Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency

Yi Gu, Baoqing Jia, Feng Chun Yang, Maria D'Souza, Chad E. Harris, Caroline W. Derrow, Yi Zheng, David A. Williams

Research output: Contribution to journalArticle

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Abstract

The Rho GTPase, Rac2, is expressed only in hematopoietic cell lineages, suggesting a specific cellular function in these cells. Genetic targeting studies in mice showed that Rac2 is an essential regulator of neutrophil chemotaxis, L-selectin capture and rolling, and superoxide production. Recently, a dominant negative mutation of Rac2, D57N, has been reported to be associated with a human phagocytic immunodeficiency. To understand further the cellular phenotypes associated with this D57N Rac2 mutant we examined its biochemical characteristics and functional effects when expressed in primary murine bone marrow cells. When compared with wild type (WT) Rac2, D57N Rac2 displayed ∼10% GTP binding ability resulting from a markedly enhanced rate of GTP dissociation and did not respond to the guanine nucleotide exchange factors. These results suggest that D57N Rac2 may act in a dominant negative fashion in cells by sequestering endogenous guanine nucleotide exchange factors. When expressed in hematopoietic cells, D57N Rac2 reduced endogenous activities of not only Rac2, but also Rac1 and decreased cell expansion in vitro in the presence of growth factors due to increased cell apoptosis. Unexpectedly, D57N expression had no effect on proliferation. In contrast, expansion of cells transduced with WT Rac2 and a dominant active mutant, Q61L, was associated with significantly increased proliferation. Transplantation of transduced bone marrow cells into lethally irradiated recipients showed that the percentage of D57N-containing peripheral blood cells decreased markedly from 40% at 1 month to <5% by 3 months postinjection. Neutrophils derived in vitro from the transduced progenitor cells containing D57N demonstrated markedly impaired migration and O2- responses to formyl-methionyl-leucyl-phenylalanine, reflecting the same cellular phenotype in these differentiated cells as those described previously in patient cells. These data suggest that the phenotypic abnormalities associated with D57N Rac2 may involve not only neutrophil cellular functions, but also abnormal cell survival in other hematopoietic cells and that overexpression of Rac leads to increased proliferation of normal cells in vitro, whereas deficiency of Rac leads to increased apoptosis.

Original languageEnglish (US)
Pages (from-to)15929-15938
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number19
DOIs
StatePublished - May 11 2001

Fingerprint

GTP Phosphohydrolases
Cells
Guanine Nucleotide Exchange Factors
Guanosine Triphosphate
Bone
methionyl-leucyl-phenylalanine
Apoptosis
L-Selectin
rho GTP-Binding Proteins
Neutrophils
Superoxides
Intercellular Signaling Peptides and Proteins
Blood
Phenotype
Aptitude
Cell Lineage
Chemotaxis
Bone Marrow Transplantation
Bone Marrow Cells
Blood Cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Gu, Y., Jia, B., Yang, F. C., D'Souza, M., Harris, C. E., Derrow, C. W., ... Williams, D. A. (2001). Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency. Journal of Biological Chemistry, 276(19), 15929-15938. https://doi.org/10.1074/jbc.M010445200

Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency. / Gu, Yi; Jia, Baoqing; Yang, Feng Chun; D'Souza, Maria; Harris, Chad E.; Derrow, Caroline W.; Zheng, Yi; Williams, David A.

In: Journal of Biological Chemistry, Vol. 276, No. 19, 11.05.2001, p. 15929-15938.

Research output: Contribution to journalArticle

Gu, Y, Jia, B, Yang, FC, D'Souza, M, Harris, CE, Derrow, CW, Zheng, Y & Williams, DA 2001, 'Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency', Journal of Biological Chemistry, vol. 276, no. 19, pp. 15929-15938. https://doi.org/10.1074/jbc.M010445200
Gu, Yi ; Jia, Baoqing ; Yang, Feng Chun ; D'Souza, Maria ; Harris, Chad E. ; Derrow, Caroline W. ; Zheng, Yi ; Williams, David A. / Biochemical and Biological Characterization of a Human Rac2 GTPase Mutant Associated with Phagocytic Immunodeficiency. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 19. pp. 15929-15938.
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abstract = "The Rho GTPase, Rac2, is expressed only in hematopoietic cell lineages, suggesting a specific cellular function in these cells. Genetic targeting studies in mice showed that Rac2 is an essential regulator of neutrophil chemotaxis, L-selectin capture and rolling, and superoxide production. Recently, a dominant negative mutation of Rac2, D57N, has been reported to be associated with a human phagocytic immunodeficiency. To understand further the cellular phenotypes associated with this D57N Rac2 mutant we examined its biochemical characteristics and functional effects when expressed in primary murine bone marrow cells. When compared with wild type (WT) Rac2, D57N Rac2 displayed ∼10{\%} GTP binding ability resulting from a markedly enhanced rate of GTP dissociation and did not respond to the guanine nucleotide exchange factors. These results suggest that D57N Rac2 may act in a dominant negative fashion in cells by sequestering endogenous guanine nucleotide exchange factors. When expressed in hematopoietic cells, D57N Rac2 reduced endogenous activities of not only Rac2, but also Rac1 and decreased cell expansion in vitro in the presence of growth factors due to increased cell apoptosis. Unexpectedly, D57N expression had no effect on proliferation. In contrast, expansion of cells transduced with WT Rac2 and a dominant active mutant, Q61L, was associated with significantly increased proliferation. Transplantation of transduced bone marrow cells into lethally irradiated recipients showed that the percentage of D57N-containing peripheral blood cells decreased markedly from 40{\%} at 1 month to <5{\%} by 3 months postinjection. Neutrophils derived in vitro from the transduced progenitor cells containing D57N demonstrated markedly impaired migration and O2- responses to formyl-methionyl-leucyl-phenylalanine, reflecting the same cellular phenotype in these differentiated cells as those described previously in patient cells. These data suggest that the phenotypic abnormalities associated with D57N Rac2 may involve not only neutrophil cellular functions, but also abnormal cell survival in other hematopoietic cells and that overexpression of Rac leads to increased proliferation of normal cells in vitro, whereas deficiency of Rac leads to increased apoptosis.",
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