Sustained phosphorylation of cytosolic phospholipase A2 accompanies cycloheximide- and adenovirus-induced susceptibility to TNF.
In this report we examine the phosphorylation state of cytosolic phospholipase A2 (cPLA2) in C3HA fibroblasts that have been treated with TNF, cycloheximide (CHI), or a combination of both compounds. Our experiments show that TNF and CHI, when used independently, caused the rapid phosphorylation of cPLA2 (within 10 min). In both cases, cPLA2 was subsequently dephosphorylated to pretreatment levels by 40 min. In addition, under these conditions [3H]arachidonic acid was not released, and we could not detect a change in the activity of cPLA2 in vitro. In contrast, in cells treated with a combination of TNF and CHI, we found that the dephosphorylation of cPLA2 was inhibited, and cPLA2 remained phosphorylated for up to 2 h. In vitro we found that sustained phosphorylation of cPLA2 was accompanied by a 60 to 80% increase in the activity of cPLA2. The sustained phosphorylation of cPLA2 also occurred in cells infected with the adenovirus mutant dl309, suggesting that sustained phosphorylation may be a general requirement for the activation of cPLA2 in apoptotic cells. We also found that sustained phosphorylation of phosphoproteins is not a general consequence of apoptotic death, since the phosphorylation of p42 mitogen-activated protein kinase was not sustained. Finally, we show that the phosphatase inhibitor orthovanadate acts as does CHI to render cells susceptible to TNF, suggesting that resistance to TNF may depend on TNF's ability to induce the expression of tyrosine or dual specificity phosphatase(s).
Department of Microbiology, North Carolina State University, Raleigh 27695, USA.
J B
JB
O'Brien
D L
DL
Piddington
C
C
Voelkel-Johnson
D J
DJ
Richards
L A
LA
Hadley
S M
SM
Laster
D016475:3T3 Cells; D000256:Adenoviridae / Q000276:immunology*; D000257:Adenoviridae Infections / Q000201:enzymology / Q000276:immunology; D000818:Animals; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000276:immunology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D007113:Immunity, Innate; D016130:Immunophenotyping; D051379:Mice; D008809:Mice, Inbred C3H; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology* / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium