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Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1021/acs.biochem.7b00689
Eric Sheng-Wen Chen,Jui-Hung Weng,Yu-Hou Chen,Shun-Chang Wang,Xiao-Xia Liu,Wei-Cheng Huang,Tsutomu Matsui,Yoshiaki Kawano,Jiahn-Haur Liao,Liang-Hin Lim,Yoshitaka Bessho,Kai-Fa Huang,Wen-Jin Wu,Ming-Daw Tsai

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

中文翻译:

磷酸化底漆赋予Rad53激酶自磷酸化功能相关的特异性

蛋白质激酶激活机制的绝大多数体外结构和功能研究仅使用激酶结构域。对于丝氨酸/苏氨酸激酶而言,调节域或变构因子的充分证明的作用是稀缺的。在这里,我们使用丝氨酸/苏氨酸激酶Rad53的位点特异性磷酸化SCD1-FHA1-激酶三结构域构建体来显示体内磷酸引发的作用调节机制,对自身磷酸化的中间性和特异性。未磷酸化的Rad53是溶液中的柔性单体,但是被晶体中的两个FHA结构域彼此分离的不对称酶:底物复合物捕获。磷酸引发可通过溶液中的分子间pT-FHA结合诱导形成稳定的二聚体。重要的是,未引物和磷酸引物Rad53的自磷酸化分别主要产生无活性的pS350-Rad53和有活性的pT354-Rad53。后者的机制也在体内得到证实。我们的结果表明,尽管Rad53可以在各种条件下显示活性构象,但体内调节条件的模拟赋予了功能上相关的自磷酸化作用。
更新日期:2017-09-15
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