The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases.
Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox/Nox2 (cytochrome b-245, beta polypeptide; also known as Cybb), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes. Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 (NADPH oxidase 1) and Nox3 (NADPH oxidase 3), each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123-135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. In the present study we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. The insert region of Rac2 is also dispensable for gp91phox/Nox2 activation at the cellular level. Although Rac2, as well as Rac1, is capable of enhancing superoxide production by Nox1 and Nox3, the enhancements by the two GTPases are both independent of the insert region. We also demonstrate that Rac3, a third member of the Rac family in mammals, has an ability to activate the three oxidases and that the activation does not require the insert region. Thus the insert region of the Rac GTPases does not participate in regulation of the Nox family NADPH oxidases.
Miyano
Kei
K
Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Koga
Hirofumi
H
Minakami
Reiko
R
Sumimoto
Hideki
H
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D004251:DNA Transposable Elements; D013481:Superoxides; D019255:NADPH Oxidases; D020558:GTP Phosphohydrolases; D020744:rac GTP-Binding Proteins