©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Protein-coupled Chemoattractant Receptors Regulate Lyn Tyrosine KinaseShc Adapter Protein Signaling Complexes (*)

(Received for publication, May 9, 1995)

Andrzej Ptasznik (1) Alexis Traynor-Kaplan (2) Gary M. Bokoch (1)(§)

From the  (1)Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037 and the (2)Department of Medicine, University of California San Diego, San Diego, California 92103

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Receptors for chemoattractants that direct the migration of phagocytic leukocytes to sites of injury/infection also modulate many other leukocyte functions that are critical to the inflammatory response. These chemoattractant receptors, members of the G protein-coupled heptahelical receptor family, have been classically linked to cell activation via phospholipase C, calcium, and protein kinase C. We show here that activation of the N-formyl peptide chemoattractant receptor stimulates an additional protein kinase C-independent pathway through the Src-related tyrosine kinase, Lyn, in human neutrophils. We demonstrate that activation of Lyn is associated with binding to the Shc adapter protein, which becomes phosphorylated on tyrosine residues. This interaction appears to be mediated via the Shc SH2 domain. Complexes of phosphorylated Lyn and Shc with phosphatidylinositol 3-kinase are rapidly formed in stimulated neutrophils, correlating with phosphatidylinositol 1,4,5-trisphosphate formation and cell activation. This signaling pathway involving a Src-related kinase and the Shc adapter protein provides a potential mechanism linking chemoattractant receptors to downstream events involving Rac activation and NADPH oxidase. Regulation of Shc by G protein-coupled receptors may also allow these receptors to modulate the activity of the Ras/mitogen-activated protein kinase cascade.


INTRODUCTION

The regulation of leukocyte function through the action of chemoattractants and chemokines constitutes a critical aspect of the body's immune response against microbial pathogens. Chemoattractant receptors are members of the seven transmembrane-spanning receptor family that utilizes heterotrimeric GTP-binding proteins to transduce signals to the interior of the cell(1) . The chemoattractant receptors of neutrophils, including those for the N-formyl peptides (NFP), (^1)C5a, leukotriene B(4), interleukin-8, etc., have been shown to couple to ``classic'' signal transduction pathways involving the activation of phospholipase C, formation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, and the subsequent mobilization of Ca and activation of protein kinase C(2, 3) . It has only been recently that evidence pointing to the existence of additional signaling pathways has been obtained.

Phosphatidylinositol 3-kinase, which generates the putative signaling molecule PIP(3), is activated by chemoattractants(4, 5, 6) . PIP(3) formation is required for the stimulation of oxidant production by the phagocyte respiratory burst (NADPH) oxidase(7, 8) , and has been implicated in assembly of the neutrophil actin cytoskeleton(9) . Neutrophils contain both the p85/p110 PI 3-kinase, whose regulation by chemoattractant receptors is not understood(10, 11) , as well as a novel G protein beta subunit-regulated PI 3-kinase whose contribution to neutrophil activation has not yet been established(11) . The mitogen-activated protein kinase cascade also becomes stimulated during the course of acute neutrophil activation by chemotactic agents(12, 13) . Levels of Ras GTP have been shown to increase, and activity of the downstream kinases MEK and mitogen-activated protein kinase (MAPK) is stimulated in response to C5a and NFP(14, 15, 16) . The mechanisms utilized by chemoattractant receptors to activate these pathways is unknown.

Recently published reports have shown that a number of G protein-coupled receptors can activate the MAPK cascade through Ras-dependent mechanisms(14, 15, 16, 17, 18, 19) . Since these receptors are not known to couple to previously defined pathways for Ras activation, the means by which these receptors stimulate Ras function remain to be determined. In the present paper, we describe a novel pertussis toxin-sensitive, staurosporine-insensitive signaling mechanism used by chemoattractant receptors in which a nonreceptor tyrosine kinase, Lyn, and an SH2 domain-containing adapter protein, Shc, couple the N-formyl peptide receptor to PI 3-kinase and, potentially, to activation of the Ras/MAPK cascade and the respiratory burst oxidase.


