Johnson James M, Hebert Alexander S, Drane Quentin H, Lera Robert F, Wan Jun, Weaver Beth A, Coon Joshua J, Burkard Mark E
Department of Medicine, Division of Hematology/Oncology, University of Wisconsin, 1111 Highland Avenue, WIMR 6059, Madison, WI 53705, USA; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI 53705, USA.
Genome Center, University of Wisconsin, Madison, WI 53705, USA; Morgridge Institute for Research, Madison, WI 53705, USA.
Cell Chem Biol. 2020 Mar 19;27(3):350-362.e8. doi: 10.1016/j.chembiol.2020.01.007. Epub 2020 Feb 3.
Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1 (L197F) and Plk1 (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1 but not Plk1 sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.
Polo样激酶1有数百种底物和多种功能，这些功能在约60分钟的有丝分裂过程中发挥作用。在此，我们描述了一种化学遗传系统，该系统允许利用工程化的磷酸化受体选择性将特定底物“切换”为受化学控制或不受化学控制。对有丝分裂细胞提取物的生化分析和磷酸化蛋白质组分析表明，Plk1（L197F）和Plk1（L197S/L211A）分别选择性地磷酸化丝氨酸和苏氨酸。Plk1而非Plk1能维持有丝分裂进程至后期，这为在丝氨酸和苏氨酸之间切换底物残基以使其受化学控制提供了机会。利用该系统，我们评估了Kif2b，它是Plk1的一个已知底物，参与调节染色体排列。将Kif2b上的丝氨酸切换为苏氨酸会使这些磷酸化位点处于可逆的化学控制之下，这表现为染色体排列错误的频率急剧增加以及前中期停滞。因此，我们证明了通过遗传的丝氨酸/苏氨酸切换对单个底物进行化学控制的能力。
