Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase - Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at othersites and is required for activity
Jl. Johnson et al., Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase - Phosphorylation of Ser-359 or Ser-370 precedes phosphorylation at othersites and is required for activity, J BIOL CHEM, 273(52), 1998, pp. 35147-35152
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide
(O-2(radical anion) at the expense of NADPH in phagocytes and B lymphocyte
s, The enzyme is dormant in resting cells but becomes active when the cells
are exposed to appropriate stimuli. During oxidase activation, the highly
basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on sever
al serines and migrates to the plasma membrane. We report here that p47(PHO
X)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47PHOX
S359K/S370K double mutation show dramatically reduced levels of enzyme act
ivity and phosphorylation of p47PHOX as compared with the same cells expres
sing wild type p47PHOX. I, addition, these mutant p47PHOX proteins fails to
translocate to the plasma membrane when the cells are stimulated. In contr
ast, normal phosphorylation and translocation are seen in mutants containin
g aspartate or glutamate at positions 359 and 370, but oxidase activity is
still greatly reduced. These results imply that a negative charge at positi
on 359 and/or 370 is sufficient to allow the phosphorylation and translocat
ion of p47(PHOX) to take place but that features unique to a phosphorylated
hydroxyamino acid are required to support O-2(radical anion) production. T
hese findings, plus those from an earlier study (Inanami, O., Johnson, J, L
., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P, E., and Babior
, B, M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activat
ion requires 1) the sequential phosphorylation of at least two serines on p
47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the tr
anslocation of p47(PHOX) to the membrane at some point after the first phos
phorylation takes place.