Neutrophils play a central role in host protection against infection, killing pathogens by a series of rapid and highly regulated responses. These include chemotaxis, phagocytosis, secretion of anti-microbial agents, and generation of reactive oxygen intermediates (ROI) ()(reviewed by Sha'afi and Molski(1988)). Production of ROI is mediated by a multicomponent enzyme complex, the NADPH oxidase, present in the membranes of neutrophils and other leukocytes (Morel et al., 1991). Functional assembly of the oxidase facilitates the transfer of one electron from cytosolic NADPH to molecular oxygen, producing superoxide. Dismutation of superoxide in turn generates hydrogen peroxide, and both of these molecules can further generate other reactive oxygen intermediates, including hypochlorous acid, hydroxyl radical, and peroxynitrite ion (Halliwell and Gutteridge, 1990). Although the mechanisms whereby NADPH oxidase-derived ROI attack microbial targets are not completely understood, their importance in host defence is highlighted by a rare genetic disorder, chronic granulomatous disease. Patients afflicted with this disorder lack the ability to produce ROI and, as a result, suffer from chronic and recurring infections that can be lethal (Smith and Curnutte, 1991).

Although neutrophils are probably the most efficient source of superoxide, virtually all eukaryotic cells produce ROI, primarily as side products of electron transfer reactions in mitochondria and the endoplasmic reticulum (Halliwell and Gutteridge, 1985). In addition to their microbicidal role in phagocytes, ROI have been suggested to act as signaling molecules in other cells (Schreck and Baeuerle, 1991). In principle, ROI constitute good candidate signaling molecules because they are small, rapidly diffusible, and highly reactive. Moreover, both intra- and extracellular concentrations of ROI can be rapidly scavenged by several enzymes, including superoxide dismutase, catalase, and the glutathione peroxidase system, allowing tight control of ROI concentrations and rapid termination of signals. The notion that reactive, small inorganic molecules can function as intracellular signals is supported by the well established role of nitric oxide in the regulation of vascular tone, neurotransmission, and cell-mediated immune responses (Nathan and Xie, 1994). By comparison, much less is known about the role of ROI in signaling, but suggested targets include the transcription factor NFB (Schreck et al., 1991), tyrosine phosphatases (Hecht and Zick, 1992, Fialkow et al., 1994), and phospholipase A (Zor et al., 1993).

Recent observations suggested a role for ROI in neutrophil signal transduction (Fialkow et al., 1993). Neutrophils stimulated to produce ROI were reported to undergo increased tyrosine phosphorylation of several proteins. Exogenous oxidants were able to mimic this response, whereas anti-oxidants could block it. Several lines of evidence suggested that ROI generated by the NADPH oxidase were responsible for the effect, including the finding that the increased tyrosine phosphorylation failed to occur in neutrophils from patients with chronic granulomatous disease. Inasmuch as tyrosine phosphorylation is an important mediator in the regulation of anti-microbial responses (Berkow and Dodson, 1990; Grinstein and Furuya, 1991), ROI may play an important role in the control of auto/paracrine signaling at sites of inflammation.

The extent of tyrosine phosphorylation is determined by the activity of two competing enzyme families, tyrosine kinases and phosphatases. Earlier in vitro (Hecht and Zick, 1992) and in vivo (Zor et al., 1993) studies have suggested that ROI can inhibit the activity of certain tyrosine phosphatases by oxidation of a conserved cysteine residue within their catalytic domain. Although the inhibition of tyrosine phosphatases may account for the elevated tyrosine phosphorylation induced by ROI, increased activity of tyrosine kinases could conceivably contribute to the response. Indeed, tyrosine kinases have been reported to be activated in lymphocytes by oxidizing agents (Bauskin et al., 1991; Nakamura et al., 1993). For these reasons, we investigated whether endogenous ROI generated by the NADPH oxidase affected the activity of tyrosine kinases in human neutrophils.

