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A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases

Nature Methods volume 2, pages 435–441 (2005)Cite this article

Abstract

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3′)-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the β sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

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Figure 1: Natural variation of the gatekeeper residue and its tolerability of substitution with glycine.
Figure 2: Protein kinase–like structure of APH(3′)-IIIa, and its intolerance of gatekeeper mutation to glycine.
Figure 3: Suppressor mutations selected from a random mutant library of APH(3′)-IIIaM90G.
Figure 4: A structure-based sequence alignment of kinases within the β sheet in the N-terminal lobe.
Figure 5: Effects of the designed suppressor mutations on four intolerant protein kinases.

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Acknowledgements

We thank G.D. Wright for providing the APH(3′)-IIIa expression plasmid. We also thank M. Simon, Z. Knight and S. DiMagno for critical reading of the manuscript. This work was supported by the National Institutes of Health (R01EB001987 & AI44009 to K.M.S.) and Binational Science Foundation (Grant 2001124 to G.S.).

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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California San Francisco, 600 16th Street, San Francisco, 94143-2280, California, USA

    Chao Zhang, Denise M Kenski, Jennifer L Paulson & Kevan M Shokat

  2. Department of Plant Sciences, Tel-Aviv University, Tel-Aviv, 69978, Israel

    Arale Bonshtien & Guido Sessa

  3. Department of Pathology, University of Virginia, Charlottesville, 22908, Virginia, USA

    Janet V Cross & Dennis J Templeton

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  1. Chao Zhang
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Correspondence to Kevan M Shokat.

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Supplementary information

Supplementary Fig. 1

Viability of yeast strains carrying different Cdc5 alleles based on colony-forming efficiency. (PDF 248 kb)

Supplementary Fig. 2

Enzymatic activity of Pto proteins revealed in an in vitro autophosphorylation kinase assay. (PDF 127 kb)

Supplementary Fig. 3

Catalytic activity of MEKK1 proteins using N6-phenethyl-ATP revealed in an in vitro kinase assay. (PDF 423 kb)

Supplementary Table 1

Mutagenic primers used in the paper. (PDF 85 kb)

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Zhang, C., Kenski, D., Paulson, J. et al. A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases. Nat Methods 2, 435–441 (2005). https://doi.org/10.1038/nmeth764

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