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 a bundant in phagocyte membranes. Although Rap1A copurifies with cytochr ome b(558), a component of the superoxide-generating NADPH oxidase com plex of human phagocytes and B lymphocytes, the involvement of Rap1A i n the regulation of the oxidative burst in these cells has not been cl early 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-bo und (63E) and GDP-bound (17N) state. Both the 17N and 63E mutants of R ap1A inhibited phorbol ester-stimulated O-2(radical-anion) production by 50 and 80%, respectively, while transfection with cDNA for wild-typ e Rap1A had no effect on the respiratory burst. No effects of the Rap1 A mutants on cell viability proliferation, expression of cell-surface markers, or phorbol 12-myristate 13-acetate stimulated interleukin-8 g eneration were detected. These data demonstrate that Rap1A is a regula tor of O-2(radical-anion) formation in intact cells. Furthermore, the inhibitory effect of both GTP as well as GDP-bound mutants indicates t hat Rap1A functions in a dynamic cycle as opposed to a unidirectional pathway, as is the case for the other NADPH oxidase regulatory GTP-bin ding protein, Rac.