Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils.
Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.
Department of Medicine, University of Louisville, Louisville, KY 40202, USA.
Qingdan
Q
Chen
David W
DW
Powell
Madhavi J
MJ
Rane
Saurabh
S
Singh
Waseem
W
Butt
Jon B
JB
Klein
Kenneth R
KR
McLeish
D000730:Androstadienes / Q000494:pharmacology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D010455:Peptides / Q000494:pharmacology; D010587:Phagocytosis / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D016897:Respiratory Burst / Q000502:physiology*; D000077191:Wortmannin
D000730:Androstadienes; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; C060505:neutrophil cytosolic factor 1; C494918:AKT1 protein, human