Abstract
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.
Highlights
Members of the AGC superfamily of kinases move about cells phosphorylating key proteins on serine and threonine
We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on protein kinases C (PKC), [3H]phorbol dibutyrate (PDB), increased binding of [3H]PDB within 15 s of exposure to >10 –30 nM Arachidonic acid (AA)
HEK 293 cells transfected with vectors encoding PKCI or PKC␦ fused to the reporter enhanced green fluorescent protein (EGFP) were studied
Summary
Reagents and Buffers—The following were purchased: [3H]phorbol dibutyrate ([3H]PDB) (19.1 Ci/mmol) and polyvinylidene difluoride membranes (PerkinElmer Life Sciences); PDB, phorbol 12-myristate 13-acetate, dioctanoylglycerol, N-formyl-methionyl-leucyl-phenylalanine (FMLP), nordihydroguaiaretic acid, sphinganine, 0.01% poly-Llysine plates, and fatty acid-free BSA (Sigma); intracellular Ca2ϩ chelators (Molecular Probes); fatty acids (NuChek Prep, Elysean, MN); PKC isoform standards and rabbit antibodies to these isoforms (PanVera, Madison WI); horseradish peroxidase-linked rabbit anti-IgG (Transduction Laboratories); pPKC-EGFP (CLONTECH); rabbit antibody to enhanced green fluorescent protein (EGFP) (Santa Cruz Biotechnology); Supersignal chemiluminescence kits (Pierce); silicone oil (Versilube F50; General Electric Corp., Westview NY); DH5␣-competent cells, LipofectAMINE kits, Maxi-prep DNA isolation kits (Promega), and LB medium (Life Technologies, Inc.); and HEK 293 cells (ATCC). Human PMN were suspended in a modified Hanks’ balanced solution [33]; HEK 293 cells were grown in Dulbecco’s modified essential medium (DMEM) with streptomycin (100 g/ml) and penicillin (100 units/ ml), Ϯ 10% fetal calf serum. To assay [Ca2ϩ]i, PMN loaded with fura-2 were incubated at 1 ϫ 107/ml in Hanks’ buffer Ϯ 1.4 mM CaCl2 at 37 °C, excited alternately at 340 and 380 nm, and monitored at 510 nm with an Aminco-Bowman spectrofluorometer. Final colonies, which expressed appropriate molecular weight proteins that reacted with antibody to EGFP and either PKCI or PKC␦ on Western blots, were maintained in this medium and transferred to serum-free DMEM supplemented with insulin, transferrin, and selenium for 18 h. Cell Toxicity—As little as a 20 M concentration of a UFA caused PMN to leak preloaded fura-2 or Quin, leak cytosolic LDH, and take up trypan blue (assayed as in Ref. 34). The data indicate that AA produces morphology changes that are not due to overt alterations in plasma membrane integrity
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