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Inhibitor hijacking of Akt activation

Nature Chemical Biology volume 5, pages 484–493 (2009)Cite this article

Abstract

The kinase Akt plays a central role as a regulator of multiple growth factor input signals, thus making it an attractive anticancer drug target. A-443654 is an ATP-competitive Akt inhibitor. Unexpectedly, treatment of cells with A-443654 causes paradoxical hyperphosphorylation of Akt at its two regulatory sites (Thr308 and Ser473). We explored whether inhibitor-induced hyperphosphorylation of Akt by A-443654 is a consequence of disrupted feedback regulation at a pathway level or whether it is a direct consequence of inhibitor binding to the ATP binding site of Akt. Catalytically inactive mutants of Akt revealed that binding of an inhibitor to the ATP site of Akt is sufficient to directly cause hyperphosphorylation of the kinase in the absence of any pathway feedback effects. We conclude that ATP-competitive Akt inhibitors impart regulatory phosphorylation of their target kinase Akt. These results provide new insights into both natural regulation of Akt activation and Akt inhibitors entering the clinic.

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Figure 1: Chemical genetic strategy for achieving Akt-specific inhibition.
Figure 2: Cellular effects of asAkt transfection and asAkt-specific inhibitor treatment.
Figure 3: Pharmacological and genetic dissection of upstream regulators of Akt inhibitor-induced Akt hyperphosphorylation.
Figure 4: Hyperphosphorylation is independent of Akt signaling and results from inhibitor binding to Akt.
Figure 5: The Akt inhibitor A-443654 induces Akt membrane localization.
Figure 6: Hyperphosphorylated Akt is hyperactive in vitro after dissociation of Akt inhibitor.

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Acknowledgements

We thank B. Houseman (University of California, San Francisco) for PIK90 synthesis. We thank A. Dar, M. Feldman, A. Garske and A. Statsuk for helpful comments on the manuscript. T.O. was supported by Ajinomoto Co., Inc. D.C.G. is supported by a training grant from the University of California Systemwide Biotechnology Research & Education Program GREAT (Graduate Research and Education in Adaptive bio-Technology) and by a US National Institutes of Health Chemistry and Chemical Biology Training Grant. D.F. thanks the Ernst Schering Stiftung for funding.

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Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    Tatsuya Okuzumi, Dorothea Fiedler, Chao Zhang & Kevan M Shokat

  2. Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, San Francisco, California, USA

    Daniel C Gray

  3. Cell Systems Division, SelectScreen Services, Discovery Assays and Services, Invitrogen Corporation, Madison, Wisconsin, USA

    Brian Aizenstein & Randy Hoffman

  4. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA

    Kevan M Shokat

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  1. Tatsuya Okuzumi
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Contributions

T.O. designed and synthesized PrIDZ, determined in vitro IC50 values of PrIDZ, 3-IB-PP1 and A-443654, prepared the variants of Akt, measured in vitro kinase activity of Akt variants, performed all cell-based assays for HEK 293, HEK 293T, MiaPaCa-2, MCF-7 and PC-3 cells followed by immunoblotting, and carried out immunofluorescence microscopy experiments. C.Z. designed and synthesized 3-IB-PP1. D.F. synthesized A-443654 and BX-795. D.C.G. and D.F. synthesized DG2, measured in vitro IC50 values of DG2 and conducted cell-based assays for L6 cells followed by immunoblotting. B.A. and R.H. performed Invitrogen SelectScreen kinase profiling assays. T.O. and K.M.S. conceived the experiments and wrote the manuscript, with help from and editing by all the co-authors.

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Correspondence to Kevan M Shokat.

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Supplementary Figures 1–11, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 10339 kb)

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Okuzumi, T., Fiedler, D., Zhang, C. et al. Inhibitor hijacking of Akt activation. Nat Chem Biol 5, 484–493 (2009). https://doi.org/10.1038/nchembio.183

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