磷酸化是一种可逆的翻译后修饰,可改变蛋白质的功能以控制各种细胞事件。包括细胞信号。激酶催化磷酰基转移到丝氨酸的羟基残基上,苏氨酸,和酪氨酸,而磷酸酶催化去除。在各种癌症和神经退行性疾病中已经观察到不调节的激酶和磷酸酶活性。尽管它们在细胞生物学中很重要,磷酸酶在细胞事件中的作用尚未完全表征,部分原因是缺乏在生物学背景下鉴定磷酸酶-底物对的工具。开发了一种称为激酶催化生物素化的方法来鉴定磷酸酶底物(K-BIPS),以弥补磷酸酶生物学信息的缺乏。特别关注底物识别。在K-BIPS方法中,γ-磷酰基修饰的腺苷5'-三磷酸(ATP)类似物,ATP-生物素,激酶用于生物素标记磷蛋白。因为磷酸酶必须首先去除磷酰基,以便随后通过ATP-生物素进行生物素化,通过在存在和不存在活性磷酸酶的情况下比较生物素化的蛋白质来鉴定K-BIPS中的磷酸酶底物。K-BIPS已用于发现丝氨酸/苏氨酸和酪氨酸磷酸酶的新型底物。本章介绍了K-BIPS方法,以鉴定任何感兴趣的磷酸酶的底物,这将加强磷酸酶生物学的研究。
Phosphorylation is a reversible post-translational modification that alters the functions of proteins to govern various cellular events, including cell signaling. Kinases catalyze the transfer of a phosphoryl group onto the hydroxyl residue of serine, threonine, and tyrosine, while phosphatases catalyze the removal. Unregulated kinase and phosphatase activity have been observed in various cancers and neurodegenerative diseases. Despite their importance in cell biology, the role of phosphatases in cellular events has yet to be fully characterized, partly due to the lack of tools to identify phosphatase-substrate pairs in a biological context. The method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) was developed to remedy the lack of information surrounding phosphatase biology, particularly focused on substrate identification. In the K-BIPS method, the γ-phosphoryl modified adenosine 5\'-triphosphate (ATP) analog, ATP-biotin, is used by kinases to biotin-label phosphoproteins. Because phosphatases must initially remove a phosphoryl group for subsequent biotinylation by ATP-biotin, phosphatase substrates are identified in K-BIPS by comparing biotinylated proteins in the presence and absence of active phosphatases. K-BIPS has been used to discover novel substrates of both serine/threonine and tyrosine phosphatases. This chapter describes the K-BIPS method to enable the identification of substrates to any phosphatases of interest, which will augment studies of phosphatase biology.