CHEMOTACTIC FACTOR-INDUCED RECRUITMENT …mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol

CHEMOTACTIC FACTOR-INDUCED RECRUITMENT AND ACTIVATION OF

TEC FAMILY KINASES IN HUMAN NEUTROPHILS.

IMPLICATION OF PI 3KINASES

Lachance, G., Levasseur, S. and *Naccache, P.H.

CIHR Group on the Molecular Mechanisms of Inflammation, Centre de recherche

en rhumatologie et immunologie, Centre de recherche du CHUL, Department of

Medicine, Laval University, Québec, Canada

*To whom correspondence should be addressed:

CHUL du CHUQ, Room T 1-49

2705 Boulevard Laurier

Québec, Québec

Canada, G1V 4G2

Tel: (418) 654-2772

Fax:(418) 654-2765

Email: [email protected]

Running title: Activation of Tec kinases in human neutrophils

Supported in part by grants from the Canadian Institutes for Health Research.

ABSTRACT

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

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Lachance, G. et al. Activation of Tec kinases in human neutrophils Page 2

hand, the link between the G protein-coupled receptors (to which belong the receptors for

chemotactic factors) 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 PI 3kinase inhibitor wortmannin, and

the recent description of PH domain-containing 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 kinase assays.

The activation of Tec was inhibited by pertussis toxin as well as by wortmannin. The

results of this study provide direct evidence for the implication of Tec family kinases in

the responses of human neutrophils to chemotactic factors. They also suggest that one of

the link between G protein-coupled receptors and tyrosine kinases depends on the

activation of PI 3kinases and the generation of phosphatidylinositol 3,4,5-trisphosphate.

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INTRODUCTION

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 which have been shown to negatively modulate various responses to

these agonists including adherence and locomotion (2) and the stimulation of the NADPH

oxidase (3-6). Additionally, the stimulation of several tyrosine kinases following the

ligation of chemoattractant receptors has also been reported (7-14). 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 protein-coupled receptors (17-19). 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 Gi subfamily) and link them to the modulation of

the activity of tyrosine kinases and phosphatases remain undefined in spite of 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) 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

3kinase isoform, p110γ, 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).


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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)P3 ) (25,26). The recent

characterization of a novel family of tyrosine kinases containing PH domains, the Tec

family (27-32) provided a potential novel link between the PI 3kinase and the tyrosine

phosphorylation-dependent pathways. This hypothesis was corroborated in several

tyrosine kinase-dependent systems including the TCR, the BCR 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-kinases (33-44). 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.


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The present investigation was initiated in order to examine the possible 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 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-coupled family to downstream

tyrosine phosphorylation-dependent signalling events.

MATERIALS AND METHODS

Antibodies

Anti-Bmx (N-16), anti-Btk (C-20) and anti-Tec (M-20) and immunizing peptides were

obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Tec (06-561),

used for immunoprecipitations, and anti-phosphotyrosine (05-321, clone 4G10) were

obtained from Upstate Biotechnology, Inc (Lake Placid, NY, USA).

Reagents

Adenosine-5'-triphosphate (ATP) magnesium salt, diisopropylfluorophosphate (DFP),

phenylmethylsulfonyl fluoride (PMSF), dimethyl sulfoxide (DMSO), fMet-Leu-Phe and

wortmannin were from Sigma-Aldrich Canada (Oakville, Ontario, Canada). The

enhanced chemiluminescence reagents used for Western blotting were purchased from

DuPont Pharmaceuticals (Missisauga, Ontario, Canada). Dextran T-500, Glutathion-

Sepharose and protein A-Sepharose were obtained from Pharmacia Biotech (Dorval,

Québec, Canada). Ficoll-Paque and Mg2+-free Hanks’ balanced salt solution (HBSS)

were from Wisent Canadian Laboratories (St-Bruno, Québec, Canada) and pertussis toxin

was from List Biologicals (Campbell, CA, USA). 3-[(3-Cholamidopropanyl) dimethyl-

ammonio]-1-propanesulfonate (CHAPS) was obtained from Boehringer Mannheim,

(Germany). SAM68-GST (sc-4249) was obtained from Santa Cruz (Santa Cruz, CA,

USA).

Neutrophil purification


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Venous blood was collected in isocitrate anticoagulant from healthy adult volunteers and

neutrophils were purified sterilely as previously described (47). Neutrophils at 2x107

cells/ml were resuspended in HBSS containing 1.6 mM CaCl2, but no magnesium, and

pre-treated with 1mM DFP for 10 minutes at room temperature before any additional

manipulation.

