Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes.
Rap1A is a GTP-binding protein of the Ras superfamily that is highly abundant in phagocyte membranes. Although Rap1A copurifies with cytochrome b558, a component of the superoxide-generating NADPH oxidase complex of human phagocytes and B lymphocytes, the involvement of Rap1A in the regulation of the oxidative burst in these cells has not been clearly established. Therefore, we have stably transfected human Epstein-Barr virus-transformed B lymphocytes that possess an activable NADPH oxidase complex with cDNAs for mutants of Rap1A "locked" in a GTP-bound (63E) and GDP-bound (17N) state. Both the 17N and 63E mutants of Rap1A inhibited phorbol ester-stimulated O2-. production by 50 and 80%, respectively, while transfection with cDNA for wild-type Rap1A had no effect on the respiratory burst. No effects of the Rap1A mutants on cell viability, proliferation, expression of cell-surface markers, or phorbol 12-myristate 13-acetate-stimulated interleukin-8 generation were detected. These data demonstrate that Rap1A is a regulator of O2-. formation in intact cells. Furthermore, the inhibitory effect of both GTP- as well as GDP-bound mutants indicates that Rap1A functions in a dynamic cycle as opposed to a unidirectional pathway, as is the case for the other NADPH oxidase regulatory GTP-binding protein, Rac.
Maly
F E
FE
Physiologisches Institut, Universität Zürich, Switzerland.
Quilliam
L A
LA
Dorseuil
O
O
Der
C J
CJ
Bokoch
G M
GM
D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000378:metabolism*; D002472:Cell Transformation, Viral; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004854:Herpesvirus 4, Human; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000096:biosynthesis; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D016897:Respiratory Burst; D020668:rap GTP-Binding Proteins
D000954:Antigens, Surface; D016209:Interleukin-8; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins