scholarly journals Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

2003 ◽  
Vol 162 (4) ◽  
pp. 683-692 ◽  
Author(s):  
Zeev Shmelzer ◽  
Nurit Haddad ◽  
Ester Admon ◽  
Itai Pessach ◽  
Thomas L. Leto ◽  
...  

Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

1997 ◽  
Vol 326 (3) ◽  
pp. 867-876 ◽  
Author(s):  
Inbal HAZAN ◽  
Raya DANA ◽  
Yoseph GRANOT ◽  
Rachel LEVY

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibitor genistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.


2000 ◽  
Vol 118 (4) ◽  
pp. A732-A733
Author(s):  
Gerardo Nardone ◽  
Eileen Holicky ◽  
Jim R. Uhl ◽  
Vittorio Colantuoni ◽  
Lina Sabatino ◽  
...  

1999 ◽  
Vol 59 (1-6) ◽  
pp. 37
Author(s):  
Takao Shimizu ◽  
Naonori Uozumi ◽  
Noriaki Nakatani ◽  
Tetsuya Hirabayashi ◽  
Kazuhiko Kume

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