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Bioluminescent imaging of Cdk2 inhibition in vivo

Nature Medicine volume 10, pages 643–648 (2004)Cite this article

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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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Figure 1: p27Luc mimics p27 with respect to regulation by Skp2 and the cell cycle.
Figure 2: Inhibition of Cdk2 induces p27Luc.
Figure 3: Induction of p27Luc by Cdk2 inhibitory drugs in vitro.
Figure 4: Monitoring Cdk2 inhibition in vivo using bioluminescent imaging.

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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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Authors and Affiliations

  1. Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Guo-Jun Zhang, Michal Safran, Wenyi Wei, Geoffrey Shapiro & William G Kaelin Jr

  2. Novartis Institutes for Biomedical Research, Cambridge, 02139, Massachusetts, USA

    Erik Sorensen & Peter Lassota

  3. Cyclacel Ltd James Lindsay Pl, Dundee, DD1 5JJ, Scotland

    Nikolai Zhelev

  4. CMCBR, SIMBIOS, School of Contemporary Sciences, University of Abertay, Bell Str., Dundee, DD1 1HG, Scotland

    Nikolai Zhelev

  5. Department of Biostatistical Science, Dana-Farber Cancer Institute, Boston, 02115, Massachusetts, USA

    Donna S Neuberg

  6. Howard Hughes Medical Institute, Chevy Chase, 20815, Maryland, USA

    Guo-Jun Zhang & William G Kaelin Jr

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Correspondence to William G Kaelin Jr.

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Competing interests

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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