Department of Chemistry, Wayne State University, Detroit, Michigan.
Curr Protoc. 2023 Aug;3(8):e851. doi: 10.1002/cpz1.851.
Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation-mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS). As described in these protocols, K-BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ-phosphate-modified ATP analog ATP-biotin for kinase-catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K-BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K-BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. © 2023 Wiley Periodicals LLC. Basic Protocol 1: FSBA treatment of lysates to inactivate kinases Basic Protocol 2: Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS).
蛋白质磷酸化是由激酶催化的，以调节包括生长和信号转导在内的多种细胞活动。鉴定激酶底物的方法对于充分了解磷酸化介导的细胞事件和疾病状态至关重要。在这里，我们报告了一组使用激酶催化的失活裂解物中的生物素化用于发现底物（K-BILDS）来鉴定靶激酶底物的方案。如这些方案中所述，K-BILDS 涉及在裂解物中失活内源性激酶，然后加入活性外源性激酶和γ-磷酸化修饰的 ATP 类似物 ATP-生物素，以进行细胞底物的激酶催化生物素化。亲和素富集分离活性激酶的生物素化底物，可通过 Western blot 进行监测。还可以使用质谱分析发现靶激酶的底物。K-BILDS 的主要优点包括与任何裂解物、组织匀浆或任何感兴趣的活性激酶的相关复杂混合物兼容。K-BILDS 是一种研究或发现感兴趣的激酶的底物的通用方法，可彻底表征生物途径。©2023Wiley Periodicals LLC. 基本方案 1：用 FSBA 处理裂解物以失活激酶 基本方案 2：激酶催化的失活裂解物中的生物素化用于发现底物（K-BILDS）。
