A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities

Abstract

GTPases play a major role in various cellular functions such as cell 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 has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg2+ plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z′-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add–mix–read, and applicable to high-throughput screening.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

“…We found no significant difference in the k cat /K m of DrrA 340-533 toward WT Rab1 and Rab1T75E, and only a slight difference with Rab1T75A; thus, we infer that it is unlikely phosphorylation of T75 prevents Rab1 interaction with GEFs or interferes with activation. Phosphomimetic mutant Rab1T75E yields similarly insignificant effects on the ability of a Legionella GAP, LepB (33), to stimulate Rab1 hydrolysis of GTP, as measured with the Promega GTPase-Glo system (34). Briefly, increasing concentrations of LepB with excess GTP held at a constant concentration are added to wells containing a constant concentration of Rab1 and allowed to react for 1 h. The amount of remaining, unhydrolyzed GTP in each condition is detected by luminescencecoupled assay, plotted against GAP concentration, and a LepB EC 50 is determined.…”

Sar1 GTPase Activity Is Regulated by Membrane Curvature

“…The GTPase/GAP/GEF-Glo system (Promega) was used to measure the concentration of GTP remaining after incubation with Sar1 isoforms ( 24 ). Sar1 (3.1 μ m , unless otherwise noted) was added to the reaction before the addition of GTP (5 μ m ) in a 25-μl reaction (Corning Costar 3912 solid white 96-well plate) in the supplied GEF buffer containing MgCl 2 .…”

Cryo-EM structure of C9ORF72–SMCR8–WDR41 reveals the role as a GAP for Rab8a and Rab11a

“…The CSW complex has been reported to function as a GEF of Rab7a, Rab8a, Rab11a, Rab39a, and Rab39b (14,17,31). Hence, a bioluminescence-based GTPase activity assay was carried out to assess whether the CSW complex stimulates these Rabs (38). In this type of assay, unhydrolyzed GTP can be transformed into a bioluminescence signal.…”