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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 791-797
 -Chemokine Receptor CCR5 Signals Via the Novel Tyrosine Kinase RAFTK
By
Ramesh K. Ganju,
Parmesh Dutt,
Lijun Wu,
Walter Newman,
Hava Avraham,
Shalom Avraham, and
Jerome E. Groopman
From the Divisions of Experimental Medicine and Hematology/Oncology,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
MA and LeukoSite, Inc, Cambridge, MA.
 |
ABSTRACT |
Chemokine receptors are coupled to G-proteins and their activation
results in prominent changes in cell migration and growth. The
downstream signaling pathways that mediate these effects of chemokines
are largely uncharacterized. Macrophage inflammatory protein 1 (MIP
1) binding to its cognate receptor CCR5 resulted in activation of
the related adhesion focal tyrosine kinase (RAFTK), with subsequent
activation of the cytoskeletal protein paxillin and the downstream
transcriptional activators, c-Jun N-terminal kinase
(JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase
mutant markedly attenuated JNK/SAPK activity. Thus, RAFTK appears to
provide a functional "bridge" for the transmission of CCR5
receptor signaling to the cytoskeleton and nucleus, primary sites of
chemotaxis and growth regulation.
| |
INTRODUCTION |
CONSIDERABLE ATTENTION has recently
focused on chemokines and their receptors as important mediators of the
inflammatory response1-5 and of human immunodeficiency
virus (HIV) pathogenesis.6-9 As inflammatory mediators,
chemokines act to direct cell migration10 and to modulate
cell proliferation as part of the host response to microbial and
allergic stimuli.4-11 In HIV pathogenesis, certain chemokine receptors bind to HIV strains and facilitate target cell
infection.6-9 Recent observations indicate that a human herpes virus called Kaposi's sarcoma herpes virus type 8 (KSHV/HHV-8) encodes functional homologues of certain chemokines and chemokine receptors, suggesting that chemokines may contribute to the growth and
spread of neoplasms seen in acquired immunodeficiency syndrome (AIDS).12-16 Despite the prominent roles of chemokines in
inflammatory and infectious processes, relatively limited information
is available about chemokine receptor signaling.2-3,17-23
The chemokine superfamily has been subdivided into the  (C-X-C), (C-C),  (C), and recently identified delta (C-X-X-X-C) groups based
on the arrangement of the first two of four conserved cysteine
residues.5,18 Both - and -chemokine receptors are members of the G-protein-coupled receptor superfamily.1-5
Previous reports have shown that chemokine receptors transmit
information through G-proteins, resulting in intracellular changes in
adenylate cyclase and phosphoinositol lipid metabolism and in the
ras/raf/map kinase pathway.2,5,20,21,23-27 It has also been
suggested that G-protein-coupled receptors or receptor protein
tyrosine kinases (RTKs) may result in a common signaling pathway
leading to the activation of nuclear transcription
factors.25-27 Recently, JNK/SAPK has been shown to be
activated by transforming G-protein-coupled
receptors.26,27 Importantly, the mechanisms by which the
effects of chemokines on cell migration and proliferation may be
functionally linked through signaling pathways have not yet been
elucidated.
The CCR5 receptor binds the -chemokines macrophage inflammatory
protein 1 (MIP1), MIP1, and RANTES, and is a major attachment protein for the macrophage-tropic strains of HIV-1.1,6-9 We now report that signaling via the CCR5 receptor leads to the
phosphorylation and activation of a recently discovered protein kinase,
termed RAFTK (also known as Pyk2 or CAK-), of the focal adhesion
kinase (FAK) family.28-32 Chemokine activation of RAFTK
resulted in the downstream modulation of the JNK/SAPK kinase system.
Chemokine stimulation via the CCR5 receptor also resulted in
the phosphorylation of the cytoskeletal protein paxillin and its
association with RAFTK. Our observations provide a possible mechanism
by which intracellular tyrosine kinases may coordinate chemokine
receptor signaling to alter chemotaxis and cell growth.
| |
MATERIALS AND METHODS |
Reagents and materials.