MATERIALS AND METHODS

Immunoprecipitation Experiments

Neutrophils (>98% pure) were prepared from blood freshly drawn from human volunteers by isolation on Percoll cushions, as described previously(20) . Cells (1 10^8/ml) were suspended in Krebs-Ringer Hepes, pH 7.4, containing 5.5 mM glucose, divided into 1-ml incubations, and stimulated with either buffer (0.1 mg/ml bovine serum albumin in phosphate-buffered saline) or 1 µM fMetLeuPhe for the indicated times or routinely for 1 min in single-time point determinations. Incubations were terminated by rapid centrifugation in a table-top Microfuge E (Beckman) and placed on ice after resuspension in 500 µl of lysis buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na(3)VO(4), 1% Nonidet P-40 with 1 mM phenylmethylsulfonyl fluoride, 0.25 units/ml aprotonin, 10 µg/ml leupeptin, 1 µM pepstatin). Lysates (10 min on ice) were cleared by centrifugation, and the supernatant was incubated with the indicated antibody: 5 µl of anti-Lyn (Upstate Biotechnology Inc.), 10 µl of anti-Shc, and 10 µl of anti-Grb2 (both from Signal Transduction Laboratories) for 4 h at 4 °C. 30 µl of a 1:1 suspension of Protein A-Sepharose beads was added for 30 min, and then the beads were washed twice with lysis buffer and once with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM Na(3)VO(4). In the experiments of Fig. 3, antibodies were precoupled to Protein A-Sepharose beads as described in (21) prior to use.


Figure 3: Shc is present in Lyn precipitates from activated cells. Neutrophils were incubated for 1 min with 1 µM NFP (+) or control buffer(-) and then immunoprecipitated with anti-Lyn or anti-Shc antibody, as described under ``Materials and Methods.'' Precipitates were blotted for Shc as described. +/- in lane 1 indicates that an identical result was obtained +/- NFP. The position of Shc is indicated by the arrows. Results are representative of at least two similar experiments.



Assay of Kinase Activity

To measure kinase activity, Lyn precipitates were washed as indicated and then incubated in 25 mM Hepes, pH 7.1, 10 mM MnCl(2), 1 µM ATP, and 10 µCi [-P]ATP (DuPont NEN, 6000 mCi/mmol). 5 µg of acid-denatured enolase was added as an exogenous substrate in some experiments. Kinase assays were carried out for 2 min at room temperature, and then the reaction was stopped by the addition of Laemmli sample buffer and boiling. Samples were analyzed by 8% SDS-polyacrylamide gel electrophoresis, with autoradiography on Kodak X AR-5 film with enhancing screen at -70 °C and quantitated using the Personal Densitometer from Molecular Dynamics.

Treatment with Inhibitors

Neutrophils were incubated at 37 °C for 1.5-2.5 h with or without 1 µg/ml pertussis toxin (List Biological Laboratories) prior to stimulation with 1 µM NFP for 1 min. The effectiveness of toxin treatment was monitored by determining the ability of the cells to respond to NFP with O generation during the course of the toxin incubation(22) . Cells were treated with 50 nM staurosporine for 5 min at 37 °C prior to stimulation to inhibit protein kinase C activity. These conditions had been established to totally block oxidant production in prior experiments(22) .

Preparation/Use of GST-Shc SH2

The Shc SH2 domain expressed as a GST fusion protein (a kind gift of T. Pawson, University of Toronto) was purified on glutathione-Sepharose beads, per manufacturer's recommendation (Pharmacia Biotech Inc.). Lysates from stimulated (1 µM fMetLeuPhe) or unstimulated cells were incubated with 30 µl of GST-coated bead controls or Shc-SH2 beads for 2 h at 4 °C, and then the incubation was stopped and treated as described for immunoprecipitations.

Assay of PI 3-Kinase

PI 3-kinase activity was measured in immunoprecipitates directly on the washed Sepharose beads. The reaction was carried out for 10 min at room temperature in a buffer containing 40 mM Hepes, pH 7.2, 6 mM MgCl(2), and 1 mM EDTA, 20 µg of PI (Avanti Polar Lipids), 8 µg of PIP(2) (Calbiochem), 10 µM ATP, and 10 µCi [-P]ATP (6000 Ci/mmol, DuPont NEN). Following the incubation, the reaction was stopped with methanol plus 2.4 N HCl, 1/1 (v/v), and lipids were extracted and analyzed as described previously(5, 9) .


