White Colin D, Toker Alex
Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.
Curr Protoc Mol Biol. 2013 Jan;Chapter 18:Unit 18.20.. doi: 10.1002/0471142727.mb1820s101.
Phosphorylation of substrates by protein kinases regulates a myriad of cellular processes, ranging from proliferation and migration to autophagy, senescence, and apoptosis. Kinase substrate selectivity is largely dependent on the amino acid sequence surrounding the phosphorylation site; therefore, substrate-directed, phosphorylation-state-sensitive, motif-specific ("phospho-motif") antibodies represent powerful tools to identify novel kinase substrates and to investigate mechanisms of substrate phosphorylation in many signaling pathways typically associated with human malignancies. Phospho-motif antibodies are engineered to recognize proteins that contain a phosphorylated residue in the context of a specific motif. They are raised against a library of phospho-peptides comprising both the phosphorylated residue and the surrounding residues that determine kinase specificity, with degenerate residues taking up the remaining positions. Currently, several categories of phospho-motif antibody are commercially available, which may be used to specifically detect Ser, Thr, Ser/Thr, or Tyr residues phosphorylated by different protein kinase families. These antibodies are commonly used in immunoprecipitation and/or immunoblotting protocols to determine kinase-induced substrate phosphorylation. This unit describes the use of phospho-motif antibodies to elucidate the kinase(s) responsible for phosphorylating substrate proteins.
蛋白激酶对底物的磷酸化作用调控着众多细胞过程，从细胞增殖、迁移到自噬、衰老及凋亡。激酶底物选择性很大程度上取决于磷酸化位点周围的氨基酸序列；因此，针对底物、对磷酸化状态敏感、具有基序特异性（“磷酸化基序”）的抗体是鉴定新型激酶底物以及研究许多通常与人类恶性肿瘤相关的信号通路中底物磷酸化机制的有力工具。磷酸化基序抗体经设计可识别在特定基序背景下含有磷酸化残基的蛋白质。它们是针对一个磷酸化肽文库产生的，该文库包含磷酸化残基以及决定激酶特异性的周围残基，其余位置由简并残基占据。目前，有几类磷酸化基序抗体可通过商业途径获得，可用于特异性检测由不同蛋白激酶家族磷酸化的丝氨酸、苏氨酸、丝氨酸/苏氨酸或酪氨酸残基。这些抗体常用于免疫沉淀和/或免疫印迹实验方案中，以确定激酶诱导的底物磷酸化情况。本单元介绍了如何使用磷酸化基序抗体来阐明负责磷酸化底物蛋白的激酶。
