PDF(Pubmed)
Abstract：
Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multispecificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.
摘要：
激酶的蛋白质磷酸化调节哺乳动物细胞功能，比如增长，司,和信号转导。在人类激酶中，NME1和NME2与转移性肿瘤抑制有关，但由于缺乏监测其细胞底物的工具，仍未得到充分研究。特别是,NME1和NME2是磷酸化丝氨酸的多特异性激酶，苏氨酸,组氨酸,和底物蛋白的天冬氨酸残基，磷酸组氨酸和磷酸天冬氨酸的热和酸敏感性使底物发现和验证变得复杂。为了提供新的底物监测工具，我们建立了γ-磷酸修饰的ATP类似物，ATP-生物素,作为NME1和NME2细胞底物的磷酸化生物素化的共底物。建立在这种ATP-生物素相容性的基础上，激酶催化的用失活裂解物发现底物的生物素化（K-BILDS）方法能够验证已知底物并发现7种NME1和3种NME2底物。鉴于缺乏研究激酶底物的方法，ATP-生物素和K-BILDS方法是表征NME1和NME2在人类细胞生物学中的作用的有价值的工具。
