KINASE-DEPENDENT ACTIVATION OF THE LEUKOCYTE NADPH OXIDASE IN A CELL-FREE SYSTEM - PHOSPHORYLATION OF MEMBRANES AND P47(PHOX) DURING OXIDASE ACTIVATION

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxyg en to O-2(-) at the expense of NADPH: 2 O-2 + NADPH --> 2 O-2(-) + NAD P(+) + H+. The oxidase is dormant in resting cells but acquires activi ty when the cells are stimulated with a suitable agent, Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), oxidase subunit located in the cytosol of resting cells that during ox idase activation migrates to the plasma membrane to complex with cytoc hrome b(558), an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent, We now report a cell-free system in which the oxidas e can be activated in two stages using phosphorylated p47(PHOX). The f irst stage, which effects a change in the membrane, requires ATP and G TP and is blocked by the protein kinase inhibitor GF-109203X, suggesti ng a protein kinase requirement, The second stage requires phosphoryla ted p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; as sembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not p rotein kinase A or mitogen-activated protein kinase, We believe that a ctivation by phosphorylated p47(PHOX) is more physiological than activ ation by amphiphiles, because the mutant p47(PHOX) S379A, which is ina ctive in whole cells, is also inactive in this system but works in sys tems activated by amphiphiles.