(Received for publication, April 19, 1996, and in revised form, October 16, 1996)
From the Division of Cell Biology, Research
Institute, Hospital for Sick Children,
Toronto, Ontario M5G-1X8, Canada, Departments of
§ Biochemistry,
Medicine,
§§ Immunology, and ¶¶ Molecular and
Medical Genetics, University of Toronto,
Toronto, Ontario M5S-1A8, Canada, ** Granulocyte Research Laboratory,
Department of Haematology L, Rigshospitalet, Copenhagen DK-2100,
Denmark,
Samuel Lunenfeld Research
Institute, Mount Sinai Hospital, Toronto, Ontario M5G-1X5, Canada,
and c Respiratory Division, Toronto Hospital, Toronto, Ontario
M5G 2C4, Canada
The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.
Polymorphonuclear (PMN)1 leukocytes, particularly neutrophils, destroy pathogenic microorganisms via a series of rapid and coordinated responses that include chemotaxis, phagocytosis, secretion of a variety of granules and vesicles, and production of reactive oxygen intermediates. These responses are mediated by the interaction of cell surface receptors with specific ligands found on microbial targets or in the inflammatory milieu. These receptor-ligand interactions, in turn, activate intracellular signal transduction cascades that couple the activating stimulus to physiological responses (1, 2).
One of the earliest biochemical events that follows receptor engagement is the accumulation of phosphate on tyrosine residues of cellular proteins. Increases in tyrosine phosphorylation can be initiated by a variety of soluble and particulate stimuli and temporally correlate with the appearance of cellular responses (3, 4, 5). The importance of tyrosine phosphorylation to neutrophil function is further underlined by the finding that inhibitors of protein tyrosine kinases block many neutrophil responses, including adherence (6), chemotaxis (7), phagocytosis (8), and production of reactive oxygen intermediates (9, 10).
Phosphotyrosine accumulation is regulated by the competing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Increased activities of tyrosine kinases have been demonstrated in neutrophils treated with chemotactic peptides (11), cytokines (12, 13), and other ligands (14) and have been postulated to account for the increased tyrosine phosphorylation observed following stimulation with these agents. The subcellular localization of tyrosine kinases and their substrates may also play a role in regulating tyrosine phosphorylation. For example, neutrophils contain at least four types of secretory granules within their cytoplasm (15), some of which have been shown to contain tyrosine kinases. Following stimulation, granular fusion with the plasma membrane and/or phagosome may allow associated kinases access to their substrates (16, 17).
In addition to the effects of tyrosine kinases, decreases in the activity of tyrosine phosphatases may also lead to an increase in cellular tyrosine phosphorylation. In support of this notion, overall neutrophil phosphotyrosine phosphatase activity has been shown to decrease following stimulation with the chemoattractant fMLP or with phorbol esters, although the identities of the particular phosphatases responsible for this effect were not determined (18, 19). Similarly, the inhibition of tyrosine phosphatases with vanadate or its peroxides has been shown to potentiate fMLP-induced superoxide production in whole cells (20) and to activate a respiratory burst in electroporated cells, providing further evidence that a reduction of phosphatase activity may lead to antimicrobial responses in PMN leukocytes (21, 22).
Although the spectrum of tyrosine phosphatases responsible for regulating neutrophil responses requires further definition, one PTP implicated in neutrophil signaling is CD45. A member of the transmembrane class of tyrosine phosphatases, CD45 has been found on secretory granules and the plasma membranes of neutrophils (23). Stimulation with a variety of agents has been shown to enhance the expression of CD45 on the plasma membrane (24), a phenomenon thought to contribute to the desensitization of neutrophils following further stimulation with fMLP (25). The intrinsic activity of CD45 is also modulated in neutrophils following production of reactive oxygen intermediates by the NADPH oxidase (26). The latter finding is thought to reflect the oxidation of critical cysteine residues within the catalytic domain of CD45 (which is conserved in all tyrosine phosphatases; see Ref. 27) and may represent a unique mode of autocrine and paracrine signaling (28). Inhibition of CD45 activity may also contribute to oxidant-induced activation of mitogen-activated protein kinase (26), an enzyme thought to represent a substrate of CD45 (29).
