Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.
Purification of Cdk-CyclinB-Kinase-Targeted Phosphopeptides from Nuclear Envelope.
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从核膜中纯化 Cdk-CyclinB-激酶靶向磷酸肽。
DOI：
10.1007/978-1-0716-2337-4_18
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Glavy,JosephS
中科院分区：
文献类型：
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作者：
Blethrow,JustinD;DiGuilio,AmandaL;Glavy,JosephS
我们描述了一种快速鉴定核膜内蛋白激酶底物的方法。在高等真核生物中，开放式有丝分裂的特征是核膜破裂（NEBD），伴随着核纤层的解体和核孔复合体（NPC）的解离，形成单个的亚复合体。有证据表明，可逆的磷酸化事件在很大程度上驱动这种有丝分裂NEBD。这些翻译后修饰可能破坏核孔蛋白（NUPS），核被膜（NE）的纤层和膜蛋白之间的结构上重要的相互作用。因此，确定这些底物何时何地被磷酸化至关重要。一种可能的调节因子是有丝分裂激酶：Cdk 1-Cyclin B。我们采用“类似物敏感”的Cdk 1进行生物正交，并用磷酸盐类似物标签独特地标记NE中的底物。随后，用磷酸盐类似物共价修饰的肽通过标签特异性共价捕获和释放方法快速纯化。通过这种方式，我们能够确认NE中已知Cdk 1靶点的身份，并发现通过有丝分裂磷酸化进行调节的其他候选者。
We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an “analog-sensitive” Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.