title: Rac2D57N, a dominant inhibitory Rac2 mutant that inhibits p38 kinase signaling and prevents surface ruffling in bone-marrow-derived macrophages.
abstract: Rac2 is a Rho GTPase that is expressed in cells of hematopoietic origin, including neutrophils and macrophages. We recently described an immunodeficient patient with severe, recurrent bacterial infections that had a point mutation in one allele of the Rac2 gene, resulting in the substitution of aspartate 57 with asparagine. To ascertain further the effects of Rac2D57N in leukocytes, Rac2D57N was expressed in primary murine bone-marrow-derived macrophages (cells that we show express approximately equal amounts of Rac1 and Rac2). Rac2D57N expression in macrophages inhibited membrane ruffling. Rac2D57N expression inhibited the formation of macropinosomes, demonstrating a functional effect of the loss of surface membrane dynamics. Surprisingly, Rac2D57N induced an elongated, spread morphology but did not affect microtubule networks. Rac2D57N also inhibited lipopolysaccharide-stimulated p38 kinase activation. Examination of guanine nucleotide binding to recombinant Rac2D57N revealed reduced dissociation of GDP and association of GTP. Coimmunoprecipitation studies of Rac2D57N with RhoGDI alpha and Tiam1 demonstrated increased binding of Rac2D57N to these upstream regulators of Rac signaling relative to the wild type. Enhanced binding of Rac2D57N to its upstream regulators would inhibit Rac-dependent effects on actin cytoskeletal dynamics and p38 kinase signaling.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262, USA. amy_abdell@med.unc.edu
authors.forename: Amy N
authors.initials: AN
authors.lastname: Abell
authors.forename: Aimee M
authors.initials: AM
authors.lastname: DeCathelineau
authors.forename: Scott A
authors.initials: SA
authors.lastname: Weed
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Ambruso
authors.forename: David W
authors.initials: DW
authors.lastname: Riches
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D019556:COS Cells; D048429:Cell Size; D022081:Cell Surface Extensions / Q000378:metabolism; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism; D004790:Enzyme Induction; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D048051:p38 Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D000097766:RAC2 GTP-Binding Protein
chemical_list: D000199:Actins; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D020928:Mitogen-Activated Protein Kinases; D011506:Proteins; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D048051:p38 Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C513260:Tiam1 protein, mouse