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A semisynthetic epitope for kinase substrates.

Research paper by Jasmina J JJ Allen, Manqing M Li, Craig S CS Brinkworth, Jennifer L JL Paulson, Dan D Wang, Anette A Hübner, Wen-Hai WH Chou, Roger J RJ Davis, Alma L AL Burlingame, Robert O RO Messing, Carol D CD Katayama, Stephen M SM Hedrick, Kevan M KM Shokat

Indexed on: 09 May '07Published on: 09 May '07Published in: Nature Methods



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.