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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3713-3722
CrkL Activates Integrin-Mediated Hematopoietic Cell Adhesion
Through the Guanine Nucleotide Exchange Factor C3G
By
Ayako Arai,
Yurika Nosaka,
Hitoshi Kohsaka,
Nobuyuki Miyasaka, and
Osamu Miura
From the First Department of Internal Medicine, Tokyo Medical and
Dental University, Tokyo, Japan.
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ABSTRACT |
CrkL is a member of the Crk family of adapter proteins consisting
mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in
hematopoietic cells and has been implicated in pathogenesis of chronic
myelogenous leukemia. However, its function has not been precisely
defined. Here, we show that overexpression of CrkL enhances the
adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced
increase in cell adhesion was blocked by antibodies against VLA-4
( 4 1) and VLA-5 (51) but was observed without changes in
surface expression levels of these integrins. Studies using CrkL
mutants demonstrated that the SH2 domain is partially required for
enhancing cell adhesion, whereas the C-terminal SH3 domain as well as
the tyrosine phosphorylation site (Y207) is dispensable. In contrast,
the N-terminal SH3 domain, involved in binding C3G and other signaling
molecules, was showed to play a crucial role, because a mutant
defective of this domain showed an inhibitory effect on the cell
adhesion to fibronectin. Furthermore, overexpression of C3G also
increased the adhesion of hematopoietic cells to fibronectin, whereas a
C3G mutant lacking the guanine nucleotide exchange domain abrogated the
CrkL-induced increase in cell adhesion. On the other hand, a dominant
negative mutant of H-Ras or that of Raf-1 enhanced the basal and
CrkL-induced cell adhesion and that of R-Ras modestly decreased the
adhesion. Taken together, these results indicate that the CrkL-C3G
complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the
guanine nucleotide exchange activity of C3G.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS takes place in close
contact with the bone marrow microenvironment, which is composed of
stromal cells and extracellular matrix components, including
fibronectin. Members of the integrin superfamily of adhesion molecules
mainly mediate adherence of hematopoietic cells to both the
extracellular matrix and stromal cells. Integrins are heterodimers of
 and subunits that can pair to form more than 20 receptors.1,2 Integrins of the 1 subfamily, mostly VLA-4
(41) and VLA-5 (51), have been identified on most of the
hematopoietic progenitor cells as well as on various hematopoietic cell
lines3-5 and shown to bind ligands especially when these
cells are stimulated with growth-stimulating cytokines such as
interleukin-3 (IL-3), granulocyte-monocyte colony-stimulating factor,
erythropoietin (Epo), and stem cell factor.6-9 One of the
ligands involved in the adhesion of hematopoietic cells through VLA-4
and VLA-5 is fibronectin, which preferentially mediates adhesion of
primitive progenitor cells to the bone marrow
microenvironment.10 Recently, accumulating evidence has
suggested that adhesive interaction mediated by integrins of 1
subfamily and fibronectin plays a critical role in controling
proliferation, apoptosis, migration, and mobilization of hematopoietic
cells.4,8,11-18 Thus, knowledge of the mechanisms by which
the functional states of these integrins are regulated is critical to
our understanding of the physiologic mechanisms responsible for the
regulation of normal hematopoiesis.
The Crk proteins, originally identified as homologues of the product of
the v-crk oncogene,19 are adapter proteins composed of SH2 and SH3 domains with very short intervening sequences. Three
forms of cellular Crk proteins have been found; both Crk II and CrkL
(for Crk-like) have the domain structure SH2-SH3-SH3, although Crk I,
the alternatively spliced form of Crk II, lacks the C-terminal SH3
domain.20,21 The N-terminal SH3 domain of CrkL has been
shown to bind Sos1 and C3G, two guanine nucleotide exchange proteins
for the Ras family of small GTPases.22,23 Interestingly,
recent studies have established that CrkL, which is most abundantly
expressed in hematopoietic cells,24 also binds through its
N-terminal SH3 domain to the BCR-ABL fusion protein expressed in
chronic myelogenous leukemia cells and becomes phosphorylated at
Y-207.25-28 CrkL also becomes tyrosine phosphorylated in
hematopoietic cells stimulated with stem cell factor,29
thrombopoietin,30 Epo,31 IL-3,31
and IL-2.32 In addition, CrkL, through its SH2 domain,
forms complexes with tyrosine-phosphorylated signaling molecules,
including c-Cbl,29,31-33 Shc,31 and
SHP-231 in hematopoietic cells stimulated with cytokines.
