Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
J Biol Chem. 2024 Aug;300(8):107588. doi: 10.1016/j.jbc.2024.107588. Epub 2024 Jul 18.
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.
蛋白激酶通过磷酸化调控哺乳动物细胞的功能，如生长、分裂和信号转导。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，研究较少。特别是，NME1 和 NME2 是多特异性激酶，可磷酸化丝氨酸、苏氨酸、组氨酸和天冬氨酸残基的底物蛋白，而磷酸组氨酸和磷酸天冬氨酸的热和酸敏感性使底物的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 细胞底物磷酸化生物素化的共底物。在此 ATP-生物素兼容性的基础上，激酶催化的失活裂解物生物素化用于底物发现的方法能够验证已知的底物，并发现七个 NME1 和三个 NME2 底物。鉴于研究激酶底物的方法很少，ATP-生物素和激酶催化的失活裂解物生物素化用于底物发现的方法是表征 NME1 和 NME2 在人类细胞生物学中作用的有价值的工具。