EXPERIMENTAL PROCEDURES

Materials

Antibodies

Solutions

Cell Isolation and Permeabilization

SDS-PAGE and Immunoblotting

Immunoprecipitation and Immune Complex Kinase Assay

The kinase activity of immune complexes was determined essentially as described (Burkhardt and Bolen, 1993). In brief, immunoprecipitates were washed with 1 ml of kinase buffer (5 mM MnCl, 20 mM MOPS, pH 7.0), and autophosphorylating activity was assayed by incubation with 25 µl of kinase buffer containing 12.5 µCi of [-P]ATP and 1 µM K-ATP. Where specified, 1 µg of rabbit muscle enolase was included as an exogenous substrate. Samples were incubated at 25 °C in an Eppendorf Thermomixer, and reactions were stopped by the addition of boiling 2 concentrated Laemmli sample buffer. The samples were subjected to SDS-PAGE, and the gels were stained with Coomassie Blue and dried in gel wrap (Biodesign Inc.). Dried gels were used for direct quantitation of radioactivity with a Molecular Dynamics PhosphorImager using Imagequant software or were subjected to radiography with an intensifying screen.

To study the effect of oxidizing agents on the tyrosine kinase activity of hck, immunoprecipitates of this kinase were isolated from untreated, electroporated cells. After washing, immune complexes were treated for 30 min with 1 mM diamide or 1 mM hydrogen peroxide at 30 °C while shaking in a Thermomixer. As above, identical aliquots were used in parallel for immunoblotting and in vitro kinase assay. To study the effect of reducing agents on hck, immune complexes from GTPS-stimulated cells were treated with 20 mM NAC or 1 mM dithiothreitol for 30 min at 37 °C and processed as above.

To study the role of tyrosine phosphorylation in hck activation, immunoprecipitates obtained from GTPS-treated cells were incubated at 37 °C for 30 min with or without 2 µg/ml of T-cell phosphatase. Aliquots of the beads were used for kinase assays and for immunoblotting with anti-phosphotyrosine antibodies to confirm the effectiveness of dephosphorylation by T-cell phosphatase.

Other Methods

RESULTS

NADPH Oxidase-derived ROI Induce Tyrosine Phosphorylation

As shown in Fig. 1A, the addition of GTPS and NADPH to permeabilized cells induced the accumulation of phosphotyrosine on a number of proteins, as determined by immunoblotting (cf. lanes 1 and 4). Treatment of the electroporated cells with GTPS and NADPH alone was found to have little effect (lanes 2 and 3). The stimulatory effect of GTPS or NADPH was moderated by the presence of active tyrosine phosphatases. This is indicated by the pronounced enhancement in phosphotyrosine accumulation noted when vanadate, a phosphatase inhibitor, was included during stimulation (Fig. 1A). For this reason, 10 µM sodium orthovanadate was included routinely in subsequent assays to minimize dephosphorylation, thereby magnifying the responses. At the concentration used, vanadate itself had negligible effects on tyrosine phosphorylation (see lanes 1 and 2 in Fig. 1C), consistent with earlier findings (Bourgoin and Grinstein, 1992). Moreover, whereas vanadate increased the extent of phosphotyrosine accumulation, the phosphorylated substrates and the time course of phosphorylation were similar in the presence and the absence of the phosphatase inhibitor. As illustrated in Fig. 1B, phosphotyrosine accumulation induced by GTPS stimulation was rapid (evident after 1 min) and time-dependent, with a maximal response seen after 10 min.

Figure 1: Effect of GTPS on tyrosine phosphorylation. A, NADPH dependence and potentiation by sodium vanadate. Electroporated neutrophils were incubated at 37 °C without(-) or with (+) the following agents for 5 min: 10 µM GTPS, 2 mM NADPH, and 10 µM NaV, as indicated. Cells were then rapidly sedimented, boiled in sample buffer, and subjected to SDS-PAGE. Analysis was performed by immunoblotting with a monoclonal antibody to phosphotyrosine. B, time course of phosphotyrosine accumulation. Electroporated neutrophils were treated without(-) or with 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for the indicated time (min) and processed as in A. C, dependence of tyrosine phosphorylation on NADPH oxidase-derived ROI. Electroporated neutrophils were treated without(-) or with (+) 10 µM GTPS and/or 2 mM NADPH for 5 min at 37 °C. Where specified, the cells were treated with 2 mM diphenylene iodonium (DPI) or 2 mM NAC for 2 min at 37 °C prior to GTPS stimulation. The presence of 10 µM NaV during treatment is indicated. The results shown are representative of three separate experiments.

The effect of GTPS on tyrosine phosphorylation was entirely dependent on the presence of NADPH. As shown in Fig. 1C (as well as in Fig. 1A), treatment of electroporated cells with GTPS had little effect when the nucleotide was omitted (cf. lanes 3 and 4). This finding suggests that generation of superoxide by the NADPH oxidase is required for the increase in tyrosine phosphorylation following stimulation with GTPS. In support of this hypothesis, we found that NAC, a powerful anti-oxidant that has been shown to scavenge ROI and increase cytosolic levels of reduced glutathione (Halliwell and Gutteridge, 1985), effectively attenuated the tyrosine phosphorylation produced by GTPS in the presence of NADPH. Moreover, diphenylene iodonium, an inhibitor of the flavoprotein component of the NADPH oxidase (Ellis et al., 1988), had a comparable effect (lane 6). These findings are in agreement with those of Fialkow et al.(1993) and indicate that NADPH oxidase-derived ROI promote tyrosine phosphorylation in neutrophils.

Tyrosine Kinases Present in Neutrophils

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Figure 2: Identification of tyrosine kinases present in neutrophils. A, immune complex kinase assays were performed in vitro using immunoprecipitates of the tyrosine kinases indicated, prepared from lysates of electroporated neutrophils treated with 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 2 min. Kinase reactions were stopped, and the material was subjected to SDS-PAGE and autoradiography of the dried gel. The assay was also performed using a rabbit nonimmune serum (cont). B, whole neutrophil lysates were immunoblotted with antisera to the tyrosine kinases indicated. The closed arrowheads point to the tyrosine kinase. The open arrowhead indicates an unidentified protein of 65 kDa that cross-reacts with the syk antibody.

In good agreement with the kinase assays of Fig. 2A, the presence of lyn, hck, fgr, syk, and btk in neutrophils was confirmed by immunoblotting whole cell lysates with the same antisera used for precipitation (Fig. 2B). Both the full-length (72 kDa) syk protein as well as its 40-kDa degradation product were observed upon immunoblotting (Fig. 2B, closed arrowheads). A band of 65 kDa was also recognized by the syk anti-serum. It is not presently clear whether this polypeptide is related to syk or is merely a fortuitously cross-reacting protein. It is noteworthy, however, that a band of similar mobility was often found to be phosphorylated in syk immune complex assays (Fig. 3A), suggesting that the 65-kDa polypeptide co-immunoprecipitates and can be phosphorylated by syk.

Figure 3: Modulation of tyrosine kinase activity by ROI. A, immune complex kinase assays were performed in vitro using immunoprecipitates of the tyrosine kinases indicated, prepared from lysates of electroporated neutrophils treated without (-) or with (+) 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 1 min. B, immunoprecipitates of hck were prepared from lysates of electroporated neutrophils treated without or with 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for the indicated time (min) and subjected to immune complex kinase assays in the presence of enolase. The kinase reactions were stopped, and the samples were subjected to SDS-PAGE followed by autoradiography of the dried gels. A representative experiment is shown in the inset. Bands that correspond to autophosphorylation (closed arrow) and enolase phosphorylation (open arrow) were quantified with a PhosphorImager, and the results are presented as the percentage of maximal response in the main panel. C, in vitro autophosphorylation and enolase phosphorylation activities of lyn were determined as in B for hck. The data in B and C are the means ± S.E. of three experiments.

Modulation of Tyrosine Kinase Activity by ROI

Although production of ROI stimulated some tyrosine kinases, others were seemingly inhibited. The autophosphorylating abilities of lyn and fgr were diminished (73 ± 14 and 48 ± 16% of control activity, respectively; n = 3) following 1 min of GTPS stimulation (Fig. 3A). As for hck, the detailed time course of the effects of ROI on lyn activity was analyzed with enolase as substrate (see Fig. 3C). Interestingly, quantitation of the auto- and enolase-phosphorylating activities of lyn immune complexes revealed a discrepancy. Phosphorylation of the exogenous substrate was markedly increased, whereas autophosphorylation decreased. These findings suggest that nonradioactive phosphate is incorporated into lyn in the cells, prior to immunoprecipitation, precluding subsequent incorporation of radiolabel into these sites. The reduced autophosphorylation is therefore an inaccurate indication of the enzymatic activity of lyn, which is at least transiently stimulated by GTPS.

Role of Direct Oxidation in hck Activation by ROI

Figure 4: Effect of oxidizing/reducing agents on hck activity. hck immunoprecipitates were obtained from lysates of electroporated neutrophils treated without (-) or with (+) 10 µM GTPS, 2 mM NADPH, and 10 µM NaV (5 min) were incubated at 30 °C for 30 min with either 1 mM diamide, 1 mM HO, 20 mM NAC, 1 mM dithiothreitol, or with buffer alone (none), as indicated. An aliquot of the immunoprecipitate was used for in vitro kinase assay (A), and another was used for anti-phosphotyrosine immunoblotting (B). The closed arrows indicate the position of immunoprecipitated hck, whereas the open arrow indicates the position of the exogenous substrate, enolase.

The inability of oxidants and reducing agents to affect hck autophosphorylation was confirmed by immunoblotting the immunoprecipitates with anti-phosphotyrosine antibodies (Fig. 1B). The kinase was found to be tyrosine-phosphorylated only after stimulation of the cells with GTPS, and the phosphotyrosine content was unaffected by oxidizing and reducing agents. Together, these results suggest that ROI do not directly activate hck in GTPS-stimulated neutrophils.

Role of Tyrosine Phosphorylation in the Regulation of Kinase Activity

Figure 5: Phosphotyrosine-associated kinase activity. Anti-phosphotyrosine immunoprecipitates were obtained from lysates of electroporated neutrophils treated without(-) or with (+) 10 µM GTPS, 2 mM NADPH, and 10 µM NaV (2 min) and used to perform in vitro kinase assays as described under ``Experimental Procedures.'' The closed arrowheads indicate bands that displayed a prominent increase in phosphorylation following stimulation. The results are representative of three separate experiments.

The size of some of the phosphoproteins in phosphotyrosine immunoprecipitates correspond to that of the active kinases detailed in Fig. 2. To establish more directly whether the active kinases are tyrosine phosphorylated, immunoprecipitates of lyn, hck, fgr, syk, and btk were prepared from control and GTPS-treated cells and probed by immunoblotting with anti-phosphotyrosine antibodies. As illustrated in Fig. 6A, endogenous generation of ROI is accompanied by tyrosine phosphorylation of all the kinases studied (indicated with closed arrowheads), with the notable exception of btk, which remained unaffected. The figure also shows that both the intact form of syk as well as its 40-kDa proteolytic fragment (open arrowhead) was phosphorylated on tyrosine.

Figure 6: A, phosphorylation of tyrosine kinases in situ. Anti-phosphotyrosine immunoblotting was performed on immunoprecipitates of the specified tyrosine kinases, obtained from electroporated neutrophils treated without(-) or with (+) 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 5 min. The solid arrows indicate the position of the immunoprecipitated kinase. The open arrowhead indicates the 40-kDa proteolytic fragment of syk, also observed in anti-syk immunoblots (see Fig. 2B). B, time course of tyrosine phosphorylation. Cells were stimulated for the times indicated (min), and immunoprecipitates of the indicated kinases were obtained and subjected to SDS-PAGE and anti-phosphotyrosine immunoblotting. The extent of tyrosine phosphorylation was quantified in each case by densistometry and is presented as the percentage of the maximum. The data are the means ± S.E. for three experiments.

The correlation between the occurrence of tyrosine phosphorylation and the activation of the tyrosine kinases is further stressed by the similarity of the time courses of both events. In Fig. 6B, the degree of tyrosine phosphorylation was quantified in immunoprecipitates from cells stimulated for varying periods of time with GTPS. All four kinases undergo rapid and progressive phosphorylation, which is detectable by 1 min and maximal between 5 and 10 min. This pattern closely resembles the time course of activation of hck determined in Fig. 3C using enolase as the substrate. It therefore appears likely that tyrosine phosphorylation of the kinases regulates their activity.

This notion was directly addressed by treatment of immunoprecipitates with an active tyrosine phosphatase, the truncated T-cell phosphatase. hck was precipitated from GTPS-stimulated neutrophils and incubated for 30 min in the presence or the absence of T-cell phosphatase. The effectiveness of the phosphatase was ascertained by immunoblotting the precipitates with anti-phosphotyrosine antibodies. Exposure to T-cell phosphatase led to complete dephosphorylation of hck (indicated by the solid arrowhead in Fig. 7A). Immunoblotting confirmed that equal amounts of hck were present before and after treatment with T-cell phosphatase (data not shown). The autophosphorylating (solid arrowhead) and exogenous kinase activity (open arrowhead) of stimulated and dephosphorylated hck was next compared. Fig. 7B demonstrates that treatment with T-cell phosphatase eliminated the kinase activity of hck stimulated by ROI.

