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Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Abstract

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry–based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

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Figure 1: Workflow for a single-reaction, 90-substrate in vitro kinase assay.
Figure 2: Sensitivity and reproducibility of the single-reaction KAYAK assay.
Figure 3: Induced core pathway phosphorylation changes in human cell lines are faithfully reported by profiling using the single-reaction KAYAK assay.
Figure 4: Profiling of 11 human cell lines using the single-reaction KAYAK assay demonstrates heterogeneity in basal kinase activities and activation state of core pathways.
Figure 5: Cancer cell lines with elevated Akt activity differ markedly in their response to inhibitors of Akt and PDK1.
Figure 6: Identification of Cdc2/Cyclin B1 complex as an activated kinase in mitosis.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health (NIH; HG3456 and GM67945) and an industry-sponsored research project to S.P.G. from Merck and ThermoFisher. K.K. was supported by Daiichi Sankyo Co., Ltd. Y.Y. was partly supported by J. Blenis through grants from the NIH (GM51405). J.V. was supported by a grant from the Spanish Ministry of Education and Science. We thank C. Zhou, S. Elledge and N. Dephoure for help with cell cycle sample preparation, J. Elias and D. Kolippakkam for bioinformatics support, M. Rodriguez-Falcon for establishing Stagetip-IMAC protocol, and S. Sando and T. Robbins for peptide purification. We appreciate the advice and encouragement of many members of the Gygi lab and Mayumi Kubota.

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K.K. and R.A. are co-first authors. K.K., R.A., Y.Y., R.C.K., J.V. and S.P.G. participated in the planning, data generation and data interpretation. J.N.A., M.K., H.K., K.N., S.K., C.P. and R.C.H. prepared cell lysates using Akt and PDK1 inhibitors. A.S.F. and C.-L.W. prepared human renal carcinoma samples. J.R. synthesized all peptides. K.K. and S.P.G. wrote the manuscript and all authors edited it.

Corresponding author

Correspondence to Steven P Gygi.

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Kubota, K., Anjum, R., Yu, Y. et al. Sensitive multiplexed analysis of kinase activities and activity-based kinase identification. Nat Biotechnol 27, 933–940 (2009). https://doi.org/10.1038/nbt.1566

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