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.
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08 May 2007
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.
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Acknowledgements
We thank D. Randle and D. Morgan for wild-type Cdc5, D. Kenski for performing GRK2 kinase assays, H. Luecke for helpful suggestions, D. Maly for helpful comments on the manuscript, and V. Ohman for excellent administrative assistance. Erk1−/− mice were generously provided by G. Pages and J. Pouyssegur (Centre National de la Recherche Scientifique, Nice, France). HeLa cells were provided by the National Cell Culture Center. This work was supported by the US National Institutes of Health (NIH) National Institute of Biomedical Imaging and BioEngineering R01 EB001987, and in part by NIH grant AA013588 and funds provided by the State of California for medical research on alcohol and substance abuse through the University of California at San Francisco (to R.O.M.). J.J.A. was funded by an Achievement Rewards for College Scientists fellowship, Genentech, and the Sandler Foundation. Mass spectrometry was made possible by NIH grants NCRR RR015804 and NCRR RR001614.
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Contributions
J.J.A. and K.M.S. wrote the manuscript. J.J.A. performed all experiments except those mentioned below. M.L. performed Erk2 substrate labeling reactions, C.S.B. performed mass spectrometry of modified peptides, J.L.P. expressed Cdc5, D.W. provided technical assistance for PKCδ experiments, A.H. constructed JNK1 plasmids, C.D.K. constructed the AS Erk2 mouse, W.C. constructed PKCδ plasmids, A.L.B. advised C.S.B., R.O.M. advised D.W. and W.C., and assisted in manuscript revision, R.J.D. advised A.H., S.M.H. advised M.L. and C.D.K. and K.M.S. advised J.J.A.
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Antibody 51-8 was made for a fee paid by grant NIH EB001987 to Epitomics Inc., under an academic-institution agreement with Epitomics Inc.. University of California San Francisco is presently negotiating with Epitomics to commercialize this antibody along with the necessary reagents for labeling kinase substrates.
Supplementary information
Supplementary Fig. 1
Western blot analysis of α-hapten–IgY specificity and full length western blots for main text figures. (PDF 545 kb)
Supplementary Fig. 2
Screen for thiophosphate ester specific IgG. (PDF 397 kb)
Supplementary Fig. 3
Specificity and yield of PNBM alkylation. (PDF 99 kb)
Supplementary Fig. 4
DELFIA for thiophosphorylation kinetics. (PDF 92 kb)
Supplementary Fig. 5
Synthesis of hapten and thioether control. (PDF 51 kb)
Supplementary Fig. 6
Synthesis and purification of A*TPγS analogs. (PDF 150 kb)
Supplementary Table 1
Generality of AS kinase engineering. (PDF 70 kb)
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Allen, J., Li, M., Brinkworth, C. et al. A semisynthetic epitope for kinase substrates. Nat Methods 4, 511–516 (2007). https://doi.org/10.1038/nmeth1048
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DOI: https://doi.org/10.1038/nmeth1048
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