Immunoblotting

Neutrophil suspensions (250 µl of 2x107 cells/ml) were added to an equal volume of

boiling 2x Laemmli sample buffer (1x is 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 5% β-

mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-

phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol blue)

and boiled for 7 minutes. Samples were then subjected to 8% SDS-polyacrylamide gel

electrophoresis and transferred to Immobilon polyvinylidene difluoride membranes

(Millipore Corp, Bedford, MA, USA). Immunoblotting was performed using anti-Bmx

(1/1000), anti-Btk (1/1000) and anti-Tec (1/1000) in TBS-Tween with 2% gelatin.

Immunoprecipitation under native conditions

After stimulation, neutrophils were centrifuged and the cell pellets 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 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml soybean trypsin

inhibitor, 1 mM PMSF, 1.5 mM DFP), for 3 minutes on ice. The insoluble material was

discarded after centrifugation at 13,000 g at 4°C during 10 minutes and the lysate was

harvested. The supernatants were precleared with protein A-Sepharose at 4°C for 30

minutes. The lysates were then incubated at 4°C with 4 µg of anti-Tec antibodies for 2

hours followed by 1 hour incubation with protein A-Sepharose beads. The beads were

collected and washed three times with cold lysis buffer. Laemmli sample buffer (2X) was

added to the beads, which were boiled for 7 minutes.


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In vitro autophosphorylation assay

Neutrophils (2x107 cells/ml) were stimulated for 60 seconds with 10-7 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 MnCl2, 2 mM MgCl2, 1 mM paranitrophenylphosphate and 50 µM 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 (2X) was added to the beads which were boiled for 7 minutes. 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 towards SAM68-GST

This assay was carried out exactly like the in vitro autophosphorylation assay described

above, except that 0.5 µg SAM68-GST was added to the kinase buffer. The supernatants

of the kinase assays were collected and incubated for 1 hour at 4°C in lysis buffer in 1:2

proportions with glutathion-Sepharose beads. The glutathion Sepharose beads were

washed twice and boiled for 7 minutes in Laemmli sample buffer (2X). 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 µl at 4x107 cells/ml) were incubated or not with 200 nM wortmannin

for 10 minutes or 1 µg/ml pertussis toxin for 2 hours. The cell suspensions were pre-

warmed at 37°C for 5 minutes and stimulated with 10-7M fMet-Leu-Phe or an equal

volume of diluent (DMSO) for different times. Stimulations were stopped by sonication

for 20 seconds. One ml of cold KCl-HEPES relaxation buffer (100 mM KCl, 50 mM

HEPES, 5 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 5 µg/ml aprotinin, 5 µg/ml

leupeptin, 1 mM orthovanadate, 2.5 mM PMSF, 1 mM DFP, pH 7.2) was added rapidly.

The lysates were centrifuged for 10 minutes at 13,000 g. Unbroken cells and nuclei were

discarded and supernatants were then ultracentrifuged for 45 minutes at 180,000 x g in a

Beckman TL-100 ultracentrifuge using a TL-100.4 rotor. The membrane pellets were

resuspended in 80 µl of solubilisation buffer (250 mM phosphate buffer, pH 6.8, 300 mM

NaCl, 2.5% SDS, 0.25 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM


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orthovanadate). 5 µl of each sample was discarded for protein quantification and 75 µl of

2x Laemmli sample buffer was added to each sample and boiled for 7 min. Proteins were

quantified by ELISA with Coomassie Protein Assay Reagent (Pierce, Rockford, IL,

USA). 10 µg of each condition was subjected to 8% SDS-polyacrylamide gel

electrophoresis and transferred to Immobilon polyvinylidene difluoride membranes.

RESULTS

1/ 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 Figure 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 weights of 58 and

66 kDa. Alternatively spliced Tec isoforms of these molecular weights have previously

been described (31).

2/ 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)P3 (27,28,50,51). We had

previously shown that the stimulation of human neutrophils by chemotactic factors was

associated with the translocation of p110γ 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 Figure 2. The chemotactic factor was found to induce a

rapid translocation of the 66 kDa isoform of Tec which was evident within 5 seconds of

stimulation with fMet-Leu-Phe. Maximal levels of membrane-associated Tec was

reached at 10-15 seconds post-stimulation (Figure 2, panel A). It should be noted that the


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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 (Figure 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 (Figure 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 Figure 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 seconds of the addition of fMet-Leu-Phe and was maintained for the next 60

seconds. 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 seconds, lysed under native

conditions as described in 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 (Figure 4, panel B), or an exogenous

substrate, SAM68-GST (Figure 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 Figure 4, panel B and C show that stimulation

of neutrophils by fMet-Leu-Phe increased the activity of Tec towards itself as well as


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towards SAM68-GST as evidenced by a time-dependent increase in tyrosine

phosphorylation.

3/ 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 3kinases, we examined next whether it was affected by preincubation of

the cells with the PI 3-kinase inhibitor wortmannin. In these experiments, neutrophils

were pre-treated for 10 minutes with 200 nM wortmannin and then stimulated with fMet-

Leu-Phe. The effects of wortmannin on the stimulated translocation of Tec are illustrated

in Figure 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 towards itself and SAM68-GST observed following the addition of

fMet-Leu-Phe to neutrophils (Figure 5, panels B, C and D, respectively).

