is controlled by the phosphorylation of a critical tyrosine residue in the activation loop. Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) autophosphorylate on this tyrosine residue but phosphorylate substrates on aliphatic amino acids. This study addresses the mechanism of dual specificity kinase activity in DYRK1A and related kinases. Tyrosine autophosphorylation of DYRK1A occurred rapidly during invitro translation and did not depend on the non-catalytic domains or other proteins. Expression in bacteria as well as in mammalian cells revealed that tyrosine kinase activity of DYRK1A is not restricted to the co-translational autophosphorylation in the activation loop. Moreover, mature DYRK1A was still capable of tyrosine autophosphorylation. Point mutants of DYRK1A and DYRK2 lacking the activation loop tyrosine showed enhanced tyrosine kinase activity. A series of structurally diverse DYRK1A inhibitors was used to pharmacologically distinguish different conformational states of the catalytic domain that are hypothesized to account for the dual specificity kinase activity. All tested compounds inhibited substrate phosphorylation with higher potency than autophosphorylation but none of the tested inhibitors differentially inhibited threonine and tyrosine kinase activity. Finally, the related cyclin-dependent kinase-like kinases (CLKs), which lack the activation loop tyrosine, autophosphorylated on tyrosine both invitro and in living cells. We propose a model of DYRK autoactivation in which tyrosine autophosphorylation in the activation loop stabilizes a conformation of the catalytic domain with enhanced serine/threonine kinase activity without disabling tyrosine phosphorylation. The mechanism of dual specificity kinase activity probably applies to related serine/threonine kinases that depend on tyrosine autophosphorylation for maturation.