Activation of the Mitogen-activated Protein Kinase Pathway by fMet-Leu-Phe in the Absence of Lyn and Tyrosine Phosphorylation of SHC in Transfected Cells*

The chemotactic peptide f-Met-Leu-Phe (fMLP) stimu- lates leukocyte functions through binding and activation of a specific G-protein-coupled formyl peptide re- ceptor (FPR). Recent studies have shown that stimulation of neutrophils with fMLP induces the acti- vation of two members of the mitogen-activated protein kinase (MAP kinase) family, ERK1 and ERK2, through mechanisms that are not completely understood but may involve the phosphorylation of the adapter protein SHC by the Src-related kinase Lyn. In this study, transfected fibroblasts expressing the rabbit FPR were used to investigate further the role of Lyn and SHC phosphorylation in fMLP-stimulated MAP kinase activation. Stimulation of transfected cells with fMLP resulted in the time- and dose-dependent increase in tyrosine phos- phorylation and activation of ERK1 and ERK2 and the activation of MEK, the MAP kinase/ERK kinase. The activation of both ERKs and MEK was inhibited by pre-incubation of the cells with pertussis toxin, indicating that activation was dependent upon a G i /G o -like protein that couples to the receptor. Our data also show that, unlike neutrophils, FPR-transfected fibroblasts do not express the Src-related kinase Lyn. In the absence of Lyn, fMLP stimulation did not result in an increased tyrosine phosphorylation of the adapter protein SHC, whereas it was still able to induce MAP kinase activation.

The chemotactic peptide f-Met-Leu-Phe (fMLP) stimulates leukocyte functions through binding and activation of a specific G-protein-coupled formyl peptide receptor (FPR). Recent studies have shown that stimulation of neutrophils with fMLP induces the activation of two members of the mitogen-activated protein kinase (MAP kinase) family, ERK1 and ERK2, through mechanisms that are not completely understood but may involve the phosphorylation of the adapter protein SHC by the Src-related kinase Lyn. In this study, transfected fibroblasts expressing the rabbit FPR were used to investigate further the role of Lyn and SHC phosphorylation in fMLP-stimulated MAP kinase activation.

Stimulation of transfected cells with fMLP resulted in the time-and dose-dependent increase in tyrosine phosphorylation and activation of ERK1 and ERK2 and the activation of MEK, the MAP kinase/ERK kinase. The activation of both ERKs and MEK was inhibited by preincubation of the cells with pertussis toxin, indicating that activation was dependent upon a G i /G o -like protein
that couples to the receptor. Our data also show that, unlike neutrophils, FPR-transfected fibroblasts do not express the Src-related kinase Lyn. In the absence of Lyn, fMLP stimulation did not result in an increased tyrosine phosphorylation of the adapter protein SHC, whereas it was still able to induce MAP kinase activation. These data suggest that Lyn and SHC are not the only upstream signals for activation of the MAP kinase/ ERK pathway by fMLP and demonstrate the potential application of the FPR-transfected cells for the delineation of additional signaling mechanisms stimulated by fMLP.
Binding of an agonist to its receptor leads to activation and expression of specific responses via triggering of a cascade of signaling events that most likely include the activation of kinases and phosphorylation of regulatory proteins. Formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (fMLP), 1 which are potent chemoattractants for polymorpho-nuclear neutrophils (PMN), bind to seven transmembrane helical G-protein-coupled receptors on the surface of these cells (1). Early responses induced by fMLP are mediated by activation of the functionally associated G-protein(s), which in turn activate effectors such as phospholipase C␤ for the generation of inositol 1,4,5-trisphosphate and diacylglycerol. Mobilization of Ca 2ϩ from intracellular stores and activation of various isoforms of protein kinase C are then observed (2,3). Recently, evidence was provided that additional signaling pathways involving kinases other than protein kinase C are triggered by fMLP (4,5). We and others (6 -9) demonstrated that fMLP stimulates the increase in tyrosine phosphorylation of several proteins. Two of these proteins that became rapidly and transiently activated were identified as members of the family of mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs) (7,8,10). Activation of the MAP kinases requires dual phosphorylation on tyrosine and threonine by a family of multifunctional MAP kinase kinases or MAP kinase/ERK kinases (MEK), which are themselves activated by MEK kinases, a cascade of kinase events collectively known as the MAP kinase pathway (11). In several cell types, including in neutrophils (12), activation of the MAP kinase cascade has been shown to occur through the GTP-binding protein, p21 ras (13). The molecular mechanisms leading to Ras activation are best understood with tyrosine kinase growth factor receptors (RTK) such as that for epidermal growth factor or nerve growth factor and have been shown to involve the participation of adapter proteins such as SHC (14) and Grb2 (15). This novel class of molecules lacks enzymatic activity but bears src homology (SH) domains that confer them the ability to bind phosphotyrosine (SH2) or proline-rich region (SH3), resulting in coupling to other signaling molecules (16). Binding of a ligand to the RTK induces dimerization and transphosphorylation of the RTK, resulting in the tyrosine phosphorylation of SHC isoforms and their concomitant association with the activated RTK (17). Tyrosine-phosphorylated SHC isoforms subsequently bind to the SH2 domain of Grb2, which is itself constitutively associated with the guanine nucleotide exchange factor, Sos (18,19). This results in the formation of a SHC-Grb2-Sos complex at the plasma membrane where Sos catalyzes the Ras guanine nucleotide exchange (17). The activation of the MAP kinase pathway by G i -protein-coupled receptors is less well understood and has been shown to occur through Ras-dependent (11,12,20) and -independent pathways (21,22). Several studies identified the G ␤␥ subunits of the hetero-trimeric G proteins as the primary mediator of Ras activation (11,23,24), and, recently, the tyrosine phosphorylation of SHC through a tyrosine phosphorylation event mediated by the G ␤␥ subunits and formation of the SHC-Grb2-Sos complex were also implicated in Ras activation by G i -coupled receptors (25)(26)(27). Expression of a specific G ␤␥ inhibitor, i.e. a G ␤␥ binding peptide derived from ␤ARK1 (23, 28), reduced MAP kinase activation and abolished SHC tyrosine phosphorylation stimulated by the activation of the ␣2-adrenergic receptor (25). The G ␤␥ -mediated activation of MAP kinase was also blocked by disruption of the SHC-Grb2-Sos complex and by tyrosine kinase inhibitors (23,25). In addition, thyrotropin-releasing factor and endothelin-1 induced the tyrosine phosphorylation of SHC and SHC-Grb2-Sos complex formation (26,27), and the ␣-thrombin receptor activated p60 src tyrosine kinase (29). Recent studies in neutrophils demonstrated that fMLP induced the activation of Lyn and the association and tyrosine phosphorylation of SHC (30), suggesting that SHC and Lyn might play a role in the activation of the Ras/MAP kinase pathway. Transfected cells have recently been used by others (23,24,(31)(32)(33) for the identification of signaling pathways that are otherwise difficult to characterize in the native receptor-expressing cells. The rabbit fMLP receptor (FPR) has been cloned and transfected into mouse fibroblasts. Stably transfected cells displayed high affinity fMLP binding, Ca 2ϩ mobilization, sensitivity to pertussis toxin (PT), and homologous desensitization, demonstrating that the transfected FPR could mediate transmembrane signaling and was coupled to a G i -like protein (34,35). In this report, we used these cells to determine whether fMLP triggered the increased tyrosine phosphorylation and activation of the MAP kinase pathway and to investigate the role of Lyn and SHC phosphorylation in activation of this pathway. We show that ERK1, ERK2, and MEK are activated through a G-protein-coupled pathway that does not involve the Src-related kinase Lyn and the adapter protein SHC.

EXPERIMENTAL PROCEDURES
Cell Culture, Preparation of Neutrophils, and Treatment with Pertussis Toxin-Formyl peptide receptor (FPR) and vector (V)-transfected mouse fibroblasts were maintained in the presence of 175 g/ml G418 (Geneticin, Life Technologies, Inc.) (34). PMN were purified from the peripheral blood of normal volunteer adults as approved by the Committee on Human Investigation at Childrens Hospital Los Angeles, as described (7). FPR cells were incubated for 16 h in medium containing 100 ng/ml PT (List Biological Laboratories, Campbell, CA) while PMN (5 ϫ 10 6 /ml) were treated with 1 g/ml PT for 2 h at 37°C.
