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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3967-3973
The Related Adhesion Focal Tyrosine Kinase (RAFTK) Is Tyrosine
Phosphorylated and Participates in Colony-Stimulating
Factor-1/Macrophage Colony-Stimulating Factor Signaling in
Monocyte-Macrophages
By
William C. Hatch,
Ramesh K. Ganju,
Dananagoud Hiregowdara,
Shalom Avraham, and
Jerome E. Groopman
From the Divisions of Experimental Medicine and Hematology/Oncology,
Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA.
 |
ABSTRACT |
RAFTK, a novel nonreceptor protein kinase, has been shown to be
involved in focal adhesion signal transduction pathways in neuronal
PC12 cells, megakaryocytes, platelets, and T cells. Because focal
adhesions may modulate cytoskeletal functions and thereby alter
phagocytosis, cell migration, and adhesion in monocyte-macrophages, we
investigated the role of RAFTK signaling in these cells. RAFTK was
abundantly expressed in THP1 monocytic cells as well as in primary
alveolar and peripheral blood-derived macrophages. Colony-stimulating factor-1 (CSF-1)/macrophage colony-stimulating factor
(M-CSF) stimulation of THP1 cells increased the tyrosine
phosphorylation of RAFTK; similar increases in phosphorylation were
also detected after lipopolysaccharide stimulation. RAFTK was
phosphorylated with similar kinetics in THP1 cells and peripheral
blood-derived macrophages. Immunoprecipitation analysis showed
associations between RAFTK and the signaling molecule
phosphatidylinositol-3 (PI-3) kinase. PI-3 kinase enzyme activity also
coprecipitated with the RAFTK antibody, further confirming this
association. The CSF-1/M-CSF receptor c-fms and RAFTK appeared
to associate in response to CSF-1/M-CSF treatment of THP1 cells.
Inhibition of RAFTK by a dominant-negative kinase mutant reduced
CSF-1/M-CSF-induced MAPK activity. These data indicate that RAFTK
participates in signal transduction pathways mediated by CSF-1/M-CSF, a
cytokine that regulates monocyte-macrophage growth and function.
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INTRODUCTION |
THE MACROPHAGE IS a cell that serves
critical functions in immune system defense, including the phagocytosis
of microbial pathogens, the proteolytic processing and presentation of
foreign antigens, and the elaboration of a repertoire of
cytokines.1,2 The regulation of monocyte-macrophage (MM)
production, maturation, and survival is subserved primarily through the
growth factor colony-stimulating factor-1 (CSF-1)/macrophage
colony-stimulating factor (M-CSF).3 CSF-1/M-CSF interacts
with its cognate receptor, c-fms, a member of the protein
tyrosine kinase family, and its activation leads to its rapid
autophosphorylation and dimerization.4,5 Other downstream
molecules that appear to participate in c-fms signal
transduction and are phosphorylated after CSF-1/M-CSF treatment of MMs
include Shc, Raf-1, c-cbl, phosphatidylinositol-3 (PI-3) kinase, and
the protein tyrosine phosphatase 1C.5-11
Functional changes induced in MMs that may be important in host defense
include alterations in the expression of surface molecules that mediate
adhesion.12-14 Particular attention has been focused on the
integrin family of surface receptors that facilitates the formation of
focal adhesion contacts upon binding to certain extracellular ligands.
Such focal adhesion contacts represent the interaction sites of
intracellular signaling molecules and cytoskeletal
proteins.15 CSF-1/M-CSF, which modulates  V 5 integrin
expression, enhanced the formation of focal contacts involving the
cytoskeletal protein paxillin in human macrophages bound to
vitronectin.16,17 The mechanism of this phenomenon is still
obscure, because the focal adhesion kinase (FAK), which has been
reported to serve as a critical molecule in forming such focal
contacts, was not detected in human macrophages.16,18
We have recently identified and characterized a novel signaling
molecule, the related adhesion focal tyrosine kinase (RAFTK). RAFTK,
also termed Pyk2, CAK-, and CADTK, appears to be a member of the FAK
family based on its deduced amino acid sequence.19-22 RAFTK
resembles FAK in that it has similar consensus motifs in the central
kinase catalytic domain and also lacks a transmembrane region,
myristylation sites, and SH2 and SH3 domains. Both of these signaling
molecules have a proline-rich region in the carboxy-terminal domain
that may function as binding domains for SH3-containing signaling
proteins.23 Studies to date indicate that RAFTK may participate in several signaling pathways, including those involving calcium ion channels in neuronal cells, integrin activation in megakaryocytes, the Ras/MAPK and JNK pathways in response to UV irradiation, T-cell receptor (TCR) cross-linking, G
protein stimulation, and via the cytokine tumor necrosis factor-
(TNF-).20,24-27
Based on this background, we investigated whether RAFTK was expressed
in the cells of MM lineage and whether it participated in
CSF-1/M-CSF-induced signaling. In parallel, we studied the effects of
treatment of MMs with the potent physiological activator of
macrophages, bacterial lipopolysaccharide (LPS). We observed that RAFTK
was expressed in the THP1 monocytic cell line and in peripheral
blood-derived MMs as well as tissue-derived alveolar macrophages.
Moreover, RAFTK was phosphorylated upon the treatment of mononuclear
phagocytes with CSF-1/M-CSF or LPS and associated with PI-3 kinase and
the CSF-1/M-CSF receptor, c-fms. These observations provide new
data on CSF-1/M-CSF signaling.
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MATERIALS AND METHODS |
Cells and cell culture.
The permanent human monocytic cell line THP1 was obtained from the
American Type Culture Collection (ATCC; Rockville, MD) and
shown to be mycoplasma-free before expansion in culture. The cells were
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal calf serum, 2 mmol/L glutamine, 100 µmol/mL sodium pyruvate, 1 mmol/mL nonessential amino
acids, 50 µg/mL penicillin, and 50 µg/mL streptomycin. Primary
human peripheral blood MMs were obtained by the phlebotomy of normal
volunteers, after obtaining their informed consent, and isolated by
Ficoll Hypaque density centrifugation, as previously
described.28 MMs were plated on 24-well tissue culture
plates (Costar, Cambridge, MA) for 24 hours, shaken at 150 RPM for 15 minutes and washed three times with Hank's balanced salt solution
(HBSS) to remove the nonadherent cells. The adherent cells were
cultured for an additional 7 days before their use. MM cultures were
determined to contain greater than 95% macrophages by nonspecific
acetate esterase staining (Sigma, St Louis, MO). All cells used in
these studies were maintained in DMEM supplemented with 10% fetal
bovine serum (FBS), 100 µmol/mL sodium pyruvate, 1 mmol/mL
nonessential amino acids, 50 µg/mL penicillin, and 50 µg/mL
streptomycin at 37°C, 5% CO2 under humidified
atmosphere.
RAFTK transfectants.
Dominant-negative RAFTK kinase mutants were produced by electroporation
of THP1 cells with 10 µg purified plasmids, and cells were maintained
in G418 selection medium (DMEM, 10% FBS, 0.5 mg/mL G418). Controls
consisted of a pcDNA vector without the RAFTK construct. The
dominant-negative kinase mutant RAFTKm457 was generated by
replacing Lys-(457) with Ala by site-directed mutagenesis.
Reagents and materials.
LPS from Escherichia coli was obtained from Sigma Chemical Co
and recombinant human CSF-1/M-CSF was kindly provided by Genetics Institute (Cambridge, MA). The monoclonal antibodies against
phosphotyrosine (4G10), the PI-3 kinase p85 regulatory subunit, and the
polyclonal rabbit antisera to the human c-fms receptor were
obtained from Upstate Biotechnology, Inc (Lake Placid, NY) and Santa
Cruz Biotechnology (Santa Cruz, CA). Specific polyclonal antibodies to
RAFTK were generated by immunizing New Zealand White rabbits with a
bacterially expressed fusion protein consisting of GST and the carboxy
terminus (amino acids 681-1,009) of human RAFTK cDNA subcloned into the pGEX-2T expression vector as described.19 High-titer RAFTK
antiserum (R-4250) was used in the subsequent experiments, because it
was shown to be specific and not cross-reactive with FAK in prior experiments.19,24
Electrophoresis reagents and nitrocellulose membranes were obtained
from Bio-Rad Laboratories (Hercules, CA). All other chemicals, including the protease inhibitors pepstatin, antipain, chymostatin, leupeptin, aprotinin, sodium vanadate, and sodium fluoride, were obtained from Sigma. Because bacterial endotoxin is a potent regulator of MM function,29 all media and reagents were shown to be
free of endotoxin contamination by the Limulus endotoxin assay (Sigma) before their use in cell cultures (<1 ng/mL).
Cell treatment and processing.
Cells were initially starved in serum-free DMEM for 16 hours and
stimulated in HBSS at a density of 5 × 106/mL for the
indicated time periods at 37°C with either LPS (2 µg/mL) or
CSF-1/M-CSF (1,000 U/mL). For each timepoint, 20 × 106 cells were lysed in 1 mL of ice-cold modified RIPA
buffer (50 mmol/L Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium
deoxycholate, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl
fluoride, 10 µg/mL of pepstatin, antipain, chymostatin,
leupeptin, aprotinin, 10 mmol/L sodium vanadate, 10 mmol/L sodium
fluoride, and 10 mmol/L sodium pyrophosphate) for 30 minutes at
4°C. Detergent-insoluble material was removed by centrifugation at
18,000g for 10 minutes at 4°C. Protein concentrations were
determined by BioRad DC protein assay (Bio-Rad Laboratories). Cell
lysates for the PI-3 kinase assays were performed using RIPA lysis
buffer as previously described, without sodium deoxycholate.
Immunoprecipitation and Western blot analysis.
For the immunoprecipitation studies, identical amounts of protein from
each sample were clarified by incubation with protein sepharose-A CL-4B
(Pharmacia Biotech, Piscataway, NJ) for 1 hour at 4°C. After the
removal of protein sepharose-A by brief centrifugation, the solution
was incubated with different primary antibodies as detailed below for
each experiment for 4 hours or overnight at 4°C.
Immunoprecipitations of the antibody-antigen complexes were performed
by incubation for 3 hours at 4°C with 75 µL of protein sepharose-A (10% suspension). Nonspecific bound proteins were removed
by washing the sepharose beads three times with the modified RIPA
buffer and three times with phosphate-buffered saline (PBS). The bound
proteins were solubilized in 30 µL of 2× Laemmli buffer and
boiled for 5 minutes. Samples were then run on 7.5% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to nitrocellulose membranes. The membranes were blocked with 5% nonfat
milk protein and probed with primary antibody for 3 hours at room
temperature or overnight at 4°C. Immunoreactive bands were
visualized using horseradish peroxidase-conjugated secondary antibody
and the enhanced chemiluminescent system (Amersham Corp, Arlington
Heights, IL). Blots were stripped (2% SDS, 62.5 mmol/L Tris, and 100 mmol/L -mercaptoethanol) for 30 minutes at 50°C and washed in
Tris-buffered saline/0.5% Tween 20 (TBS-T) for 60 minutes before
blocking and reprobing with primary antibodies.
In vitro PI-3 kinase assay.
Aliquots of cell lysates were normalized for protein concentration and
then incubated overnight at 4°C with antibodies against RAFTK, PI-3
kinase, or control normal rabbit serum. Immune complexes were absorbed
to sepharose-A beads for 3 hours at 4°C. Nonspecific binding was
removed by washing three times with PBS 1% NP-40 and three times with
0.5 mol/L LiCl/0.5 mol/L Tris, followed by washing three times with TE
buffer. Samples were resuspended in 20 µL TE buffer, 20 µL
phosphoinositol (10 µg; Avanti Polar Lipids, Alabaster, AL), and 10 µL ATP mix (1 mmol/L HEPES, 10 µmol/L ATP, 1 µmol/L
MgCl2, 5 µCi  32 P-ATP) and incubated at
room temperature for 10 minutes. The reaction was stopped by adding 60 µL of 2 mol/L HCL and 160 µL chloroform:methanol (1:1 vol/vol).
Lipids were separated on oxalate impregnated silica TLC plates using a
solvent system of chloroform:methanol:water:ammonium hydroxide (28%)
(35:35:3.5:7). TLC plates were dried and subjected to autoradiography
at  80°C.
Immune complex kinase assay for MAPK activity.
Aliquots of cell lysates normalized for protein concentration were
incubated overnight at 4°C with antibodies against ERK1 and ERK2
(Santa Cruz Biotechnology). Immune complexes were then absorbed to
sepharose-A beads for 3 hours at 4°C. Nonspecific binding was
removed by washing three times with RIPA buffer followed by washing
three times with kinase buffer (50 mmol/L HEPES, pH 7.4, 5 mmol/L
MgCl2, and 20 mmol/L ATP). The complex was incubated in 30 µL kinase buffer containing 7 µg myelin basic protein (MBP; Upstate
Biotechnology) and 5 µCi 32P-ATP for 20 minutes at
30°C. The reaction was terminated by adding 4× Laemmli sample
buffer and boiling samples for 5 minutes. Proteins were separated on
15% SDS-PAGE and detected by autoradiography.
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RESULTS |
RAFTK is expressed and phosphorylated in human MMs.
To further characterize the signaling pathways in human MMs that are
involved in their growth, differentiation, and function, we used as a
model the permanent monocytic cell line THP1 as well as primary
peripheral blood-derived MMs. Analysis by immunoprecipitation showed an
abundance of RAFTK protein in these cells
(Fig 1A through C). There
appeared to be low levels of constitutive phosphorylation of RAFTK
under unstimulated culture conditions. Depending on the resolution of
the gels, RAFTK was seen to migrate either as a single band or as a
doublet (Fig 1A through C).
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| Fig 1.
Tyrosine phosphorylation of RAFTK in THP1
monocytic cells and primary MMs. THP1 cells (20 × 106)
were stimulated with either (A) 1,000 U/mL CSF-1/M-CSF or (B) 2 µg/mL
LPS for the indicated time periods. (C) MMs (20 × 106)
were allowed to adhere and mature in culture for 7 to 14 days before
their stimulation with 1,000 U/mL CSF-1/M-CSF. Cell lysates prepared in
RIPA buffer were subjected to immunoprecipitation with anti-RAFTK
antibody or normal rabbit serum as a control. Anti-RAFTK
immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to
nitrocellulose membranes, and immunoblotted with antiphosphotyrosine
antibody (4G10) (top panel). The same blot was subjected to serial
immunoblotting with anti-RAFTK antibody (bottom panel). TCL, total cell
lysates; NRS, normal rabbit serum control.
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We then addressed whether certain stimuli associated with mononuclear
phagocyte activation modulated RAFTK phosphorylation. Our preliminary
experiments determined that 1,000 U/mL CSF-1/M-CSF and 2 µg/mL LPS
were optimal concentrations for the stimulation of RAFTK in THP1 cells
and primary macrophage cultures (data not shown). As seen in Fig 1A and
B (top panels), an increase in the tyrosine phosphorylation of RAFTK
was specifically observed in THP1 cells after their treatment with
either CSF-1/M-CSF or LPS. The membrane was then stripped and reprobed
with anti-RAFTK antibody to confirm that equivalent amounts of RAFTK
were loaded in each lane (Fig 1A and B, bottom panels).
To determine the time course of the tyrosine phosphorylation of RAFTK,
THP1 cells or MMs were stimulated with CSF-1/M-CSF or LPS and harvested
at the times indicated in Fig 1. The phosphotyrosine levels in the
RAFTK immunoprecipitates of the CSF-1/M-CSF-treated THP1 cells showed
a strong peak at 2.5 minutes that appeared to decrease in intensity at
later time points (Fig 1A, top panel). The membrane was then stripped
and reprobed with anti-RAFTK antibody to confirm that equivalent
amounts of RAFTK were loaded in each lane (Fig 1A, bottom panel). There
did not appear to be any changes in the levels of RAFTK protein to
explain these fluctuations in the degree of tyrosine phosphorylation.
However, we routinely see a slight difference in mobility between RAFTK
detected in immunoprecipitates and that in total cell lysates that
appears to migrate at a slightly faster rate.
LPS treatment of THP1 cells resulted in a maximum tyrosine
phosphorylation of RAFTK within 2.5 minutes that then decreased with
increasing time of stimulation (Fig 1B, top panel). The phosphotyrosine levels of RAFTK after LPS stimulation appear to have similar kinetics to those we observed after CSF-1/M-CSF stimulation.
CSF-1/M-CSF stimulation of primary MMs resulted in a peak tyrosine
phosphorylation of RAFTK by 1 minute, which gradually decreased over
time (Fig 1C, top panel). Anti-RAFTK immunoblotting of RAFTK immunoprecipitates showed that the approximately 120-kD phosphoprotein corresponded to the RAFTK protein and remained constant between samples
(Fig 1A through C, bottom panels).
RAFTK associates with the PI-3 kinase.
Because RAFTK, like FAK, is believed to act as a platform kinase site
for the coalescence of signaling and adaptor molecules at sites of
focal adhesions, we examined RAFTK immunoblots for associating
coprecipitating proteins. Using immunoprecipitation analysis, we
observed a specific association of RAFTK with PI-3 kinase, an important
enzyme in the modulation of phosphoinositol signaling30,31
(Fig 2A and B). Time course studies after
either CSF-1/M-CSF or LPS treatment of THP1 cells demonstrated that the PI-3 kinase-RAFTK association increased over time of stimulation and
gradually decreased to background levels at longer stimulation times
(data not shown). Similar findings were observed after phorbol 12-myristate 13-acetate (PMA) treatment (not shown). This
association between RAFTK and PI-3 kinase was confirmed by an in vitro
kinase assay (Fig 3). These studies
demonstrated that PI-3 kinase activity increased and migrated with
RAFTK immunoprecipitates after CSF-1/M-CSF or LPS stimulation of THP1
cells (Fig 3). We did not detect PI-3 kinase activity in the normal
rabbit serum control immunoprecipitates.
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| Fig 2.
Association of RAFTK with PI-3 kinase in THP1 cells. THP1
cells (20 × 106) were stimulated with either (A)
CSF-1/M-CSF (1,000 U/mL) or (B) LPS (2 µg/mL) for the indicated time
periods. Cell lysates prepared in RIPA buffer were subjected to
immunoprecipitation with anti-RAFTK antibody. Anti-RAFTK
immunoprecipitates were resolved by 7.5% SDS-PAGE, transferred to
nitrocellulose membranes, and immunoblotted with anti-PI-3 kinase
antibody. TCL, total cell lysates.
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| Fig 3.
PI-3 kinase activity associates with RAFTK in THP1 cells.
THP1 cells were stimulated with either 1,000 U/mL or 2 µg/mL LPS for
2 minutes and lysed in RIPA buffer without sodium deoxycholate. Lysates
were immunoprecipitated with either anti-RAFTK, normal rabbit serum
control, or anti-PI-3 kinase p85 antibody. Immune complexes were
absorbed to sepharose-A beads for 3 hours, washed, and subjected to
PI-3 kinase assay. Lipids were extracted using methanol:chloroform
(1:1) and spotted on oxalate-impregnated silica gel TLC plates. Samples
were subjected to ascending chromatography using a
methanol:chloroform:water:ammonium hydroxide solvent system. TLC plates
were dried and samples were visualized by autoradiography.
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RAFTK associates with the c-fms receptor upon mononuclear
phagocyte cell activation with CSF-1/M-CSF.
Because CSF-1/M-CSF stimulation of THP1 cells and primary macrophages
appeared to have rapid effects on RAFTK phosphorylation, we examined
whether RAFTK may directly associate with the c-fms receptor.
We observed a specific association of RAFTK with the c-fms
receptor upon CSF-1/M-CSF treatment of the cells
(Fig 4A and B). Associations were detected
in blotting experiments of the THP1 cell lysates that were
immunoprecipitated with RAFTK antisera followed by c-fms
immunoblotting. We identified immunoreactive bands at both 135 and 150 kD that correspond to the mobility of the c-fms receptor (Fig
4B). The reciprocal experiment of c-fms immunoprecipitation
followed by RAFTK immunoblotting identified a prominent 120-kD molecule
(Fig 4A). Interestingly, the RAFTK and c-fms association
appeared to increase with longer times of CSF-1/M-CSF stimulation, but
did not respond to PMA stimulation (data not shown).
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| Fig 4.
Association of RAFTK with the c-fms receptor in
THP1 cells. THP1 cells (20 × 106) were stimulated with
1,000 U/mL CSF-1/M-CSF for the indicated time periods. Cell lysates
prepared in RIPA buffer were subjected to immunoprecipitation with
either the anti-c-fms antibody (A) or anti-RAFTK antibody (B).
Immune complexes were absorbed onto sepharose-A beads, washed and
resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes,
and immunoblotted with either anti-RAFTK (A) or anti-c-fms
antibody (B). NRS, normal rabbit serum control.
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Dominant-negative RAFTK kinase mutant reduces MAPK activity.
The RAFTK protein has been identified as an upstream mediator of the
Ras pathway via Grb2-SOS interactions.20 THP1 cells expressing a dominant-negative kinase mutant, RAFTKm457,
were used to determine if RAFTK participates in c-fms signaling through the ERK1/ERK2 pathway.5 MAP kinase activity was
strongly activated after CSF-1/M-CSF treatment of THP1 cells expressing the RAFTKpcDNA control vector alone. However, MAP kinase
activity in THP1 cells expressing the dominant-negative kinase mutant
RAFTKm457 was decreased when compared with the control THP1
cells expressing the RAFTKpcDNA vector
(Fig 5). Although MAPK activity was
routinely reduced in RAFTKm457-expressing THP1 cells, there
were no detectable differences in cell viability between the
RAFTKm457- or RAFTKpcDNA-expressing THP1 cells
to account for this finding.
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| Fig 5.
Reduction of MAP Kinase activity by overexpression of a
RAFTK dominant-negative kinase mutant. THP1 cells were stably
transfected with the RAFTKpcDNA vector control or with
RAFTKm457 dominant-negative kinase mutant. THP1
transfectants (20 × 106) were stimulated with CSF-1/M-CSF
(1,000 U/mL), and then cell lysates were prepared in RIPA buffer.
Lysates were subjected to immunoprecipitation with anti-ERK1 and ERK2
antibodies. Immune complexes were absorbed with sepharose-A beads and
then washed and subjected to in vitro kinase assay for 30 minutes.
Samples were subjected to 15% SDS-PAGE and to autoradiography at
80°C.
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DISCUSSION |
Our studies indicate that human mononuclear phagocytes, including
peripheral blood-derived MMs, express RAFTK, a recently identified
signaling molecule that is a member of the FAK family. RAFTK appeared
to participate in certain previously described signaling pathways after
the activation of these cells. Treatment with CSF-1/M-CSF showed an
increased phosphorylation of RAFTK in both the model THP1 monocytic
cell line as well as in primary blood-derived MMs. Parallel studies
using the potent macrophage stimulators (LPS) and the chemical
activator PMA, an activator of protein kinase C (PKC), also showed
RAFTK phosphorylation in macrophages in a time and concentration
dependent manner (data not shown). In these studies, phosphorylated
RAFTK appears to migrate either as a single band or as a doublet. It is
likely that one of the doublet bands represents either a phosphorylated form or a degradation product of RAFTK produced by endogenous proteases
found in abundance in cells of the macrophage/monocyte lineage. The
degradation of the RAFTK protein by endogenous proteases has been
previously described in platelets in an integrin-independent mechanism.32
The phosphorylation of RAFTK has been reported to result in its
association with several well-characterized components of cellular
signaling pathways, including src kinases, paxillin, and the adaptor
molecule Grb2. In this study, we observed that RAFTK can associate with
the enzyme PI-3 kinase and the CSF-1/M-CSF receptor
c-fms.20,24,26,27,31 Grb2 is an adaptor protein that has the capacity to link with a number of kinases and substrates and functions by facilitating signaling through the creation of physical associations of such partners in enzymatic
reactions.23 PI-3 kinase appears to modulate
phosphoinositol metabolism in a variety of cell types, including
mononuclear phagocytes, and is an important component of the tyrosine
kinase-regulated signaling pathways that lead to cell
proliferation.33,34 CSF-1/M-CSF has been reported to induce
the direct association of the p85- subunit of PI-3 kinase with the
SH2 domain of Grb2 and Grb2-SOS complexes, thus supporting its role
upstream of the Ras signaling pathway in monocytes.5,11 In
addition, PI-3 kinase activation and the production of its metabolites
have been suggested to be an upstream activator of the
calcium-independent form of PKC.35 Our data demonstrate
that PI-3 kinase associates with RAFTK and that this association
appears to increase after stimulation with either LPS or CSF-1/M-CSF.
We also found that PI-3 kinase enzymatic activity associates with RAFTK
immunoprecipitated from CSF-1/M-CSF or LPS stimulated THP1 cell
lysates. Previous reports have demonstrated that PDGF and cell adhesion
stimulate the association of PI-3 kinase, via its SH2 domain, with FAK
at Tyrosine 397.36 Future studies will focus on determining
which of the PI-3 kinase p85 SH2 or SH3 domains mediates this observed
association with RAFTK in MMs.
Our observations regarding RAFTK suggest that this recently identified
signaling molecule could play a variety of roles in the transduction of
MM signaling, particularly in light of prior studies of
CSF-1/M-CSF-induced integrin expression and the subsequent formation
of focal adhesion contacts.16 Recent studies of
CSF-1/M-CSF-induced upregulation of V5 integrin-dependent
phosphorylation of paxillin in human macrophages showed a PKC-dependent
mechanism.16 Our results complement those studies, in that
we observed that LPS, PMA, or CSF-1/M-CSF induced the phosphorylation
of RAFTK in THP1 cells and its subsequent association with
paxillin.27
Our observation that RAFTK associated with the c-fms receptor
in CSF-1/M-CSF stimulated THP1 cells contributes new information on
signaling mediated by this growth factor in mononuclear phagocytes. Our
results are of particular interest in light of the report of Kharbanda
et al,37 who did not find a direct association between
c-fms and FAK in CSF-1/M-CSF-stimulated primary macrophages. Together with these other data, our observations suggest that RAFTK and
FAK may have different associations and roles in signaling in MMs, as
they do in other cell types.20,24-27
Because RAFTK does not contain either the SH2 or SH3 binding domains
commonly found in signaling molecules,19 it is likely that
the c-fms-RAFTK association we observed may be mediated via an
adaptor molecule such as Grb2. Grb2 has been previously reported to
bind to both the c-fms receptor5 and to
RAFTK.20,26 Future studies will aim to identify the nature
of these interactions and whether Grb2 plays such a role in MMs.
Previously, c-fms has been reported to form associations with
Grb1, Shc, and SOS1 in myeloid cells, suggesting that it signals through the Ras pathway.38 Our data showing RAFTK
association with Grb2 (not shown) support the data of Lev et
al,20 who found that Pyk2 associated with the adaptor
proteins Grb2 and Shc upstream of the Ras/MAPK signaling pathway in
PC12 neuronal cells.27 Pyk2 has recently been reported to
activate the c-Jun N-terminal kinase signaling pathway after stress
signals and the MAPK pathway after G protein
stimulation.25,27 Because both c-fms and RAFTK are
upstream activators of the Ras/MAPK pathway, it is possible that RAFTK
acts as an intracellular link between divergent extracellular signals.
For example, these signals may originate from extracellular matrix
proteins that signal through integrin binding and from cytokines such
as CSF-1/M-CSF that signal through c-fms, both resulting in an
enhanced stimulation of this Ras/MAPK pathway. Alternatively, because
RAFTK function has been linked to the cytoskeleton through its
association with paxillin and focal adhesions,19,24 its
association with c-fms may be due, in part, to c-fms
internalization after CSF-1/M-CSF binding, which may also involve
cytoskeletal interactions.39 Whereas our MAPK data (Fig 5)
suggest that RAFTK may play an intermediary role linking c-fms
to the MAPK pathway and that MAPK activity was only partially reduced
by the RAFTKm457 dominant-negative mutant, these data
suggest that RAFTK is only one of several signaling molecules that act
upstream to activate MAPK and contribute to the complex cascade leading
to MAPK activation.
In summary, RAFTK appears to function in LPS and CSF-1/M-CSF signaling
pathways in MMs through multiple downstream pathways, including PI-3
kinase, Ras/MAPK, and JNK.20,24,25,36 Although further
studies are needed to characterize the sites and mechanisms of
interaction among the currently identified molecules that associate with RAFTK, it seems clear that macrophages, like megakaryocytes and T
cells in our prior work, prominently use RAFTK in cytokine-mediated pathways of activation that are linked to focal contact formation. The
confluence of RAFTK, other kinases, and cytoskeletal molecules may
provide a platform for the interactions of signaling molecules and
adaptor proteins that regulate downstream signaling pathways and affect
cell morphology by finely controlling certain components of the immune
response, like adhesion or migration. It will be of interest to
characterize how such cellular responses are triggered in the
macrophage host defense against extracellular pathogens that are often
coated with extracellular matrix proteins that bind to integrins, such
as Pneumocystis carinii.
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FOOTNOTES |
Submitted March 10, 1997;
accepted December 26, 1997.
This manuscript is dedicated to the memory of Dananagoud Hiregowdara.
Supported in part by National Institutes of Health Grants No. HL
43510-07, HL 53745-02, HL 55187-01, and HL 51456-02. W.C.H. is
supported by a David Geffen Foundation fellowship.
Address reprint requests to Jerome E. Groopman, MD, Chief, Division of
Experimental Medicine, Harvard Institutes of Medicine, Beth Israel
Deaconess Medical Center, Harvard Medical School, 4 Blackfan
Circle, Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Janet Delahanty for her editing and the
preparation of the figures as well as Jennifer McGrath and Nancy
DesRosiers for their assistance with the figures. We thank our
colleagues Zhong-Ying Liu and Jian-Feng Wang for their technical
assistance. Finally, we appreciate Tee Trac and Youngsun Jung for their
typing assistance and Delroy Heath for facilitating our receipt of the
needed reagents for the experiments. We also thank the Cantley Lab for
their help with the PI-3 kinase assays and the Genetics Institute for
supplying CSF-1/M-CSF.
| |
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Abstract
Introduction
Abstract