RAFTK antibodies were generated using GST-fusion proteins by immunizing
New Zealand rabbits as previously described.31 Serum R-4250
was chosen for further studies based on its titer in enzyme-linked immunosorbent assay (ELISA). This antisera did not crossreact with FAK
and recognized both human and murine forms of RAFTK. Antibodies to
paxillin, JNK, p38 kinase and recombinant GST-c-Jun amino-terminal
protein (1-79 amino acids) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal
antiphosphotyrosine antibody (4G10) was a generous gift from Dr Brian
Druker (Oregon Health Sciences University, Portland).
Electrophoresis reagents were obtained from Bio-Rad Laboratories
(Hercules, CA). The protease inhibitors leupeptin and 1 antitrypsin
and all other reagents were obtained from Sigma Chemical Co (St Louis,
MO). The nitrocellulose membrane was obtained from Bio-Rad
Laboratories. Indo-1 AM was purchased from Molecular Probes (Eugene,
OR).
Construction of CCR5 stable transfectants.
We used a murine pre-B lymphoma cell line, L1.2, for transfection
studies. CCR5 cDNA, tagged at the N-terminus with a Flag epitope
(Asp.Tyr.Lys. Asp.Asp.Asp.Asp.Lys), was subcloned to the HindIII-Xba I site of the expression vector pMRB101
(kindly provided by Martin Robinson, CellTech, Slough, UK),
in which the inserted gene was driven by a CMV promoter.
The DNA was stably transfected into L1.2 cells as
described33-35 except that the mycophenolic acid-selective
medium, instead of G418-selective medium, was used to select for
transfectants. The cell-surface expression of CCR5 was monitored by
FACS analysis. These cells express CCR5 at a high level (80,000 sites/cell) and bind the -chemokines MIP1, MIP1, and
RANTES with high affinity (kd = 0.2 to 1.0 nmol/L). Additionally, these cells were shown to bind M-tropic HIV-1 gp120 in
the presence of soluble human CD4, a characteristic of the native CCR5
receptor.35
Cell culture.
The L1.2 cells were grown at 37°C in 5% CO2 in RPMI-1640
with 10% fetal calf serum (FCS), 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 55 µmol/L 2-mercaptoethanol. CCR5 transfectants were grown in RPMI-1640 media containing HT supplements (100 nmol/L sodium hypoxanthine and 16 nmol/L thymidine), 2.5 µg/mL mycophenolic acid, and 125 µg/mL
xanthine. For selection of RAFTK mutants, 0.8 mg/mL Geneticin (G418)
(GIBCO-BRL, Grand Island, NY) was used in the media.
Generation of activated T cells.
Peripheral blood mononuclear cells (PBMCs) were isolated, and activated
T cells were generated as described.33,36 Briefly, 2 x
106 PBMCs/mL in RPMI containing 10% fetal bovine serum
(FBS) were added to tissue culture plates coated with anti-CD3 antibody
TR77. T cells were removed to fresh media supplemented with recombinant human interleukin-2 (IL-2) after 4 to 6 days. Three- to 4-week-old activated T cells were used for chemokine stimulation.
Calcium flux assay.
L1.2 or CCR5 transfectants were washed with RPMI-1640 and resuspended
at 10 × 106 cells/mL in RPMI. The cells were loaded with
Indo-1 acetoxymethyl esters (Indo-1 AM; Molecular Probes) by adding 5 µL of working Indo-1 solution to 10 × 106 cells
suspended in 1 mL of RPMI solution and incubated for 45 minutes at
37°C. Cells were diluted to 1 × 106/mL, treated with
MIP1, and analyzed for calcium mobilization by flow cytometry
(Coulter Electronics, Hialeah, FL) as
described.34 Calcium flux assay and all other subsequent
signaling assays were repeated at least three times.
RAFTK transfectants.
Wild-type and kinase dead RAFTK mutants were produced by transfection
of the CCR5-L1.2 cells. Controls consisted of a pcDNA vector without an
RAFTK construct. Plasmids carrying the control vector, wild-type RAFTK
(RAFTKwt), or dominant-negative mutant
(RAFTKm457) were transfected by electroporation into the
CCR5-L1.2 cells using Bio-Rad electroporation equipment. The
dominant-negative kinase mutant RAFTKm457 was generated by
replacing Lys-(457) with Ala by site-directed mutagenesis.37,38
Stimulation of cells.
Cells were washed twice with RPMI-1640 (GIBCO-BRL) and resuspended at
10 × 106 cells/mL in RPMI-1640 medium. Cells were starved
for 4 hours at 37°C and were stimulated with different concentrations
of MIP1 at 37°C for various time periods. After stimulation, cells
were lysed in modified RIPA buffer (50 mmol/L Tris-HC1, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L
phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL of aprotinin,
leupeptin and pepstatin, 10 mmol/L sodium vanadate, 10 mmol/L sodium
fluoride, and 10 mmol/L sodium pyrophosphate). Total cell lysates (TCL)
were clarified by centrifugation at 10,000g for 10 minutes.
Protein concentrations were determined by protein assay (Bio-Rad
Laboratories). Cell lysis, RAFTK immunoprecipitation, immunoblotting,
kinase assays, and autophosphorylation assays were performed as
described below.
Immunoprecipitation and Western blot analysis.
For immunoprecipitation studies, identical amounts of protein from each
sample were clarified by incubation with protein A-Sepharose CL-4B
(Pharmacia Biotech) for 1 hour at 4°C. After the removal of protein
A-Sepharose 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 2 hours at
4°C with 50 µL of protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads
three times with modified RIPA buffer and one time with phosphate-buffered saline (PBS). Bound proteins were solubilized in 40 µL of 2× Laemmli buffer and further analyzed by immunoblotting. Samples were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and
probed with primary antibody for 3 hours at room temperature (RT) for
4°C overnight. Immunoreactive bands were visualized using horseradish
peroxidase (HRP)-conjugated secondary antibody and the enhanced
chemiluminescent (ECL) system (Amersham Corp, Arlington Heights, IL).
Monoclonal antibody (4G10, IgG2a) was used for Western blot analysis of
phosphotyrosine protein.
Kinase assays.
In vitro kinase assays were performed as described
earlier.39 The cell lysates immunoprecipitated with RAFTK
antiserum were washed twice with RIPA buffer and once in kinase buffer
(20 mmol/L HEPES, pH 7.4; 50 mmol/L NaCl; 5 mmol/L MgCl2; 5 mmol/L MnCl2; 100 mmol/L Na3VO4.
For the in vitro kinase assays, the immune complex was incubated in
kinase buffer containing 25 µg of poly (Glu:Tyr) (4:1); 20 to 50 kD; Sigma and 5 µCi 32P-ATP at RT for 30 minutes. The reaction was stopped by adding 2× SDS sample buffer and
boiling the sample for 5 minutes at 100°C. Proteins were then
separated on 7.5% SDS-PAGE and detected by autoradiography. Normal
rabbit serum was used as a negative control. An autophosphorylation
assay was performed by incubating the immune complex in a kinase buffer
containing 5 µCi 32P-ATP at RT for 30 minutes. The
reaction was stopped by adding 4× SDS sample buffer and by boiling
the sample for 5 minutes. Proteins were then separated on SDS-PAGE and
detected by autoradiography.39
JNK and p38 MAP kinase assays.