Preparation of Cell Extracts and Immunoblotting-Confluent cells were harvested with trypsin-free dissociation buffer (Life Technologies, Inc.), washed in phosphate-buffered saline and used directly. FPR and V cells or PMN (1 ϫ 10 7 /ml) were suspended in Krebs-Ringer phosphate glucose buffer, warmed to 37°C, and stimulated for various times. The reaction was stopped by a quick spin, and total cellular extracts were prepared as described (7). Proteins (30 g) were subjected to SDSpolyacrylamide gel electrophoresis (PAGE), electrotransferred to nitrocellulose membranes, and analyzed by immunoblotting with anti-phosphotyrosine (PY) (4G10, Upstate Biotechnology, Inc., Lake Placid, NY) and MAP kinase (SC-94, Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibodies, as described (7).
MAP Kinase Immunoprecipitation and MBP Kinase Activity-MAP kinases were immunoprecipitated under native conditions with agarose-conjugated peptide antibodies to either ERK1 (SC-93) or ERK2 (SC-154) (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After stimulation, cells were lysed on ice for 20 min (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 100 mM NaF, 25 mM ␤-glycerophosphate, 10 g/ml leupeptin and aprotinin) and centrifuged (15,000 ϫ g, 20 min), and the cleared lysates, adjusted for protein content (400 -600 g), were incubated for 2 h with the antibodies. The agarose beads were subsequently washed with high salt (2 M NaCl) and lysis buffers. The ERK immune complexes were either released with 2 ϫ sample buffer (36) for analysis by SDS-PAGE and immunoblotting with PY or MAP kinase antibodies as above or used to determine kinase activity with myelin basic protein (MBP) as substrate (37). After washing in kinase buffer (10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 10 mM MgCl 2 ), agarose beads were incubated for 30 min at 30°C with 50 l of kinase buffer containing 1 mg/ml MBP, 50 M ATP, 10 mM p-nitrophenyl phosphate, and 5 Ci of [␥-32 P]ATP (10 Ci/l). The reaction was stopped by addition of 5 ϫ Laemmli buffer and boiling for 5 min. The 32 P-labeled MBP was resolved on 16% SDS-PAGE, and gels were exposed for autoradiography or the MBP protein band was excised from the gels and incorporated radioactivity was measured by Cerenkov counting.
MEK Immunoprecipitation and Kinase Activity-Cells were stimulated and lysed on ice as above, and cleared lysates (500 g of protein) were incubated with 10 l of MEK1 antibody (Transduction Laboratories, Lexington, KY) at 4°C for 2 h prior to incubation with 30 l of protein A-Sepharose (Pharmacia Biotech Inc.) for 1 h. Immune complexes were recovered by centrifugation, washed several times with lysis buffer, and released in 2 ϫ sample buffer for analysis by immunoblotting or used for kinase activity measurement using kinase-inactive recombinant ERK1 protein (K71A)ERK1 (rERK Ϫ , Upstate Biotech. Inc., Lake Placid, NY) as substrate (38). Immune complexes were incubated for 30 min at 30°C with 20 l of agarose-conjugated rERK Ϫ and 50 l of kinase buffer (50 mM Tris-HCl, pH 8, 10 mM MgCl 2 , 5 mM dithiothreitol, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM NaF, 25 M ATP, and 10 Ci of [␥-32 P]ATP) with frequent and strong agitation. The reaction was stopped by addition of 5 ϫ Laemmli buffer, and the amount of 32 P incorporated into rERK Ϫ was determined after SDS-PAGE by autoradiography and Cerenkov counting.
SHC Immunoprecipitation-PMN or FPR cells were lysed as above after stimulation with fMLP for 1 or 5 min, respectively, and the cleared lysates were incubated overnight with 15 l of polyclonal SHC antibody (Transduction Laboratories) and 1 h with 30 l of protein A-Sepharose. Immune complexes were recovered by centrifugation, washed with lysis buffer, and analyzed by SDS-PAGE and immunoblotting with monoclonal antibodies to PY (Upstate Biotechnology, Inc.), SHC, Grb2, and Lyn (Transduction Laboratories).

RESULTS
fMLP Induces the Pertussis Toxin-sensitive Activation of ERK1 and ERK2 in FPR Cells-Immunoblotting of whole protein extracts from FPR and V cells with an anti-phosphotyrosine antibody demonstrated that several protein bands were phosphorylated on tyrosine in the absence of stimulation. No substantial difference in the basal level of tyrosine phosphorylation was observed between FPR and vector cells, except for a protein band migrating in the 36 -37-kDa area that exhibited a higher level of tyrosine phosphorylation in FPR cells than in V cells where it appeared to be constituted of a doublet band. Stimulation of FPR cells with 10 Ϫ7 M fMLP did not alter the tyrosine phosphorylation of this protein band but clearly resulted in the rapid and transient increase in the tyrosine phosphorylation state of a protein band with an approximate molecular mass of 42 kDa that peaked by 5 min (Fig. 1A). A slight increase in a 45-kDa protein band that also peaked at 5 min was observed, although it was more difficult to assess since this band was not always well resolved from a tyrosine-phosphorylated band that migrated slightly slower and did not change with stimulation (compare Fig. 1, A and B). Low levels of tyrosine phosphorylation of the p45 and p42 protein bands were noted in the absence of stimulation in both FPR and V cells that varied between experiments and increased with incubation at 37°C; however, these levels were consistently increased in FPR cells stimulated with fMLP whereas no discernible change was ever observed in V cells under similar conditions (Fig. 1A). In contrast, two protein bands with similar molecular masses exhibited increased tyrosine phosphorylation after treatment of V cells with phorbol myristate acetate, supporting the notion that the lack of response of V cells to fMLP was due to the lack of the receptor. None of the protein bands previously reported to exhibit increased tyrosine phosphorylation after fMLP stimulation in PMN (6 -9) were observed in fMLP-stimulated FPR cells, suggesting that these proteins are either only expressed in myeloid cells or that their fMLP Receptor Signaling and MAP Kinase Pathway phosphorylation is dependent upon a myeloid-specific pathway. The increase in tyrosine phosphorylation of the 45-and 42-kDa proteins was dose-dependent and appeared to peak between 10 Ϫ8 and 10 Ϫ7 M fMLP (Fig. 1B). Based on our previous studies, these proteins were likely to be the two members of the MAP kinase family, ERK1 and ERK2. Immunoblotting with a MAP kinase antibody recognizing both isoforms showed the presence of two proteins co-migrating with the tyrosine-phosphorylated proteins and exhibiting a shift in migration after fMLP stimu-lation of FPR cells, more readily detected with ERK2 (Fig. 1A). No such shift was seen in V cells, unless the cells were stimulated with phorbol myristate acetate. Incubation with pertussis toxin (PT) inhibited the increase in tyrosine phosphorylation of the MAP kinase/ERKs, similarly in FPR cells as in PMN (Fig.  1C). This is consistent with previous reports showing inhibition of the fMLP-stimulated increase in intracellular Ca 2ϩ by PT (34,39). To more clearly demonstrate that the 45-and 42-kDa proteins with increased tyrosine phosphorylation were the activated MAP kinase/ERKs, we performed immunoprecipitation with two antibodies that more specifically recognize either ERK1 or ERK2, as seen in Fig. 2A. Immunoblotting of the immunoprecipitates with the PY antibody showed low levels of

FIG. 2. The MAP kinase/ERKs are activated by fMLP in transfected cells.
A, immunoprecipitation with ERK1 and ERK2 antibodies and immunoblotting of the immunoprecipitates with anti-MAP kinase and anti-PY antibodies. FPR cells were treated with buffer or 10 Ϫ7 M fMLP for various times prior to lysis, and immunoprecipitation was performed as described under "Experimental Procedures." The positions of the heavy (H) chain of the immunoglobulin and that of the MAP kinase/ERKs are indicated by an arrow. Note that the antibodies recognized more specifically either ERK1 or ERK2 and that resting cells demonstrated a low level of tyrosine phosphorylation of these proteins while stimulation with fMLP induced a time-dependent increase in their tyrosine phosphorylation and a shift in the migration of ERK2. B, MBP kinase activity of ERK1 and ERK2 immunoprecipitates from FPR and V cells. Immunoprecipitates were prepared from V and FPR cells stimulated with fMLP for various times and incubated in kinase buffer containing MBP as described under "Experimental Procedures." Shown is a representative autoradiogram of the radioactivity incorporated into MBP, after separation by SDS-PAGE. C, Cerenkov counting of the radioactivity incorporated into MBP after excision of the protein from the gel.
