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A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

Abstract

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase–associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.

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Figure 1: Design of fluorescent chemosensors of Akt, MK2 and PKA activity.
Figure 2: Akt-S1 kinase activity is quantitatively linear and preferential for Akt.
Figure 3: MK2-S1 kinase activity is quantitatively linear and preferential for MK2.
Figure 4: PKA-S3 kinase activity is quantitatively linear and preferential for PKA.
Figure 5: The multiplex kinase assay reveals differential responses of Akt, MK2 and PKA in response to stimulation by EGF and insulin.

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Acknowledgements

This research was supported by the US National Institutes of Health (GM64346 Cell Migration Consortium, P50-068762 Cell Decision Processes Center), the National Science Foundation (CHE-9996335), the Whitaker Foundation and the Department of Defense Institute for Collaborative Biotechnologies. We thank I.A. Manke and M.B. Yaffe for providing the MK2tide sequence ahead of publication.

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Correspondence to Barbara Imperiali.

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Competing interests

B.I. and M.D.S. are among the filers of a US patent application (see below) that describes the assay for measuring kinase activity using the fluorescent amino acid Sox. Imperiali, B. and Shults, M.D. "Versatile and continuous fluorescence assay for kinase activity utilizing the fluorescent amino acid Sox," US Patent Application filed 8 October, 2003.

Supplementary information

Supplementary Fig. 1

Comparison of Akt-S1 kinase assay sensitivity at 10 μM and 1 mM ATP. (PDF 35 kb)

Supplementary Fig. 2

Effect of kinase inhibitors GF109203X, PKC inhibitor peptide, PKItide and calmidazolium on recombinant Akt1, MK2, PKA and PKCα. (PDF 52 kb)

Supplementary Fig. 3

Quenched-point fluorescence assays with immunopurified Akt and unfractionated HT-29 lysates. (PDF 47 kb)

Supplementary Fig. 4

Comparison of Akt-S1 activity in CHO cell lysates with a radioactivity-based assay. (PDF 46 kb)

Supplementary Fig. 5

Quenched-point fluorescence assays with immunopurified MK2 and unfractionated HT-29 lysates. (PDF 49 kb)

Supplementary Fig. 6

Comparison of MK2-S1 activity in CHO cell lysates with a radioactivity-based assay. (PDF 46 kb)

Supplementary Fig. 7

Dose-response curves for H89 and PKItide inhibition of recombinant PKA. (PDF 54 kb)

Supplementary Methods (PDF 97 kb)

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Shults, M., Janes, K., Lauffenburger, D. et al. A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates. Nat Methods 2, 277–284 (2005). https://doi.org/10.1038/nmeth747

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