Abstract
Post-translational K63-linked poly-ubiquitination of AKT is required for its membrane recruitment and phosphorylation dependent activation in response to growth-factor stimulation. Current assays for target specific poly-ubiquitination involve cumbersome enzymatic preparations and semi-quantitative readouts. We have engineered a reporter that can quantitatively and in a target specific manner report on AKT-directed K63-polyubiquitination (K63UbR) in live cells. The reporter constitutes the AKT-derived poly-ubiquitination substrate peptide, a K63 poly-ubiquitin binding domain (UBD) as well as the split luciferase protein complementation domains. In cells, wherein signaling events upstream of AKT are activated (e.g. either EGFR or IGFR), poly-ubiquitination of the reporter leads to a stearic constraint that prevents luciferase complementation. However, upon inhibition of growth factor receptor signaling, loss of AKT poly-ubiquitination results in a decrease in interaction between the target peptide and the UBD, allowing for reconstitution of the split luciferase domains and therefore increased bioluminescence in a quantitative and dynamic manner. The K63UbR was confirmed to be suitable for high throughput screen (HTS), thus providing an excellent tool for small molecule or siRNA based HTS to discover new inhibitors or identify novel regulators of this key signaling node. Furthermore, the K63UbR platform could be adapted for non-invasive monitoring of additional target specific K63-polyubiquitination events in live cells.
Highlights
Eukaryotic cells employ a wide repertoire of ubiquitin (Ub) post-translational modifications to regulate biological processes
Based on the split luciferase complementation technology, wherein AKT-directed K63-polyubiquitination activity leads to polyubiquitination of K63UbR which results in the interaction of the target peptide with the adjacent ubiquitin binding domain (UBD)
A critical role of K63-linked ubiquitination and deubiquitination in the activity of oncogenic kinases is appreciated in tumorigenesis [10,11,12,13,14], technologies to monitor this key event under physiological conditions have been missing
Summary
Eukaryotic cells employ a wide repertoire of ubiquitin (Ub) post-translational modifications to regulate biological processes. The Ser/Thr kinase AKT (PKB) is a key hub in the cellular signaling response to growth factor and cytokine receptor activation and mediates biological functions including cell growth, cell survival and therapeutic resistance. Based on the split luciferase complementation technology, wherein AKT-directed K63-polyubiquitination activity leads to polyubiquitination of K63UbR which results in the interaction of the target peptide with the adjacent ubiquitin binding domain (UBD). This interaction restricts the complementation of luciferase, inhibition of target specific ubiquitination activity relieves this stearic constraint, allowing for luciferase complementation and enhanced bioluminescence activity. The reporter provides a sensitive, quantitative and dynamic non-invasive imaging of AKT poly-ubiquitination in live cells, and can be used as a research tool to delineate novel mechanisms that regulate the AKT signaling hub and as a platform for high-throughput screening
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