Chemotactic Factor-induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASES*

The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established. On the other hand, the link between the G pro-tein-coupled receptors (to which the receptors for chemotactic factors belong) and the activation of tyrosine kinases is very poorly characterized. Based on previous observations indicating that the stimulation of tyrosine phosphorylation was sensitive to inhibition by the phosphatidylinositol 3-kinase inhibitor wortmannin and the recent description of pleckstrin homology domain-con-taining tyrosine kinases (the Tec family), we have examined the potential implication of the latter in the responses of human neutrophils to chemotactic factors. The results obtained indicate firstly that several members of the Tec family of tyrosine kinases are expressed in human neutrophils, including Tec, Btk, and Bmx. Stimulation of the cells with fMet-Leu-Phe led to a rapid activation of Tec as indicated by its translocation to a membrane fraction and to increases in its in situ level of tyrosine phosphorylation and its capacity to tyrosine phosphorylate itself or an exogenous substrate (SAM68-GST) in in vitro sible involvement of Tec family kinases in the responses of human neutrophils to chemotactic factors. The results obtained demonstrate that several Tec family kinases are expressed in mature blood neutrophils (Tec, Btk, Bmx). Stimulation of human neutrophils by chemotactic factors led to the G protein-and PI 3-kinase-dependent activation of multiple members of the Tec family kinases. These results provide evidence for a novel pathway linking chemotactic factor receptors of the G protein-coupledfamilytodownstreamtyrosinephosphorylation-dependent signaling events.

Stimulation of human polymorphonuclear neutrophil leukocytes (neutrophils) by chemoattractants is accompanied by a distinct tyrosine phosphorylation signature (1). The functional significance of this response has been established using a variety of tyrosine kinase inhibitors that have been shown to negatively modulate various responses to these agonists including adherence and locomotion (2) and the stimulation of the NADPH oxidase (3)(4)(5)(6). Additionally, the stimulation of several tyrosine kinases following the ligation of chemoattrac-tant receptors has also been reported (7)(8)(9)(10)(11)(12)(13)(14). Finally, the absence of Src kinases in mice knock-out models has been shown to result in impaired signaling and responsiveness in neutrophils (15,16).
Chemoattractant receptors, including those to chemokines and to lipid mediators, belong to the superfamily of G proteincoupled receptors (17)(18)(19). Accordingly, the stimulation of the tyrosine phosphorylation induced by the occupation of these receptors is known to be sensitive to inhibition by pertussis toxin (1). The steps that follow the activation of the G proteins (presumably members of the G i subfamily) and link them to the modulation of the activity of tyrosine kinases and phosphatases remain undefined despite the critical importance of this knowledge to the development of a complete understanding of G protein-coupled receptor signaling and of the regulation of the functional responsiveness of human neutrophils.
The results of a previous study have provided evidence that the stimulation of tyrosine phosphorylation by chemoattractants in human neutrophils was sensitive to the phosphatidylinositol 3-kinase (PI 3-kinase) 1 inhibitor, wortmannin (20). This observation was originally difficult to reconcile with the characteristics of PI 3-kinases known at the time, which were themselves controlled by the tyrosine phosphorylation of their regulatory p85 subunit (21). Since then, the existence of a G protein-dependent PI 3-kinase isoform, p110␥, has been established (22,23). Its presence and activation in human neutrophils, in preference over that of p85/p110, by chemoattractants has also been reported (24).
The end result of the activation of PI 3-kinases in situ is the generation of phosphatidylinositol (3, 4, 5)-trisphosphate (PtdIns(3,4,5)P 3 ) (25,26). The recent characterization of a novel family of tyrosine kinases containing PH domains, the Tec family (27)(28)(29)(30)(31)(32), provided a potential novel link between the PI 3-kinase and the tyrosine phosphorylation-dependent pathways. This hypothesis was corroborated in several tyrosine kinase-dependent systems including the T cell receptor, the B cell receptor, and CD32 in which the activation of various members of the Tec family was found to be secondary to that of the p85/p110 PI 3-kinase (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The report of the involvement of Btk in thrombin-stimulated platelets (45,46) represents the only evidence of the relationship of the Tec family of tyrosine kinases with the activation of G protein-coupled receptors to date.
The present investigation was initiated to examine the pos-sible involvement of Tec family kinases in the responses of human neutrophils to chemotactic factors. The results obtained demonstrate that several Tec family kinases are expressed in mature blood neutrophils (Tec, Btk, Bmx). Stimulation of human neutrophils by chemotactic factors led to the G proteinand PI 3-kinase-dependent activation of multiple members of the Tec family kinases. These results provide evidence for a novel pathway linking chemotactic factor receptors of the G protein-coupled family to downstream tyrosine phosphorylationdependent signaling events.
