期刊
NATURE METHODS
卷 4, 期 6, 页码 511-516出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nmeth1048
关键词
-
资金
- Howard Hughes Medical Institute Funding Source: Medline
- NCRR NIH HHS [RR015804, P41 RR001614, RR001614, S10 RR015804] Funding Source: Medline
- NIAAA NIH HHS [AA013588, R37 AA013588, R01 AA013588] Funding Source: Medline
- NIBIB NIH HHS [R01 EB001987] Funding Source: Medline
The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPcS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38 alpha MAPK, Erk1, Erk2, Akt1, PKC delta, PKC epsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3b, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据