Rac2D57N, a dominant inhibitory Rac2 mutant that inhibits p38 kinase signaling and prevents surface ruffling in bone-marrow-derived macrophages

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 Rac2(D57N) in leukocytes, Rac2(D57N) was expressed in primary murine bone-marrow-derived macrophages (cells that we show express approximately equal amounts of Rac1 and Rac2). Rac2(D57N) expression in macrophages inhibited membrane ruffling. Rac2(D57N) expression inhibited the formation of macropinosomes, demonstrating a functional effect of the loss of surface membrane dynamics. Surprisingly, Rac2(D57N) induced an elongated, spread morphology but did not affect microtubule networks. Rac2(D57N) also inhibited lipopolysaccharide-stimulated p38 kinase activation. Examination of guanine nucleotide binding to recombinant Rac2(D57N) revealed reduced dissociation of GDP and association of GTP. Coimmunoprecipitation studies of Rac2(D57N) with RhoGDIalpha and Tiam1 demonstrated increased binding of Rac2(D57N) to these upstream regulators of Rac signaling relative to the wild type. Enhanced binding of Rac2(D57N) to its upstream regulators would inhibit Rac-dependent effects on actin cytoskeletal dynamics and p38 kinase signaling.