Thus, it is implied that CrkL may play a role in growth control and
leukemic transformation of hematopoietic cells. However, the function
of CrkL in hematopoietic cells has not been precisely defined. In the
present study, we show that overexpression of CrkL activates adhesion
of hematopoietic cells to fibronectin through VLA-4 and VLA-5. CrkL was
further shown to transduce the signal to activate these integrins
through the guanine nucleotide exchange activity of C3G.
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MATERIALS AND METHODS |
Cells and reagents.
A clone of IL-3-dependent 32D cells expressing the wild-type murine
Epo receptor (32D/EpoR-Wt) was previously described34 and
maintained in RPMI 1640 medium supplemented with 10% fetal calf serum
(FCS) and 1 U/mL human recombinant Epo. COS7 cells were cultured in
Dulbecco's modified Eagle medium (Nissui Seiyaku, Tokyo,
Japan) supplemented with 10% FCS. Recombinant human Epo was kindly
provided by Chugai Pharmaceutical Co Ltd (Tokyo, Japan). Recombinant
murine IL-3 was purchased from PeproTech Inc (Rocky Hill, NJ).
Antibodies against CrkL, C3G, and R-Ras were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Monoclonal antibodies (MoAbs) against
murine 4 (428) and 5 (5H10-27) integrin subunits were from
Seikagaku Corp. (Tokyo, Japan) and from PharMingen (San Diego, CA), respectively. Fluorescein (DTAF)-conjugated goat
anti-rat IgG secondary antibody was obtained from Immunotech
(Marseille, France). Human plasma fibronectin was purchased from
GIBCO-BRL (Grand Island, NY).
Expression plasmids.
An expression plasmid for human CrkL, pSG-CrkL,35 was
kindly provided by Dr John Groffen (Childrens Hospital Los Angeles, Los
Angeles, CA). Expression plasmids for various CrkL mutants were
constructed by deletion of the following fragments from the CrkL cDNA:
dSH2, a Cfr101 fragment (nucleotides 560 to 752); dSH3N, a
DraI-AluI fragment (nucleotides 941 to 1049); dY, an
AvaIII-PstI fragment (nucleotides 1127 to 1163); dSH3C,
an AvaIII-BalI fragment (nucleotides 1127 to 1327).
For construction of pTet-CrkL, in which the CrkL cDNA is placed
downstream of a tetracycline operator (tetO)-controlled
promoter, a 5' portion of the CrkL cDNA (nucleotides 514 to 844) was
amplified by the polymerase chain reaction (PCR) with 5' and 3' primers of 5'-CCGGATCCTCCGCCAGGTTCGACTC-3' and
5'-CCGAATTCATCCCATTGGTGGGCTTGGAT-3', respectively. The primer
sequences were designed to add the BamHI and EcoRI
recognition sequences at the 5' and 3' ends, respectively, of the
amplified fragment. These sites were then used for subcloning of the
amplified fragment into the multiple-cloning site of an expression
plasmid, pJ3H,36 obtained from American Type Culture Collection (Rockville, MD). The SalI-ClaI fragment,
encompassing the amplified region, was then excised from this plasmid
and subcloned between the SalI and ClaI sites of
pTet-Splice (GIBCO-BRL). This plasmid was then digested with
CpoI and EcoRV to subclone the CpoI-BglII fragment, containing nucleotides 536 to the
3' end of the CrkL cDNA, from pSG-CrkL to replace the PCR-amplified
region, thus giving pTet-CrkL.