Figure 7: Effect of tyrosine dephosphorylation on hck activity. hck was immunoprecipitated from lysates of neutrophils treated with 10 µM GTPS, 2 mM NADPH, and 10 µM NaV for 5 min. Immunoprecipitates were then treated for 30 min without(-) or with (+) recombinant T-cell phosphatase (TC-PTP) at 30 °C while shaking. An aliquot of the immunoprecipitated material was subjected to anti-phosphotyrosine immunoblotting (A), and another was used to perform the in vitro kinase assay (B). The closed arrowheads indicate immunoprecipitated hck, whereas the open arrow indicates the location of the exogenous substrate, enolase. The results shown are representative of three separate experiments.

DISCUSSION

In this report, we analyzed the mechanism leading to increased phosphotyrosine accumulation following ROI production in neutrophils. In electroporated cells treated with GTPS, we detected an elevated activity of several kinases, measured in vitro. The activation of these kinases was rapid and correlated well with the increase in tyrosine phosphorylation observed under these conditions.

Kinases of three separate families were found to be activated by ROI, as determined by autophosphorylation and phosphorylation of an exogenous substrate, enolase. hck, a member of the src family of tyrosine kinases and highly expressed in granulocytes and macrophages (Ziegler et al., 1987), displayed little activity in untreated cells but was rapidly stimulated following the addition of GTPS. syk, which belongs to a separate family of kinases, also displayed increased activity following ROI production. In contrast, the closely related ZAP-70 tyrosine kinase, thought to be important in B- and T-cell receptor signaling (Sefton and Taddie, 1994) was not detectable in active neutrophils using our immune complex kinase assay. btk, a member of the tec family of tyrosine kinases, is expressed in cells of myeloid and lymphoid lineage (Yamada et al., 1993) and was also activated by ROI. To our knowledge, activation of btk in neutrophils had not been reported previously.

Although ROI production led to the activation of some tyrosine kinases, it appeared to have an opposite effect on the activity of others when estimated from autophosphorylation in immune complex kinase assays. Thus, lyn and fgr displayed high activities in untreated, electroporated neutrophils, which decreased following GTPS stimulation. However, at least in the case of lyn, the apparent decrease in activity likely reflected occupancy of substrate sites by nonradioactive phosphate, which may have occurred in situ, prior to immunoprecipitation. Indeed, the ability of the enzyme to phosphorylate exogenous substrates was increased following stimulation of the respiratory burst. Therefore, caution must be exercised when equating the autophosphorylating and catalytic activities of tyrosine kinases.

None of the other tyrosine kinases tested were found to be activated following generation of ROI. These included yes, which is reported to be present in neutrophils, where it can be stimulated by granulocyte macrophage colony-stimulating factor (Corey et al., 1993). Clearly, although 11 different antisera were used, our survey was incomplete, because other tyrosine kinases are likely to exist in neutrophils.

The mechanism underlying the activation of the kinases by ROI was explored in some detail using hck as a prototype. Although ROI production in situ appeared to activate hck, oxidizing agents could not mimic this effect when applied to hck immunoprecipitates in vitro. Moreover, reducing agents failed to reverse the activation of hck isolated from GTPS-treated cells. We conclude that hck activity is not regulated directly by ROI but rather by some other post-translational modification. Though not tested directly, we suggest by extension that activation of the other kinases is similarly indirect.

Because tyrosine phosphorylation of lyn, hck, fgr, and syk was found to occur upon stimulation by GTPS, this post-translational modification was considered as a possible mechanism of regulation. This notion was evaluated using T-cell phosphatase to dephosphorylate activated hck. This procedure was found to eliminate the activity of the kinase, suggesting that tyrosine phosphorylation mediates the effect of ROI on hck activation.

