Development of a Kinase Translocation Reporter Assay for High-Throughput Screening of Novel PAK1 Inhibitors

David Harbaugh, Scott Troutman, and Dr. Joseph Kissil, Department of Molecular Medicine, Scripps Research, 120 Scripps Way, Jupiter FL 33458

The project aims to develop a cell-based reporter that tracks kinase activity in intact cells or phenotypic assays, to ascertain novel inhibitors of the P21-activated kinases. PAK1 is one of six serine/threonine kinases of the p21-activated kinases (PAKs) that play roles in cell proliferation, invasion, motility, and survival. Dysregulation of PAK1 stimulates the growth of breast, colon, ovarian, and brain cancer cells. Although extensive work has been carried out to identify PAK1 inhibitors, challenges related to efficacy, toxicity, and selectivity of the inhibitors have prevented progression into therapeutic development. To overcome the issues, we are developing a novel cell-based reporter to identify PAK1-specific inhibitors that are potent and exhibit limited toxicity in vitro and in vivo. The cell-based assay utilizes a kinase translocation reporter (KTR) approach that tracks the nucleocytoplasmic shuttling of a green fluorescent protein (GFP). The GFP is fused to another protein that contains a nuclear localization signal and is thus localized to the cell nucleus. In addition, the fused reporter contains a PAK1 phosphorylation site that when phosphorylated by PAK1, leads to the reporter to be excluded from the nucleus. Thus, by tracking the localization of GFP in the cell, the level of PAK1 activity can be ascertained. Currently, the generation of a number of GFP-fusion reporters has been completed and their subcellular localization to the nucleus determined. In the next step, I will determine whether PAK1 mediated phosphorylation results in translocation of the reporter from the nucleus and whether additional modifications to the KTR reporter are required. The assay will allow the generation of stable cell lines that express the KTR reporter which will then be used for high-throughput screening of PAK1 inhibitors. 

Additional Abstract Information

Presenter: David Harbaugh

Institution: Florida Atlantic University

Type: Poster

Subject: Biological & Chemical Engineering

Status: Approved

Time and Location

Session: Poster 2
Date/Time: Mon 3:00pm-4:00pm
Session Number: 2547