In contrast to CD45, little is known about the role of soluble tyrosine
phosphatases in neutrophil signal transduction. One such phosphatase,
SHP-1 (also known as PTP1C, hemotopoetic cell phosphatase, PTP-N6, and
SHPTP-1), has recently been extensively studied as a potential
regulator of the action of growth promoters in hemopoietic cells
(reviewed in Ref. 30). SHP-1 has been shown to dephosphorylate and
inactivate the erythropoietin (31, 32), stem cell factor (33),
interleukin 3 (34), interferon (35), epidermal growth factor (36),
and B-cell antigen receptors (37). In addition, loss of function
mutations in SHP-1 are known to represent the genetic defect
responsible for serious autoimmune and immunodeficiency defects in
motheaten mice (38, 39, 40, 41). To further delineate the role of
tyrosine phosphatases in the initiation and/or modulation of neutrophil
responses, we have studied the localization, activity, and possible
modes of regulation of SHP-1 in these cells.
RPMI 1640 medium, HEPES, phenylmethylsulfonyl
fluoride, pepstatin A, leupeptin, aprotinin, and the 4 form of
12-O-tetradecanoylphorbol 13-acetate (TPA) were from
Sigma. Bisindolylmaleimide I (BIM, also known as GF
109203X) was purchased from Calbiochem. Diisopropylfluorophosphate was
obtained from Aldrich. Polyvinylidene difluoride filters were from
Millipore. Donkey serum was obtained from Jackson ImmunoResearch. Diphenylene iodonium was synthesized in our laboratory according to the
method of Collette et al. (42). Bovine serum albumin (BSA)
and alkaline phosphatase were from Boerhinger Mannheim. [32P]H2PO4 and
[
-32P]ATP were from ICN. Monoclonal
anti-phosphotyrosine antibodies (4G-10) and purified PKC from rat brain
were purchased from U. S. Biochemical Corp. Goat anti-rabbit and donkey
anti-mouse secondary antibodies coupled to horseradish peroxidase used
for immunoblotting were from Jackson ImmunoResearch and Amersham Corp.,
respectively. The secondary antibody used for immunofluorescence,
fluorescein isothiocyanateconjugated donkey anti-rabbit antibody,
was from Jackson ImmunoResearch.
A glutathione S-transferase fusion protein of wild-type murine SHP-1 encompassing its two SH2 domains (amino acids 1-296) was generated as described previously (40). The recombinant protein was used to generate polyclonal antibodies to SHP-1, which were affinity purified and have been shown to be suitable for immunoblotting and immunoprecipitation (37, 40). A monoclonal antibody to SHP-1 was obtained from Transduction Laboratories.
SolutionsBicarbonate-free RPMI 1640 medium was buffered to pH 7.3 with 25 mM Na-HEPES. Powdered phosphate-buffered saline (PBS) was obtained from Pierce. The Na+-rich medium used for incubation of intact cells contained (in mM) 140 NaCl, 5 KCl, 10 glucose, 1 MgCl2, 1 CaCl2, and 10 Na-HEPES (pH 7.3). All media were adjusted to 290 ± 5 mosm with the major salt.
Cell Isolation and ManipulationNeutrophils were isolated from fresh heparinized blood of healthy human volunteers by dextran sedimentation, followed by Ficoll-Hypaque gradient centrifugation. Contaminating red cells were removed by NH4Cl lysis. Neutrophils were counted using a Coulter ZM counter, resuspended in HEPES-buffered RPMI 1640 medium at 107 cells/ml, and maintained in this medium at room temperature until use. To minimize proteolysis following cell lysis, the cells were pretreated with 2.5 mM diisopropylfluorophosphate for 30 min at room temperature.