An expression plasmid for C3G, pcDNA-C3G, was constructed by subcloning
the HindIII-BamHI fragment (nucleotides 66 to 3377) of
C3G cDNA,22 obtained through the Riken Gene Bank (Ibaraki, Japan) with the permission from Dr Michiyuki Matsuda (National Institute of Health, Tokyo, Japan), into pcDNA3 (Invitrogen, San Diego,
CA). An expression plasmid for mutant C3G, pcDNA-C3G-dSS, was created
by deletion of the SmaI-ScaI fragment of C3G cDNA (nucleotides 2609 to 2999) from pcDNA-C3G. Tetracycline responsive expression plasmid for C3G and the C3G mutant, pTet-C3G and
pTet-C3G-dSS, were constructed by subcloning the
HindIII-AvrII fragments (nucleotides 66 to 3360) from
pcDNA-C3G and pcDNA-C3G-dSS, respectively, between the HindIII
and SpeI sites of pTet-Splice.
For construction of an expression plasmid for mutant Raf-1,
pcDNA-Raf-dSE, the Raf-1 cDNA was excised from p627,37
obtained from the Riken Gene Bank, by digestion with EcoRI and
XbaI and subcloned into pcDNA3 to give pcDNA-Raf-1. The
StuI-EcoRV fragment (nucleotides 1122 to 2028) was then
deleted to give pcDNA-Raf-dSE. An expression plasmid for a dominant
negative mutant of R-Ras, pcDNA-R-Ras43N,38 was kindly
provided by Dr Erkki Ruoslahti (La Jolla Cancer Research Center, La
Jolla, CA). An expression plasmid for dominant negative H-Ras,
pcDNA-H-Ras17N, was constructed by subcloning cDNA coding for H-Ras17N
(Upstate Biotechnology, Lake Placid, NY) into the pcDNA3 vector.
Transfection.
Transfection for stable expression was performed essentially as
described previously.34 In brief, 32D/EpoR-Wt cells were transfected with or without 5 µg of pTet-CrkL along with 5 µg of
pTet-tTAk (GIBCO-BRL), which is an expression plasmid for the tetracycline transactivator (tTA),39 and 1 µg of pSV-Zeo
(Invitrogen) by electroporation at 960 µF and 300 V, followed by
selection in medium containing Zeocine (Invitrogen) and 500 ng/mL
tetracycline. Six clones transfected with pTet-CrkL were isolated by
limiting dilution and examined for the induction of CrkL expression by anti-CrkL immunoblotting of cell lysates prepared after withdrawal from
tetracycline for 24 hours. The clone inducibly expressing the highest
level of CrkL, 32DE/Tet-CrkL, was selected for the subsequent studies.
Clones transfected with pTet-tTAk and pSV-Zeo alone were similarly
selected and examined for the expression of tTA by the luciferase assay
by using the Dual-Luciferase Reporter Assay System (Promega, Madison,
WI), the pUHC13-3 (GIBCO-BRL) and a control Renilla luciferase plasmid,
pRL-SV (Promega), a reporter plasmid, as described
previously.40 The clone inducibly expressing the highest
level of tTA, which was comparable with that expressed by
32DE/Tet-CrkL, was designated as 32DE/TA and used for the subsequent
studies. 32DE/Tet-C3G and 32DE/Tet-C3G-dSS clones were similarly
obtained by transfecting pTet-C3G and pTet-C3G-dSS, respectively, into
32DE/TA cells along with pMAM2-BSD41 (Funakoshi, Tokyo,
Japan) followed by selection in medium containing blasticidin-S (Funakoshi).
Transfection of expression plasmids into COS7 cells was performed with
the Lipofectamin reagent (GIBCO-BRL), as described previously.42 Cells were harvested for analysis with
immunoprecipitation and immunoblotting 2 days after transfection.
