Nature Methods
- 4, 511 - 516 (2007)
Published online: 7 May 2007; Corrected online: 8 2007 | doi:10.1038/nmeth1048
A semisynthetic epitope for kinase substratesJasmina J Allen1, Manqing Li2, Craig S Brinkworth3, 7, Jennifer L Paulson3, 7, Dan Wang4, Anette Hübner5, Wen-Hai Chou4, Roger J Davis5, Alma L Burlingame3, Robert O Messing4, Carol D Katayama2, Stephen M Hedrick2 & Kevan M Shokat61
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94143, USA. 2
Divisions of Biological Sciences, and Cellular and Molecular Medicine, University of California, San Diego, California 92093, USA. 3
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94143, USA. 4
Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California 94608, USA. 5
Howard Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. 6
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA. 7
Present addresses: Defense Science and Technology Organization, Fishermans Bend, VIC 3207, Australia (C.S.B.); Oregon Health and Science University, Portland, Oregon 97239, USA (J.L.P.).
Correspondence should be addressed to Kevan M Shokat shokat@cmp.ucsf.edu 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 ATP S 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 MAPK, Erk1, Erk2, Akt1, PKC , PKC , Cdk1/cyclinB, CK1, Cdc5, GSK3 , 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*TPS analogs; N6-phenethyl ATPS 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.
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