Protein kinases C translocation responses to low concentrations of arachidonic acid.
Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.
O'Flaherty
J T
JT
Department of Internal Medicine, Section on Infectious Diseases, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27156, USA. joflaher@wfubmc.edu
Chadwell
B A
BA
Kearns
M W
MW
Sergeant
S
S
Daniel
L W
LW
D016718:Arachidonic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology; D002630:Chemotactic Factors / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007975:Leukotriene B4 / Q000494:pharmacology; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; D014162:Transfection
D002630:Chemotactic Factors; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D008024:Ligands; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007975:Leukotriene B4; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C497258:PRKCD protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; D002118:Calcium