RESULTS

Activation of Lyn Tyrosine Kinase by NFP Receptor

Stimulation of the neutrophil respiratory burst by chemoattractants is blocked by tyrosine kinase inhibitors, suggesting that one or more tyrosine kinase regulates the activity of critical signaling pathways utilized by such agents(23, 24, 25) . The Rac2 GTP-binding protein is a direct regulator of the respiratory burst oxidase(26) , and we have recently shown that blockade of Rac activation can account for the inhibitory effect of tyrosine kinase inhibitors on the NADPH oxidase in N-formyl peptide-stimulated cells(22) . To evaluate the possible role of nonreceptor tyrosine kinases in the oxidase activation process, we screened NFP-stimulated human neutrophils for the activities of known Src-like kinases(27) . We observed that activity of the Lyn tyrosine kinase was significantly increased in NFP-stimulated cells, as previously reported(28) . Fig. 1A shows a representative time course for Lyn activation by NFP. Both Lyn autophosphorylation and the ability of Lyn to stimulate phosphorylation of the exogenous substrate protein enolase were increased within seconds, with a maximum activity detected at 1 min. Lyn activity declined slowly after the 1 min peak and returned to base line by 10 min. Autophosphorylation of both of the two major Lyn isoforms (29) present in neutrophils (53 and 56 kDa) was detected. There was no change in the actual levels of either Lyn protein during this time period, as determined by Western blotting. We also determined that there was no detectable Lck kinase activity in our cell preparations, ruling out mononuclear cell contamination as a possible source of Lyn activity. Lyn was phosphorylated on tyrosine residues, as determined by blotting by anti-phosphotyrosine antibodies, and the level of Lyn phosphotyrosine increased by 3-fold within 1 min of stimulation by NFP.


Figure 1: Time course of phosphorylation of Lyn and Shc, and of PI 3-kinase activity in Lyn, Shc, and Grb2 immunoprecipitates in NFP-stimulated neutrophils. PanelA, bullet, Lyn autophosphorylation; an identical time course was obtained using phosphorylation of enolase as the assay; up triangle, filled, Shc tyrosine phosphorylation; immunoprecipitates were blotted for phosphotyrosine using the 4G10 monoclonal antibody (UBI) at 1:1000 dilution. PanelB, , , , PI 3-kinase activity in Lyn, Shc, and Grb2 precipitates, respectively. The initial activity at t = 0 min was set to 100% in each panel. The results shown are representative of four experiments.



Lyn Binds to PI 3-Kinase in Stimulated Cells

The time course for Lyn activation correlates well with the some of the earliest signaling events known to occur after chemoattractant stimulation. In particular, this time course was identical to that previously determined for activation of PIP(3) formation in human neutrophils(5, 9) . Lyn has been shown to directly couple to PI 3-kinase in B lymphocytes activated through the B cell antigen receptor (30, 31, 32, 33) . We observed (Fig. 1B) that we could detect PI 3-kinase activity in Lyn immunoprecipitates from NFP-stimulated human neutrophils. Similarly, we could detect the presence of the p85 subunit of PI 3-kinase in these precipitates by Western blotting (not shown). The amount of PI 3-kinase specifically associated with Lyn ranged from 5 to 25% in various experiments, comparing the amount of PI 3-kinase activity in the Lyn precipitates with the total activity measured in p85 subunit antibody precipitates. The time course of the association of PI 3-kinase with Lyn was identical to that observed for activation of Lyn tyrosine kinase activity (Fig. 1A). In contrast, we observed no PI 3-kinase activity in control immunoprecipitates with Lck antibody. These data indicate that there is a close temporal association between Lyn activation and the binding of Lyn to PI 3-kinase in NFP-stimulated neutrophils.

