PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-4 (4)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
1.  Phosphorylation of p47phox is required for receptor-mediated NADPH oxidase/NOX2 activation in Epstein-Barr virus-transformed human B lymphocytes 
The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA. GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and 345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines, p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced NADPH oxidase activation in these cells.
PMCID: PMC3484414  PMID: 23119229
NADPH oxidase; NOX2; p47phox; B lymphocytes; pansorbin; ROS; phosphorylation
2.  Francisella Acid Phosphatases Inactivate the NADPH Oxidase in Human Phagocytes 
Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [ΔABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the ΔABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida ΔABCH strain stimulated a significant level of reactive oxygen species. The ΔABCH mutant, but not the wild-type strain, strongly colocalized with p47phox and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47phox knockout mice. Finally, purified AcpA strongly dephosphorylated p47phox and p40phox, but not p67phox, in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.
doi:10.4049/jimmunol.0903413
PMCID: PMC2952287  PMID: 20348422
3.  p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases 
Experimental & Molecular Medicine  2009;41(4):217-225.
Phagocytes such as neutrophils play a vital role in host defense against microbial pathogens. The anti-microbial function of neutrophils is based on the production of superoxide anion (O2•-), which generates other microbicidal reactive oxygen species (ROS) and release of antimicrobial peptides and proteins. The enzyme responsible for O2•- production is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox, also called NOX2, which together form the cytochrome b558) and four cytosolic proteins (p47phox, p67phox, p40phox and a GTPase Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate agents. This process is dependent on the phosphorylation of the cytosolic protein p47phox. p47phox is a 390 amino acids protein with several functional domains: one phox homology (PX) domain, two src homology 3 (SH3) domains, an auto-inhibitory region (AIR), a proline rich domain (PRR) and has several phosphorylated sites located between Ser303 and Ser379. In this review, we will describe the structure of p47phox, its phosphorylation and discuss how these events regulate NADPH oxidase activation.
doi:10.3858/emm.2009.41.4.058
PMCID: PMC2679237  PMID: 19372727
CYBB protein, human; NADPH oxidase; neutrophil cytosolic factor 1; neutrophils; phosphorylation; reactive oxygen species; review
4.  A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites  
Journal of Clinical Investigation  2006;116(7):2033-2043.
Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-α prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-α induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF–induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-α–induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF– and TNF-α–induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF– and TNF-α–induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy.
doi:10.1172/JCI27544
PMCID: PMC1479423  PMID: 16778989

Results 1-4 (4)