Bioluminescent imaging of ubiquitin ligase activity: measuring Cdk2 activity in vivo through changes in p27 turnover.

Abstract:

: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.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Zhang GJ,Kaelin WG Jr

doi

10.1016/S0076-6879(05)99036-8

subject

Has Abstract

pub_date

2005-01-01 00:00:00

pages

530-49

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(05)99036-8

journal_volume

399

pub_type

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