Éva Wisniewski1, Fanni Kovács1, Enikő Bahurek1, Roland Csépányi-Kömi1, Erzsébet Ligeti1
1Semmelweis University, Department of Physiology, Budapest, Hungary
Introduction: Small G-proteins regulate numerous immune cell functions. ARHGAP25, a Rac-specific GTPase activating protein is a key factor in the regulation of phagocytosis, leukocyte migration and extravasation according to our previous results.
Aims: To further investigate its precise enzymatic function and effect on Rac, finding a reliable GTPase assay with high temporal resolution was crucial. Since the currently available methods were not suited for our purposes, we considered that using bioluminescence resonance energy transfer (BRET), the active, GTP-bound Rac could be monitored by measuring the distance between Rac and CRIB, a domain which binds active Rac only.
Method: Luciferase-tagged CRIB (CRIB-Rluc) and Venus-tagged Rac (Venus-Rac) as well as ARHGAP25 were produced in E.coli. BRET ratio was measured every 30 s under in vitro conditions in 96-well plates for 15 minutes. (Optimal donor (CRIB-Rluc) to acceptor (Venus-Rac) ratio was determined in advance.) GTPγS or GDPβS-loaded Rac were used to determine BRETmax and BRETmin, resp. Phosphorylation of ARHGAP25 was carried out with activated and heat-inactivated neutrophil cytosol. All results were validated by both radioactive and commercially available endpoint GTPase activity assays.
Results: BRET signal between Venus-Rac and CRIB-Rluc displayed a continuous decrease demonstrating the endogenous GTPase activity of Rac. BRET signal decrease was accelerated by ARHGAP25 in a dose-dependent manner, verifying its effect as a RacGAP. Importantly, phosphorylation of ARHGAP25 with neutrophil cytosol attenuated its GAP activity.
Conclusion: We developed a simple and cost-effective in vitro BRET-based assay, which measures Rac GTPase activity in a real-time manner and without the use of radioactive labelling. Using this novel approach we demonstrated that phosphorylation of ARHGAP25 with neutrophil cytosol reduces its GAP activity.
Funding sources: NKFIH, Hungary (K108382, FK128376), ÚNKP-18-3-III-SE-37
Doctoral School: Molecular Medicine
Program: Cellular and Molecular Physiology
Supervisor: Erzsébet Ligeti, Roland Csépányi-Kömi
E-mail address: email@example.com