fMLP Receptor Signaling and MAP Kinase Pathway tyrosine phosphorylation of ERK1 and ERK2 in control cells; however, both isoforms increased their tyrosine phosphorylation after treatment with fMLP with a time course similar to that observed with whole cell extracts ( Fig. 2A). A shift in the electrophoretic mobility of ERK2 was also observed when the immunoprecipitates were probed with the MAP kinase antibody, consistent with phosphorylation and activation of the kinase activity of the MAP kinase/ERKs. To confirm this, we determined the kinase activity of the ERK1 and ERK2 immunoprecipitates, using MBP as substrate. No increase in 32 P incorporation into MBP was observed in V cells while a timedependent increase was detected after fMLP stimulation of FPR cells. The activity peaked at 5 min for both ERK1 and ERK2 and was greater in the ERK2 immunoprecipitates (Fig.  2, B and C). This time course of activation was slightly more prolonged than that observed in PMN where the activity peaked at 1 min and was close to basal levels by 5 min (7,8). Thus, these data demonstrate that transfection of the FPR conferred to the fibroblasts the ability to activate ERK1 and ERK2 by coupling the FPR to a PT-sensitive G-protein, probably of the G i type.
The MAP Kinase Kinase/MEK Is Activated by fMLP in the Transfected Cells-To determine whether MEK was activated by fMLP in FPR cells, lysates were efficiently immunoprecipitated with a MEK1 antibody, as determined by immunoblotting (Fig. 3A), and the kinase activity of the immunoprecipitated MEK was determined using its specific substrate, a kinase-negative ERK (rERK Ϫ ). fMLP induced the time-dependent activation of MEK, with the activity peaking between 1 and 5 min and decreasing thereafter (Fig. 3, B and C). These data confirm previous reports with PMN, showing activation of MEK by fMLP using different methodologies (12,40,41). In addition, treatment with PT inhibited the activation of MEK, as demonstrated by the return to basal levels of rERK Ϫ phosphorylation. As with the MAP kinases, a low level of activity was observed both in V and FPR cells in the absence of stimulation. Thus, these data indicate that a G-protein-linked receptor can trigger the activation of the MAP kinase pathway.
Absence of Lyn Does Not Prevent MAP Kinase Activation-Recent studies showed that the Src-related kinase Lyn becomes activated upon PMN treatment with fMLP (30,42) and associates with the adapter protein SHC (14), which undergoes tyrosine phosphorylation (30). It was suggested that the Lyn-SHC signaling pathway might provide a link between the FPR and the Ras/MAP kinase pathway. We compared the expression of SHC and Lyn in FPR cells and PMN. As expected, immunoblotting with an antibody against Lyn demonstrated the presence of Lyn in PMN as two strong protein bands migrating approximately at 54 and 56 kDa; however, no immunoreactivity was detected with two different antibodies in FPR lysates, indicating that Lyn is not expressed in these cells (Fig.  4A). Strong expression of the three SHC isoforms (p46, p52, and p66) was observed in FPR cells while PMN had very low level of expression of mainly p52 SHC (Fig. 4A). SHC was immunoprecipitated from PMN and FPR lysates with a polyclonal antibody, and the immunoprecipitates were probed with

FIG. 3. MEK activation by fMLP in transfected cells is inhibited by pertussis toxin.
A, FPR cells were immunoprecipitated with a monoclonal antibody to MEK1, and the immunoprecipitates were analyzed by immunoblotting with a polyclonal MEK1 antibody. B, MEK activation after stimulation of V and FPR cells with 10 Ϫ7 M fMLP for various times and treatment in the presence or absence of pertussis toxin (PT). The immunoprecipitates were incubated in kinase buffer in the presence of recombinant kinase negative ERK1 (rERK Ϫ ) as described under "Experimental Procedures." The rERK Ϫ was separated by SDS-PAGE (10%), and gels were exposed to x-ray films. C, Cerenkov counting of the radioactivity incorporated into the rERK Ϫ after excision of the protein band from the gel. fMLP Receptor Signaling and MAP Kinase Pathway PY, SHC, Lyn, and Grb2 antibodies. Low amount of p52SHC was found in immunoprecipitates from PMN lysates. While consistent with the low level expression of SHC in PMN, the amount in the immunoprecipitates appears lower. This is due to the use of a monoclonal antibody to probe the blot that was not as efficient as the polyclonal antibody used for the immunoprecipitation and expression in lysates. The latter could not be used because of strong interference with the immunoglobulin heavy chain. A phosphotyrosine protein that appeared to co-migrate with p52 SHC and exhibited an increase in tyrosine phosphorylation after fMLP treatment was observed when immunoblotting the SHC immunoprecipitates with the PY antibody, indicating that fMLP stimulates the tyrosine phosphorylation of SHC in PMN. Another tyrosine phosphorylated protein, migrating at 44 kDa, was also detected when longer