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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3967-3973
By
From the Divisions of Experimental Medicine and Hematology/Oncology,
Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA.
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.
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 We have recently identified and characterized a novel signaling
molecule, the related adhesion focal tyrosine kinase (RAFTK). RAFTK,
also termed Pyk2, CAK- 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.
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
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 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 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 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).
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.
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).
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.
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
Submitted March 10, 1997;
accepted December 26, 1997.
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
V
5 integrin
expression, enhanced the formation of focal contacts involving the
cytoskeletal protein paxillin in human macrophages bound to
vitronectin.
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.
-kinase SH2 domains: A model for SH2-mediated receptor-target interactions.
EMBO J
11:1365,
1992
