A semisynthetic epitope for kinase substrates

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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 ATPγS 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, p38α MAPK, Erk1, Erk2, Akt1, PKCδ, PKCε, Cdk1/cyclinB, CK1, Cdc5, GSK3β, 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. NOTE: In the version of this article initially published online, a sentence was missing a word and did not make sense. The corrected sentence now reads, “Erk2 was immunoprecipitated from each of these cell lines and assayed with A*TPγS analogs; N6-phenethyl ATPγS was a preferred nucleotide substrate for AS Erk2 and was not accepted by wild-type Erk2 (data not shown).” The error has been corrected for all versions of the article.