Abstract C204: GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities.

Abstract

Abstract It is well known that GTPases play major roles in cell functions such as GPCR signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. GTPases act as molecular switches cycling between an activated GTP-bound state (“ON”-state) and an inactive GDP-bound state (“OFF”-state). They have very high affinity for guanine nucleotides GDP or GTP and are extremely slow acting GTP-hydrolases. To mediate the process of shuttling between the active and inactive forms, GTPases are modulated by two groups of enzymes, guanine nucleotide exchange factors (GEF) and guanine nucleotide activating proteins (GAPs). The GEFs proteins substitute GTP for the bound GDP and thus activating them while the GAP family activates the hydrolysis of GTP and terminating their effector function. We have developed a homogenous bioluminescent assay for monitoring the enzyme activity of GTPase, GAP, and GEF proteins. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP, and manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The assay system for measuring GTPase/GAP/GEF-Glo™ is enabling to monitoring the intrinsic GTPase-, GAP-stimulated GTPase-, GAP-, and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C204. Citation Format: Subhanjan Mondal, Said Goueli, Kevin Hsiao. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C204.