Abstract
Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle–dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle–dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
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Acknowledgements
We thank A. Kung for advice, S. Ren for help with the Cdk2 kinase assays, G. Enders and J. Roberts for critical reading of the manuscript, and members of the Kaelin laboratory for useful discussions. This work was supported by a National Institutes of Health RO1 to W.G.K. W.G.K. is a Howard Hughes Medical Institute Investigator.
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W.G.K. owns more than 5% of Imigen Systems Inc., a biotechnology company devoted to developing molecular imaging tools that might enhance preclinical and clinical drug development. Imigen Systems Inc. owns certain rights surrounding the use of bioluminescent fusion proteins.
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Zhang, GJ., Safran, M., Wei, W. et al. Bioluminescent imaging of Cdk2 inhibition in vivo. Nat Med 10, 643–648 (2004). https://doi.org/10.1038/nm1047
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DOI: https://doi.org/10.1038/nm1047
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