The JNK assay was performed as has been described
earlier.40 Briefly, cell lysates were immunoprecipitated
with JNK antibody (Santa Cruz Biotechnology). The immune complexes were
washed twice with RIPA buffer and once in kinase buffer (50 mmol/L
HEPES, pH 7.4, 10 mmol/L MgCl2, 20 µmol/L ATP). The
complex was then incubated in kinase buffer containing recombinant GST
c-jun 0.2 µg/µL (1-79 amino acids) (Santa Cruz Biotechnology) and 5 µCi 32P-ATP for 10 minutes at RT. The reaction was
terminated by adding 2× SDS sample buffer and boiling the sample for
5 minutes at 100°C. Proteins were separated on 12% SDS-PAGE and
detected by autoradiography. Rabbit IgG was used as a negative control.
For the p38 MAP kinase assay, cell lysates from unstimulated or
stimulated cells were immunoprecipitated with anti-p38 MAP kinase
antibody (Santa Cruz Biotechnology). The immune complexes were washed
twice with RIPA buffer and once in kinase buffer (50 mmol/L HEPES, pH
7.4; 10 mmol/L MgCl2; and 20 µmol/L ATP). The complex was
incubated in kinase buffer containing 7 µg myelin basic protein (MBP;
Upstate Biotechnology, Lake Placid, NY) and 5 µCi
32P-ATP for 20 minutes at 30°C. Proteins were
separated on 15% SDS-PAGE and detected by autoradiography. Rabbit IgG
was used as a negative control.
| |
RESULTS |
MIP1 stimulates Ca2+ flux in CCR5
transfectants.
Ligand binding to chemokine receptors causes characteristic fluxes in
intracellular calcium. To verify that the expressed human CCR5 receptor
in the CCR5-L1.2 cells retained this fundamental signaling property, we
treated these cells with MIP1. Calcium fluxes were observed only in
the CCR5-L1.2 cells and not in the untransfected L1.2 cells (Fig
1).
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| Fig 1.
Calcium mobilization in response to MIP1 stimuli.
Ca2+ flux was performed in (a) untransfected L1.2 or (b)
CCR5-transfected L1.2 cells in the presence or absence of chemokine
treatment. Treatment of CCR5-L1.2 cells with 10 nmol/L MIP1 resulted
in an increase in Ca2+ flux. No response was observed in
the untransfected L1.2 cells.
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RAFTK is phosphorylated and activated upon MIP1
stimulation of CCR5 transfectants.
To characterize the signaling pathways in the cells expressing
the chemokine receptor CCR5, the CCR5 transfectants were stimulated with MIP1 and lysates were analyzed for RAFTK phosphorylation. We
observed rapid phosphorylation of endogenous murine RAFTK under these
conditions (Fig 2A). This observation
showed that the transfected human CCR5 receptor could signal to
downstream endogenous murine substrates in response to its specific
ligand, MIP1, similar to native murine chemokine receptors. We also
observed an increase in the tyrosine phosphorylation of RAFTK in
activated T cells upon MIP1 stimulation (Fig 2B). Activated T cells
have been shown to express high levels of CCR5.35,36 The
slower activation in T cells may be due to differences in cell type
compared with L1.2 cells and/or differences in numbers of
cell-surface receptors.
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| Fig 2.
Tyrosine phosphorylation of RAFTK in CCR5-L1.2 cells and
activated T cells in response to chemokines. (A) CCR5-L1.2 cells or (B)
T cells were unstimulated (UN) or stimulated with 100 nmol/L or 250 nmol/L MIP1 for varying time intervals. Cells were lysed and
immunoprecipitated with anti-RAFTK, and analyzed by immunoblotting with
antiphosphotyrosine (anti-pTyr). The same immunoblot was stripped and
immunoblotted with anti-RAFTK (lower panel).
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The autophosphorylation and kinase activities of protein tyrosine
kinases can be activated upon their tyrosine phosphorylation, which is
essential for their role in signal transduction. Therefore, we
performed an autophosphorylation assay and an in vitro kinase assay in
which poly (Glu:Tyr) (4:1) was used as an exogenous substrate to
determine the intrinsic tyrosine kinase activity of RAFTK. As shown,
the stimulation of CCR5 cells with MIP1 resulted in an increase in
the autophosphorylating (Fig 3A) as well as
the kinase activity of RAFTK (Fig 3B). The in vitro kinase activity observed in the RAFTK immune complexes using exogenous substrate could
be the result of RAFTK and other co-associated kinases. The background
levels observed in untransfected L1.2 cells likely reflect low levels
of the endogenous CCR5 receptor.
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| Fig 3.
RAFTK activation upon chemokine stimulation. Total cell
lysates from L1.2 or CCR5-L1.2 cells unstimulated (UN) or stimulated with MIP1 (25 nmol/L) were immunoprecipitated with RAFTK antibody. The immune complexes were subjected to (A) autophosphorylating activity
or (B) in vitro kinase assays using poly (Glu/Tyr, 4:1) substrate. The
32P-incorporated proteins were resolved on 7.5% SDS-PAGE
followed by autoradiography. Both autokinase and total kinase activity were increased upon stimulation of the CCR5-L1.2 cells with MIP1.
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RAFTK acts as a mediator of MIP1-stimulated JNK/SAPK
activity.
Treatment of CCR5-L1.2 cells with MIP1 resulted in the activation of
JNK/SAPK activity compared with untransfected L1.2 cells (Fig
4A). To address whether RAFTK participates
in this process, we created double transfected L1.2 cells that
expressed human CCR5 and wild-type RAFTK (CCR5-RAFTKwt)
or human dominant-negative RAFTK lacking kinase activity
(CCR5-RAFTKm457). An increase of ~80% to 90% was
observed in JNK/SAPK activation in chemokine-treated
CCR5-RAFTKwt cells compared with pcDNA-transfected control
CCR5-L1.2 cells. This activity in the dominant-negative
CCR5-RAFTKm457 cells was reduced by 40% to 50% compared
with the pcDNA control (Fig 4B). Similar results were observed in three
different clones of each transfectant and thus do not reflect clonal
variation.
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| Fig 4.
Activation of JNK and p38 kinase upon chemokine
stimulation. (A) L1.2 and CCR5-L1.2 cells were stimulated with MIP1
and were immunoprecipitated with JNK antibody and subjected to in vitro kinase assay using GST-c-jun (1-79 amino acids) as substrates. The
experiment was repeated three times with similar results. (B) CCR5-L1.2
cells were stably transfected with vector control pcDNA,
RAFTKwt or RAFTKm457. The transfectants were
stimulated with MIP1 (25 nmol/L) for 15 minutes and the cells were
lysed, immunoprecipitated with JNK antibody, and subjected to kinase
assay. (C) CCR5-L1.2 cells were stimulated with MIP1 or anisomycin
and cell lysates were immunoprecipitated with p38 kinase antibody. The
immune complexes were subjected to kinase assay using MBP as a
substrate.
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Chemokine stimulation of CCR5-transfected cells enhances their p38
kinase activity.
To assess whether p38 MAP kinase also participates in -chemokine
signaling, lysates from MIP1-stimulated CCR5-L1.2 cells were
immunoprecipitated with anti-p38 MAP kinase and subjected to kinase
assay. As shown in Fig 4C, MIP1 treatment resulted in an increase in
p38 MAP kinase activity. Anisomycin which was used as a positive
control also stimulated p38 MAP kinase activity (Fig 4C).
Paxillin is phosphorylated and associated with RAFTK upon
MIP1 stimulation.
Since chemokines potently effect cell migration which involves
alterations in cytoskeletal elements, we assessed changes in paxillin,
a major cytoskeletal component of focal adhesions. As shown in Fig
5A, MIP1 treatment of CCR5-L1.2 cells
resulted in the tyrosine phosphorylation of paxillin. In these
immunoprecipitates, we also observed a tyrosine phosphorylated protein
of ~115 kd which associated with paxillin and was shown to be RAFTK
(Fig 5A). However, this complex may also contain other tyrosine
phosphorylated proteins of a similar molecular weight. To further
confirm the association between RAFTK and paxillin, CCR5-L1.2 cells
were stimulated with MIP1 and the cell lysates were
immunoprecipitated with RAFTK antibodies and subjected to Western
blotting with anti-paxillin antibodies. As shown, paxillin was observed
to be associated with RAFTK (Fig 5B).
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| Fig 5.
Phosphorylation of paxillin and its association with
RAFTK. (A) CCR5-L1.2 cells were stimulated with 100 nmol/L MIP1 for varying time intervals, and stimulated or unstimulated cell lysates were immunoprecipitated with anti-paxillin antibody. The immune precipitates were then run on SDS-PAGE and subjected to Western blotting with phosphotyrosine antibody, followed by antipaxillin and
anti-RAFTK antibodies. (B) Stimulated or unstimulated cell lysates were
immunoprecipitated with RAFTK antibody and blotted with paxillin
antibody, followed by RAFTK antibody.
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| |
DISCUSSION |
Although chemokines have been shown to play important biological roles,
relatively little information is available regarding their signaling
mechanisms.1,5,19-22 Because chemokine stimulation has
prominent effects on both chemotaxis and
proliferation,1,2,5,10 we focused on RAFTK, a
recently identified member of the focal adhesion kinase family, that
has been found to link growth factors and stress signals (such as UV
and osmotic shock) to the cytoskeleton and to the nucleus in
hematopoietic and neuronal cells.28-32,37-39
We have observed that the chemokine MIP1, which binds specifically
to the CCR5 receptor, brings about an increase in intracellular calcium
in cells overexpressing the cognate CCR5 receptor (CCR5-L1.2). Stimulation of CCR5-L1.2 cells with MIP1 resulted in an increased tyrosine phosphorylation of the RAFTK molecule. MIP1 stimulation of
activated T cells also led to the increased tyrosine phosphorylation of
RAFTK. The observed increase in phosphorylation was associated with
RAFTK activation, a result apparent both in the enhanced autophosphorylation of RAFTK as well as in the increase in its in vitro
kinase activity. Chemokine stimulation also enhanced JNK/SAPK activity,
which is known to activate the AP-1 transcriptional complex.41,42 Furthermore, we observed that overexpression of wild-type RAFTK (CCR5-RAFTKwt) enhanced JNK/SAPK
activity, whereas overexpression of a dominant-negative form of RAFTK
lacking kinase activity (CCR5-RAFTKm457) reduced JNK/SAPK
activity. This suggests that RAFTK may mediate chemokine-stimulated
JNK/SAPK activation. Pyk2/RAFTK has previously been shown to mediate
stress-induced JNK/SAPK activation in neuronal cells.32
Another recently discovered pathway mediating transcriptional
activation is via the p38 MAP kinases. These kinases are activated by
physical and chemical stresses as well as bacterial lipopolysaccharides and various cytokines.43-48 The p38 MAP kinase plays an
important role in the phosphorylation and activation of transcription
factors including CHOP, ELK-1, and ATF-2.46,47 JNK/SAPK and
p38 kinases are known to be activated by SPRK, a mixed-lineage
kinase.49 We also observed an increase in p38 MAP kinase
activity upon MIP1 stimulation. Recently, it has been shown that
signaling via the -chemokine receptor for IL-8 appeared to involve
p38 MAP kinase, but not JNK/SAPK.50 Our results suggest
that there may be important differences in the pathways of
transcriptional activation used by - and -chemokines.