Neutrophil Purification-Venous blood was collected in isocitrate anticoagulant from healthy adult volunteers, and neutrophils were purified sterilely as described previously (47). Neutrophils at 2 ϫ 10 7 cells/ml were resuspended in Hanks' balanced salt solution containing 1.6 mM CaCl 2 but no magnesium and pretreated with 1 mM DFP for 10 min at room temperature before any additional manipulation.
Immunoprecipitation under Native Conditions-After stimulation, neutrophils were centrifuged, and the cell pellets were lysed by adding cold lysis buffer (10 mM Tris-HCl, pH 7.4, 137.2 mM Nacl, 1 mM EDTA, 0.6% CHAPS, 2 mM orthovanadate, 10 g/ml leupeptin, 10 g/ml aprotinin, 50 g/ml soybean trypsin inhibitor, 1 mM PMSF, 1.5 mM DFP) for 3 min on ice. The insoluble material was discarded after centrifugation at 13,000 ϫ g at 4°C during 10 min, and the lysate was harvested. The supernatants were precleared with protein A-Sepharose at 4°C for 30 min. The lysates were then incubated at 4°C with 4 g of anti-Tec antibodies for 2 h followed by 1 h incubation with protein A-Sepharose beads. The beads were collected and washed three times with cold lysis buffer. Laemmli sample buffer (2ϫ) was added to the beads, which were boiled for 7 min.
In Vitro Autophosphorylation Assay-Neutrophils (2 ϫ 10 7 cells/ml) were stimulated for 60 s with 10 Ϫ7 M fMet-Leu-Phe. Tec was then immunoprecipitated as described above. The Tec immunoprecipitates were washed three times in lysis buffer, resuspended at 4°C in kinase buffer (50 mM HEPES, pH 7.6, 10 mM MnCl 2 , 2 mM MgCl 2 , 1 mM paranitrophenylphosphate, and 50 M ATP) and transferred at 37°C for different times. The reactions were stopped by a quick spin in a microcentrifuge, and the beads were washed twice in cold lysis buffer. Laemmli sample buffer (2ϫ) was added to the beads, which were boiled for 7 min. The samples were then electrophoresed and transferred to Immobilon polyvinylidene difluoride membranes, which were blotted with the anti-phosphotyrosine or with the anti-Tec antibodies.
In Vitro Kinase Activity toward SAM68-GST-This assay was carried out exactly like the in vitro autophosphorylation assay described above except that 0.5 g of SAM68-GST was added to the kinase buffer. The supernatants of the kinase assays were collected and incubated for 1 h at 4°C in lysis buffer in 1:2 proportions with glutathione-Sepharose beads. The glutathione-Sepharose beads were washed twice and boiled for 7 min in Laemmli sample buffer (2ϫ). The samples were then electrophoresed and transferred to Immobilon polyvinylidene difluoride membranes, which were blotted with the anti-phosphotyrosine or with the anti-Tec antibodies.
Membrane Preparation and Translocation Assays-Neutrophils (500 l at 4 ϫ 10 7 cells/ml) were incubated or not with 200 nM wortmannin for 10 min or 1 g/ml pertussis toxin for 2 h. The cell suspensions were prewarmed at 37°C for 5 min and stimulated with 10 Ϫ7 M fMet-Leu-Phe or an equal volume of diluent (Me 2 SO) for different times. Stimulations were stopped by sonication for 20 s. One ml of cold KCl-HEPES relaxation buffer (100 mM KCl, 50 mM HEPES, 5 mM NaCl, 1 mM MgCl 2 , 0.5 mM EGTA, 5 g/ml aprotinin, 5 g/ml leupeptin, 1 mM orthovanadate, 2.5 mM PMSF, 1 mM DFP, pH 7.2) was added rapidly. The lysates were centrifuged for 10 min at 13,000 ϫ g. Unbroken cells and nuclei were discarded, and supernatants were then ultracentrifuged for 45 min at 180,000 ϫ g in a Beckman TL-100 ultracentrifuge using a TL-100.4 rotor. The membrane pellets were resuspended in 80 l of solubilization buffer (250 mM phosphate buffer, pH 6.8, 300 mM NaCl, 2.5% SDS, 0.25 mM PMSF, 5 g/ml aprotinin, 5 g/ml leupeptin, 1 mM orthovanadate). 5 l of each sample was discarded for protein quantification, and 75 l of 2ϫ Laemmli sample buffer was added to each sample and boiled for 7 min. Proteins were quantified by enzyme-linked immunosorbent assay with Coomassie protein assay reagent (Pierce). 10 g of each condition was subjected to 8% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon polyvinylidene difluoride membranes.

Presence of Tec Family Members in Human Neutrophils-
As of yet, the only evidence for the expression in granulocytes of Tec family members is that of the mRNA for Bmx, one member of this family of kinases (48,49). The data shown in Fig. 1 illustrate that Tec, Bmx, and Btk are expressed at the protein level in human peripheral blood neutrophils. These immunoblots indicate that specific staining of proteins of the expected apparent molecular weight was seen with the three antibodies and that this staining was displaced by the appropriate immunizing peptides. It should be noted that two isoforms of Tec were detected with apparent molecular masses of 58 and 66 kDa. Alternatively spliced Tec isoforms of these molecular weights have been described previously (31).
Activation of Tec Family Members by Chemotactic Factors in Human Neutrophils-One of the characteristic features of the activation of Tec family tyrosine kinases is their stimulated translocation to membrane fractions, an event that is thought to result from high affinity interactions of their PH domains with PtdIns(3,4,5)P 3 (27,28,50,51). We had shown previously that the stimulation of human neutrophils by chemotactic factors was associated with the translocation of p110␥ to a crude membrane fraction (24). We therefore examined whether the distribution of Tec family kinases in this fraction was altered upon stimulation of neutrophils by fMet-Leu-Phe. The results of these experiments are illustrated in Fig. 2. The chemotactic factor was found to induce a rapid translocation of the 66-kDa isoform of Tec which was evident within 5 s of stimulation with fMet-Leu-Phe. Maximal levels of membrane-associated Tec were reached at 10 -15 s post-stimulation (Fig. 2, panel A). It should be noted that the 58-kDa isoform of Tec did not appear to be sensitive to stimulation by fMet-Leu-Phe as its distribution in the membrane fraction did not change upon stimulation. Equivalent translocations of Btk and Bmx were also observed ( Fig. 2, panels B and C). Preliminary experiments indicated that interleukin-8 also induced a translocation of Tec, Btk, and Bmx to the membrane fraction (data not shown).
The translocation of Tec family kinases to the membrane fraction induced by stimulation by fMet-Leu-Phe was sensitive to inhibition by pertussis toxin (Fig. 3). Preincubation of the cells with the bacterial toxin decreased to a large extent the stimulated increases in the levels of Tec, Bmx, and Btk that could otherwise be detected in the membrane fractions derived from fMet-Leu-Phe-stimulated cells.
Another index of the activation of tyrosine kinases is their level of in situ tyrosine phosphorylation. The latter can be increased upon stimulation due to autophosphorylation events or secondarily to transphosphorylation by other tyrosine kinases (31). The data shown in Fig. 4, panel A, indicate that stimulation of human neutrophils by fMet-Leu-Phe led to the in situ phosphorylation of Tec. This phosphorylation was detected within 5-15 s of the addition of fMet-Leu-Phe and was maintained for the next 60 s. A similar increase in the tyrosine phosphorylation of Btk was also observed (results not shown). The inability of the available anti-Bmx antibodies to precipitate Bmx (under native or denaturing conditions) prevented us from determining whether the latter was similarly affected upon the stimulation of the cells (data not shown).
We examined next whether the translocation of Tec and the stimulation of its in situ levels of tyrosine phosphorylation were associated with an increase in kinase activity. Neutrophils were stimulated with fMet-Leu-Phe for 60 s and lysed under native conditions as described under "Materials and Methods," and Tec was immunoprecipitated. The immunoprecipitates were then resuspended in kinase buffer, and the kinase activity of Tec was monitored by its ability to phosphorylate itself (Fig. 4, panel B) or an exogenous substrate, SAM68-GST (Fig. 4, panel C). The latter was chosen as it has previously been shown to associate with members of the Tec family of tyrosine kinases (52,53). The results of these experiments, illustrated in Fig. 4, panels B and C, show that stimulation of neutrophils by fMet-Leu-Phe increased the activity of Tec toward itself as well as toward SAM68-GST as evidenced by a time-dependent increase in tyrosine phosphorylation.
PI 3-Kinase Dependence of the Activation of Tec Family Tyrosine Kinases by Chemotactic Factors-The pertussis toxin sensitivity of the stimulated translocation of Tec, Btk, and Bmx indicated that the recruitment of these kinases was secondary to the activation of heterotrimeric G proteins. To test whether this response was up-or downstream of the activation of PI 3-kinases, we examined next whether it was affected by prein- FIG. 4. Phosphorylation status and enzymatic activity of Tec in fMet-Leu-Phe-stimulated human neutrophils. Neutrophil suspensions were stimulated with fMet-Leu-Phe (10 Ϫ7 M) for the indicated times (A). They were then lysed under native conditions, Tec was immunoprecipitated, and the precipitates were processed for immunoblotting (IB) with anti-phosphotyrosine (upper panel) or anti-Tec antibodies as described under "Materials and Methods." Neutrophil suspensions were stimulated with fMet-Leu-Phe (10 Ϫ7 M) for 1 min (B). They were then lysed under native conditions, Tec was immunoprecipitated, and the precipitates were transferred to a kinase buffer as described under "Materials and Methods," and the assays were carried out for the indicated times. The level of tyrosine phosphorylation of Tec was then monitored by immunoblotting. The membranes were reblotted with an anti-Tec antibody. Neutrophil suspensions were stimulated with fMet-Leu-Phe (10 Ϫ7 M) for 1 min (C). They were then lysed under native conditions, Tec was immunoprecipitated, and the precipitates were transferred to a kinase buffer to which SAM68-GST was added as described under "Materials and Methods," and the assays were carried out for the indicated times. SAM68-GST was then isolated, and its level of tyrosine phosphorylation was monitored as described under "Materials and Methods." The membranes were reblotted with an anti-SAM68 antibody. The data shown are from three separate representative experiments, each repeated at least three times, with identical results, on separate cell preparations. In these experiments, the cells (10 7 cells/ml) were incubated with 1 g/ml pertussis toxin for 2 h in the presence of 1 mg/ml bovine serum albumin before being stimulated with fMet-Leu-Phe for the indicated times as indicated under "Materials and Methods." The data shown are from two separate representative experiments, each repeated at least three times, with identical results, on separate cell preparations.
cubation of the cells with the PI 3-kinase inhibitor wortmannin. In these experiments, neutrophils were pretreated for 10 min with 200 nM wortmannin and then stimulated with fMet-Leu-Phe. The effects of wortmannin on the stimulated translocation of Tec are illustrated in Fig. 5, panel A. The results of these experiments demonstrate that wortmannin inhibited to a significant extent the ability of the chemotactic factor to increase the levels of the 66-kDa isoform of Tec associated with membranes. Similar results were obtained with Btk and Bmx (data not shown). Wortmannin also inhibited the increased in situ phosphorylation of Tec as well as the in vitro activity of Tec toward itself and SAM68-GST observed following the addition of fMet-Leu-Phe to neutrophils (Fig. 5, panels B-D, respectively).

DISCUSSION
Tyrosine phosphorylation-dependent signaling cascades play critical roles in the initiation and regulation of the activation of various cell types including that of peripheral blood human neutrophils. The results of the present investigation provide direct evidence for a participation of Tec family kinases in the initial events leading to the activation of the tyrosine phosphorylation signaling pathways in human neutrophils. The recruitment and activation of the Tec kinases was also found to be secondary to the activation of heterotrimeric G proteins and PI 3-kinases.
Various lines of evidence have accumulated during the last decade in support of the role of the tyrosine phosphorylation cascade in the initiation of the functional responsiveness of human neutrophils (see references in the introduction). These include the demonstration of the presence and activation of several classes of tyrosine kinases (54) and phosphatases (55) upon the stimulation of neutrophils by a variety of agonists. This results in a transient increase in the overall level of tyrosine phosphorylation in these cells due to the phosphorylation of a large number of substrates, only some of which have been identified. Additional support for the functional relevance of these biochemical responses was derived from the effects of tyrosine kinase inhibitors, which dramatically affect several critical neutrophil responses such as adhesion, chemotaxis, superoxide generation, and phagocytosis. Finally, knock-out cell lines have provided further confirmation of the role of various tyrosine kinases in the regulation of neutrophil responsiveness in addition to identifying relevant kinase families and establishing the redundant nature of several of these kinases, and in particular, the members of the Src family.
Neutrophil function is critically dependent on their migration to the proper sites (infection, injury). This directed locomotion is dictated by the generation of gradients of a series of chemotactic factors. The latter interact for the most part with members of the G protein-coupled, seven-transmembranespanning surface receptors. All of the known neutrophil chemotactic factors stimulate an increase in the level of tyrosine phosphorylation in these cells (1). This response is known to be mediated by heterotrimeric G proteins as it is inhibited by pertussis toxin. On the other hand, little else is known about the steps that couple the relevant G protein to the tyrosine phosphorylation cascade.
