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The reverse in-gel kinase assay to profile physiological kinase substrates

Nature Methods volume 4, pages 957–962 (2007)Cite this article

Abstract

Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the α catalytic subunit of protein kinase CK2 (CK2α) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2α and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.

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Figure 1: CK2α and PKA substrates can be revealed by RIKA.
Figure 2: Comparison of KESTREL and RIKA.
Figure 3: Substrates revealed by CK2α RIKA are phosphorylated by CK2 in vivo.
Figure 4: Proteins phosphorylated in a CK2α RIKA can be phosphorylated by CK2α in vitro.
Figure 5: CK2α RIKA sensitivity.

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Acknowledgements

We thank D. Litchfield, (University of Western Ontario) for providing the CK2α clones, P. Jayachandran for cloning the PGES gene, and L. Mutton for technical assistance. This work was supported by grant DAMD17-03-1-0091 from the Congressionally Directed Medical Research Program, and by grant R21-CA122884 from the US National Institutes of Health, National Cancer Institute Innovative Technologies for the Molecular Analysis of Cancer program.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, 21250, Maryland, USA

    Xiang Li, Bin Guan, Minu K Srivastava, Achuth Padmanabhan & Charles J Bieberich

  2. Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, 21201, Maryland, USA

    Brian S Hampton

  3. University of Maryland Marlene and Stewart Greenebaum Cancer Center, 22 S. Greene Street, Baltimore, 21201, Maryland, USA

    Charles J Bieberich

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  1. Xiang Li
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Correspondence to Charles J Bieberich.

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Competing interests

C.J.B. and X.L. are named as inventors on US and PCT patent applications entitled “Devices and Methods for Profiling Enzyme Substrates” which were filed on June 6, 2006 (PCT publication number WO 2007 046882).

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Supplementary Figures 1–5, Supplementary Tables 1–2, Supplementary Methods (PDF 2740 kb)

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Li, X., Guan, B., Srivastava, M. et al. The reverse in-gel kinase assay to profile physiological kinase substrates. Nat Methods 4, 957–962 (2007). https://doi.org/10.1038/nmeth1106

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