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A clickable inhibitor reveals context-dependent autoactivation of p90 RSK

Nature Chemical Biology volume 3, pages 156–160 (2007)Cite this article

Abstract

p90 ribosomal protein S6 kinases (RSKs) integrate upstream signals through two catalytic domains. Autophosphorylation of Ser386 by the regulatory C-terminal kinase domain (CTD) is thought to be essential for activation of the N-terminal kinase domain (NTD), which phosphorylates multiple downstream targets1. We recently reported fmk, an irreversible inhibitor of the CTD of RSK1 and RSK22. Here we describe fmk-pa, a propargylamine variant that has improved cellular potency and a 'clickable' tag for assessing the extent and selectivity of covalent RSK modification. Copper-catalyzed conjugation of an azidoalkyl reporter (the click reaction) revealed that fmk-pa achieves selective and saturable modification of endogenous RSK1 and RSK2 in mammalian cells. Saturating concentrations of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide. RSK autoactivation by the CTD is therefore context dependent, which suggests that NTD and CTD inhibitors should have distinct physiological effects.

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Figure 1: Mechanism of RSK activation.
Figure 2: fmk-BODIPY irreversibly targets RSK but has only modest selectivity and potency in cells.
Figure 3: Irreversible, selective and potent inhibition of RSK by fmk-pa.
Figure 4: fmk-pa reveals a RSK CTD–dependent pathway for rpS6 phosphorylation.
Figure 5: fmk-pa and click chemistry reveal a CTD-independent pathway for RSK Ser386 phosphorylation.

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Acknowledgements

This work was supported by the US National Institutes of Health (GM71434) and a University of California at San Francisco President's Dissertation-Year Fellowship to M.S.C. We thank M. Pak and E. Brown (University of California at San Francisco) for the primary mouse macrophages. We thank H. Luecke, M. Simon and members of the Taunton laboratory for many helpful discussions and for critical reading of the manuscript.

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Author notes

  1. Michael S Cohen

    Present address: Present address: Department of Pharmacology, Weill Medical College, Cornell University, New York, New York 10021, USA.,

Authors and Affiliations

  1. and Department of Cellular and Molecular Pharmacology, Program in Chemistry and Chemical Biology, University of California, San Francisco, 94158-2280, California, USA

    Michael S Cohen, Haralambos Hadjivassiliou & Jack Taunton

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  1. Michael S Cohen
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  2. Haralambos Hadjivassiliou
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Contributions

J.T. and M.S.C. designed the experiments; M.S.C. and H.H. executed the experiments; J.T. and M.S.C. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Jack Taunton.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Inhibition of PMA-stimulated RSK Ser386 phosphorylation by fmk. (PDF 104 kb)

Supplementary Fig. 2

fmk-pa covalently modifies RSK1 and RSK2 in intact cells as determined by click chemistry. (PDF 130 kb)

Supplementary Fig. 3

Covalent modification of RSK by fmk-pa is unaffected by serum in the incubation medium, whereas covalent modification by fmk-BODIPY is significantly decreased. (PDF 333 kb)

Supplementary Fig. 4

fmk-pa does not inhibit S6K phosphorylation at Thr389, an essential activation site. (PDF 124 kb)

Supplementary Fig. 5

Inhibition of rpS6 Ser235/236 phosphorylation by rapamycin and fmk-pa. (PDF 189 kb)

Supplementary Table 1

Half-maximal inhibitory concentrations (IC50s) for fmk, fmk-BODIPY and fmk-pa against the kinase activity of RSK2 CTD. (PDF 116 kb)

Supplementary Methods

Synthetic schemes and procedures. (PDF 332 kb)

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Cohen, M., Hadjivassiliou, H. & Taunton, J. A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat Chem Biol 3, 156–160 (2007). https://doi.org/10.1038/nchembio859

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