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Active site profiling reveals coupling between domains in SRC-family kinases

Nature Chemical Biology volume 9, pages 43–50 (2013)Cite this article

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Abstract

Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that have a distinct preference for the autoinhibited forms of these kinases. Furthermore, we show that ligands that have this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.

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Figure 1: An active site–directed probe for ratiometric profiling of protein kinases.
Figure 2: Characterization of 1 in cell lysate and live cells.
Figure 3: Photocrosslinking to SFKs that have diverse regulatory domain interactions.
Figure 4: Photocrosslinking competition assays.
Figure 5: The catalytic domain of SRC is in the SRC/CDK-like inactive conformation when bound to 9.
Figure 6: ATP-competitive SFK inhibitors modulate the SH3 domain accessibilities of SRC and HCK.

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Acknowledgements

Several ATP-competitive inhibitors were provided by R. Murphy (University of Washington, Seattle) and G. Perera (University of Washington, Seattle). This research was supported by US National Institutes of Health grants R01GM086858 (D.J.M.) and R01AI089441 (E.A.M.) and the Alfred P. Sloan Foundation (D.J.M.).

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Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, Washington, USA

    Ratika Krishnamurty, Jennifer L Brigham, Stephen E Leonard, Pratistha Ranjitkar, Edward J Dale & Dustin J Maly

  2. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Eric T Larson & Ethan A Merritt

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  1. Ratika Krishnamurty
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Contributions

R.K. and D.J.M. conceived the idea and designed the study. R.K. conducted crosslinking experiments. R.K. and S.E.L. performed biochemical studies with SFK variants. R.K. and E.J.D. synthesized compounds. J.L.B. designed, expressed and purified HaloTag fusion constructs. P.R. constructed, expressed and purified SFK variants. E.T.L. and E.A.M. carried out crystallographic studies. R.K. and D.J.M. wrote the paper.

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Correspondence to Dustin J Maly.

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Krishnamurty, R., Brigham, J., Leonard, S. et al. Active site profiling reveals coupling between domains in SRC-family kinases. Nat Chem Biol 9, 43–50 (2013). https://doi.org/10.1038/nchembio.1118

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