Abstract
We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44erk1 and p42erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p2lras activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt2cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt2cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt2cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p2lras-, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.
Highlights
We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44erk1 and p42erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors
In the present study we utilize intact neutrophils and neutrophil cytoplasts to demonstrate that MAPK activation by FMLP is associated with activation of p21ras and translocation of Raf-1 to the plasma membrane (PM) and that cAMP acts via protein kinase A (PKA) to inhibit FMLP-stimulated MAPK activation in cytoplasts but not neutrophils
Granule-depleted neutrophil cytoplasts retain the capacity to respond to chemoattractants [30] and represent a simplified system useful in studying chemoattractant signaling through G proteins
Summary
Materials—Except where otherwise noted, reagents were purchased from Sigma. AccuprepTM was from Accurate Scientific, Inc. Neutrophil lysate (106 cell eq) or cytoplast lysate (107 cell eq) was analyzed using the gel renaturation method of Kameshita and Fujisawa [23] except that prepared gels contained 0.25 mg/ml myelin basic protein (MBP) or bovine serum albumin (BSA), and phosphorylation buffer contained 2.5 Ci/ml [32P]ATP. Lysates were heated for 5 min at 100 °C and diluted 1:5 in ice-cold Nonidet P-40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM sodium vanadate, 25 mM sodium fluoride, 2 mM PMSF, 10 trypsin inhibitor units/ml aprotinin, and 10 g/ml each chymostatin, antipain, and pepstatin) and kept on ice for 30 min, followed by immunoprecipitation with antiserum specific for p44erk, p42erk, both, or isotype control, and capture on protein A-Sepharose beads. Aggregation curves were quantitated as the area under the curve in the first 2 min following stimulation
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