DISCUSSION

Tyrosine phosphorylation-dependent signaling cascades play critical roles in the initation

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.


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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 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, 7-transmembrane spanning 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 (p110γ) (22,23)

responsive to stimulation by fMet-Leu-Phe (24) and by the identification of a PH domain-

containing tyrosine kinase family (the Tec kinases) (27-32).


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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 domain

of Btk (28,50) and Tec (56) exhibit a high degree of specificity towards PtdIns(3,4,5)P3, it

is tempting to speculate that the membrane recruitment of the Tec kinases stimulated by

fMet-Leu-Phe is a consequence of the activation of PI 3-kinase(s) and the formation of

PtdIns(3,4,5)P3.

It is noteworthy in this respect to point out that stimulation of human neutrophils causes a

very rapid formation of PtdIns(3,4,5)P3 (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 seconds), p110γ was the predominant PI 3-kinase species activated by fMet-

Leu-Phe (24). The kinetics of the activation of p110γ, of the formation of PtdIns(3,4,5)P3

and of the Tec kinases are strikingly similar, the three of which being close to maximal

within 5 to 10 seconds of the addition of chemotactic factors. While not a proof of a

direct causal relationship exists between the activation of p110γ 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


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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 (57-61) and the

observation that neutrophil recruitment into inflammatory sites was defective in p110γ

knock-out mice (62-64). They suggest that these findings may be accomodated by a

model in which Tec kinases play a role downstream from the activation of p110γ.

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 protein-coupled receptors to the tyrosine phosphorylation-dependent

signalling pathways, a poorly understood element of cell biology.

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FIGURE LEGENDS

Figure 1: Presence of Tec family kinases in human neutrophils. Whole neutrophil

extracts were processed for immunoblotting as described in Materials and Methods. The

membranes were blotted with the indicated antibodies, pre-neutralized (+) or not (-) with

the immunizing peptides. The data shown are from a representative experiment, repeated

at least three times, with identical results, on separate cell preparations.

Figure 2: Stimulated translocation of Tec family kinases upon stimulation by fMet-Leu-

Phe in human neutrophils. Neutrophil suspensions were stimulated with 10-7 M fMet-

Leu-Phe for the time indicated in the legends following which the cells were processed

for membrane preparation and immunoblotting as detailed in 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.

Figure 3: Pertussis toxin-sensitivity of the translocation of Tec family kinases induced by

fMet-Leu-Phe. In these experiments, the cells (107 cells/ml) were incubated with 1_g/ml

pertussis toxin for 2 hours in the presence of 1 mg/ml bovine serum albumin before being

stimulated with fMet-Leu-Phe for the indicated times as indicated in 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.

Figure 4: Phosphorylation status and enzymatic activity of Tec in fMet-Leu-Phe-

stimulated human neutrophils. Panel A: Neutrophil suspensions were stimulated with

fMet-Leu-Phe (10-7 M) for the indicated times. They were then lysed under native

conditions, Tec was immunoprecipitated and the precipitates processed for

immunoblotting with anti-phosphotyrosine (upper panel) or anti-Tec antibodies as

described in Materials and Methods. Panel B: Neutrophil suspensions were stimulated

with fMet-Leu-Phe (10-7 M) for 1 minute. They were then lysed under native conditions,

Tec was immunoprecipitated and the precipitates transferred to a kinase buffer as

described in Materials and Methods and the assays carried out for the indicated times.


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The level of tyrosine phosphorylation of Tec was then monitored by immunoblotting. The

membranes were reblotted with an anti-Tec antibody. Panel C: Neutrophil suspensions

were stimulated with fMet-Leu-Phe (10-7 M) for 1 minute. They were then lysed under

native conditions, Tec was immunoprecipitated and the precipitates transferred to a

kinase buffer to which SAM68-GST was added as described in Materials and Methods

and the assays carried out for the indicated times. SAM68-GST was then isolated and its

level of tyrosine phosphorylation was monitored as described in 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.

Figure 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

minutes as indicated. They were then stimulated with fMet-Leu-Phe (10-7 M) for 1 minute

following which the membrane translocation, in situ phosphorylation and in vitro

autophosphorylation and tyrosine kinase activity towards SAM68-GST were monitored

as described in Materials and Methods using a 3 minute 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.


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Figure 1


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Figure 2


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Figure 3


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Figure 5


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Geneviève Lachance, Sylvain Levasseur and Paul H Naccachehuman neurophils. Implications of PI 3kinases

Chemotactic factor-induced recruitment and activation of TEC family kinases in

published online April 8, 2002J. Biol. Chem.

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CHEMOTACTIC FACTOR-INDUCED RECRUITMENT …mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol