From the University of North Carolina Lineberger
Comprehensive Cancer Center, § Departments of Medicine and
Pharmacology, and Department of Microbiology and
Immunology and Comprehensive Center for Inflammatory Disorders,
University of North Carolina,
Chapel Hill, North Carolina 27599-7295
Received for publication, August 1, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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Freshly isolated peripheral blood monocytes lack
focal adhesion kinase (p125FAK) but activate a second
member of this kinase family, calcium-dependent tyrosine
kinase (CADTK; also known as Pyk2/CAK Integrin-mediated monocyte adhesion to extracellular matrix or
endothelial monolayers results in a dramatic alteration of the
cytoskeleton and rapid transcriptional activation of numerous genes
required in the inflammatory response (1-6). Monocytes adhered in the
absence of any chemotactic stimuli fail to polarize and maintain a
radial symmetry while exhibiting continuous but moderate membrane
ruffling (7).1 In contrast,
monocytes adhered in the presence of either chemotactic stimuli or
autologous serum will rapidly polarize and migrate (9, 10). Migration
requires both contractile and protrusive events, mediated by changes in
the actin cytoskeleton (7). Although several lamellipodia are extended
simultaneously, directional movement is obtained by the development of
a dominant lamellipodium protruding in response to a concentration
gradient (7).
Focal adhesion kinase
(p125FAK)2 often
coordinates integrin-mediated cell migration in nonhematopoetic cells
(10, 11). After adhesion, p125FAK is targeted to focal
adhesions, where it colocalizes with integrins and becomes activated
and phosphorylated on multiple tyrosine residues. p125FAK
serves as both a kinase and as a structural protein capable of binding
other proteins in a phosphotyrosine-dependent
(e.g. src or Grb2) and -independent (e.g.
paxillin) manner (11, 12). In an alternatively spliced form of
p125FAK, FRNK, the C-terminal noncatalytic domain has been
used to identify the role of p125FAK in motility,
presumably because its experimental overexpression displaces
p125FAK from its site of action. For example, FRNK
expression in endothelial cells blocked migration (13). In addition,
fibroblasts isolated from p125FAK A second member of the p125FAK family has been identified,
the calcium-dependent tyrosine kinase CADTK (17), also
known as Pyk2, CAK A number of recent studies report CADTK/Pyk2 tyrosine phosphorylation
in cells of hematopoetic origin, e.g. T and B lymphocytes (37-39), monocytes (23, 29), NK cells (34, 40, 41),
granulocytes (42), bone marrow progenitors (43), mast cells (44),
megakaryocytes (45), and platelets (46, 47). The stimuli that trigger
CADTK in these cells are often associated with cell motility or at
least cytoskeletal rearrangement. However, the downstream targets of CADTK activation and its role in hematopoetic cell cytoskeletal rearrangement are unknown. In other cell types, CADTK has been implicated in the activation of ERK, JNK, and p38 MAP kinases (18, 26,
48). Monocytes constitutively express a detectable level of activated
p38 MAP kinase and rapidly activate ERK and JNK kinases upon adhesion
(6). However these activations are transient in motile monocytes
adhered in the presence of autologous serum.1
The present work supports a role for CADTK in monocyte motility first
by showing that CADTK immunolocalized to the leading edge ruffling
lamellipodia. Taking a cue from studies of the role of
p125FAK in adhesion and motility and its inhibition by FRNK
(36), we inhibited CADTK function by introducing a homologous protein
referred to as CRNK (36, 49), beginning at an initiating methionine (CADTK Met-685) in a position similar to that of FRNK (36). This
encompasses areas of the proline-rich region to the C terminus. In
freshly isolated monocytes, GST-CRNK blocked adhesion-induced CADTK
autophosphorylation and also resulted in dramatic inhibition of cell
spreading, a loss of cell motility, and blockade of tyrosine phosphorylation redistribution. Although GST-CADTK had no effect on JNK
activation, GST-CRNK introduction did block adhesion-induced ERK
activation. Importantly, introduction of other GST proteins did not
produce the same results, and GST-CRNK inhibited monocyte motility
without altering monocyte phagocytosis or
adhesion-dependent transcriptional activation of cytokine
genes. These results suggest that CADTK regulates cytoskeletal
reorganization and motility but is not required for all
adhesion-dependent functions.
Isolation of Monocytes and Culture Conditions--
Human
monocytes were isolated from healthy volunteer donors as described
previously (5) by centrifugation through Ficoll/Histopaque 1077 (Sigma)
and a Percoll (Amersham Pharmacia Biotech) step gradient (50).
Monocytes were adherently cultured in endotoxin-free RPMI 1640 medium
at 37 °C and 5% CO2. To produce polarized cells,
monocytes were cultured in 5% autologous serum (9). Monocyte motility was assessed using 20-s frame-time lapse microphotometry with a 40×
phase objective and the NIH Image Movie program. Monocytes were studied
for 70 frames.
Immunostaining--
Cells were adhered for 30 min, fixed in 4%
paraformaldehyde in PBS (pH 7.5) for 10 min, and permeabilized with
0.2% Triton X-100 for 5 min at room temperature. Cells were blocked
with 25% normal goat serum in PBS for 30 min at room temperature,
washed in PBS, and incubated with BodipyFL-phallacidin
(Molecular Probes) diluted 1:20 with 25% goat serum in PBS for 30 min
at room temperature. For the detection of CADTK, cells were stained
with purified rabbit polyclonal anti-CADTK antibody (1:50-1:200). The
anti-CADTK #72 antiserum was raised against amino acids 660-880 of
CADTK expressed as a GST fusion protein. Cells were washed in PBS and
incubated with a rhodamine (tetramethylrhodamine B
isothiocyanate)-conjugated affinity-purified goat anti-rabbit IgG
(Molecular Probes) 1:400 dilution with 25% goat serum in PBS. After
extensive washing in PBS, cells were mounted in 50% glycerol in PBS.
Specificity of the anti-CADTK antisera was determined by blocking with
purified GST-CRNK for 1 h at room temperature before use.
Preparation GST Fusion Proteins--
GST fusion proteins were
grown and isolated from the expression plasmids pGEX3X (Amersham
Pharmacia Biotech), pGEX3X-C3 (generously provided by Dr. K. Burridge,
University of North Carolina), or pGEX3X-CRNK as described (6). Protein
concentrations were determined by the Bradford method (Bio-Rad).
GST-CRNK was labeled with Oregon green 488 (Molecular Probes) according
to the manufacturer's recommendations.
Electroporation of GST Fusion Proteins into
Monocytes--
Monocytes (1 × 107/ml) were
resuspended in intracellular electroporation buffer, which consisted of
125 mM KOH, 4 mM NaOH, 73 mM PIPES,
10 mM NaHCO3, 5 mM
K2HPO4, 5 mM
KH2PO4, 5 mM D-glucose, 4 mM MgCl2, 1 mM MgSO4,
10 µM CaCl2 (pH 7.0) (47). Approximately 2 × 106 monocytes were electroporated in the presence
of 250 µg of purified GST, GST-CRNK, or GST-C3 proteins in a total
volume of 400 µl in a 0.4-cm sterile cuvette at 750 V/cm2. After electroporation, cells were incubated on ice
for 5 min in 1 ml of RPMI 1640 containing 10 µg/ml polymyxin-B before
use. Purified GST has a molecular mass of ~24 kDa. Purified GST-CRNK and GST-C3 have molecular masses of ~42 and 46 kDa, respectively.
Immunoprecipitation--
Cells, 5-8 × 106,
were adhered, rinsed, and lysed on ice for 30 min in 50 mM
Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM sodium vanadate, 10 µg/ml
leupeptin, 1 µg/ml aprotinin, and 10 µg/ml phenylmethylsulfonyl
fluoride. Lysates were clarified at 10,000 rpm for 30 min at 4 °C.
Equivalent protein concentrations were incubated with 5 µl of
anti-CADTK antibody for 1 h at 4 °C in the presence of 20 µl
of protein-A/protein G-agarose beads. Beads were washed 3 times in
lysis buffer and resuspended in 30 µl of 2× Laemmli sample buffer,
boiled, and frozen until use.
Western Transfer Analysis--
Whole cell extracts were prepared
from 2 × 106 cells by direct lysis into Laemmli
buffer. Proteins were resolved by SDS-10% polyacrylamide gel
electrophoresis and analyzed by immunoblotting using antibodies that
detect the active, phosphorylated forms of the ERK, JNK, and p38 MAP
kinases. The p38 MAP kinase antibody was obtained from New England
Biolabs. Phospho-specific ERK and JNK antibodies were from Promega and
used according to the manufacturer's recommendations. Horseradish
peroxidase-RC20 (anti-phosphotyrosine) was obtained from Transduction Labs.
Phagocytosis Assays--
Monocytes, 1 × 106,
were adhered in the presence of autologous serum for 30 min and then
fed 3 × 105/ml FITC-conjugated zymosan A
(Saccharomyces cerevisiae) bioparticles (Molecular Probes)
for 45 min. Extracellular fluorescence was quenched by the addition of
0.4% trypan blue. The percentage of phagocytosing cells was determined
as the fraction of monocytes with internalized fluorescent particles
from 500 monocytes counted by fluorescence microscopy. Experiments were
performed in triplicate and repeated at least twice.
CADTK Concentrates toward the Leading Edge in Migrating, Polarized
Monocytes--
To begin tests of the role of CADTK in monocyte
motility, we immunolocalized CADTK under several conditions. When
monocytes were adhered under serum-free conditions, they failed to
polarize and maintained radial symmetry (Fig.
1A, M-NM). In
contrast, monocytes adhered in the presence of 5% autologous serum
rapidly polarized, generated multiple small lamellipodia, but then
migrated after development of a single large lamellipodium (Fig.
1A, M-M). Retraction fibers and filapodia were
only observed in motile monocytes. Indirect immunofluorescence revealed
that CADTK localized in the leading edge of motile monocytes and to the
actin ruffles of both nonpolarized and polarized monocytes (Fig. 1,
B and E). CADTK was detected diffusely throughout
the monocyte cytoplasm in nonmotile cells, although it also
concentrated in the nuclear cleft. Regions of intense actin staining
showed increased localization of CADTK. Although some monocytes
demonstrated increased CADTK localization at the adhered surfaces,
CADTK immunoreactivity was most prominent in the cortical ruffles of
both polarized and nonpolarized cells (Fig. 1, B and
E). CADTK did not apparently localize to podosomes or points
of focal contact, retraction fibers, or filapodia. In contrast, as
previously shown, (28), CADTK did localize to points of contact in the
large enucleated structures, which are presumably derived from the
platelet precursor, megakaryocytes (Fig. 1B). CADTK
localization was highly specific, as immunostaining with antisera that
was absorbed with purified, baculoviral-expressed and purified CRNK,
which contains the amino acids used to construct the immunizing GST
fusion peptide, failed to detect any immunoreactivity within the
ruffles (data not shown). Another group has also recently published
data showing that CADTK/Pyk2 localized in the ruffles of
lipopolysaccharide-stimulated human monocytes and murine macrophages (51).
CADTK Is Required for Monocyte Spreading, Adhesion-induced
Phosphorylation and ERK Activation--
Localization of CADTK in the
leading edge ruffles reinforced a possible role in migration and
monocyte recruitment. To test this hypothesis, a GST fusion protein was
generated by cloning the C-terminal region of CADTK, CRNK, (nucleotides
2050-3027) into the expression vector pGEX3X (36, 49). Purified
GST-CRNK has a molecular mass of ~42 kDa. Coexpression of CADTK and
CRNK in 293T cells (49) or expression in rat liver epithelial or rat
smooth muscle cells inhibits CADTK
activation.4 Thus, CRNK has
dominant negative activity.
Monocytes cannot be transfected by conventional methods. However, high
efficiency introduction of recombinant proteins into cells can be
achieved by electroporation (6, 50). We have recently shown that
electroporation is adaptable for primary monocytes with proteins
retaining functional activity over time (6).
Monocytes were electroporated with ~300 µg of fluorescently labeled
GST-CRNK to assess the uptake efficiency. Electroporated monocytes were
extensively washed in cold medium and cytospin preparations made
immediately or cells held at 37 °C for various time periods.
Although uptake distribution was heterogenous, greater than 90% of
monocytes were positive (Fig.
2A). Monocytes remained viable
and retained expression of the fusion protein even after incubation for
90 min. Similar results were obtained with other FITC-labeled proteins
including GST-C3 or with FITC-conjugated dextran (data not shown).
Western blot analysis of monocytes lysed 30 min after electroporation
and washing revealed comparable protein uptake for multiple GST fusion
proteins (Fig. 2B), GST at ~21 kDa, GST-CRNK at ~42 kDa,
and the GST fusion protein including the RhoA inhibitor, C3
transferase, at ~46 kDa. Both GST-CRNK and GST-C3 show some protein
degradation after introduction and adhesion. This was not surprising,
as we noted intense lysosomal staining after the monocytes were
electroporated with the FITC-labeled GST-CRNK.
To test the functionality of GST-CRNK, we assessed both a direct effect
on CADTK phosphorylation and a downstream consequence of CADTK
activation. As previously shown (23), CADTK is not phosphorylated in
freshly isolated nonadherent monocytes. Adhesion rapidly induced
tyrosine phosphorylation of several proteins including CADTK (Fig.
2C). Electroporation per se did not interfere
with adhesion-induced CADTK tyrosine phosphorylation; monocytes
electroporated with GST alone showed CADTK phosphorylation comparable
with that of unelectroporated monocytes (compare adherence,
ADH, with GST, Fig. 2D).
Electroporation of GST-CRNK, however, inhibited both total
adhesion-induced tyrosine phosphorylation as well as tyrosine phosphorylation of endogenous CADTK without affecting total CADTK levels (Fig. 2, C and D). The inhibition of tyrosine
phosphorylation was similar to that seen with 20 µM genistein.
We recently showed that adhesion rapidly activated ERK and JNK kinases
in monocytes (6). As CADTK/Pyk2 has been shown to activate ERK, JNK,
and p38 MAP kinases in various cell types (18, 26, 48), we assessed the
effects of GST-CRNK on adhesion-induced MAP kinase activation. GST-CRNK
clearly blocked adhesion-induced ERK activation (Fig. 2E).
Although GST-CRNK had a slight but inconsistent effect on p38 MAP
kinase activation, there was no effect on activated JNK expression
(Fig. 2E). Similar results were obtained whether the
inhibitory CRNK protein electroporated was from bacteria (GST or
GST-CRNK) or from baculovirus-infected insect cells (GFP or CRNK) (Fig.
2E). The effect was specific for CRNK as we detected no
modulation of ERK, JNK, or p38 MAP kinase activation in monocytes electroporated with GST-C3 despite profound differences in cell morphology and cell spreading (see below).
We recently showed that inhibition of adhesion-induced tyrosine
phosphorylation with either genistein or the cellular phosphatase, PTP1B, introduced as GST-PTP1B, can inhibit monocyte spreading (6).
Because GST-CRNK also inhibited total tyrosine phosphorylation, we
assessed the effects on monocyte adhesion and spreading. Monocytes were
adhered and immunostained for tyrosine phosphorylation (Fig. 3A). In control GST
electroporated monocytes, tyrosine phosphorylation was seen as a
pattern of intense staining around the nucleus and a distinct
continuous ring of peripheral staining in the ruffling lamellipodia.
Phosphotyrosine staining correlated with that of filamentous actin. In
contrast, tyrosine phosphorylation appeared random and diffuse in
GST-CRNK monocytes, and these cells lacked both the continuous
peripheral edge staining seen in control monocytes and the intense
perinuclear phosphotyrosine staining (Fig. 3A). There was
little correlation between phosphotyrosine staining and actin
localization.
One striking difference between GST-, GST-C3-, and
GST-CRNK-electroporated monocytes was the effect on cell spreading.
Although GST-CRNK electroporated cells adhered with similar efficiency to those electroporated with GST, spreading was markedly compromised (Fig. 3B). Using the NIH Image software, we quantitated
spreading by measuring the total surface area occupied by each cell.
Approximately 50% of monocytes electroporated with GST occupied a
surface area of at least 2000 pixels. In contrast, less than 20% of
GST-CRNK monocytes were well spread (>2000 pixels), with the majority
of GST-CRNK monocytes occupying a surface area of less than 2000 pixels. As a positive control for modifying cell spreading, we electroporated monocytes with the RhoA inhibitor C3 transferase (52).
Monocytes electroporated with GST- C3 demonstrated profound cell
flattening and increased cell spreading, with almost 70% of cells
having an area of >2000 pixels (Fig. 3B). Thus,
electroporation of a GST protein does not, by itself, inhibit cell
spreading. In other experiments we showed that cell spreading was
neither inhibited by the MEK1/2 (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase 1/2) inhibitor PD
98059 nor the p38 MAP kinase inhibitor SB 203580, suggesting that the
absence of activation of these kinases is not directly responsible for the alteration in the cytoskeletal organization seen in GST-CRNK monocytes (data not shown).
CADTK Is Critical for Monocyte Motility but Not Phagocytosis nor
Adhesion-induced Cytokine Gene Expression--
Monocytes exhibit
chemokinetic motility when cultured in the presence of autologous serum
(9, 10). Because CADTK localized to the leading edge lamellipodia and
membrane ruffles and is activated by chemokines (23), we assessed the
motility by time-lapse photography of monocytes electroporated with
either GST, GST-CRNK, or GST-C3 proteins and adhered to glass slides in
the presence of 5% autologous serum. Data from an experiment
superimposing the first and last frames over 25 min of monocyte
motility are displayed in Fig. 4.
GST-electroporated cells exhibited motility for greater than 20 min and
covered distances of several cell diameters during this time. In
contrast, although continuing to ruffle and bleb, GST-CRNK-electroporated monocytes failed to migrate. Quantification of
5 independent experiments revealed that only an average 26.0 ± 17.9% (range 10-50%) of monocytes electroporated with GST-CRNK exhibited motility. Motility in monocytes electroporated with GST was
demonstrable in 82.3 ± 12.5% (range 70-100%) of cells. Although the leading edges of GST-C3-electroporated monocytes were
highly motile, allowing the majority of the cell to move up to several
diameters, the tails failed to retract. This indicates that GST
proteins that have distinct effects on monocyte cytoskeletal organization also have distinct effects on motility.
To determine whether GST-CRNK was inhibiting other monocyte functions,
we examined the role of CADTK in phagocytosis and adhesion-induced gene
induction. Electroporated cells were adhered in the presence of
autologous serum for 1 h and then fed fluorescent zymosan yeast particles for 45 min, fixed, and analyzed. Random fields were chosen by
light microscopy and then subjected to fluorescence to avoid bias in
data collection. Although GST-CRNK had marked effects on monocyte
spreading and motility, phagocytosis was unimpaired by the
electroporated protein (Fig. 5). There
was no significant difference between GST-, GST-C3-, and
GST-CRNK-electroporated monocytes in the percentage of cells capable of
phagocytosis.
Monocyte adhesion and spreading are associated with the rapid (within 5 min) transcriptional activation of a variety of cytokine genes (4-6).
Because these events occur consequential to profound changes in
cytoskeletal organization, we were interested in determining whether
GST-CRNK modulation of monocyte spreading also interferes with
adhesion-induced cytokine gene induction. As previously reported (5,
6), nonadhered monocytes do not spontaneously express interleukin-1 Freshly isolated peripheral blood monocytes do not make typical
focal adhesions or actin stress fibers nor do they express p125FAK. Monocytes do express the closest known orthologue
tyrosine kinase, CADTK. These highly related signaling molecules are
expressed together in many cell types and presumably have distinct but
perhaps complementary functions. The specific biology of monocytes
apparently allow it to dispose with p125FAK and rely on a
single cytoskeletal-associated kinase from this family, CADTK. In
monocytes, CADTK does not localize to contact points upon adherence
but, instead, localizes to the ruffling lamellipodial structures. As
shown in Fig. 1 and as recently published by Williams and Ridley (51),
CADTK/Pyk2 localized to the leading lamellipodia and ruffling membranes
of adhered motile monocytes (Fig. 1) and lipopolysaccharide-stimulated
monocytes and macrophages.
CADTK/Pyk2/CAK CADTK is rapidly phosphorylated upon adhesion in monocytes,
megakaryocytes, and platelets and when overexpressed in COS cells (23,
28, 46-47). Additionally, CADTK can be tyrosine-phosphorylated after
engagement of integrins (23, 44, 53), integrin-dependent platelet aggregation (46), or by activation of either the T cell
receptor complex or the B cell antigen receptor complex (37, 39, 53,
54). Activation of CADTK in platelets results in its translocation from
the cytosol to the cytoskeleton (46). In most hematopoetic cells,
cytoskeletal organization is critical to CADTK tyrosine phosphorylation
because treatment with cytochalasin D to disrupt the actin cytoskeleton
inhibits CADTK phosphorylation (27, 30, 53). We have recently shown
that monocyte cytoskeletal organization can be dramatically disrupted
by the introduction of the phosphatase PTP1B (GST-PTP1B) or by
treatment with tyrosine kinase inhibitor, genistein (6). Here we have
identified adhesion-induced tyrosine phosphorylation of CADTK as having
a regulatory role in monocytic cytoskeletal organization. Inhibition of
adhesion-induced CADTK autophosphorylation by the introduction of
GST-CRNK blocked cell spreading and disrupted the distribution of
tyrosine-phosphorylated proteins in adhered monocytes, presumably by
displacing CADTK. We had previously shown that cotransfection of CRNK
can block CADTK activation (49). However, transfection is not suitable for introduction of genes into monocytes, and we used electroporation as a means to introduce GST-CRNK into freshly isolated monocytes. FITC-labeling of GST-CRNK and other GST proteins showed the utility and
efficiency (>90% of cells) of this method. The amount of GST-CRNK electroporated into monocytes is easily detectable in lysates and
exceeds the amount of CADTK. When immunofluorescence was performed on
GST-CRNK electroporated cells, the protein was widely distributed and
did not just localize to structures binding CADTK. However, introduction of GST, GST-C3, or GST-PTP1B (6) produced similar or
greater levels of protein than GST-CADTK; all served as controls showing the specificity of the GST-CRNK biologic effect. The specific changes in motility are only seen with CRNK and are clearly not simply
due to introduction of a range of proteins.
Preliminary experiments indicate that introduction of GST FRNK also
blocks monocyte motility. FAK is not expressed in monocytes; however,
we have shown using liver epithelial cells, rat smooth muscle cells,
and human embryonic kidney cells that over expression of FRNK disrupts
both FAK and CADTK signaling. Therefore, it was not a surprise that the
high level of GST-FRNK obtained by electroporation blocked CADTK
function. We believe that FRNK disrupts the "cytoskeletal engagement" step (23), which we have hypothesized is involved in
CADTK activation in cells. Since FAK has no direct role in these cells,
FRNK introduction may inhibit some processes simply by binding to
CADTK-binding proteins with motifs similar to its other cell binding partners.
The present data, in addition to our previous observations with
GST-PTP1B and genistein, highlight the interrelation between adhesion-dependent tyrosine phosphorylation and
cytoskeletal organization in monocytes. They suggest that CADTK
activation is important in triggering these tyrosine phosphorylation
events but do not prove that CADTK alone is responsible. Decreases in
adhesion-dependent monocyte tyrosine phosphorylation are
also seen in Hck and Fgr double knockout macrophage cells (55). Thus
src family members could be upstream or downstream of CADTK in monocyte
adhesion-signaling events.
Introduction of GST-CRNK into monocytes not only inhibited CADTK
tyrosine phosphorylation but also led to a more global loss and lack of
cellular redistribution of other phosphotyrosine-containing proteins.
GST-CRNK but not GST interfered with monocyte cytoskeletal reorganization, as indicated by lack of spreading and motility. The
identity of the CADTK tyrosine-phosphorylated proteins key to these
events is unknown. However, CADTK can interact with and/or phosphorylate several signaling-associated proteins in a variety of
cells; these include Grb2, Src family tyrosine kinases,
p130Cas, Crk (56), paxillin (30), and SHPS-1 (57) in
numerous cell types including THP-1 promonocytic cells and epithelial
cells (29, 30). A particularly interesting substrate is the abundant paxillin family member leupaxin, which is present in monocytes and
which has been demonstrated to bind to CADTK (32). Leupaxin as a
tyrosine-phosphorylated monocyte cytoskeletal protein may be an
important target disrupted by the introduction of GST CRNK.
CADTK has also been linked to other signaling pathways including
activation of the MAP kinases. Monocyte adhesion resulted in the
activation of ERK and JNK kinases (6). Introduction of GST-CRNK blocked
ERK activation but not JNK activation, suggesting that in monocytes,
CADTK is an upstream mediator of ERK activation. This has also been
suggested for other cell types including NK cells (41). The
consequences of inhibiting ERK activation in monocytes are not readily
apparent. We have previously shown that a loss of ERK activation
rapidly effects adhesion-induced cytokine mRNA stability without
significantly inhibiting gene transcription (6). Here we have confirmed
the observation that ERK activation is dispensable for adhesion-induced
transcriptional activation of interleukin-1 We have observed that the pharmacologic inhibitor of
MEK1/2(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2), PD 98059, inhibited neither cell spreading nor cell
motility despite a loss of ERK activation in primary monocytes5 or primary bone
marrow CD34+ progenitor cells (8). Thus in monocytes,
CADTK-induced activation of ERK is probably not a primary mechanism by
which CADTK is involved in cell motility.
CADTK overexpression in fibroblasts (cells that do not normally express
CADTK) does not result in cytoskeletal association and often results in
cell rounding and apoptosis (25). This is despite the facts that CADTK
contains a focal adhesion targeting region that closely resembles
p125FAK and that CADTK binds paxillin with similar affinity
to that of p125FAK (30) as well as Hic-5 and leupaxin
(31-32). Clearly there are other complexities to CADTK cytoskeletal
association. For example, in rat smooth muscle cells, which normally
express both p125FAK and CADTK, CADTK is localized to the
actin cytoskeleton. In mouse embryo fibroblasts derived from FAK In summary, our results implicate CADTK as a critical intermediary of
monocyte motility. Our previous results that introduction of a tyrosine
phosphatase PTP1B also inhibits motility (6) and the fact that
macrophages derived from mice deficient in the related Src kinases, Hck
and Fgr, also exhibited severely retarded motility (56) add to the
evidence that tyrosine phosphorylation regulates monocyte motility. The
macrophages from Hck and Fgr /RAFTK/FAK2), upon adhesion or
stimulation with chemokines. To study the role of CADTK in monocyte
adherence and motility, we performed immunocytochemical localization
that showed CADTK at the leading edge and ruffling lamellipodial
structures in freshly isolated, adhered human monocytes. We next
introduced CADTK/CAK
-related non-kinase (CRNK), the C-terminal noncatalytic domain of CADTK, into monocytes by electroporation and
showed that it inhibited CADTK autophosphorylation. Introduction of the
fusion protein glutathione S-transferase (GST)-CRNK also reduced (i) cell spreading, as reflected in a reduced cell area 30 min
after adhesion, (ii) adhesion-induced phosphotyrosine increases and
redistribution into lamellipodia, and (iii) adhesion-induced extracellular signal-regulated protein kinase (ERK) activation. In
control experiments, introduction of GST or GST-C3 transferase (an
inhibitor of RhoA GTPase activity) by electroporation did not affect
these parameters. Monocytes adhered in the presence of autologous serum
were highly motile even after introduction of GST (83% motile cells).
However, only 26% of monocytes with introduced GST-CRNK were motile.
In contrast, GST-CRNK-treated monocytes were fully capable of
phagocytosis and adhesion-induced cytokine gene induction, suggesting
that CADTK is not involved in these cellular activities and that
GST-CRNK introduction does not inhibit global monocyte functions. These
results suggest that CADTK is crucial for the in vitro
monocyte cytoskeletal reorganization necessary for cell motility and is
likely to be required in vivo for recruitment to sites of inflammation.
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MATERIALS AND METHODS
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/
knockout mice had
reduced motility (14), whereas ectopic expression of
p125FAK in p125FAK-null cells dramatically
enhanced cell spreading and migration (15, 16). The mechanism by which
p125FAK promotes cell migration is, however, still not clear.
, RAFTK, and FAK2 (18-21). CADTK and p125
FAK are coexpressed in many epithelial, some mesenchymal, and
hematopoetic cells and presumably have complementary functions. In
contrast to fibroblasts and epithelial cells, freshly isolated
peripheral blood monocytes do not form typical focal adhesions and
totally lack p125FAK transcripts and protein (22, 23).
Monocytes do express CADTK, (23) a cytoskeletal associated kinase of
this family, as a splice variant that deletes 42 amino acids in the
first proline-rich domain, reducing the approximate molecular mass from
115 to 110 kDa. This variant has been observed in other cell types as
well (24, 25). Unlike p125FAK, which is constitutively
phosphorylated in adherent cells, in resting adherent epithelial and
mesenchymal cells CADTK is predominantly unphosphorylated. Agonists,
which raise intracellular calcium, activators of G-protein-coupled
receptors, and other signals such as protein kinase C induce CADTK
tyrosine autophosphorylation (17, 18, 26-28). We and others (23, 29)
show that adhesion or other stimuli activate monocyte CADTK tyrosine
phosphorylation. There are complexities to adherence-induced activation
of CADTK, as adherence to tissue culture dishes provides a stronger
stimulus than adhesion to fibronectin-coated surfaces (23). CADTK
associates with cytoskeletal proteins such as paxillin, Hic-5 (31), and leupaxin (32) through conserved "LD" motifs, (33), found in all
three proteins. CADTK activation results in paxillin tyrosine phosphorylation in rat hepatic epithelial (30) and other cells (34, 35)
and may well be involved in tyrosine phosphorylation of leupaxin (32).
CADTK immunolocalization varies by cell type. Immunolocalization
demonstrated CADTK in a concentric pattern, like that of actin in rat
hepatic epithelial cells,3
along actin stress fibers and in focal adhesions in rat smooth muscle
cells (27) and diffusely throughout the cell when overexpressed in
fibroblasts (36).
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ABSTRACT
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Fig. 1.
Lamellipodial immunolocalization of CADTK in
adhered monocytes. Freshly isolated human monocytes were adhered
to glass coverslips for 30 min in the presence of 5% autologous serum.
Cells were rinsed in PBS, fixed in 4% paraformaldehyde, and processed
for immunofluorescence as described under "Materials and Methods"
A, actin immunofluorescence of monocytes adhered in the
absence (left) or presence of serum (right).
P, giant platelet; M-NM, monocyte
nonmotile; M-M, monocyte motile; L, lymphocyte.
B, CADTK immunofluorescence of cells shown in A. C, basal surface of adhered monocytes stained for actin.
D, apical surface of monocytes shown in C stained
for actin. E, apical surface of monocytes shown in
C stained for CADTK.
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Fig. 2.
High efficiency uptake of GST-CRNK inhibits
adhesion-induced CADTK autophosphorylation and ERK activation.
A, monocytes were electroporated with 300 µg of purified
FITC-labeled GST-CRNK protein and sequentially washed 4× in cold
serum-free media, and cytospin preparations were made. Electroporation
of FITC-labeled GST-CRNK resulted in >90% positive cells detectable
within minutes. A representative experiment is shown (n = 3). B, monocytes were electroporated with GST,
GST-CRNK, or GST-C3 and adhered for 30 min. Western blot
analysis was performed on whole cell lysates for expression of the
GST-tag. CTRL, control. C, monocytes were
electroporated, and Western blot analysis was performed on both
nonadhered (NAD) and adhered monocytes
(GST-CRNK, Genistein) for total
phosphotyrosine. As a control, monocytes were treated with genistein
(20 µM) for 20 min before adhesion. D,
monocytes were electroporated with GST or GST-CRNK and adhered
for 30 min, and CADTK was immunoprecipitated (IP) as
described previously (20). Equivalent protein concentrations were
loaded, and proteins were separated by 10% SDS-polyacrylamide gel
electrophoresis. Blots were sequentially probed for phosphotyrosine and
CADTK. NAD, nonadherent monocytes; ADH, adherent
monocytes; GN/A, GN4 rat smooth muscle cells stimulated with
angiotensin II. pTyr, phosphorylated tyrosine. E,
adhesion-induced ERK activation was inhibited by GST-CRNK. Monocytes
were electroporated with either GST fusion proteins (GST,
GST-CRNK, or GST-C3), or with baculoviral-produced recombinant
proteins, adhered for 30 min in serum-free medium. Western blot
analysis was performed with phospho-specific antibodies for ERK, p38,
or JNK MAP kinases. E. coli, Escherichia
coli.
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Fig. 3.
GST-CRNK alters phosphotyrosine localization
and cell spreading. A, GST-CRNK alters phosphotyrosine
localization in monocytes away from the peripheral edge.
Monocytes were electroporated with GST or GST-CRNK, adhered for 30 min,
fixed in 3% paraformaldehyde-sucrose, and immunostained for
phosphotyrosine as described under "Materials and Methods."
B, monocytes electroporated with either GST, GST-C3, or
GST-CRNK were adhered for 30 min, fixed in 3% PFA-S, and
stained with hematoxylin-eosin. The area was determined by phase light
microscopy using a 40× lens and the NIH Image Scion 1.6 software. At
least 150 cells were measured per sample. Bars = mean ± S.D. of triplicate samples. A representative experiment of
five experiments is shown. pTyr, phosphorylated
tyrosine.
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Fig. 4.
GST-CRNK inhibits monocyte motility.
Monocytes were electroporated either with GST, GST-C3, or
GST-CRNK and adhered in the presence of 5% autologous serum.
Motility was monitored by light microscopy using a 40× objective and
the NIH Image 1.6 Movie software. Monocytes were followed over 70 frames. The first and last frames were combined to show cell movement.
The arrow denotes direction of motility from the first frame
to the last. The asterisk denotes nonmotile cell. A
representative of five experiments is shown.
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Fig. 5.
GST-CRNK does not inhibit monocyte
phagocytosis or adhesion-induced gene expression. A,
monocytes were adhered in the presence of 5% autologous serum for 45 min and then fed FITC-labeled zymosan bioparticles for 45 min. Cells
were washed twice in PBS and incubated with 0.4% trypan blue to quench
extracellular fluorescence from unphagocytosed particles. At least 100 cells were counted per sample. Bars represent mean ± S.D. of triplicate samples. A representative of three experiments is
shown. B, monocytes were electroporated and adhered in the
presence of autologous serum for 45 min, and total RNA was isolated.
Northern analysis was performed on 1 µg of total RNA/lane, and the
blot was probed sequentially for interleukin-1 and
-actin.
mRNA transcripts (Fig. 5B). Adhesion resulted in the
induction of interleukin-1
mRNA after electroporation with GST,
GST-CRNK, or GST-C3. These results suggest that CADTK, whereas important in monocyte cytoskeletal organization and motility, plays a
limited, if any, role in the functional activities of phagocytosis or
adhesion-induced gene induction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/RAFTK/FAK2 and p125FAK are members of a
kinase family, at least one of which is expressed in virtually all cell types. For example, most fibroblasts express only p125FAK,
epithelial and neuronal cells express both p125FAK and
CADTK, and monocytes express only CADTK. CADTK is expressed throughout
the hematopoetic lineages including monocytes (23, 24, 29),
megakaryocytes (45), platelets (46-47), NK cells (34, 40-41),
granulocytes (42), and both T and B lymphocytes (37-39). In monocytic
cells, CADTK is predominately expressed as a splice variant that
deletes one of the proline-rich regions, yielding a protein of ~110
kDa (23). CADTK shares topological similarities with
p125FAK (49), although one striking difference is the lack
of localization to focal adhesions when overexpressed in chicken and
other fibroblasts (36). Monocytes lack traditional focal adhesions but
form focal contacts sites, termed podosomes, when cultured under
conditions that stimulate motility. Whereas CADTK localized to the
monocyte cortical ruffles, it did not localize to the podosome structures.
mRNA by inhibiting
its activation through CRNK introduction. Thus, CADTK activation
appeared to be dispensable for adhesion-induced gene induction but not
for adhesion-induced ERK activation.
/
knockout animals, CADTK/Pyk2 protein is expressed, an unusual
occurrence in fibroblasts, and yet motility is not restored by the
compensatory CADTK expression (15, 16); furthermore, in these cells,
CADTK/Pyk2 is localized not to the cytoskeleton but to perinuclear
bodies.3 Thus the ability of CADTK to participate or govern
motility appears to require a cell type in which it is capable of
localizing to cytoskeletal elements, perhaps through the expression of
a specific CADTK cytoskeletal linker protein.
/
mice failed to localize paxillin,
talin, and filamentous actin to the leading lamellipodia, all
contributing to reduced motility (56). CADTK is activated by adhesion,
autophosphorylates on tyrosine 402 (49), a site that binds src family
SH2 groups, and might regulate Hck and Fgr activity. Taken together,
our results suggest that the in vivo consequences of
preventing CADTK activation and/or function, like that of Hck and Fgr,
would be defective localization of monocytes to sites of inflammation.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants 1-P60-DE13079 (NIDCR), AI26774 (to J. S. H.), and CA81503 (to H. S. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 919-966-1573; Fax: 919-966-3015; E-mail: hse@med.unc.edu.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M006916200
1 O. I. Sirenko, U. Bocker, J. Morris, S. Haskill, and J. M. Watson, submitted for publication.
3 T. W. Harding and H. S. Earp, unpublished observations.
4 T. W. Harding, L. M. Graves, and H. S. Earp, unpublished observations.
5 J. M. Watson and J. S. Haskill, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
p125FAK, p125 focal adhesion kinase;
CADTK, calcium-dependent
tyrosine kinase;
CRNK, CADTK/CAK-related non-kinase;
FRNK, FAK-related non-kinase;
JNK, c-Jun N-terminal kinase;
ERK, extracellular signal-regulated protein kinase;
GST, glutathione
S-transferase;
PTP1B, protein tyrosine phosphatase 1B;
PBS, phosphate-buffered saline;
MAP kinase, mitogen-activated protein
kinase;
PIPES, 1,4-piperazinediethanesulfonic acid;
FITC, fluorescein
isothiocyanate.
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