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Nat Methods. 2007 Jun;4(6):511-6. Epub 2007 May 7.

A semisynthetic epitope for kinase substrates.

Author information

1
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94143, USA.

Abstract

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 ATPgammaS 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, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, 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.

PMID:
17486086
PMCID:
PMC2932705
DOI:
10.1038/nmeth1048
[Indexed for MEDLINE]
Free PMC Article

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