Regulation of Lyn by the B cell receptor and other cytokine receptors involves the interaction of Lyn with a specific Src kinase binding motif present on these receptors(31, 32, 33) . This so-called ARH1 motif is not present in the NFP receptor or other chemoattractant receptors, suggesting that stimulation of Lyn does not result from direct binding to receptor. Consistent with this, we could detect no Lyn activity in NFP receptor precipitates from stimulated neutrophils(34) . Stimulation of Lyn activity was dependent upon activation of the G(i) protein coupled to the NFP receptor, as pertussis toxin effectively blocked Lyn activation (Fig. 2). In contrast, this response was not inhibitable by the protein kinase C inhibitor staurosporine, indicating that this was not a downstream effect resulting from protein kinase C activation by the receptor (Fig. 2). Coupled with the fact that the Ca ionophore A23187 did not stimulate Lyn activity (not shown), these data indicate that Lyn activation is not a secondary response resulting from activation of the classic chemoattractant signaling mechanisms.


Figure 2: Effect of inhibitors on activation of the Lyn and Shc pathways. Cells were treated with pertussis toxin or staurosporine prior to stimulation with NFP as described under ``Materials and Methods.'' PanelA, effects on Lyn-stimulated phosphorylation of enolase; panelB, effects on tyrosine phosphorylation of Shc. Results shown are averages of two experiments.



Lyn Interacts with the Shc Adapter Protein

We examined whether any of the recently described ``adapter'' molecules involved in growth factor signaling could potentially participate in linking the NFP receptor to Lyn and perhaps other signaling pathways. The mammalian Shc proteins (46-, 52-, and 66-kDa forms) are such molecules, containing a carboxyl-terminal Src homology 2 (SH2) domain and an adjacent proline-rich SH3 binding motif(35) . The Shc proteins are substrates for a considerable number of receptor tyrosine kinases, and are also phosphorylated by nonreceptor tyrosine kinases that are recruited (via the ARH1 motif) to receptors that have no intrinsic tyrosine kinase activity themselves (i.e. the T-cell, interleukin-2 and Steel factor receptors)(36) . The tyrosine phosphorylation of Shc induces the formation of a stable complex between Shc and the SH2 domain of the Grb2 adapter protein, which results in the recruitment of the Ras exchange factor Sos to the plasma membrane where it can stimulate Ras activation(37, 38, 39) . We observed that Shc became tyrosine-phosphorylated after stimulation of neutrophils by NFP (Fig. 1A). Maximal phosphorylation occurred within 1 min of leukocyte activation. Treatment of neutrophils with pertussis toxin to block NFP receptor coupling to G(i) effectively inhibited Shc phosphorylation, while the protein kinase C inhibitor staurosporine did not inhibit this response (Fig. 2). As with Lyn activation, the calcium ionophore A23187 had no influence on Shc phosphorylation.

We evaluated whether tyrosine-phosphorylated Shc interacted with other signaling proteins in NFP-stimulated neutrophils. The possible association of Lyn with Shc was probed by looking for the presence of Shc in Lyn immunoprecipitates and vice versa. The presence of Shc in anti-Lyn immunoprecipitates was observed by Western blotting (Fig. 3). A large percentage of total immunoprecipitatable Shc appeared to associate with Lyn in NFP-stimulated cells. In contrast, the presence of Shc could not be detected in Hck immunoprecipitates from activated cells (not shown). Consistent with the data of Fig. 3, we observed phosphorylated 53- and 56-kDa forms of Lyn when kinase assays were performed in Shc immunoprecipitates from NFP-activated neutrophils, but not in precipitates from unactivated cells (data not shown).

Shc Binds to Lyn through its SH2 Domain

In order to determine if the interaction of Lyn and Shc occurred through the SH2 domain of Shc, we purified a GST-ShcSH2 domain fusion protein that we used as a probe for Lyn in neutrophil lysates. Fig. 4shows that we could specifically precipitate phosphorylated 53- and 56-kDa proteins with the ShcSH2 fusion protein and that this association only took place in NFP-stimulated cells. The phosphorylated 53- and 56-kDa proteins comigrated with Lyn, and the identity of these bands as Lyn was confirmed by preclearing the activated neutrophil lysate with anti-Lyn antibody: this eliminated over 90% of the phosphorylated 53- and 56-kDa proteins associated with GST-ShcSH2. In contrast, preclearing with an antibody against another Src-like kinase of similar size (Hck) had no effect. Approximately 30% of the total Lyn activity in the lysates bound to the GST-ShcSH2 beads under these conditions. These findings suggest that the ShcSH2 domain binds to a tyrosine-containing motif on Lyn that becomes phosphorylated after stimulation of Lyn by NFP receptor activation.