Immunoprecipitation and immunoblotting.
Cells were solubilized with a lysis buffer composed of 1% Triton
X-100, 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin. Cell lysates were
subjected to immunoprecipitation and immunoblotting as described previously.34
Cell adhesion assays.
For stable transfectants, cell adhesion assay was performed essentially
as described with some modifications.38 In brief, 96-well,
flat-bottom tissue culture plates were coated with indicated concentrations of fibronectin overnight at 4°C. Plates were then blocked with 1% bovine serum albumin (BSA) at 37°C for 1 hour followed by washing three times with RPMI 1640 containing 0.2% BSA,
referred to as cell adhesion medium. Cells were washed twice and
resuspended in cell adhesion medium supplemented with 5 ng/mL IL-3,
unless indicated otherwise. Cells (5 to 10 × 104/well)
were added to each well in triplicate and incubated for 30 minutes at
37°C. In some experiments, cells were incubated with indicated
concentrations of anti-integrin antibodies or irrelevant MoAb for 15 minutes at room temperature before plating on fibronectin coated wells.
Plates were then washed three times with cell adhesion medium to remove
unbound cells. Cells remaining attached to the plates were measured by
the sodium
3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) colorimetric assay
(Boehringer-Mannheim, Indianapolis, IN) according to the
manufacturer's recommendation. After subtraction of background cell
binding to BSA-coated wells, the percentage of adherent cells was
determined by dividing the optical density of the adherent cells by
that of the initial cell input.
For adhesion assay of transiently transfected cells, 32D/EpoR-Wt cells
were electroporated at 960 µF and 300 V with indicated amounts of
relevant plasmids and 1 µg of a control Renilla luciferase plasmid,
pRL-SV. After a recovery period of 1 day, cells were subjected to the
cell adhesion assay described above except that 4 × 105
cells were plated on fibronectin-coated 24-well plates in duplicate, and the adhesion was assayed by the luciferase activity of cell lysates.
All the cell adhesion assays in Results were repeated at least three
times, and the results were reproducible.
Flow cytometry.
To analyze the surface expression of VLA-4 and VLA-5, 32DE/Tet-CrkL
cells were cultured in the presence or absence of tetracycline for 24 hours and stained with anti-4 or anti-5 antibody or left unstained as control. Cells were further stained with
fluorescein-labeled secondary antibody and analyzed with an Epics Elite
flow cytometer (Coulter Electronics, Miami, FL).
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RESULTS |
Overexpression of CrkL increases adhesion of cells to fibronectin.
To explore the functions of CrkL in hematopoietic cells, we established
a clone of 32D/EpoR-Wt cells, 32DE/Tet-CrkL, which overexpresses CrkL
when withdrawn from tetracycline, as described in Materials and
Methods. As shown in Fig 1A,
32DE/Tet-CrkL cells, which grow in suspension, became highly adherent
when cultured in the absence of tetracycline and extended long
protrusions. 32DE/TA cells, which express only tTA at a comparable
level with 32DE/Tet-CrkL when withdrawn from tetracycline, did not show
this change. To confirm that overexpression of CrkL activates cell adhesion, 32DE/Tet-CrkL cells as well as 32DE/TA cells were cultured with or without tetracycline for 24 hours and allowed to attach to
wells coated with a defined substrate, fibronectin, for 30 minutes in
the presence of IL-3. As shown in Fig 1B, 32DE/Tet-CrkL cells attached
dramatically better to fibronectin when tetracycline was removed from
culture medium, whereas 32DE/TA cells, cultured with or without
tetracycline, attached poorly to this substrate. Ant-CrkL
immunoblotting of lysates obtained from cells cultured under the same
conditions confirmed that CrkL was overexpressed in 32DE/Tet-CrkL cells
in the absence of tetracycline (Fig 1B, upper panel). When removed from
tetracycline to overexpress CrkL, 32DE/Tet-CrkL cells were also shown
to attach better to wells coated with various concentrations of
fibronectin (Fig 1C). In accordance with previous
reports,6,9 32DE/Tet-CrkL cells starved from Epo and IL-3
for 16 hours barely attached to fibronectin, whereas the adhesion was
remarkably activated when cultured in the presence of IL-3 (Fig 1D).
Although Epo also activated the adhesion of 32DE/Tet-CrkL or
32D/EpoR-Wt cells in repeated experiments, the Epo-induced increase in
cell adhesion was always only moderate and much less than that induced
by IL-3 (Fig 1D; data not shown). As shown in Fig 1D, the
overexpression of CrkL induced by withdrawal from tetracycline
dramatically increased the low adhesion levels of 32DE/Tet-CrkL cells
starved from cytokines or cultured in Epo, while the IL-3-induced,
high level of adhesion was only moderately increased by the CrkL
overexpression.
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| Fig 1.
Overexpression of CrkL increases adhesion of 32D cells.
(A) Morphology of 32D cells overexpressing CrkL. A clone of 32D/EpoR-Wt
cells stably transfected with the expression plasmid for tetracycline
transactivator alone (32DE/TA) or a clone also transfected with
pTet-CrkL (32DE/Tet-CrkL) was cultured in the presence (+) or absence
( ) of 100 ng/mL of tetracycline (Tet), as indicated, for 24 hours
and photographed under an inverted microscope (Olympus, Tokyo, Japan).
(B) Adhesion of CrkL-overexpressing 32D cells to fibronectin. 32DE/TA
and 32DE/Tet-CrkL cells were cultured in the presence (+) or absence
() of Tet, as indicated, for 24 hours and allowed to attach to wells
coated with 10 µg/mL fibronectin for 30 minutes at 37°C in the
presence of IL-3. The extent of cell adhesion was quantitated as
described in Materials and Methods. The data represent averages ± SD
of triplicate determinations. Anti-CrkL immunoblotting of cell lysates
obtained under the same conditions is also shown. (C) Effect
of fibronectin concentration on adhesion of CrkL-overexpressing 32D
cells. 32DE/Tet-CrkL cells, cultured with or without tetracycline, as
indicated, were allowed to attach to wells coated with indicated
concentrations of fibronectin for the cell adhesion assay. (D) Effect
of cytokines on adhesion of CrkL-overexpressing 32D cells.
32DE/Tet-CrkL cells were cultured with or without tetracycline, as
indicated, for 24 hours. During the last 16 hours, cells were cultured
with 1 U/mL Epo (Epo), 5 ng/mL IL-3 (IL-3), or without any cytokine
(), as indicated. Cells were allowed to attach to wells coated with
10 µg/mL fibronectin for the cell adhesion assay in the presence or
absence of cytokine, as indicated.
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CrkL increases cell adhesion to fibronectin by activating VLA-4 and
VLA-5.
Integrins of the 1 family, mostly VLA-4 (41) and VLA-5
(51), have been shown to mediate adhesion of hematopoietic cells, including 32D cells38 to fibronectin. Therefore, to
identify the receptors involved in the CrkL-induced increase of cell
adhesion to fibronectin, we examined the effects of antibodies against VLA-4 and VLA-5 on adhesion of CrkL-overexpressing cells to
fibronectin. As shown in Fig 2A, a
function-blocking anti-4 or anti-5 integrin antibody, when added
alone, partially inhibited the adhesion of 32DE/Tet-CrkL cells cultured
without tetracycline to fibronectin. Notably, when the two antibodies
were added in combination, adherent cells were reduced to less than 5%
of the total cells added to fibronectin-coated wells. These results
agree with the previous report38 that 32D cell attachment
to fibronectin is mediated by VLA-4 and VLA-5 integrins and further
indicate that overexpression of CrkL enhanced 32D cell attachment to
fibronectin through these integrins. As shown in Fig 2B, the flow
cytometry analysis using anti-4 and anti-5 antibodies confirmed
the presence of these integrins on the cell surface of 32DE/Tet-CrkL
cells and further showed that the expression levels of these integrins
were not significantly altered by withdrawal from tetracycline. Thus,
these results suggest that CrkL enhances cell adhesion by increasing the activities of VLA-4 and VLA-5.
