Bioluminescent imaging of ubiquitin ligase activity: measuring Cdk2 activity in vivo through changes in p27 turnover.
Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by different ubiquitin ligases are often modular and can be used to target foreign proteins for destruction in cis. We recently fused the Cdk inhibitor p27, which is polyubiquitylated by a Skp2-containing ubiquitin ligase if phosphorylated by cdk2 to firefly luciferase. The resulting fusion protein, p27-Luc, was induced by cdk2 inhibitors in living cells grown in culture or in nude mice. This article describes protocols for validation of p27-Luc in cell culture using siRNA against cdk2 (or its partner cyclin A) and for imaging cells producing p27-Luc grown in transparent hollow fibers after treatment with cdk2 inhibitory drugs in vivo. These approaches should be generalizable to other ubiquitin-ligase substrate pairs.
Zhang
Guo-Jun
GJ
Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Brighan and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Kaelin
William G
WG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D049449:Luminescence; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D017931:DNA Primers; D044767:Ubiquitin-Protein Ligases; D051357:Cyclin-Dependent Kinase 2