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*
Ludwig Institute for Cancer Research, Royal Free and University College Medical School, London, United Kingdom; and
Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
TNF, IL-6, and arachidonic acid metabolites recruit and activate other
leukocytes to help fight infection (5). High levels of LPS
are a major cause of Gram-negative septic shock, in which LPS induces
numerous changes including endothelial injury and activation, leukocyte
adhesion to endothelial cells, and generation of free radicals. Lower
levels of LPS produced during chronic infections may contribute to the
development of other diseases: for example, the pathological changes
involved in the initiation and development of atherosclerosis resemble
those induced by endotoxin (6), and accumulating data
suggest that persistent Gram-negative periodontal disease can lead to
the development of cardiovascular disease (7).
The major cell surface receptor for LPS on macrophages is CD14, a
55-kDa glycosylphosphatidylinositol-linked membrane protein
(8), although at higher concentrations LPS can bind to
other receptors such as the ß2 integrin
CD11b/CD18 (9). Recently, the transmembrane protein
Toll-like receptor 2 has also been implicated in LPS signaling in
macrophages (10). A number of signaling pathways can be
activated by LPS, including phospholipases A (11) and C
(12), protein kinase C isoforms (13, 14), and
the Src family tyrosine kinases Hck, Lyn, and Fgr (15, 16). In monocytes/macrophages, LPS induces the tyrosine
phosphorylation of many proteins, including Vav (17) and
the mitogen-activated protein kinase
(MAPK)3 family members
p42/p44 MAPK (18), stress-activated protein kinase 1/c-Jun
N-terminal kinase (19), and p38/stress-activated protein
kinase 2 (20). LPS also stimulates phosphatidylinositide
3-kinase (PI 3-K) activity in monocytes, resulting in a transient
increase in the level of phosphatidylinositol
3,4,5-trisphosphate (21). This
process was reported to involve the association of PI 3-K with the Src
kinase Lyn and to act upstream of protein kinase C-
activation
(22).
Of the signaling pathways activated by LPS, the p42/p44 MAPK and the
p38 pathways have both been shown to be directly involved in the
production of the cytokines TNF- and IL-1 (20, 23). The
functions of other signaling pathways induced by LPS have not been
established, and in particular the signaling pathways involved in
LPS-induced adhesion have yet to be characterized. In other cell types,
focal adhesion kinase (FAK), initially identified as a unique
cytoplasmic tyrosine kinase involved in focal adhesions, is thought to
play a key role in integrin-mediated cell adhesion (24).
Proline-rich tyrosine kinase 2 (Pyk2), also known as related adhesion
focal tyrosine kinase or cell adhesion kinase ß
(25, 26, 27), is a cytoplasmic tyrosine kinase related to FAK.
A Pyk2 splice variant has been identified, termed Pyk2-H or monocyte
calcium-dependent tyrosine kinase (28, 29). Pyk2 shows
considerable sequence homology and structural similarity to FAK,
including consensus motifs in the catalytic domain. Pyk2 expression is
more limited than FAK, and has been detected in epithelial cells,
neuronal cells, T and B cells, megakaryocytes, platelets, mast cells,
and monocytes and macrophages (26, 30, 31), whereas Pyk2-H
is only expressed in hemopoietic cells (28, 29). The
stimulation of many types of cell surface receptors results in the
tyrosine phosphorylation of Pyk2, including integrins, cytokines, and
immune receptors (30, 32, 33, 34); and diverse stimuli such as
membrane depolarization, stress stimuli, angiotensin, and PMA have all
been shown to induce Pyk2 tyrosine phosphorylation, resulting in the
activation of its kinase activity (26, 31).
Many proteins localize to focal adhesion sites, in which transmembrane integrins are clustered and link the extracellular matrix with the cytoskeleton (35). Paxillin, a vinculin-binding protein, colocalizes with FAK and integrins in fibroblast focal adhesions (36). Paxillin also binds to Pyk2 (31, 37), and PMA-dependent tyrosine phosphorylation of paxillin in megakaryocytes has been shown to be inhibited using a kinase-dead mutant form of Pyk2, suggesting a functional link between Pyk2 and paxillin (38). As yet, however, there has been no evidence that Pyk2 colocalizes with paxillin.
LPS is known to induce monocyte adhesion (3), but its effects on cytoskeletal organization have not been previously examined in detail. In this study, we have analyzed changes in the actin cytoskeleton induced by LPS in monocytes and macrophages, and correlated them with increased tyrosine phosphorylation of Pyk2 and paxillin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (Salmonella typhi and Escherichia coli serotype 026:B6) was obtained from Sigma-Aldrich (Poole, U.K.). It was reconstituted in pyrogen-free PBS (sonicated for 5 min) and stored at -20°C in glass vials. Immediately before adding to cells, LPS suspensions were sonicated for an additional 2 min before diluting to 10x final concentration in RPMI (Life Technologies, Paisley, U.K.) containing 1% heat-inactivated FCS (HIFCS) (Sigma, Dorset, U.K.) (monocytes and Bac1 cells) or 0.5% HIFCS/RPMI (J774 cells). LPS was protein free, as determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). PP1 and LY294002 were obtained from Calbiochem (Nottingham, U.K.). The mouse mAb to paxillin was purchased from Transduction Laboratories (Lexington, KY); rabbit polyclonal Pyk2 Abs (600 and 623) were a gift from Dr. I. Dikic (Ludwig Institute for Cancer Research, Uppsala, Sweden); monoclonal phosphotyrosine Py99 was from Santa Cruz Biotechnology (Santa Cruz, CA); and FITC-labeled anti-mouse IgG and tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-rabbit IgG were from Jackson ImmunoResearch (West Grove, PA).
Isolation of peripheral blood monocytes and cell culture
Single donor platelet phoresis residues were purchased from the North London Blood Transfusion Service (Collindale, U.K.). Mononuclear cells were isolated by Ficoll-Hypaque centrifugation (specific density, 1.077 g/ml) preceding monocyte separation in a Beckman JE6 elutriator. Monocyte purity was assessed by flow cytometry using directly conjugated anti-CD45 and anti-CD14 Abs (Leucogate, Becton Dickinson, San Jose, CA) and was routinely greater than 85%. Monocytes were purified in and maintained in RPMI containing 1% HIFCS. All media and sera were routinely tested for endotoxin using the Limulus amoebocyte lysate test (BioWhittaker, Walkersville, MD) and rejected if the endotoxin concentration exceeded 0.1 U/ml.
J774 cells were cultured in RPMI containing 10% HIFCS and supplemented with 25 U/ml of streptomycin and penicillin. They were cultured in RPMI containing 0.5% HIFCS for 48 h before stimulation with LPS. Bac1.2F5 cells were kindly provided by Richard Stanley (Albert Einstein College of Medicine, New York, NY). They were cultured in DMEM supplemented with 10% HIFCS and 20% L cell-conditioned media as a source of CSF-1 (39), and were starved in 1% HIFCS/RPMI (no CSF-1) for 24 h before LPS stimulation.
In some experiments, monocytes or macrophages were pretreated with 10 µM PP1 or 25 µM LY294002 for 15 min before stimulation with LPS. All cultures treated with these inhibitors were assessed for cell viability, as determined by trypan blue (0.04%) (Sigma) exclusion. In all cases, more than 99% of cells were viable.
Immunofluorescence and confocal microscopy
For immunofluorescence studies, cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. For localization of F-actin filaments, cells were incubated with 0.1 µg/ml TRITC-labeled phalloidin (Sigma) for 45 min. For localization of paxillin and Pyk2, cells were blocked with 0.2% heat-inactivated goat serum (Sigma) for 30 min, washed, and then incubated with a 1/12.5 dilution of mouse anti-paxillin Abs and/or a 1/50 dilution of rabbit anti-Pyk2 (600) Abs, washed three times with PBS, and then incubated with a 1/100 dilution of TRITC-conjugated goat anti-mouse IgG and/or FITC-conjugated goat anti-rabbit IgG. Images of cells were obtained using a Zeiss LSM 510 confocal laser-scanning microscope (Welwyn, Garden City, U.K.), using the accompanying LSM 510 software, and were processed in Adobe photoshop 4.0.
Determination of cell spreading
Monocytes (5 x 105/ml) were allowed to adhere to glass coverslips for 30 min before stimulation with LPS (100 ng/ml). Upon termination of the experiment, cells were fixed and stained with TRITC-phalloidin and imaged using confocal microscopy, as described above. Cells with a clearly visible cortical F-actin ring and a diameter equal or less than 10 µm were defined as unspread, whereas cells without a clear cortical ring and a diameter greater than 10 µm were defined as spread.
Western blotting and immunoprecipitations
Following stimulation, cells were lysed for 15 min in lysis buffer (0.5% Nonidet P-40, 30 mM sodium pyrophosphate, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin). Lysates were clarified by centrifugation at 14,000 x g for 10 min. The protein concentration of the cell extracts was determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The clarified lysates were precleared with either anti-mouse IgG (for subsequent paxillin immunoprecipitations) or rabbit serum (for Pyk2 immunoprecipitations) with protein A-Sepharose (Pharmacia, Uppsala, Sweden). Immunoprecipitations were performed using either anti-paxillin or anti-Pyk2 Abs and protein A-Sepharose. The immunoprecipitates were then washed in lysis buffer before addition of 2x gel sample buffer, heated to 95°C for 3 min, and subsequently analyzed by SDS-PAGE, followed by electrophoretic transfer to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA).
Membranes were blocked in 5% BSA and probed with a 1/4000 dilution of the anti-phosphotyrosine Ab PY99. Blots were visualized with HRP-conjugated donkey anti-rabbit Ab (1/8000) (Amersham, Little Chalfont, U.K.) and developed using enhanced chemiluminescence (ECL; Amersham). Membranes were either stripped in 0.1 mM glycine, pH 2, and reprobed with anti-paxillin Abs (Transduction Laboratories), or alternatively stripped with 2% SDS, 25 mM Tris, pH 6.8, and 100 mM 2-ME, and subsequently reprobed with anti-Pyk2 (600) Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS induced distinct changes in cell morphology and actin
organization in monocytes (Fig. 1
, a–f) and Bac1 mouse
macrophage cells (Fig. 1, g–j). When unstimulated monocytes
were allowed to adhere to glass coverslips for 90 min, the majority did
not spread significantly (Table I) and
maintained a cortical ring of actin filaments (Fig. 1, a).
This adhesion was transient, and more than 90% of cells detached again
by 24 h. In the presence of LPS, however, monocytes displayed a
considerably greater degree of spreading (Fig. 1, b–f;
Table I), and this adhesion was stable and maintained for over 24
h. LPS-induced spreading was morphologically indistinguishable whether
it was added to monocytes before or after they were allowed to adhere
to coverslips (data not shown). In addition to this increase in cell
spreading, LPS induced cell polarization indicative of cell migration
(40), as defined by the asymmetric morphology of
LPS-treated cells. Typically, one region of the cell periphery
displayed membrane ruffles (indicated by the arrowheads in Fig. 1, d and f) and lamellipodia, characteristic of the
leading edge of a motile cell, whereas the opposite edge was rounded or
displayed retraction fibers (arrowhead in Fig. 1e), typical
of the trailing edge of a motile cell. Time-lapse videomicroscopy
confirmed that actively migrating cells were present in LPS-stimulated
monocyte cultures (data not shown).
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, a–d) and heat-inactivated
(Fig. 1, e and f) LPS, indicating that the
responses observed were due to LPS itself rather than to any
heat-sensitive contaminant.
When Bac1 macrophages are deprived of CSF-1, they take on a rounded
morphology (Fig. 1g) (41). Upon LPS
stimulation, the cells responded rapidly by extending filopodia (e.g.,
arrowhead in Fig. 1h) and lamellipodia (e.g., arrowhead in
Fig. 1i). Membrane ruffles were also observed on the dorsal
surface of cells (Fig. 7, i and j). By 60 min
(Fig. 1j), the cells were more polarized and some cells
displayed punctate F-actin staining (arrowhead) similar to that
observed in monocytes (e.g., Fig. 1e), localized toward the
presumptive leading edge of the cells. Bac1 cells were less sensitive
to LPS than monocytes, and morphological changes were most clearly
observed at 1 µg/ml LPS, whereas human monocytes responded
morphologically to LPS at both 100 ng/ml (Fig. 1) and 10 ng/ml, but
showed little response at 1 ng/ml (data not shown). This difference
between Bac1 cells and monocytes could reflect receptor levels and/or
different types of receptors.
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To investigate the signaling processes involved in LPS-induced adhesion and spreading, we initially examined the ability of LPS to activate Pyk2. Pyk2 was chosen as although it was previously reported that FAK is expressed in human peripheral monocytes (42), in agreement with other workers (28), we were unable to detect any FAK expression in either human monocytes or murine macrophage cell lines. We therefore concentrated our studies to Pyk2, which is abundantly expressed in cells of the monocyte/macrophage lineage.
Freshly isolated human peripheral blood monocytes were stimulated in
suspension with 100 ng/ml LPS for different lengths of time, and then
analyzed for Pyk2 tyrosine phosphorylation (Fig. 2). The Ab used to immunoprecipitate Pyk2
(600) detects both of the Pyk2 isoforms expressed in monocytes
(29). An increase in Pyk2 tyrosine phosphorylation was
consistently induced by LPS in monocytes, and maximum Pyk2
phosphorylation was observed between 15–30 min after LPS addition. The
experiments were performed in the presence of 1% HIFCS, to facilitate
LPS binding to CD14 via LPS-binding protein present in serum
(8). In the presence of 10% HIFCS, Pyk2 tyrosine
phosphorylation was induced in the absence of LPS (data not shown).
This could be a consequence of lysophosphatidic acid present in serum,
since lysophosphatidic acid has been shown to stimulate Pyk2
phosphorylation in PC12 cells (43).
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). Reprobing with
anti-paxillin Abs demonstrated that there was an increase in lower
mobility forms of paxillin, suggesting that paxillin is also serine
phosphorylated at multiple sites in response to LPS stimulation, as has
been observed following adhesion of macrophages to vitronectin
(44).
Although CD14 is believed to be the principal receptor for LPS on
macrophages, at higher concentrations LPS has been shown to bind to
other cell surface proteins, including the ß2
integrin complement receptor type 3 (9). As engagement of
integrins has been shown to result in both Pyk2 and paxillin tyrosine
phosphorylation (30, 44), it was important to determine
the sensitivity of this response to LPS concentrations. LPS was capable
of stimulating the tyrosine phosphorylation of both Pyk2 and paxillin
at concentrations below 100 ng/ml (Fig. 3), indicating that this response is via
binding to the high affinity receptor CD14.
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). However, once the monocytes had been allowed to
adhere to tissue culture plastic (in 1% HIFCS), LPS stimulated a
further increase in the level of tyrosine phosphorylation of Pyk2 and
paxillin (Fig. 3).
To determine whether LPS could also induce paxillin and Pyk2
phosphorylation in macrophages, we investigated LPS responses in two
adherent murine macrophage cell lines. In J774 cells, some constitutive
Pyk2 tyrosine phosphorylation was observed, even at low serum
concentrations (0.5%) (Fig. 4a). However, as in adherent
monocytes, LPS enhanced the levels of tyrosine phosphorylation of both
Pyk2 and paxillin: increased tyrosine phosphorylation was detected
within 5 min and remained elevated for up to 2 h. In Bac1 cells,
both Pyk2 and paxillin were constitutively tyrosine phosphorylated, and
the level of Pyk2 tyrosine phosphorylation was not significantly
increased when stimulated with LPS, even at high concentrations of 1
µg/ml (Fig. 4b, left panels). In contrast, LPS
stimulated an increase in paxillin phosphorylation (Fig. 4b,
right panels). When Bac1 cells were gently detached from the
tissue culture plates, and kept in suspension for 3 h before
stimulation, the level of Pyk2 phosphorylation was unaltered, but
paxillin tyrosine phosphorylation was significantly decreased (Fig. 4b). LPS was incapable of stimulating increased
phosphorylation of either Pyk2 or paxillin in these suspended cells.
This suggests that Bac1 cells differ from monocytes and J774 cells in
that Pyk2 tyrosine phosphorylation is already high as a consequence of
other, unknown, signals, and cannot be further enhanced by either LPS
or adhesion. In contrast, paxillin phosphorylation is regulated by LPS,
but only in adherent Bac1 cells.
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To investigate the signaling pathways leading to Pyk2 and paxillin
phosphorylation, we have focused on two potential upstream signal
transducers. LPS has been shown to activate PI 3-K (21)
and the Src family members Hck, Lyn, and Fgr (15, 16)
within minutes of stimulation. In addition, PI 3-K physically
associates with Lyn, suggesting that they may act coordinately to
regulate downstream signaling (21). Using the Src family
kinase inhibitor PP1 (45) or the PI 3-K inhibitor LY294002
(46), we determined the roles of the targets of these
compounds in LPS responses. Preincubation of monocytes with either PP1
or LY294002 strongly inhibited the LPS-induced phosphorylation of
paxillin and Pyk2 (Fig. 5). PP1 reduced
both paxillin and Pyk2 tyrosine phosphorylation below levels detected
in unstimulated cells, suggesting that basal as well as stimulated
levels of tyrosine phosphorylation are regulated by Src family kinases.
LY294002 reduced the level of phosphorylation to that observed in
unstimulated cells. Consistent with previous reports (31, 47, 48), we observed coimmunoprecipitation of Pyk2 with paxillin
(Fig. 5a, left panel). Interestingly, LPS
stimulation did not increase the amount of Pyk2 coimmunoprecipitating
with paxillin, despite inducing tyrosine phosphorylation of Pyk2. In
addition, PP1 and LY294002 did not alter the association of Pyk2 with
paxillin, indicating that this interaction is not regulated by
tyrosine phosphorylation of Pyk2 or paxillin, in agreement with other
workers (31, 47, 48).
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a–d and f). Even cells that did spread (diameter
>10 µm) to a limited degree remained unpolarized. In addition, by 60
min after LPS stimulation, PP1 and LY294002 induced a significant
reduction in the number of adherent monocytes (Fig. 6, a–d
and e). Both inhibitors also prevented the transient
adhesion of monocytes to glass coverslips in the absence of LPS (data
not shown), consistent with a role for Src kinases and PI 3-K in
adhesion-induced signaling. The effects of PP1 and LY294002 on monocyte
morphology were reversible, as if the inhibitors were removed
from the cultures by washing; the cells were able to adhere and respond
normally to LPS, indicating that the inhibitors were not exerting some
nonspecific toxic effect upon the monocytes.
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Our observation that PP1 and LY294002 inhibited both LPS-induced
morphological changes and phosphorylation of Pyk2 and paxillin
suggested that Pyk2 and/or paxillin could be involved in the
morphological responses to LPS. We therefore investigated the
localization of Pyk2 and paxillin with respect to the actin
cytoskeleton. In unstimulated monocytes, both paxillin and Pyk2 were
distributed diffusely throughout the cytoplasm (Fig. 7, a and b). Upon
LPS stimulation and subsequent cell spreading, Pyk2 was found to
concentrate, with F-actin, at the leading edge of polarized cells (as
shown in the basal plane, Fig. 7, c and d). Pyk2
did not localize to podosomes, identified as punctate foci of F-actin
(Fig. 7c; podosomes indicated by arrows), which are adhesion
sites found in cells of monocytic origin (49, 50). Pyk2
also did not localize to podosomes in human macrophages differentiated
from monocytes (data not shown). In contrast, vinculin localized to
podosomes, as previously described (50), as did paxillin
(data not shown).
In medial sections of cells, Pyk2 localized within F-actin-rich
membrane ruffles (Fig. 7, e and f; arrowhead
indicates a membrane ruffle). Paxillin (Fig. 7g) and Pyk2
(Fig. 7h) colocalized in the perinuclear area of cells and
at the plasma membrane (e.g., arrows in Fig. 7, g and
h), consistent with their coimmunoprecipitation from cell
lysates (Fig. 5a). Paxillin also localized to small focal
complexes observed in some areas at the periphery of LPS-stimulated
monocytes (Fig. 7g; basal section, arrowheads indicate
examples of focal complexes). Pyk2 did not detectably colocalize with
paxillin in these focal complexes (Fig. 7h), indicating that
Pyk2 and paxillin are not always associated. Similarly, paxillin and
Pyk2 colocalized in protrusions of Bac1 cells (Fig. 8, a and b), but
Pyk2 clearly did not localize to paxillin-containing focal complexes.
As in monocytes, paxillin and Pyk2 colocalized in membrane ruffles of
Bac1 cells (Fig. 8, c and d).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In monocytes, Pyk2 has been shown to be tyrosine phosphorylated upon adhesion (28), and we have similarly observed that adhesion stimulates Pyk2 and paxillin tyrosine phosphorylation. When monocytes are kept in suspension, Pyk2 is not responsive to stimuli such as PMA, RANTES, and thapsigargin, although these stimuli enhance tyrosine phosphorylation of Pyk2 in adherent monocytes (28). In contrast, we have found that LPS is able to induce Pyk2 and paxillin tyrosine phosphorylation in suspension as well as in adherent monocytes. This indicates that neither Pyk2 nor paxillin requires signals provided by adhesion to become tyrosine phosphorylated, but that adhesion enhances the response to LPS. Interestingly, LPS induces morphological changes in adherent Bac1 macrophages without detectably altering Pyk2 tyrosine phosphorylation. In contrast, LPS does increase paxillin phosphorylation, and therefore it is plausible that in these cells paxillin but not Pyk2 contributes to LPS-induced morphological changes. It is also possible that Pyk2 is involved, but only in concert with paxillin and other proteins. Indeed, our observation that Pyk2 localizes to membrane ruffles and lamellipodia suggests that its site of action is in these structures, where new adhesions are formed during cell spreading and migration (40). As Pyk2 tyrosine phosphorylation remains elevated in Bac1 cells even in suspension, it is possible that these cells constitutively secrete one or more chemokine that acts in an autocrine fashion to activate Pyk2, as several chemokines have been shown to activate Pyk2 (28, 29).
We have shown that the Src family-specific inhibitor PP1 prevented LPS-induced tyrosine phosphorylation of Pyk2 and paxillin. Potential Src family members involved in this response are Hck, Lyn, and Fgr, which are all activated within minutes of LPS stimulation (15, 16). Pyk2 has been shown to associate directly with the Src kinases Fyn, Lck, and Src, and Fyn can phosphorylate Pyk2 in vitro (33, 43, 51, 52). In T cells, Pyk2 tyrosine phosphorylation is regulated by Lck or Fyn, depending on the stimulus (51, 52). Activated Pyk2 in turn may phosphorylate paxillin, as it interacts directly via its C terminus with paxillin and can phosphorylate it in vitro (37, 53). However, although a proportion of Pyk2 is constitutively associated and colocalizes with paxillin in cells, their tyrosine phosphorylation does not always correlate. For example, ligation of CD28 in Jurkat T cells stimulates Pyk2, but not paxillin tyrosine phosphorylation (54). In addition, CD3- and CD45-induced phosphorylation of paxillin, but not Pyk2, is dependent on the Src family kinase Lck in T cells (47). It is therefore likely that Pyk2 and paxillin tyrosine phosphorylation can be regulated independently of each other. Indeed, it appears that Src kinases rather than FAK are responsible for paxillin tyrosine phosphorylation during spreading of chicken embryo cells (55). Pyk2 may therefore act as an adaptor to bring paxillin and Src kinases together, as has been proposed for FAK (55). In the context of LPS signaling, it would therefore be of interest to determine whether Pyk2 or paxillin are direct substrates of Hck, Lyn, or Fgr.
Pyk2 can be activated by a diverse spectrum of stimuli, but there is substantial variation in the kinetics of its tyrosine phosphorylation. Chemokine and integrin receptor engagement leads to the phosphorylation of Pyk2 within minutes (29, 48, 56), whereas other stimuli such as IL-2 and fluoroaluminate require a longer time period (greater than 10 min) to induce the maximum level of Pyk2 phosphorylation (57, 58). The kinetics of LPS-induced Pyk2 and paxillin tyrosine phosphorylation in monocytes fall into the latter category, as maximum phosphorylation was not observed until after 15 min. This delay could be a consequence of a rate-limiting step involving auto/transphosphorylation of Pyk2, before Src kinases can bind and further stimulate tyrosine phosphorylation.
We have found that the PI 3-K inhibitor LY294002 prevents LPS-induced tyrosine phosphorylation of Pyk2 and paxillin. PI 3-K itself is activated by both receptor and nonreceptor tyrosine kinases (59). LPS-induced PI 3-K activation in monocytes is inhibited by the tyrosine kinase inhibitor herbimycin A, and Lyn associates with PI 3-K (21), suggesting that it or a related kinase mediates PI 3-K activation by LPS. The PP1 inhibitor may therefore target both a Src kinase involved in LPS-induced activation of PI 3-K and also a Src kinase(s) involved in phosphorylating Pyk2 and paxillin. Precisely how PI 3-K contributes to Pyk2 or paxillin tyrosine phosphorylation is unclear, but it is possible that it involves a member of the Tec family of Src-related tyrosine kinases, which bind to and are activated by the PI 3-K lipid product phosphatidylinositol 3,4,5-trisphosphate via their pleckstrin homology domains (59, 60). Pyk2 can interact with PI 3-K (61), and this could facilitate Pyk2 and paxillin localization to sites of PI 3-K activity, where a Tec family kinase could then tyrosine phosphorylate them. PI 3-K may also contribute to cell spreading by acting upstream of the GTPase Rac (62), which regulates the formation of lamellipodia and membrane ruffles in many cell types, including Bac1 macrophages (63, 64). Interestingly, paxillin has recently been shown to interact with two components of the Rac signaling pathway, PAK (p21-activated kinase) and PIX (PAK-interacting guanine nucleotide exchange factor) (65), and thus it is possible that Pyk2 and paxillin are part of a large complex of proteins involved in regulating lamellipodium extension and the formation of new adhesion sites.
The subcellular localization of Pyk2 has not previously been described in monocytes and macrophages. Studies in smooth muscle cells (66), fibroblasts (67), and FAK-/- fibroblasts (68) have revealed a predominantly perinuclear localization of Pyk2. Similarly, we have observed that Pyk2 and paxillin have a perinuclear distribution in monocytes and macrophages. In addition, in LPS-stimulated cells, Pyk2 was observed at the plasma membrane, colocalizing with F-actin and paxillin in lamellipodia and membrane ruffles. Ectopic expression of hemagglutinin-tagged Pyk2 in J774 cells confirmed that Pyk2 was present in membrane ruffles (our unpublished data). As lamellipodia extend over the substratum, new integrin-mediated adhesions known as focal complexes form (40), and many cytoplasmic proteins including FAK, vinculin, and paxillin localize to these complexes (63, 64). Dual staining for Pyk2 and paxillin demonstrated that although Pyk2 and paxillin colocalized in membrane ruffles, Pyk2 was not found within paxillin-containing focal complexes. Pyk2 also did not localize to podosomes, which are punctate sites of adhesion commonly observed in cells of monocytic origin (49, 50). This suggests that Pyk2-paxillin interaction occurs in membrane ruffles and/or in the perinuclear region rather than in focal complexes or podosomes. In fibroblasts, a small proportion of Pyk2 was shown to localize in the vicinity of focal adhesions (67, 68), although it had a patchy distribution unlike that of vinculin or paxillin. In contrast, Pyk2 has been reported to colocalize with vinculin in focal adhesions in neutrophils (69). It will therefore be important to determine the precise role that paxillin, and other paxillin family members such as Hic-5 and leupaxin (67, 70), plays in the localization and function of Pyk2 and associated proteins in different cell types.
In conclusion, our observations that LPS-induced monocyte adhesion and spreading are accompanied by Pyk2 and paxillin tyrosine phosphorylation, and that PP1 and LY294002 inhibit both of these responses, suggest that Pyk2 and/or paxillin play an important role in LPS-mediated morphological changes. In addition, the localization of Pyk2 and paxillin in membrane ruffles and lamellipodia is consistent with a role for these proteins in cell motility. Paxillin in particular has been implicated as playing an important role in cell spreading (55). Future studies with mutants of Pyk2 and paxillin should delineate more precisely how each protein contributes to LPS-induced monocyte and macrophage motility responses. As a variety of stimuli can induce Pyk2 and paxillin tyrosine phosphorylation in monocytes, it is likely that in vivo different signals, including LPS, cytokines, and extracellular matrix proteins, act coordinately to stimulate signaling pathways promoting monocyte motility.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anne J. Ridley, Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, U.K. E-mail address:
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; FAK, focal adhesion kinase; HIFCS, heat-inactivated FCS; PI 3-K, phosphatidylinositide 3-kinase; PP1, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Pyk2, proline-rich tyrosine kinase 2; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication September 15, 1999. Accepted for publication December 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  P. C. Patel and R. E. Harrison Membrane Ruffles Capture C3bi-opsonized Particles in Activated Macrophages Mol. Biol. Cell, November 1, 2008; 19(11): 4628 - 4639. [Abstract] [Full Text] [PDF]
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 P. F. Bradfield, C. A. Johnson-Leger, C. Zimmerli, and B. A. Imhof LPS differentially regulates adhesion and transendothelial migration of human monocytes under static and flow conditions Int. Immunol., February 1, 2008; 20(2): 247 - 257. [Abstract] [Full Text] [PDF]
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 H. Gil-Henn, O. Destaing, N. A. Sims, K. Aoki, N. Alles, L. Neff, A. Sanjay, A. Bruzzaniti, P. De Camilli, R. Baron, et al. Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2 / mice J. Cell Biol., September 7, 2007; 178(6): 1053 - 1064. [Abstract] [Full Text] [PDF]
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 K. McPhillips, W. J. Janssen, M. Ghosh, A. Byrne, S. Gardai, L. Remigio, D. L. Bratton, J. L. Kang, and P. Henson TNF-{alpha} Inhibits Macrophage Clearance of Apoptotic Cells via Cytosolic Phospholipase A2 and Oxidant-Dependent Mechanisms J. Immunol., June 15, 2007; 178(12): 8117 - 8126. [Abstract] [Full Text] [PDF]
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 D. M. Nikolic, J. Cholewa, C. Gass, M. C. Gong, and S. R. Post Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1450 - C1458. [Abstract] [Full Text] [PDF]
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 E. B. Lasunskaia, M. N. N. Campos, M. R. M. de Andrade, R. A. DaMatta, T. L. Kipnis, M. Einicker-Lamas, and W. D. Da Silva Mycobacteria directly induce cytoskeletal rearrangements for macrophage spreading and polarization through TLR2-dependent PI3K signaling J. Leukoc. Biol., December 1, 2006; 80(6): 1480 - 1490. [Abstract] [Full Text] [PDF]
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A. Lemarie, C. Morzadec, E. Bourdonnay, O. Fardel, and L. Vernhet Human macrophages constitute targets for immunotoxic inorganic arsenic. J. Immunol., September 1, 2006; 177(5): 3019 - 3027. [Abstract] [Full Text] [PDF]
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 J. G. Kay, R. Z. Murray, J. K. Pagan, and J. L. Stow Cytokine Secretion via Cholesterol-rich Lipid Raft-associated SNAREs at the Phagocytic Cup J. Biol. Chem., April 28, 2006; 281(17): 11949 - 11954. [Abstract] [Full Text] [PDF]
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 X. Cai, M. Li, J. Vrana, and M. D. Schaller Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement. Mol. Cell. Biol., April 1, 2006; 26(7): 2857 - 2868. [Abstract] [Full Text] [PDF]
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 D. Aki, R. Mashima, K. Saeki, Y. Minoda, M. Yamauchi, and A. Yoshimura Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages Genes Cells, April 1, 2005; 10(4): 357 - 368. [Abstract] [Full Text] [PDF]
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J. Melendez, C. Turner, H. Avraham, S. F. Steinberg, E. Schaefer, and M. A. Sussman Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin J. Biol. Chem., December 17, 2004; 279(51): 53516 - 53523. [Abstract] [Full Text] [PDF]
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M. B. Fessler, P. G. Arndt, S. C. Frasch, J. G. Lieber, C. A. Johnson, R. C. Murphy, J. A. Nick, D. L. Bratton, K. C. Malcolm, and G. S. Worthen Lipid Rafts Regulate Lipopolysaccharide-induced Activation of Cdc42 and Inflammatory Functions of the Human Neutrophil J. Biol. Chem., September 17, 2004; 279(38): 39989 - 39998. [Abstract] [Full Text] [PDF]
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A. Honstettre, E. Ghigo, A. Moynault, C. Capo, R. Toman, S. Akira, O. Takeuchi, H. Lepidi, D. Raoult, and J.-L. Mege Lipopolysaccharide from Coxiella burnetii Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4 J. Immunol., March 15, 2004; 172(6): 3695 - 3703. [Abstract] [Full Text] [PDF]
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 J. S. Zhou, D. S. Friend, A. M. Feldweg, M. Daheshia, L. Li, K. F. Austen, and H. R. Katz Prevention of Lipopolysaccharide-induced Microangiopathy by gp49B1: Evidence for an Important Role for gp49B1 Expression on Neutrophils J. Exp. Med., October 20, 2003; 198(8): 1243 - 1251. [Abstract] [Full Text] [PDF]
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N. J. Horwood, T. Mahon, J. P. McDaid, J. Campbell, H. Mano, F. M. Brennan, D. Webster, and B. M.J. Foxwell Bruton's Tyrosine Kinase Is Required For Lipopolysaccharide-induced Tumor Necrosis Factor {alpha} Production J. Exp. Med., June 16, 2003; 197(12): 1603 - 1611. [Abstract] [Full Text] [PDF]
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T. Pedron, R. Girard, and R. Chaby TLR4-dependent Lipopolysaccharide-induced Shedding of Tumor Necrosis Factor Receptors in Mouse Bone Marrow Granulocytes J. Biol. Chem., May 30, 2003; 278(23): 20555 - 20564. [Abstract] [Full Text] [PDF]
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R. A. Worthylake and K. Burridge RhoA and ROCK Promote Migration by Limiting Membrane Protrusions J. Biol. Chem., April 4, 2003; 278(15): 13578 - 13584. [Abstract] [Full Text] [PDF]
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 L.-Y. Chen, B. L. Zuraw, M. Zhao, F.-T. Liu, S. Huang, and Z. K. Pan Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L607 - L613. [Abstract] [Full Text] [PDF]
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 M. Ferlito, O. G. Romanenko, K. Guyton, S. Ashton, F. Squadrito, P. V. Halushka, and J. A. Cook Implication of G i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production Innate Immunity, December 1, 2002; 8(6): 427 - 435. [Abstract] [PDF]
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J. M. Dunty and M. D. Schaller The N Termini of Focal Adhesion Kinase Family Members Regulate Substrate Phosphorylation, Localization, and Cell Morphology J. Biol. Chem., November 15, 2002; 277(47): 45644 - 45654. [Abstract] [Full Text] [PDF]
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 S. R. Yan, W. Al-Hertani, D. Byers, and R. Bortolussi Lipopolysaccharide-Binding Protein- and CD14-Dependent Activation of Mitogen-Activated Protein Kinase p38 by Lipopolysaccharide in Human Neutrophils Is Associated with Priming of Respiratory Burst Infect. Immun., August 1, 2002; 70(8): 4068 - 4074. [Abstract] [Full Text] [PDF]
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C. Ameixa and J. S. Friedland Interleukin-8 Secretion from Mycobacterium tuberculosis-Infected Monocytes Is Regulated by Protein Tyrosine Kinases but Not by ERK1/2 or p38 Mitogen-Activated Protein Kinases Infect. Immun., August 1, 2002; 70(8): 4743 - 4746. [Abstract] [Full Text] [PDF]
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F. Paulhe, C. Racaud-Sultan, A. Ragab, C. Albiges-Rizo, H. Chap, N. Iberg, O. Morand, and B. Perret Differential Regulation of Phosphoinositide Metabolism by alpha Vbeta 3 and alpha Vbeta 5 Integrins upon Smooth Muscle Cell Migration J. Biol. Chem., November 2, 2001; 276(45): 41832 - 41840. [Abstract] [Full Text] [PDF]
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S. H. Diks, S. J.H. van Deventer, and M. P. Peppelenbosch Invited review: Lipopolysaccharide recognition, internalisation, signalling and other cellular effects Innate Immunity, October 1, 2001; 7(5): 335 - 348. [Abstract] [PDF]
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S. Jeyaseelan, M. S. Kannan, R. E. Briggs, P. Thumbikat, and S. K. Maheswaran Mannheimia haemolytica Leukotoxin Activates a Nonreceptor Tyrosine Kinase Signaling Cascade in Bovine Leukocytes, Which Induces Biological Effects Infect. Immun., October 1, 2001; 69(10): 6131 - 6139. [Abstract] [Full Text] [PDF]
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S. Latour, H. Tanaka, C. Demeure, V. Mateo, M. Rubio, E. J. Brown, C. Maliszewski, F. P. Lindberg, A. Oldenborg, A. Ullrich, et al. Bidirectional Negative Regulation of Human T and Dendritic Cells by CD47 and Its Cognate Receptor Signal-Regulator Protein-{alpha}: Down-Regulation of IL-12 Responsiveness and Inhibition of Dendritic Cell Activation J. Immunol., September 1, 2001; 167(5): 2547 - 2554. [Abstract] [Full Text] [PDF]
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 G. M. Scholz, S. D. Hartson, K. Cartledge, L. Volk, R. L. Matts, and A. R. Dunn The Molecular Chaperone Hsp90 Is Required for Signal Transduction by Wild-Type Hck and Maintenance of Its Constitutively Active Counterpart Cell Growth Differ., August 1, 2001; 12(8): 409 - 417. [Abstract] [Full Text] [PDF]
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Y.-S. Bae, H. Bae, Y. Kim, T. G. Lee, P.-G. Suh, and S. H. Ryu Identification of novel chemoattractant peptides for human leukocytes Blood, May 1, 2001; 97(9): 2854 - 2862. [Abstract] [Full Text] [PDF]
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S. Meconi, C. Capo, M. Remacle-Bonnet, G. Pommier, D. Raoult, and J.-L. Mege Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis Infect. Immun., April 1, 2001; 69(4): 2520 - 2526. [Abstract] [Full Text] [PDF]
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F. J. Pixley, P. S. W. Lee, J. S. Condeelis, and E. R. Stanley Protein Tyrosine Phosphatase {phi} Regulates Paxillin Tyrosine Phosphorylation and Mediates Colony-Stimulating Factor 1-Induced Morphological Changes in Macrophages Mol. Cell. Biol., March 1, 2001; 21(5): 1795 - 1809. [Abstract] [Full Text]
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 U. Kintscher, S. Wakino, S. Kim, E. Fleck, W. A. Hsueh, and R. E. Law Angiotensin II Induces Migration and Pyk2/Paxillin Phosphorylation of Human Monocytes Hypertension, February 1, 2001; 37(2): 587 - 593. [Abstract] [Full Text] [PDF]
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J. M. Watson, T. W. Harding, V. Golubovskaya, J. S. Morris, D. Hunter, X. Li, J. S. Haskill, and H. S. Earp Inhibition of the Calcium-dependent Tyrosine Kinase (CADTK) Blocks Monocyte Spreading and Motility J. Biol. Chem., January 26, 2001; 276(5): 3536 - 3542. [Abstract] [Full Text] [PDF]
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 W. Wang, M. A. Jobst, B. Bell, C.-R. Zhao, L.-H. Shang, and D. O. Jacobs Cr supplementation decreases tyrosine phosphorylation of the CreaT in skeletal muscle during sepsis Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1046 - E1054. [Abstract] [Full Text] [PDF]
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