Proteome-Wide Characterizations of N6-Methyl-Adenosine Triphosphate- and N6-Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases,Analytical Chemistry

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Proteome-Wide Characterizations of N6-Methyl-Adenosine Triphosphate- and N6-Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-09-22 , DOI: 10.1021/acs.analchem.1c02565
Xuejiao Dong ₁ , Jianan Sun ₂ , Weili Miao ₁ , Chia-En A Chang ₁ , Yinsheng Wang _{1,

2}

Affiliation

Kinases catalyze the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to their protein and small-molecule substrates, and this phosphorylation is a crucial element of multiple cell signaling pathways. Herein, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method for proteome-wide identifications of endogenous kinases that can bind to two N⁶-modified ATP derivatives, N⁶-methyl-ATP (N⁶-Me-ATP), and N⁶-furfuryl-ATP (a.k.a. kinetin triphosphate, KTP). We found that, among the ∼300 quantified kinases, 27 and 18 are candidate kinases that can bind to KTP and N⁶-Me-ATP, respectively. Additionally, GSK3α and GSK3β are among the kinases that can bind to both ATP analogues. Moreover, the in vitro biochemical assay showed that GSK3β could employ N⁶-Me-ATP but not KTP as the phosphate group donor to phosphorylate its substrate peptide. Molecular modeling studies provided insights into the differences between N⁶-Me-ATP and KTP in enabling the GSK3β-mediated phosphorylation. Together, our chemoproteomic approach led to the identification of endogenous kinases that can potentially be targeted by the two ATP analogues. The approach should be generally applicable for assessing endogenous kinases targeted by other ATP and purine analogues.

中文翻译：

N6-甲基-三磷酸腺苷和 N6-糠基-三磷酸腺苷激酶结合能力的全蛋白质组表征

激酶催化 γ-磷酸基团从三磷酸腺苷 (ATP) 转移到其蛋白质和小分子底物，这种磷酸化是多种细胞信号传导途径的关键要素。在此，我们采用同位素编码的 ATP 酰基磷酸探针，结合基于多反应监测 (MRM) 的靶向蛋白质组学方法，对可与两种N ⁶修饰的 ATP 衍生物N结合的内源激酶进行蛋白质组范围的鉴定。 ⁶ -甲基-ATP（ N ⁶ -Me-ATP）和N ⁶ -糠基-ATP（又名三磷酸激动素，KTP）。我们发现，在约 300 个定量激酶中，分别有 27 个和 18 个是可以与 KTP 和N ⁶ -Me-ATP 结合的候选激酶。此外，GSK3α 和 GSK3β 属于可以与两种 ATP 类似物结合的激酶。此外，体外生化分析表明，GSK3β可以利用N ⁶ -Me-ATP而非KTP作为磷酸基供体来磷酸化其底物肽。分子模型研究提供了关于N ⁶ -Me-ATP 和 KTP 在实现 GSK3β 介导的磷酸化方面的差异的见解。总之，我们的化学蛋白质组学方法鉴定出了可能被两种 ATP 类似物靶向的内源激酶。该方法应该普遍适用于评估其他 ATP 和嘌呤类似物靶向的内源激酶。

更新日期：2021-10-06

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