Possible clues as to one such coupling mechanism were provided by the observations that the PI 3-kinase inhibitor wortmannin significantly decreased the level of tyrosine phosphorylation induced by the chemotactic peptide fMet-Leu-Phe (20), by the recent cloning and characterization of a G protein-linked PI 3-kinase isoform (p110␥) (22,23) responsive to stimulation by fMet-Leu-Phe (24), and by the identification of a PH domaincontaining tyrosine kinase family (the Tec kinases) (27)(28)(29)(30)(31)(32).
The present data indicate that several members of the Tec family are expressed in human neutrophils. Immunoblotting revealed the presence of Btk, Bmx, and two isoforms of Tec. Preliminary experiments indicated that Rlk/Txk was not expressed in human neutrophils (data not shown). The presence of Itk in human neutrophils was not investigated. Several indices of activation of Tec kinases by fMet-Leu-Phe were obtained. These include the stimulated translocation to a membrane fraction of all but the low molecular weight Tec isoform (which was constitutively present to a significant extent in this fraction) and the increased in situ levels of tyrosine phosphorylation and in vitro kinase activity of the high molecular weight isoform of Tec and of Btk (data not shown). Taken together, these data provide strong and direct evidence for an involvement of these kinases in the responses of human neutrophils to chemotactic factors.
Of particular relevance to the specific hypothesis tested in this study, wortmannin inhibited all three indices of activation of Tec kinases by fMet-Leu-Phe. These data suggest that the recruitment and stimulation of Tec kinases by fMet-Leu-Phe was secondary to the activation of PI 3-kinases. In view of the facts that Tec kinases are unique among tyrosine kinases in that they contain a PH domain and that the PH domains of Btk (28,50) and Tec (56) exhibit a high degree of specificity toward PtdIns(3,4,5)P 3 , it is tempting to speculate that the membrane recruitment of the Tec kinases stimulated by fMet-Leu-Phe is a FIG. 5. Wortmannin sensitivity of the activation of Tec by fMet-Leu-Phe in human neutrophils. Neutrophil suspensions were pretreated or not with wortmannin for 10 min as indicated. They were then stimulated with fMet-Leu-Phe (10 Ϫ7 M) for 1 min following which the membrane translocation, in situ phosphorylation, and in vitro autophosphorylation and tyrosine kinase activity toward SAM68-GST were monitored as described under "Materials and Methods" using a 3-min incubation during the kinase assays. The data shown are from three separate representative experiments, each repeated at least three times, with identical results, on separate cell preparations. IP, immunoprecipitation.
It is noteworthy in this respect to point out that stimulation of human neutrophils causes a very rapid formation of PtdIns(3,4,5)P 3 (25,26). Furthermore, data were recently obtained indicating that, at least within the time frame examined in the present study (i.e. the first 15 s), p110␥ was the predominant PI 3-kinase species activated by fMet-Leu-Phe (24). The kinetics of the activation of p110␥, of the formation of PtdIns(3,4,5)P 3 , and of the Tec kinases are strikingly similar, and all three of these are close to maximal within 5-10 s of the addition of chemotactic factors. Although not a proof that a direct causal relationship exists between the activation of p110␥ and of Tec kinases, these data are nevertheless consistent with such an interpretation. In the absence of specific inhibitors of the different isoforms of PI 3-kinases, additional direct support for this hypothesis is likely to have to rely on the use of dominant negative mutants of the individual PI 3-kinases, a task that is particularly difficult in the terminally differentiated human neutrophils.
The above data are consistent with the previously reported inhibition of neutrophil chemotaxis by PI 3-kinase inhibitors such as wortmannin and LY294002 (57)(58)(59)(60)(61) and the observation that neutrophil recruitment into inflammatory sites was defective in p110␥ knock-out mice (62)(63)(64). They suggest that these findings may be accommodated by a model in which Tec kinases play a role downstream from the activation of p110␥.
In summary then, the results of the present data provided direct evidence for an involvement of Tec family tyrosine kinases in the responses of human neutrophils to chemotactic factors. The recruitment and activation of Tec kinases was found to be dependent on PI 3-kinase activity. These results shed new light on the link between the activation of G proteincoupled receptors to the tyrosine phosphorylation-dependent signaling pathways, a poorly understood element of cell biology.