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| Fig 2.
CrkL increases adhesion of 32D cells to fibronectin by
activating VLA-4 and VLA-5. (A) Antibodies against VLA-4 and VLA-5
inhibit adhesion of CrkL-overexpressing 32D cells to fibronectin.
32DE/Tet-CrkL cells were cultured for 24 hours in the absence of
tetracycline and allowed to attach to wells coated with 10 µg/mL
fibronectin in the absence (Control) or in the presence of indicated
anti-integrin MoAbs or irrelevant MoAb (IgG), as indicated. The extent
of cell adhesion was quantitated as described in Materials and Methods.
(B) Analysis of VLA-4 and VLA-5 expression in 32DE/Tet-CrkL cells by
flow cytometry. 32DE/Tet-CrkL cells were cultured in the presence
(upper panels) or absence (lower panels) of tetracycline for 24 hours
and stained with indicated anti-integrin MoAbs or left unstained as
control (Control), as indicated. Cells were further stained with
fluorescein-labeled secondary antibody and subjected to flow
cytometry.
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The N-terminal SH3 domain of CrkL plays a critical role in
enhancement of cell adhesion.
To explore the mechanisms by which CrkL increases the activity of VLA-4
and VLA-5, we examined the functional significance of each CrkL domain
for the enhancement of cell adhesion. For this purpose, we constructed
expression plasmids for CrkL mutants shown in Fig
3A. These mutants were first expressed
along with C3G in COS7 cells. As shown in Fig 3B, anti-CrkL
immunoblotting of transfected COS7 cell lysates showed that the CrkL
mutants with expected sizes were expressed at comparable levels.
Furthermore, anti-CrkL immunoblotting of anti-C3G immunoprecipitates
confirmed that C3G bound all the CrkL mutants except the dSH3N mutant,
which has a deletion in the N-terminal SH3 domain involved in
binding C3G (Fig 3B). These results suggest that the deletions
introduced into CrkL did not significantly affect the expression level
or the overall structure of mutant CrkL.
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| Fig 3.
The N-terminal SH3 domain of CrkL plays a critical role
in enhancement of cell adhesion. (A) Schematic representation of CrkL
and its mutants. (B) Transient expression of CrkL and its mutants with
C3G in COS7 cells. Expression plasmids for wild type and various
mutants of CrkL, as indicated, were transfected with that of C3G into
COS7 cells. Cells were harvested 2 days after transfection, and total
cell lysates (TCL) and anti-C3G immunoprecipitates were subjected to
anti-CrkL immunoblotting followed by reprobing with anti-C3G. (C)
Effects of transiently expressed CrkL mutants on adhesion of
32D/EpoR-Wt cells to fibronectin. The expression plasmids for wild type
and various mutants of CrkL, as indicated, were transfected into
32D/EpoR-Wt cells along with pRL-SV. Transiently transfected cells were
subjected to the cell adhesion assay as described in Materials and
Methods. (D) Dose-dependent effects of the CrkL dSH3N and dSH2 mutants
on 32D/EpoR-Wt cell adhesion to fibronectin. 32D/EpoR-Wt cells were
transfected with indicated amounts (microgram) of the expression
plasmids for dSH3N and dSH2 mutants of CrkL or the pSG5 vector plasmid
and subjected to the cell adhesion assay.
