Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

Abstract

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC.These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

“…Isolated neutrophils were treated with diisopropyl fluorophosphate (26), resuspended in sonication buffer (11% sucrose, 50 mM Na x PO 4 , pH 7.0, 120 mM NaCl, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 M benzamidine, 1 g/ml leupeptin, 1 M pepstatin, 1 g/ml aprotinin), and broken by sonication (36). Sonicates were centrifuged (200 ϫ g, 10 min) to remove unbroken cells and nuclei, layered onto a 15/40% (w/v) discontinuous sucrose gradient, and centrifuged (150,000 ϫ g, 30 min) (36). Cytosolic fractions were collected from the top layer down to the 15% interface, and membrane fractions were collected from the 15/40% interface.…”

Phosphorylation of p22 Is Mediated by Phospholipase D-dependent and -independent Mechanisms

“…Cells were resuspended (10 8 cells/ml) in sonication buffer (11% sucrose, 10 mM Pipes, pH 7.2, 5 mM EGTA, 5 mM EDTA, 130 mM NaCl, 1 M staurosporine, 5 mM Na 3 VO 4 , 1 M microcystin, 25 mM NaF, 1 mM p-nitrophenyl phosphate, 10 M benzamidine, 10 g/ml leupeptin, 10 M pepstatin, 1 g/ml aprotinin, and 1 M phenylmethylsulfonyl fluoride) and sonicated (41). The membrane and cytosol fractions were prepared by ultracentrifugation, using a 15/40% discontinuous sucrose gradient, as described previously (41).…”

“…The membrane and cytosol fractions were prepared by ultracentrifugation, using a 15/40% discontinuous sucrose gradient, as described previously (41). Membrane fractions were immediately solubilized with radioimmune precipitation buffer (see below) or stored at Ϫ70°C overnight for solubilization with n-octyl-␤-D-glucopyranoside.…”

Activation of p47 , a Cytosolic Subunit of the Leukocyte NADPH Oxidase

“…The labeled bands were excised from the gel, and the radioactivity was measured by Č erenkov counting. The results (Table III) (3, 24 -29) and that the target serines for this phosphorylation lie between Ser-303 and Ser-379 in the C terminus of the protein (3)(4)(5)(6)(7). Direct evidence that the phosphorylation of p47…”