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FLEXIQinase, a mass spectrometry–based assay, to unveil multikinase mechanisms

Nature Methods volume 9, pages 504–508 (2012)Cite this article

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Abstract

We introduce a mass spectrometry–based method that provides residue-resolved quantitative information about protein phosphorylation. In this assay we combined our full-length expressed stable isotope–labeled protein for quantification strategy (FLEXIQuant) with a traditional kinase assay to determine the mechanisms of multikinase substrate phosphorylation such as priming-dependent kinase activities. The assay monitors the decrease in signal intensity of the substrate peptides and the concomitant increase in the (n × 80 Da)-shifted phosphorylated peptide. We analyzed the c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity on doublecortin (DCX) revealing mechanistic details about the role of phosphorylation cross-talk in GSK3β activity and permitting an advanced model for GSK3β-mediated signaling.

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Figure 1: Two-kinase assay.
Figure 2: A theoretical approach to interpreting a serial kinase mechanism.
Figure 3: FLEXIQinase analysis of JNK and GSK3β activity on DCX.
Figure 4: Model for JNK-dependent GSK3β activity on DCX.

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Acknowledgements

We thank J. Gunawardena for his thorough reading of our manuscript and for his advice. This work is funded in part by US National Institutes of Health grants NS066973, GM094844, GM096319 and GM081578 (H.S.) and NS041021 and NS047188 (A.B.), by the Tau Consortium (J.A.S.) and by the German Academic Exchange Service (D.W.).

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Author notes

  1. Sasha A Singh & Dominic Winter

    Present address: Present addresses: Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA (S.S.), and Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany (D.W.).,

  2. Hanno Steen and Judith A Steen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA

    Sasha A Singh, Dominic Winter & Hanno Steen

  2. Proteomics Center, Children's Hospital Boston, Boston, Massachusetts, USA

    Sasha A Singh, Dominic Winter, Hanno Steen & Judith A Steen

  3. Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA

    Parizad M Bilimoria & Azad Bonni

  4. Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA

    Parizad M Bilimoria & Azad Bonni

  5. Department of Neurology, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA

    Sasha A Singh & Judith A Steen

  6. F.M. Kirby Neurobiology Center, Children's Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA

    Sasha A Singh & Judith A Steen

Authors
  1. Sasha A Singh
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  2. Dominic Winter
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Contributions

S.S. contributed to devising the strategy and qualitative and quantitative mass spectrometry experiments, and wrote the initial manuscript; D.W. contributed qualitative mass spectrometry analysis; P.M.B. contributed in vitro kinase assays and revised the manuscript; P.M.B. and D.W. made equal contributions; A.B. contributed support and revisions to the manuscript. J.A.S. and H.S. contributed to devising the strategy and experimental design, provided support and revised the manuscript.

Corresponding authors

Correspondence to Hanno Steen or Judith A Steen.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4 and Supplementary Table 1 (PDF 393 kb)

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Singh, S., Winter, D., Bilimoria, P. et al. FLEXIQinase, a mass spectrometry–based assay, to unveil multikinase mechanisms. Nat Methods 9, 504–508 (2012). https://doi.org/10.1038/nmeth.1970

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