We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44 erk1 and p42 erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucylphenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p21 ras 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 Bt 2 cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt 2 cAMP 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, Bt 2 cAMP 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 p21 ras -, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.

We earlier showed that lovastatin potentiated the chemopreventive effects of sulindac against colon neoplasia in a rodent model and augments apoptosis induced by 5-FU and cisplatin in human colon cancer cells. In the present study, we investigated effects of lovastatin in spontaneously immortalized rat intestinal epithelial cells, IEC-18 and their K-ras transformed clones. Lovastatin induced morphologic changes (cell rounding and detachment) and apoptosis that were not influenced by K-ras mutations, but were prevented by geranylgeranyl-pyrophosphate or by mevalonate. Clostridium difficile toxin B, which directly inactivates rho, induced similar morphologic changes and apoptosis. Cycloheximide prevented these effects of lovastatin, but not C. difficile toxin B. Lovastatin decreased the amounts of membrane bound rhoA and rhoB. Cycloheximide and geranylgeranyl-pyrophosphate prevented lovastatin induced morphologic changes and apoptosis but did not inhibit lovastatin-induced changes in membrane translocation of rho. Our data suggest that lovastatin induces morphologic changes and apoptosis by inhibiting geranylgeranylation of small GTPases of the rho family and thereby inactivating them. Restoration of membrane translocation of rho is not necessary for preventing lovastatin-induced morphologic changes or apoptosis.

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The y subunits of heterotrimeric guanine nucleotide-binding regulatory (G) Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric GTP-binding proteins that transduce signals from membrane receptors via their a (Ga,) and P/y (G/,y) subunits. G proteins are targeted to membranes by a series of posttranslational modifications of their Gz subunits that include prenylation, proteolysis, and carboxyl methylation of the C terminus (1). The last of these modifications, prenylcysteine a-carboxyl methyl esterification, is reversible (2, 3) and, therefore, potentially regulatory. We have demonstrated that activation of human neutrophils is associated with a similar carboxyl methylation of ras-related proteins (4) and that inhibitors of prenylcysteine-directed carboxyl methylation inhibit agonist-induced responses* in human neutrophils (4), platelets (5), and mouse peritoneal macrophages (6). We have also partially characterized (7) a phospholipid-dependent prenylcysteine-directed carboxyl methyltransferase in human neutrophil plasma membranes. Here we show that carboxyl methylation of GI is associated with activation of G proteins in human neutrophil membranes, suggesting another mechanism of G-protein regulation.from Calbiochem. N-Acetyl-S-trans, trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-trans-geranyl-L-cysteine (AGC) were synthesized and purified as described (8) (9). Neutrophil subcellular fractions (cytosol, plasma membranes, and specific granules) were produced by nitrogen cavitation followed by discontinuous sucrose density centrifugation as described (10). Cytosol was dialyzed overnight against 20 mM Tris-HCl (pH 7.4) containing 10 kallikrein inhibitor units of aprotinin per ml to remove endogenous AdoMet. Equilibrium [355]GTP[yS] binding to plasma membranes was performed as described (11). A cholate extract of bovine brain was prepared as described (12).Antibodies. Anti-Gia antiserum 8730 (13) and anti-Gp antisera 5356 and 8136 were provided by David Manning (University of Pennsylvania, Philadelphia). Anti-Gy2,3 antiserum SE (14) was the kind gift of William Simonds (National Institutes of Health, Bethesda). Anti-Gy2,3 antiserum X-263 (15) was provided by Paul Sternweis (University of Texas Southwest Medical Center, Dallas). Antiserum sc96 directed toward a C-terminal peptide of p2lrac2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Immunoblot Analysis and Immunoprecipitation. Electrophoresed proteins transferred onto nitrocellulose were blocked with 5% (wt/vol) nonfat dry milk and probed with G-protein-subunit-specific antisera followed by 125I-labeled protein A. Immunolocalized proteins were visualized by autoradiography. Immunoblots were quantitated by PhosphorImager (Molecular Dynamics). Immunoprecipitation of denatured (0.5% SDS at 100°C for 3 min) [3H]AdoMet-labeled proteins was performed as described (10).[3H]AdoMet Labeling. Neutrophil plasma membranes (25 ,g) and cytosol (100 ,ug) were suspended in 50 ,ul of 100 mM NaH2PO4/Na2HPO4 (pH 6.8)