A novel reporter for real-time, quantitative imaging of AKT-directed K63-poly-ubiquitination in living cells
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Shyam Nyati1, Nauman Chaudhry1, Areeb Chatur1, Brandon S. Gregg1, Lauren Kimmel1, Dheeraj Khare2, Venkatesha Basrur3,4, Dipankar Ray1 and Alnawaz Rehemtulla1
1Department of Radiation Oncology, University of Michigan, Ann Arbor, MI-48109, USA
2Life Sciences Institute, University of Michigan, Ann Arbor, MI-48109, USA
3UMCCC Proteomics Shared Resource, University of Michigan, Ann Arbor, MI-48109, USA
4Department of Pathology, University of Michigan, Ann Arbor, MI-48109, USA
Shyam Nyati, email: [email protected]
Alnawaz Rehemtulla, email: [email protected]
Keywords: AKT; K63-ubiquitination; reporter; luciferase imaging; real-time
Received: December 04, 2017 Accepted: January 20, 2018 Published: January 25, 2018
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.
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