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 oxygen to O-2(-) at the expense of NADPH: 2 O-2 + NADPH --> 2 O-2(-) + NADP(+) + H+. The oxidase is dormant in resting cells but acquires activity 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 oxidase activation migrates to the plasma membrane to complex with cytochrome 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 oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement, The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase, We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.