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Updated: May 6, 2026
In vivo Dual Substrate Bioluminescent Imaging
Published on: October 11, 2011
Bioluminescent imaging of Cdk2 inhibition in vivo
Guo-Jun Zhang1, Michal Safran, Wenyi Wei
1Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
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.
Insights
Researchers developed a novel bioluminescent reporter to track drug targets affecting protein degradation. This method visualizes how inhibiting cyclin-dependent kinase 2 (Cdk2) impacts the Skp2-p27 protein complex in cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Ubiquitin ligases and their substrates are crucial in pharmaceutical research.
- The cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by ubiquitin ligase Skp2.
- Phosphorylation of p27 by Cdk2 creates a binding site for Skp2, controlling its turnover.
Purpose of the Study:
- To create a p27-luciferase (p27Luc) fusion protein as a reporter for drug targets.
- To demonstrate that p27Luc abundance is regulated by Skp2 in a cell cycle-dependent manner.
- To validate the use of bioluminescent imaging for observing drug effects on protein turnover in vivo.
Main Methods:
- Constructed a p27-luciferase (p27Luc) fusion protein.
- Utilized inhibitory proteins, peptides, and small interfering RNA (siRNA) to block Cdk2 activity.
- Employed noninvasive bioluminescent imaging to monitor p27Luc accumulation in human tumor cells in vivo.
Main Results:
- p27Luc abundance mirrored p27 regulation by Skp2 in a cell cycle-dependent manner.
- Blocking Cdk2 activity led to increased p27Luc levels.
- Cdk2 inhibitory drugs (flavopiridol, R-roscovitine) caused p27Luc accumulation, visualized via bioluminescence in vivo.
Conclusions:
- The p27Luc fusion protein serves as a reliable reporter for cell cycle-dependent protein turnover.
- Noninvasive bioluminescent imaging can effectively monitor the effects of Cdk2 inhibitors on protein substrates in vivo.
- This approach offers a versatile platform for developing bioluminescent reporters for various drug targets impacting ubiquitin ligase substrate turnover.