Reverse Transcriptase-Polymerase Chain ReactionTo identify
SHP-1 in neutrophils, total RNA was isolated from fresh cells and
subjected to reverse transcriptase-polymerase chain reaction using the
Gene Amp RNA polymerase chain reaction kit from Perkin-Elmer. The
primers SHP-11002-5 (5
-CAGGAGAACACTCGTGTCAT-3
) and SHP-11122-3
(5
-TGTATGGTATTGAACAAGGACC-3
) were used to amplify a 120-base pair
mRNA fragment of SHP-1, as described previously (40). The
polymerase chain reaction conditions used were 5 min at 95 °C
followed by 25 cycles of 30 s at 94 °C, 50 °C for 30 s,
72 °C for 2 min, and 72 °C for 7 min (last cycle). Samples of the
final reaction mixture were then subjected to agarose gel electrophoresis and visualized by ethidium bromide staining. As a
positive control, the amplification of a ribonucleotide sequence encoding interleukin 1
(pAW109 RNA, Perkin Elmer) was performed (see
Fig. 1A, lane 4).
Immunofluorescence Microscopy
Neutrophil suspensions were fixed with 2% paraformaldehyde in Na+-rich medium for 30 min. Cells were allowed to adhere to poly-L-lysine-coated coverslips for 30 min and then permeabilized by treatment with a buffer containing 0.1% Triton X-100, 100 mM PIPES (pH 6.8), 5 mM EGTA, 100 mM KOH, and 2 mM MgCl2 for 15 min at room temperature. Fixed cells were preblocked with 5% donkey serum in PBS for 2-4 h, washed twice with PBS, and incubated with primary antibody for 2 h in PBS containing 1% BSA. The samples were then washed three times with PBS and incubated with secondary antibody for 2 h, also in PBS containing 1% BSA. After washing three times with PBS, the samples were treated with Slow Fade (Molecular Probes) before mounting. Samples were analyzed using a Zeiss LSM 410 laser confocal microscope with a × 63 objective. Digitized images were cropped in Adobe Photoshop and imported to Adobe Illustrator for assembly and labeling.
Subcellular FractionationNeutrophil subcellular fractionation was performed according to the method of Kjeldsen et al. (43). Briefly, neutrophils were disrupted by nitrogen cavitation in disruption buffer (100 mM KCl, 3 mM NaCl, 1 mM Na2ATP, 3.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 1 mM NaVO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µM pepstatin, and 10 mM PIPES, pH 7.2). Nuclei and unbroken cells were removed by centrifugation at 400 × g for 15 min, and the resulting postnuclear supernatant was applied on top of a three-layer Percoll gradient (1.05/1.09/1.12 g/ml). Centrifugation at 37,000 × g for 30 min yielded four separable bands. Three of these correspond to the primary, secondary, and tertiary granules. A fourth band contains a mixture of secretory vesicles and plasma membranes. One-ml fractions were collected from the gradient, and each was assayed for markers of the above subcellular compartments. Myeloperoxidase (primary granules), lactoferrin (secondary granules), gelatinase (tertiary granules), HLA class I (plasma membrane), and albumin (secretory vesicles) were all measured by enzyme-linked assay as described (44). Percoll was removed from the samples by centrifugation, and the remaining biological material was mixed with boiling, 2 × concentrated Laemmli sample buffer, and equal amounts of protein from each fraction were subjected to SDS-PAGE and immunoblotting (see below).
Isolation of Neutrophil MembranesNeutrophil suspensions
were treated with or without stimuli (107 M
TPA, 10
7 M fMLP, and a 1:100 dilution of
zymosan particles opsonized with human serum, 10 µg/ml final protein
concentration), sedimented rapidly, and resuspended (2.0 × 107 cells/ml) in disruption buffer (see above). The cells
were then disrupted by sonication, and nuclei and unbroken cells were
removed by centrifugation at 14,000 × g for 5 min. A
high speed pellet, referred to hereafter as "membranes," was
isolated by centrifugation of the lysates at 100,000 × g for 30 min, washed three times with disruption buffer, and
then boiled in Laemmli sample buffer. Remaining soluble (cytosolic)
fractions were boiled in 2 × Laemmli sample buffer, and an
identical number of cell equivalents (106) from both
membrane and cytosolic fractions were subjected to SDS-PAGE and
immunoblotting (see below). Equal protein loading was confirmed by
Coomassie Blue staining.
