Rational Redesign of a Functional Protein Kinase-Substrate Interaction.
Rational Redesign of a Functional Protein Kinase-Substrate Interaction.
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DOI：
10.1021/acschembio.7b00089
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发表时间：
2017-05-19
影响因子：
4
通讯作者：
Turk BE
中科院分区：
文献类型：
--
作者：
Chen C;Nimlamool W;Miller CJ;Lou HJ;Turk BE
真核蛋白激酶通常在特定序列基序的背景下磷酸化底物，有助于实现准确信号传输所必需的特异性。蛋白激酶通过活性位点间隙内的互补相互作用来识别其靶序列。作为构建正交激酶信号系统的一步，我们重新设计了蛋白激酶 Pim1 以改变其磷酸化共有序列。 Pim1 催化结构域中与底物中关键精氨酸残基直接相互作用的残基被取代，以产生容纳疏水性残基的激酶突变体。然后，我们在 Pim1 底物（促凋亡蛋白 BAD）中引入补偿突变，以在体外和活细胞中重建磷酸化。重新设计的激酶与其底物在细胞中的共表达可保护细胞免于凋亡。这种正交激酶-底物对提供了工具来探测活细胞中特定磷酸化事件的功能后果并设计合成信号传导途径。
Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways.