Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function
Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function
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DOI：
10.1038/nbt997
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发表时间：
2004-08-01
影响因子：
46.9
通讯作者：
Yaffe, MB
中科院分区：
文献类型：
--
作者：
Nguyen, A;Rothman, DM;Yaffe, MB
当结构域家族的其他成员补偿被敲除基因的产物时，使用经典遗传学来研究功能冗余的蛋白质家族中的模块化磷酸肽结合结构域是很困难的。在这里，我们描述了一种化学遗传学方法，通过使用紫外线激活的笼状磷酸肽克服了这一限制。通过将笼状磷酸丝氨酸残基纳入共有基序中，这些试剂以快速且暂时调节的方式同时同步地灭活所有磷酸丝氨酸/磷酸苏氨酸结合结构域家族成员。我们应用这种方法来研究 14-3-3 蛋白在细胞周期控制中的整体功能。紫外线照射激活笼状磷酸肽，使内源性蛋白质脱离 14-3-3 结合，导致细胞周期过早进入，G1 细胞从间期停滞中释放，DNA 损伤后 S 期检查点丢失，并伴有高水平的细胞死亡。当应用于其他磷酸肽结合域（包括 SH2、Polo-box 和串联 BRCT 域）时，此类试剂将极大地促进激酶依赖性信号传导通路的分子解析。
Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.