Figure 4: Lyn binds to the Shc-SH2 domain in stimulated neutrophils. Neutrophils were incubated for 1 min with 1 µM NFP or control buffer and then allowed to interact with a GST Shc-SH2 fusion protein as described, and the presence of Lyn was determined using Lyn kinase assay in the absence of exogenous enolase. Lane1, anti-Lyn antibody precipitate as a control to indicate the total amount of Lyn present; lanes2 and 3, unstimulated cells incubated with control GST-coated beads or Shc-SH2 beads, respectively; lanes4 and 5, NFP-stimulated cells with control or Shc-SH2 beads, respectively. Lane6 represents the NFP-stimulated cell lysate precleared with anti-Lyn antibody and then incubated with the Shc-SH2 beads. Results shown are representative of two experiments.



PI 3-Kinase Co-precipitates with Shc in Activated Cells

As shown in Fig. 1B, we found that anti-Shc immunoprecipitates also contained PI 3-kinase activity. The association of PI 3-kinase activity with Shc was temporally identical to that seen for interaction of PI 3-kinase with Lyn, suggesting that these events were highly correlated. By contrast, we could detect no PI 3-kinase activity in immunoprecipitates of Grb2, a second adapter protein, under the same conditions. Physical interactions between Lyn, Shc, and PI 3-kinase thus take place in a similar time frame and are correlated with tyrosine phosphorylation of Lyn and Shc.

Association of Additional Proteins with Tyrosine Phosphorylated Shc

The association of active Shc with other potential signaling molecules was examined by looking for the presence of other phosphotyrosine-containing proteins in Shc immunoprecipitates from NFP-stimulated cells (Fig. 5). We consistently observed the presence of tyrosine-phosphorylated proteins of 145 and 25 kDa in the Shc precipitates from activated human neutrophils. These proteins were not cross-reactive with antibodies to any known signaling proteins of similar size, and their identity remains to be established.


Figure 5: Tyrosine-phosphorylated Shc associates with other proteins in chemoattractant-stimulated neutrophils. Neutrophils were incubated for 1 min with 1 µM NFP (+) or control buffer(-) and then precipitated with anti-Shc antibody, as described. Precipitates were blotted for phosphotyrosine as described in Fig. 1. The positions of the unidentified associated 25- and 145-kDa proteins are indicated by * and**, respectively. Results are representative of three experiments.




DISCUSSION

We demonstrate here a pertussis toxin-sensitive interaction of the NFP chemoattractant receptor with a Src-related tyrosine kinase, Lyn, and the Shc adapter protein during acute neutrophil stimulation. Lyn, Shc, and PI 3-kinase rapidly associate in NFP-stimulated cells. This association correlates with enhanced kinase activity of Lyn, tyrosine phosphorylation of Shc, and elevation of PIP(3) levels(5) . These events are not stimulated by elevations in intracellular Ca, nor blocked by staurosporine, and they are thus not secondary to activation of protein kinase C. As a further indication of the involvement of Lyn and Shc in acute neutrophil signaling by chemoattractants, we also observed that the levels of both Lyn and Shc increased (3- and 3.5-fold, respectively, by Western blotting) in HL60 cells differentiated with Me(2)SO into neutrophil-like cells, providing circumstantial support for their role in the signaling responses of mature cells.