exposure times were used, and its identity has not been determined so far (Fig. 4B). In addition, Grb2, which associates with tyrosine-phosphorylated SHC through its SH2 domain (43), and Lyn were also detected in the SHC immunoprecipitates; however, recovery of either Grb2 or Lyn in the SHC immunoprecipitates was the same in control and stimulated PMN, suggesting that fMLP did not alter the extent of their association with SHC. Neither band of the Lyn doublet coincided with the p52 tyrosine-phosphorylated protein. Immunoblotting of the SHC immunoprecipitates from FPR lysates with the PY antibody showed a faint tyrosine-phosphorylated protein band that did not precisely co-migrate with any of the SHC isoforms and did not change its tyrosine phosphorylation after fMLP treatment, suggesting that this protein might not be SHC despite the large amount of SHC proteins immunoprecipitated from these cells (Fig. 4C). This protein did not react either with the Lyn antibody. Very little Grb2 was recovered in the SHC immunoprecipitates, consistent with the apparent lack of tyrosine phosphorylation of SHC. Thus, in the absence of Lyn, fMLP stimulation of FPR-transfected cells did not result in an increase in tyrosine phosphorylation of the SHC isoforms. DISCUSSION We demonstrate here that stimulation with fMLP of fibroblasts transfected with the formyl peptide receptor results in the pertussis toxin-sensitive activation of ERK1, ERK2, and MEK in the absence of Lyn and tyrosine phosphorylation of SHC, suggesting that Lyn and SHC are not essential for activation of the MAP kinase pathway. These data support previous reports that documented the activation of the MAP kinase pathway by G i -protein-coupled receptors (20,22,23,44,45) and suggest that more than one signal is necessary to induce maximal activation of the MAP kinase pathway by fMLP.
Previous studies have indicated that the G ␤␥ subunits might participate in the activation of the MAP kinase pathway (23,25,28). A possible scenario could be envisioned where G ␤␥ subunits activate an Src-like tyrosine kinase that phosphorylates SHC, allowing the formation of SHC-Grb2-Sos complex and the activation of the Ras/MAP kinase pathway. Our data, however, show that the lack of tyrosine phosphorylation of SHC by fMLP, which appears to be dependent upon the presence of a tyrosine kinase activity absent in FPR cells (presumably that of Lyn), did not prevent the fMLP-induced activation of ERKs and MEK in these cells. This suggests that SHC and Lyn are not the only upstream signals and that there exists an additional pathway for fMLP-induced activation of MAP kinase. This notion is supported by the observation that MAP kinase activation was only reduced by blocking SHC phosphorylation (23,25). Furthermore, expression of G ␤␥ alone produced only a 2-fold increase in MAP kinase activation compared with the severalfold increase induced by ␣2-adrenergic receptor (25). Similar observations were made when analyzing the activation of MAP kinase by the C5a receptor, another seven transmembrane domain receptor which couples to G i and G 16 , using HEK293-transfected cells (46).
While MAP kinase activation occurred in the absence of Lyn and SHC in the transfected FPR cells, we noted subtle differences in the extent and time course of activation of the MAP kinases in these cells compared with PMN, i.e. a similarly rapid activation but much slower return to basal levels and a less robust increase in activation. This suggests a differential mode of regulation of the MAP kinase cascade and supports the notion that more than one pathway might be involved. An hypothesis may be proposed where activation of the MAP kinases could occur through a pathway in which Lyn, SHC phosphorylation, and the ensuing complex do not play a role, as observed in the FPR-transfected cells, but full activation would require Ras activation through SHC. Both pathways are likely to occur after activation of the G-proteins since PT inhibited MAP kinase activation both in FPR cells and in neutrophils. Other candidate activators may include various isoforms of protein kinase C, p65 PAK (47), which has also been described in neutrophils (48) and PI 3-kinase (49). The activation of the MAP kinase pathway through G i2 was shown to involve at least in part the release of its ␤␥ subunits as well as activation of PI 3-kinase (50). Alternatively, differences at the level of the receptor and G-proteins might explain our data, even though similar deactivation was previously observed in these cells (34). At present, it is not clear whether the multifunctional phosphatases that specifically dephosphorylate the MAP kinases in some cells (51) might play a role here. Nevertheless, data presented above clearly indicate that the absence of Lyn and the lack of tyrosine phosphorylation of SHC do not abrogate the ability of the fMLP receptor to stimulate MAP kinase activation and suggest the presence of an as yet incompletely understood signaling pathway.