title: Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system.
abstract: Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.
authors: Biology Department, Bemidji State University, 1500 Birchmont Dr. NE, Bemidji, MN 56601, USA.
authors: Erik L
authors: EL
authors: Anderson
authors: Michael J
authors: MJ
authors: Hamann
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D017930:Genes, Reporter / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D019169:Jurkat Cells; D008156:Luciferases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008954:Models, Biological; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: C576602:ARHGEF4 protein, human; D002457:Cell Extracts; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D008156:Luciferases