The most likely signaling paradigm for the interactions we have just described is suggested by previous observations that Shc becomes active through tyrosine phosphorylation by c-Src(36) . We propose that the NFP receptor couples to Lyn, stimulating its autophosphorylation and kinase activity toward substrates. One of these substrates is the Shc adapter protein, which binds to phosphorylated Lyn via its SH2 domain and becomes tyrosine phosphorylated. Shc can then bind to and regulate PI 3-kinase activity. The basis for the interaction of Shc with PI 3-kinase remains to be defined, but it could involve the interaction of Shc directly with SH2 or SH3 domains on the p85 regulatory subunit of PI 3-kinase. When we evaluated whether PI 3-kinase bound to Shc-SH2 beads in stimulated neutrophils, we observed only slight increases in binding over controls, suggesting that Shc does not use this motif to bind to phosphotyrosines on the PI 3-kinase. This would be consistent with the observations of Vlahos et al.(10) , who found no stimulated PI 3-kinase activity in phosphotyrosine precipitates from activated neutrophils. Lyn has also been shown to interact directly with the PI 3-kinase p85 subunit through the binding of an SH3 domain on p85 to a proline-rich binding site on Lyn(33) . Alternatively, it is possible that the association of PI 3-kinase with Shc immunoprecipitates in stimulated cells is indirect, with Shc binding directly to Lyn, and Lyn then binding to the p85 subunit. This scenario has not been previously described and would place Shc upstream or parallel to Lyn in the transduction sequence from the NFP receptor, suggesting that Shc couples the receptor to the Lyn/PI 3-kinase pathway.

Based upon the time course and pertussis toxin sensitivity of the responses described herein, it is possible that regulation of PI 3-kinase activity through the Lyn/Shc interaction is involved with stimulation of downstream activities such as oxidant generation and chemotaxis. The requirement for PI 3-kinase activity in the signaling pathway leading to NADPH oxidase activation by chemoattractant receptors is indicated by studies with PI 3-kinase inhibitors(7, 8) . Accumulating evidence suggests that PI 3-kinase may be involved in activation of the Rac GTP-binding protein(40, 41, 42) . Rac is a critical regulator of oxidant production via the NADPH oxidase(26) . Furthermore, our results raise the probability that Shc adapter proteins play a role in linking chemoattractant receptors to the Ras pathway. The known ability of phosphorylated Shc to bind to Grb2 and the associated Ras GTP/GDP exchange protein, Sos, provides a potentially direct link between chemoattractant receptor signaling and the activation of Ras, which has been recently demonstrated in human neutrophils(14, 15) . The signaling paradigm we have described could also provide a mechanism to explain the ability of other G protein-coupled, seven transmembrane-spanning receptors to stimulate Ras activity. The endothelin and thyrotropin-releasing hormone receptors, also members of the heptahelical, G protein-coupled receptor family, have been recently reported to interact with Shc(43, 44) . Proof for the importance of the molecular interactions we have identified in intact neutrophil signaling will require the development of effective means to manipulate these cells at the molecular level; this capability is currently limited by the refractoriness of myeloid cells to conventional transfection methodologies.

In addition to possible interactions with Grb2, the presence of an unidentified 145-kDa tyrosine-phosphorylated protein was observed in Shc precipitates (Fig. 5). This protein may be the same as a 145-kDa protein recently reported to bind to Shc in response to polypeptide growth factors in hematopoietic cell lines (45) and in fibroblasts (46) . The function of this protein is not yet known, but it interacts with a unique domain on Shc that binds phosphotyrosine and is likely to be an additional mediator of chemoattractant signaling.

Activation of Lyn and Shc through the NFP receptor is pertussis toxin-sensitive, indicating that the signaling cascade is initiated at the level of the G(i) protein(s) coupled to this and other chemoattractant receptors. While the beta subunits of G(i) have been shown to mediate the transduction of chemoattractant receptor signals to phospholipase Cbeta(47, 48) , little is known about the downstream effector targets of the G(i)alpha subunit in this system. It is tempting to speculate that the G(i)alpha subunit might couple to the Lyn/Shc pathway to transduce additional downstream signals. Future studies directed at the mechanisms by which G proteins couple to Lyn and Shc activation should prove informative, as will further investigation of downstream signaling elements linked to this pathway.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM39434 (to G. M. B.) and (NIH M01RR00833) the General Clinical Research Center at The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Immunology-IMM14, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8217; Fax: 619-554-8218.

(^1)
The abbreviations used are: NFP, N-formyl peptides; PIP(3); phosphatidylinositol 1,4,5-trisphosphate; PI, phosphatidylinositol; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase.


ACKNOWLEDGEMENTS

We thank Benjamin P. Bohl and Brian Lee for excellent technical assistance and Antonette Lestelle for secretarial assistance.


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