Tyrosine kinase activity of src family members is thought to be suppressed by phosphorylation of a C-terminal residue, conserved among family members (Cooper, 1988; Liu et al., 1993; Cooper and Howell, 1993). Dephosphorylation of this residue has been shown to increase the activity of src family kinases (Cooper and King, 1987), in apparent conflict with our findings with hck, where complete dephosphorylation of the enzyme led to its inactivation. However, recent evidence has questioned this simple model of regulation. This includes the finding that a T-cell line lacking CD45 (the phosphatase that activates the src family member lck) was found to have higher lck activity even though its inhibitory C-terminal tyrosine residue was hyperphosphorylated (Burns et al. 1994). Although dephosphorylation of the C terminus may be important for the derepression of src family members, a number of unique tyrosine residues have been reported to be phosphorylated upon activation. These include the so-called autophosphorylation site within the kinase domain (Smart et al., 1981; Patchinsky et al., 1982) and sites within the N-terminal domain of some src family members (Souda et al., 1993). Evidence exists that phosphorylation of these residues is essential for kinase activity, possibly by stabilization of the active kinase (Veillette and Fournel, 1990; Mustelin, 1994). Recent evidence has also implicated serine phoshorylation in the regulation of src family members (Winkler et al., 1993; Watts et al., 1993). Whereas the regulation of src family kinases remains incompletely understood, our findings imply that tyrosine phosphorylation is necessary to maintain the activity of hck following activation by endogenous ROI.

The steps that follow ROI generation and lead to kinase phosphorylation are unknown, but some insight is provided by recent reports that (a) critical conserved cysteine residues exist in the catalytic domain of many tyrosine phosphatases (Fisher et al., 1991) and (b) that both exogenous (Zor et al., 1993, Fialkow et al., 1994) as well as endogenous oxidants ()can inactivate tyrosine phosphatases, likely by targeting their critical sulfhydryl moieties. In view of these considerations, the following scenario can be envisaged. Under basal conditions, the accumulation of tyrosine phophoproteins and the autophosphorylation and stimulation of tyrosine kinases, some of which are constitutively active, are prevented by the offsetting action of tyrosine phosphatases. This delicate balance can be disrupted when ROI diminish the rate of dephosphorylation by reaction with sulfhydryl side chains in the catalytic domain of one or more tyrosine phosphatases. Indeed, in neutrophils, CD45 has been shown to be susceptible to inactivation by oxidants (Fialkow et al., 1994), and other phosphatases present in these cells (e.g. PTP-1C) ()are likely to be similarly affected.

It is noteworthy that modulation of tyrosine kinase activity has been reported in lymphoid cells exposed to HO (Schieven et al., 1993). Treatment with the oxidant was found to activate syk but not lyn, resembling our observations in neutrophils. Like most other cells, lymphocytes can potentially generate ROI by electron transfer reactions in mitochondria and the endoplasmic reticulum. However, the magnitude of the oxidative response is far greater in phagocytes, which express high levels of the NADPH oxidase (see the Introduction). In this regard, it is important that in the present experiments activation of phosphorylation was elicited by endogenously generated ROI, lending credence to the physiological significance of the observations. It is possible to envisage that stimulation of the NADPH oxidase, one of the earliest effectors of neutrophils, could promote phosphotyrosine accumulation by the combined inhibition of phosphatases and activation of kinases. This could in turn have important consequences on more slowly developing responses such as phagocytosis and degranulation. In this context, hck has been suggested to have a role in phagocytosis (Lowell et al., 1994), and syk has also been proposed to be essential to the anti-microbial response (Asahi et al., 1993). It is also conceivable that ROI secreted by neutrophils may have paracrine effects, stimulating neighboring quiescent neutrophils or other cells present in the inflammatory milieu, including lymphocytes and macrophages.

FOOTNOTES

*

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.

§

Supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada.

¶

International Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Division of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028.

()

The abbreviations used are: ROI, reactive oxygen intermediate(s); GTPS, guanosine 5`-O-(3-thiotriphosphate); NaV, sodium orthovanadate; NAC, N-acetylcysteine; PAGE, polyacrylamide gel electrophoresis; MOPS, 8-(N-morpholino)propanesulfonic acid.

()

Hereafter, ``GTPS stimulation'' refers to treatment of electroporated neutrophils with 10 µM GTPS in the presence of 2 mM NADPH and 10 µM NaV at 37 °C.

()

We observed a phosphoprotein of about 60-62 kDa in lyn immunoprecipitates. lyn has been extensively studied and only two isoforms have been identified, suggesting that the third band may be a separate protein. Several kinases have been shown to co-precipitate with lyn in other systems, including syk (Sidorenko et al., 1995), btk (Cheng et al., 1994), and a cell cycle regulatory protein, p34cdc2 (Yuan et al., 1995).

()

L. Fialkow, C. K. Chan, and G. P. Downey, submitted for publication.

()

J. H. Brumell, unpublished observations.

ACKNOWLEDGEMENTS

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