Because chemokines potently affect cell migration which involves
alterations in cytoskeletal elements, we analyzed the phosphorylation of paxillin, a major cytoskeletal component of focal adhesions. We
observed that MIP1 stimulation results in the tyrosine
phosphorylation of paxillin. Furthermore, after paxillin was
phosphorylated by chemokines, there was an enhanced association of this
protein with RAFTK. RAFTK has previously been shown to be associated
with paxillin in hematopoietic cells following activation by growth factors.51 Activation and association of RAFTK with
paxillin may result in the formation of a cytoskeletal complex, which
contributes to enhanced chemotaxis. Interestingly, treatment of T cells
with RANTES resulted in the phosphorylation of FAK and its association with paxillin.52
Our findings provide new information on the signal transduction
pathways used by the -chemokine receptor CCR5 and show how chemokines may modulate both chemotaxis and cell proliferation. We have
observed that -chemokine stimulation results in activation of the
recently identified signaling molecule RAFTK, the cytoskeletal protein
paxillin and JNK/SAPK and p38 MAP kinase activities. Furthermore, JNK/SAPK activities were enhanced in CCR5-RAFTKwt cells and
decreased in CCR5-RAFTKm457 dominant-negative mutants.
These data suggest that RAFTK may mediate CCR5 signaling to
cytoskeletal elements such as paxillin and downstream to the nuclear
activating protein via JNK/SAPK and p38 MAP kinase pathways.
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted November 18, 1997.
The first two and last two authors of this paper contributed equally to
the work.
Supported in part by National Institutes of Health Grants No. HL 55187, HL 53745, HL 43510, and HL55445.
Address reprint requests to Jerome E. Groopman, MD, Chief, Division of
Experimental Medicine, Harvard Institutes of Medicine-BIDMC, 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 |
We thank our colleagues William C. Hatch, Zhong-Ying Liu, Jian-Feng
Wang, and Mel Ona for their help and technical assistance. We are
grateful to Janet Delahanty for editing and preparation of figures as
well as Evelyn Gould for her assistance with the figures. Finally, we
appreciate Youngsun Jung and Tee Trac for typing the manuscript. This
manuscript is submitted in honor of Ronald Ansin for his ongoing
support of our research program.
| |
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P. Dutt, J.-F. Wang, and J. E. Groopman
Stromal Cell-Derived Factor-1{alpha} and Stem Cell Factor/kit Ligand Share Signaling Pathways in Hemopoietic Progenitors: A Potential Mechanism for Cooperative Induction of Chemotaxis
J. Immunol.,
October 1, 1998;
161(7):
3652 - 3658.
[Abstract]
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R. K. Ganju, S. A. Brubaker, J. Meyer, P. Dutt, Y. Yang, S. Qin, W. Newman, and J. E. Groopman
The alpha -Chemokine, Stromal Cell-derived Factor-1alpha , Binds to the Transmembrane G-protein-coupled CXCR-4 Receptor and Activates Multiple Signal Transduction Pathways
J. Biol. Chem.,
September 4, 1998;
273(36):
23169 - 23175.
[Abstract]
[Full Text]
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R. K. Ganju, N. Munshi, B. C. Nair, Z.-Y. Liu, P. Gill, and J. E. Groopman
Human Immunodeficiency Virus Tat Modulates the Flk-1/KDR Receptor, Mitogen-Activated Protein Kinases, and Components of Focal Adhesion in Kaposi's Sarcoma Cells
J. Virol.,
July 1, 1998;
72(7):
6131 - 6137.
[Abstract]
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B. P. Lipsky, C. R. Beals, and D. E. Staunton
Leupaxin Is a Novel LIM Domain Protein That Forms a Complex with PYK2
J. Biol. Chem.,
May 8, 1998;
273(19):
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[Abstract]
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R. K. Ganju, S. A. Brubaker, R. D. Chernock, S. Avraham, and J. E. Groopman
beta -Chemokine Receptor CCR5 Signals through SHP1, SHP2, and Syk
J. Biol. Chem.,
June 2, 2000;
275(23):
17263 - 17268.
[Abstract]
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K. Kraft, H. Olbrich, I. Majoul, M. Mack, A. Proudfoot, and M. Oppermann
Characterization of Sequence Determinants within the Carboxyl-terminal Domain of Chemokine Receptor CCR5 That Regulate Signaling and Receptor Internalization
J. Biol. Chem.,
September 7, 2001;
276(37):
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[Abstract]
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Abstract
Introduction
Abstract