Neutrophil suspensions were treated with or without stimuli, sedimented, and then rapidly resuspended in lysis buffer (150 mM NaCl, 2 mM EDTA, 1 mM NaVO4, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 50 mM Tris, pH 8.0) containing 1% Triton X-100. The samples were vortexed vigorously and left on ice for a minimum of 10 min to ensure complete lysis of cells. Insoluble (cytoskeleton-associated) proteins were isolated by centrifugation at 14,000 × g for 5 min, washed three times with lysis buffer, and boiled in Laemmli sample buffer. Triton-soluble proteins were boiled with 2 × concentrated Laemmli sample buffer, and an identical number of cell equivalents (106) from both Triton-soluble and -insoluble fractions were subjected to SDS-PAGE and immunoblotting (see below).
SDS-PAGE and ImmunoblottingProteins were separated by SDS-PAGE in 12% polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes. Immunoblotting was carried out as described previously (45). Quantitation was performed by densitometry of exposed films using a Protein Databases (New York, NY) DNA 35 scanner with Discovery series 1D gel analysis software.
SHP-1 Immunoprecipitation and Phosphatase AssayNeutrophil suspensions were treated with or without stimuli, sedimented rapidly, and resuspended in ice-cold lysis buffer containing 1% Triton X-100 (see above). Lysates were centrifuged at 14,000 × g for 5 min and then precleared with 50 µl of Sepharose beads. Affinity-purified antibodies to SHP-1 were incubated with these lysates for 2 h at 4 °C while rotating end over end. Immune complexes were precipitated by addition of 100 µl of a 50% slurry of protein A-Sepharose beads, previously blocked with 10% BSA in lysis buffer, followed by incubation at 4 °C for 2 h. The immunoprecipitates were washed four to six times and then subjected to SDS-PAGE and immunoblotting or phosphatase assays. Immunoblotting confirmed that equal amounts of SHP-1 were immunoprecipitated from control and stimulated neutrophils.
For phosphatase assays, immunoprecipitates were washed once with assay buffer (0.5 mM EGTA, 25 mM HEPES, pH 7.0) and then incubated with 200 µl of assay buffer containing 10 mM p-nitrophenyl phosphate at 37 °C for 4-16 h while shaking. Reactions were stopped by addition of 800 µl of 0.2 M NaOH, beads were sedimented by brief centrifugation, and phosphatase activity was assessed by measuring the absorbance at 420 nm of the supernatant.
[32P]Orthophosphate Labeling and Phosphoamino Acid AnalysisNeutrophil suspensions (2.0 × 107 cells/ml) were incubated for 3 h at 37 °C in Na+-rich medium containing 0.5% BSA in the presence of 32P-labeled orthophosphate (2.0 mCi/ml). The cells were washed with Na+-rich medium and treated with or without stimuli before lysis and immunoprecipitation of SHP-1, as above. Following washing of immunoprecipitates, the samples were then subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes. Quantitation of in situ phosphorylation was performed with a Molecular Dynamics PhosphorImager, using the ImageQuant software. Phosphoamino acid analysis was performed as described previously (46).
In Vitro Protein Kinase C PhosphorylationNeutrophil
suspensions were treated with or without 107
M TPA for 10 min, sedimented rapidly, and then resuspended
in ice-cold lysis buffer. SHP-1 was immunoprecipitated as above, and
immune complexes were washed with kinase assay buffer (1 mM
-mercaptoethanol, 1 mM CaCl2, 1 mM MgCl2, and 20 mM MOPS, pH 7.0).
The beads were then incubated for 30 min at 30 °C in kinase buffer
in the presence of lipid activators (100 nM TPA and 100 µg/ml phosphatidylserine), purified PKC (1.6 µg/ml), 100 µM Na2ATP, and 400 µCi/ml
[
-32P]ATP. Control experiments were performed in the
absence of PKC or with immunoprecipitates obtained with nonimmune sera.
When the phosphatase activity of SHP-1 was to be determined following PKC phosphorylation, treatments were performed in the absence of
[
-32P]ATP, and 1 mM Na2ATP was
present during the incubation of immunoprecipitates with PKC.
Although previous studies have demonstrated SHP-1 expression in a variety of hematopoietic cells and cell lines (47, 48, 49), the presence of SHP-1 in neutrophils has not been documented. To address this issue, we used reverse transcriptase-polymerase chain reaction. Reactions were performed using RNA purified from neutrophils and oligonucleotide primer pairs designed for amplification of the catalytic domain of the SHP-1 cDNA (40). As shown in Fig. 1A, results of this analysis revealed amplification of a 120-base pair mRNA fragment of SHP-1 from neutrophil RNA (lane 3). No products were seen in the absence of either reverse transcriptase or neutrophil RNA (Fig. 1A, lanes 1 and 2, respectively), ensuring the specificity of these reactions.
The presence of SHP-1 protein was confirmed by immunoblotting, using a polyclonal antibody raised to a glutathione S-transferase fusion protein encompassing residues 1-296 of the phosphatase (fig. 1B). A single immunoreactive band of approximately 65 kDa is apparent in neutrophil cell lysates, and the same band was identifiable in immunoprecipitates obtained with the SHP-1 antibody. This corresponds to the reported molecular mass of one of the splice variants of SHP-1 (40, 50). To quantify the level of SHP-1 expression, increasing amounts of the fusion protein were loaded into SDS-PAGE gels along with neutrophil lysates and subjected to immunoblotting. By interpolation of the absorbance of these bands, we determined that neutrophils contain 12 ± 5 ng (n = 3) of SHP-1/106 cells or approximately 530 nM (based on a volume of 350 fl/cell, determined using a Coulter Channelyzer).
Two variants of SHP-1 have been identified, generated by the
alternative splicing of 39 amino acids within the C-terminal SH2 domain
of SHP-1 (38). To determine which splice variant is expressed in human
neutrophils, whole cell extracts were analyzed by immunoblotting with
affinity-purified antibodies to SHP-1 and compared with HL-60 cells, a
promyelocytic cell line found earlier to express only the higher
molecular weight splice variant of SHP-1 (50). As shown in Fig.
1C, the SHP-1 immunoreactive band of neutrophils co-migrated
precisely with that seen in HL-60 lysates. Similar results were
obtained using polyclonal and monoclonal antibodies to SHP-1. We
conclude that neutrophils express only the
splice variant of
SHP-1.
Immunofluorescence
staining of untreated neutrophils fixed in suspension revealed a
diffuse, predominantly cytosolic localization of SHP-1 (Fig.
2A, left panel). In these cells,
weak nuclear staining and a variable degree of punctation in the
cytosol was also noted, whereas no staining was seen in fixed cells
stained with an affinity-purified nonimmune serum (Fig. 2A, right
panel). To confirm that SHP-1 was predominantly cytosolic in
resting cells, subcellular fractions obtained by Percoll gradient
centrifugation were analyzed by immunoblotting (see "Experimental
Procedures"). As shown in Fig. 2B, the results of this
analysis also revealed SHP-1 to be almost exclusively located in the
cytosolic fraction. Minute amounts of the phosphatase were occasionally
found in the combined secretory vesicle and plasma membrane fraction,
which is known to trap cytosolic components on resealing of vesicles.
No SHP-1 was detected in fractions containing primary, secondary, or
tertiary granules.
To determine whether SHP-1 translocates between cellular compartments
on neutrophil stimulation, SHP-1 localization was examined by
separating the high speed pellet and supernatant (i.e. whole membrane and cytosolic fractions) from cells stimulated with the following agents: a chemotactic peptide (fMLP), which activates a
GTP-binding protein-coupled receptor, a phorbol ester (TPA), which
directly activates PKC, and opsonized zymosan (OPZ), a particulate stimulus that signals via Fc receptor and complement receptor 3. Immunoblotting of these fractions revealed minimal amounts of SHP-1 to
be associated with membranes (high speed pellet) in unstimulated cells,
and this amount was not substantially altered following stimulation
(data not shown). These findings were confirmed by analysis of
subcellular fractions isolated by Percoll gradient centrifugation from
cells treated with or without phorbol ester (not shown).
Although SHP-1 could not be found to associate with membranes during stimulation, a fraction of this phosphatase was found to associate with the Triton X-100-insoluble residue of activated cells. The Triton-insoluble material, thought to represent mostly cytoskeletal components, bound little SHP-1 before activation (<10%), but significant amounts were associated following stimulation with fMLP, TPA (Fig. 2C), or OPZ (not shown). Quantitation by densitometry revealed that stimulation by TPA induced a 2-3-fold increase in cytoskeletal-associated SHP-1 (Fig. 2D). A more modest increase in the cytoskeletal association of SHP-1 was noted following stimulation with OPZ and fMLP (Fig. 2D), although this change was not statistically significant. These findings suggest that SHP-1 may play a role in the cytoskeletal remodeling that occurs during neutrophil activation by dephosphorylating cytoskeletal-associated proteins.
Inhibition of SHP-1 ActivityAgonist-induced activation of
neutrophils is known to be associated with increased cellular tyrosine
phosphorylation (see the Introduction). This phenomenon is illustrated
in the experiment in Fig. 3A, in which
whole-cell lysates from control and stimulated cells were immunoblotted
with anti-phosphotyrosine antibody. This effect can be mimicked by
treatment with oxidizing agents such as diamide, which have been shown
to inhibit tyrosine phosphatases directly. Inhibition of the
phosphatases suffices to induce a massive accumulation of
phosphotyrosine on numerous cellular proteins (Fig. 3A, right
lane; the other two lanes were underexposed relative to the lanes
in the left panel, due to the high intensity of the diamide
lane).
In view of these data, we considered the possibility that inhibition of SHP-1 contributed to phosphotyrosine accumulation in cells stimulated with physiological agonists. Some of the stimuli that induced a net increase in tyrosine phosphorylation were also found to inhibit the activity of SHP-1, as determined in vitro by immune complex phosphatase assays (see "Experimental Procedures"). Shown in Fig. 3B, immunoprecipitable SHP-1 activity was inhibited by 42 ± 5.9% and 31 ± 2.1% following treatment with TPA or OPZ, respectively (n = 3). Diamide treatment of neutrophils prior to immunoprecipitation produced the greatest inhibition of SHP-1 (59.8 ± 2.7%; n = 3), whereas fMLP did not have a significant effect at any of the time points examined.2 Treatment of cells with TPA for varying times revealed a correlation between the extent of SHP-1 inhibition (Fig. 3C) and the accumulation of cellular protein phosphotyrosine (Fig. 3D). Together, these results suggest that decreased SHP-1 activity can contribute to increased tyrosine phosphorylation following stimulation of human neutrophils.
Regulation of SHP-1: Role of PhosphorylationOxidants have been shown to inhibit the activity of tyrosine phosphatases in vitro (51) and in vivo (52). We have previously demonstrated that activation of the NADPH oxidase, an endogenous source of reactive oxygen species in neutrophils, can lead to inhibition of CD45 and concomitant increased tyrosine phosphorylation (26, 53). We therefore tested whether inhibition of SHP-1 was similarly mediated by oxidation. However, under conditions that greatly inhibit CD45, SHP-1 was minimally affected (not shown), implying that these phosphatases are differentially regulated.
Phosphorylation has been shown to alter the activity of several
phosphatases, including SHP-1 (54, 55, 56). We therefore examined the
phosphorylation state of this phosphatase in resting and activated
neutrophils. As shown in Fig. 4A,
immunoprecipitation of SHP-1 from
[32P]orthophosphate-labeled cells revealed a low but
detectable amount of phosphorylation in untreated cells. By contrast,
phosphorylation was markedly increased by treatment of cells with the
agonists. Immunoprecipitates from stimulated cells obtained using
nonimmune serum displayed no signal (Fig. 4A, right lane).
As TPA induced the largest increase in phosphorylation, phosphoamino
acid analysis was then performed on immunoprecipitates of SHP-1 from
neutrophils treated with or without this PKC agonist. Illustrated in
Fig. 4B, basal and TPA-induced phosphorylation of SHP-1 was
found to be primarily on serine residues. Anti-phosphotyrosine
immunoblotting of SHP-1 immunoprecipitates confirmed that neither
TPA nor any of the other agonists studied were capable of inducing
tyrosine phosphorylation of this phosphatase (not shown).
Role of PKC
Activation of PKC is known to occur following
stimulation by either fMLP or opsonized zymosan. As both of these
agents, as well as the direct PKC activator TPA, induced
phosphorylation of SHP-1, we next examined the possibility that
phosphorylation of this phosphatase is mediated by PKC. As demonstrated
in Fig. 5A, pretreatment of
[32P]orthophosphate-labeled neutrophils with 2 µM BIM, a potent and specific inhibitor of PKC (57),
inhibited both TPA- and OPZ-induced phosphorylation of SHP-1. However,
SHP-1 phosphorylation was not entirely abrogated by BIM treatment,
suggesting that PKC was not fully inhibited or that other kinases also
contribute to SHP-1 phosphorylation.3 By
contrast, prior treatment with BIM fully prevented the agonist-induced decrease in SHP-1 activity (Fig. 5B). Thus, it appears that
PKC plays a significant role in regulating SHP-1, apparently through a
phosphorylation-dependent mechanism.
Finally, we wished to determine whether PKC was responsible for direct
phosphorylation and inhibition of SHP-1 or was instead acting upstream
of the regulatory kinase(s). To this end, immunoprecipitates of SHP-1
were prepared from resting or TPA-treated neutrophils, washed
extensively, and subjected to in vitro phosphorylation using
PKC purified from rat brain. As shown in Fig.
6A, immunoprecipitated SHP-1 was readily
phosphorylated by PKC (lane 2). Moreover, stimulation of
cells with TPA prior to immunoprecipitation decreased the amount of
in vitro PKC-mediated SHP-1 phosphorylation (Fig. 6A,
lane 3), presumably due to the incorporation in situ of
nonradioactive phosphate into sites on SHP-1 that are substrates of
PKC. Treatment of these immunoprecipitates with
[-32P]ATP alone, in the absence of PKC, did not result
in phosphorylation of SHP-1 (Fig. 6A, lane 1), confirming
that phosphorylation of SHP-1 in the in vitro assay is not
mediated by co-precipitating kinases. Similarly, no PKC-induced
phosphorylation was evident in experiments performed with
immunoprecipitates of nonimmune serum (Fig. 6A, lane 4).
We next determined the effect of in vitro phosphorylation on the activity of SHP-1. Immunoprecipitates of the phosphatase were subjected to phosphorylation by purified PKC, as in Fig. 6A, and assayed for activity. In vitro phosphorylation by PKC was found to inhibit the activity of SHP-1 to 54 ± 3.9% (three experiments with duplicate determinations) of the control level. This inhibition of phosphatase activity was comparable with that induced by TPA pretreatment of intact cells prior to immunoprecipitation of SHP-1. Together, these results suggest that direct phosphorylation of SHP-1 on serine residues by PKC mediates, at least in part, the inhibition of SHP-1 phosphatase activity following neutrophil stimulation.
Little is known about the role of tyrosine phosphatases in
regulating neutrophil antimicrobial responses. In this report, we
established that SHP-1 is expressed in human neutrophils and that the
concentration of SHP-1 in these cells is approximately 530 nM. In contrast to other hemopoietic cell types, which can express alternatively spliced forms (66 and 62 kDa) of SHP-1 (40, 50),
only the splice variant of SHP-1 was found to be expressed in human
neutrophils. Although the molecular basis for this observation is
unclear, the preferential expression of one splice variant may be of
functional relevance, as has been suggested for the related tyrosine
phosphatase SHP-2 (58).
In unstimulated cells, SHP-1 was found to be predominantly in the cytosol, although minimal amounts were found to be associated with the nucleus, the plasma membrane, and the cytoskeleton. Stimulation with TPA and, to a lesser extent, with OPZ and fMLP induced an increase in the amount of SHP-1 associated with the cytoskeleton. Cytoskeletal association of SHP-1 has been demonstrated in platelets stimulated with thrombin, and this translocation was postulated to mediate dephosphorylation of cytoskeletal-associated substrates (59). By analogy, we suggest that the functional effects of SHP-1 on neutrophil function reflect its action on both cytoskeletal and cytosolic targets.
Agents that induce tyrosine phosphorylation of neutrophil proteins, such as OPZ and TPA, were found to inhibit the activity of SHP-1. Furthermore, the time course of SHP-1 inhibition following stimulation paralleled that of cellular tyrosine phosphate accumulation. These results suggest that inhibition of SHP-1 contributes to phosphotyrosine accumulation and may play a role in the regulation of antimicrobial responses. Stimuli that inhibited the activity of SHP-1 were also found to induce its phosphorylation on serine residues. TPA-induced serine phosphorylation of SHP-1 had been reported in HL-60 cells, but inhibition of catalytic activity was not described (60). The increased SHP-1 expression induced by TPA in these cells may have masked the inhibitory effect of phosphorylation on enzyme activity (50, 60). PKC-dependent phosphorylation of SHP-1 has been demonstrated in human thymocytes, and this phosphorylation event was shown to inhibit its activity (56). That phosphorylation by PKC is responsible for the functional inhibition of SHP-1 in human neutrophils is suggested by the experiments using BIM. This PKC antagonist diminished phosphorylation of the phosphatase while precluding the inhibition of its catalytic activity. It is noteworthy that concentrations of BIM that inhibited phosphorylation incompletely resulted in almost complete reversal of the functional inhibition. This can be interpreted to mean that phosphorylation at multiple sites is required for inhibition of catalytic activity. This interpretation would also account for the observation that, although inducing phosphorylation of SHP-1, fMLP failed to significantly inhibit its activity. In accord with this notion, phosphorylation of SHP-1 on multiple sites has been described in stimulated platelets (55).
In the current study, tyrosine phosphorylation of SHP-1 was not observed with any of the agonists used. Nevertheless, tyrosine phosphorylation of SHP-1 has been observed in other cell types (61, 62) and, contrary to our findings of serine phosphorylation-mediated inhibition, is thought to increase the activity of this phosphatase (54, 55). The apparent lack of phosphotyrosine-mediated regulation of SHP-1 in human neutrophils may reflect the absence of specific tyrosine kinases capable of phosphorylating this protein. In this regard we note that Lck, which is primarily responsible for SHP-1 phosphorylation in lymphoid cells (63), is not detectable in human neutrophils (64).
In conclusion, our findings suggest that SHP-1 plays an important role in regulating the balance of protein tyrosine phosphorylation in neutrophils. Although the substrates for SHP-1 in these cells have not been defined, our data indicate a role for this enzyme in dephosphorylating both cytosolic and cytoskeleton-associated proteins. In addition, the finding that PKC acts to inhibit SHP-1 activity following neutrophil stimulation provides a new link between the serine-threonine and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.