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To examine the abilities of CrkL mutants to enhance the cell adhesion,
each mutant was then transiently expressed in 32D/EpoR-Wt cells for the
cell adhesion assay, as described in Materials and Methods. As shown in
Fig 3C, the dY mutant, which lacks a 12-amino-acid region containing
the site of tyrosine phosphorylation,28 did not show any
impairment in the ability to enhance cell adhesion. The dSH3C mutant,
which lacks most of the C-terminal SH3 domain in addition to the
tyrosine phosphorylation site, also showed the adhesion-enhancing
ability comparable with that of wild-type CrkL. On the other hand, the
dSH2 mutant, in which most of the SH2 domain is lost by deletion,
showed a significantly impaired ability to enhance the 32D cell
adhesion to fibronectin. However, repeated experiments (data not shown)
as well as a dose-dependent experiment shown in Fig 3D confirmed that
the dSH2 mutant has retained the ability, although impaired, to enhance
cell adhesion. In contrast, the dSH3N mutant, lacking the significant
portion of the N-terminal SH3 domain, not only failed to enhance but
also significantly inhibited the attachment of 32D/EpoR-Wt cells to fibronectin in repeated experiments (Fig 3C; data not shown). The
inhibitory effect of the dSH3N mutant was also demonstrated to be dose
dependent (Fig 3D). These results indicate that the N-terminal SH3
domain of CrkL, through which CrkL binds C3G and other signaling
molecules, plays a crucial role in integrin activation. Although the
SH2 domain may also play a role in integrin activation, this domain is
not crucial for this function. On the other hand, neither the tyrosine
phosphorylation site nor the C-terminal SH3 domain was shown to be
involved in integrin activation.
The guanine nucleotide exchange activity of C3G is involved in
CrkL-induced integrin activation.
Although the N-terminal SH3 domain has been shown to bind both C3G and
Grb2, we previously showed that CrkL predominantly binds C3G in 32D
cells.31 In addition, it was confirmed that an increased
amount of C3G was associated with CrkL in 32DE/Tet-CrkL cells when CrkL
was overexpressed (data not shown). Thus, we examined whether C3G is
also involved in integrin activation. As described in Materials and
Methods, we established 32D/EpoR-Wt clones, 32DE/Tet-C3G and
32DE/Tet-C3G-dSS, which overexpress wild-type C3G and the C3G-dSS
mutant, respectively, when cultured without tetracycline. When the
expression level of C3G was increased by withdrawal from tetracycline
(Fig 4A), 32DE/Tet-C3G cells showed a
moderately increased adhesion to fibronectin, as shown in Fig 4B. In
contrast, when the expression of C3G-dSS mutant, lacking the guanine
nucleotide exchange domain, was induced by withdrawal from tetracycline
(Fig 4A), the adhesion of 32DE/Tet-C3G-dSS cells was significantly inhibited, as shown in Fig 4B. These results indicate that the guanine
nucleotide exchange activity of C3G should play a role in integrin
activation.
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| Fig 4.
The guanine nucleotide exchange domain of C3G is involved
in enhancement of cell adhesion. (A) Inducible expression of C3G or its
mutant in 32D cells. A clone of 32D/EpoR-Wt cells transfected with
pTet-C3G (32DE/Tet-C3G) or pTet-C3G-dSS (32DE/Tet-C3G-dSS), coding for
C3G or its mutant lacking the guanine nucleotide exchange domain,
respectively, was cultured for 24 hours with (+) or without ()
Tet, as indicated. TCL were extracted and subjected to anti-C3G
immunoblotting. Positions of C3G and its mutant, C3G-dSS, are
indicated. (B) 32DE/Tet-C3G and 32DE/Tet-C3G-dSS cells were cultured
for 24 hours with (+) or without () Tet, as indicated, and
subjected to the cell adhesion assay as described in Materials and
Methods.
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To further confirm the involvement of C3G in CrkL-induced integrin
activation, we transiently expressed wild-type C3G or the C3G-dSS
mutant in 32D/EpoR-Wt cells and examined the effect on cell adhesion.
As shown in Fig 5A, the transient
overexpression of C3G drastically enhanced the adhesion of 32D/EpoR-Wt
cells to fibronectin, thus confirming the observation in 32DE/Tet-C3G cells. When overexpressed along with CrkL, C3G further increased the
cell adhesion enhanced by CrkL (Fig 5A). On the other hand, the C3G-dSS
mutant drastically inhibited the adhesion of transfected cells, in
accordance with the result in 32DE/Tet-C3G-dSS (Fig 5B). Importantly,
when coexpressed with CrkL, the adhesion-enhancing effect of CrkL was
also significantly inhibited by this mutant (Fig 5B). Taken together
with the result that the C3G-binding domain of CrkL is crucial for the
enhancement of cell adhesion, these data indicate that the CrkL-induced
integrin activation is mediated through the guanine nucleotide exchange
activity of C3G.
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| Fig 5.
Effects of C3G and various mutant signaling molecules on
adhesion of 32D cells. (A) Effects of overexpression of C3G on adhesion
of 32D cells to fibronectin. 32D/EpoR-Wt cells were transfected with 30 µg of pcDNA-C3G (C3G) and 10 µg of pSG-CrkL (CrkL), as indicated,
along with 1 µg of pRL-SV. The total amount of expression plasmids
for each transfection was adjusted to become equal by addition of
pcDNA3. Transiently transfected cells were subjected to the cell
adhesion assay as described in Materials and Methods. (B) Effects of
dominant negative mutants of C3G and R-Ras on adhesion of 32D cells to
fibronectin. 32D/EpoR-Wt cells were transfected with 40 µg of
pcDNA-C3G-dSS (C3G-dSS) or pcDNA-R-Ras43N (R-Ras43N) and 5 µg of
pSG-CrkL (CrkL), as indicated, along with 1 µg of pRL-SV. Transiently
transfected cells were subjected to the cell adhesion assay. (C)
Effects of dominant negative mutants of Raf-1 and H-Ras on adhesion of
32D cells to fibronectin. 32D/EpoR-Wt cells were transfected with 35 µg of pcDNA-Raf-dSE (Raf-dSE) or pcDNA-H-Ras17N (H-Ras17N) and 5 µg
of pSG-CrkL (CrkL), as indicated, along with 1 µg of pRL-SV.
Transiently transfected cells were subjected to the cell adhesion
assay.
|
|
Because C3G activates members of the Ras subfamily of small GTP binding
proteins through its guanine nucleotide exchange activity, we next
examined whether these molecules are involved in the downstream signaling pathway from the CrkL-C3G complex leading to integrin activation. First, the possible involvement of R-Ras, which activates integrin in 32D cells,38 was examined. Thus, a dominant
negative mutant of R-Ras, R-Ras43N,38 was transiently
expressed alone or along with CrkL in 32D/EpoR-Wt cells and its effect
on cell adhesion was examined. In accordance with the previous
report,38 R-Ras43N inhibited, although modestly, the basal
level of 32D/EpoR-Wt cell adhesion to fibronectin (Fig 5B). This mutant
also inhibited the cell adhesion enhanced by overexpression of CrkL to
a similar extent. However, the extent of inhibition induced by R-Ras43N was much less than that induced by C3G-dSS, although R-Ras43N was
expressed, under the same condition, at a much higher level than that
of endogenous R-Ras (data not shown). Next, the possible involvement of
H-Ras was examined by using dominant negative mutants of H-Ras and
Raf-1, an effector molecule of H-Ras. As shown in Fig 5C, these
mutants, H-Ras17N and Raf-dSE, significantly enhanced the adhesion of
32D/EpoR-Wt cells, which is in accordance with a previous report that
H-Ras and Raf-1 inhibited integrin activity.43 The enhanced
adhesion of cells overexpressing CrkL was also increased slightly by
coexpression of these mutants (Fig 5C). These results indicate that
R-Ras and H-Ras modulate the integrin activity positively and
negatively, respectively, in 32D cells and raise a possibility that the
CrkL-C3G complex may transduce the integrin activation signal, although
partly, through activation of R-Ras. However, because the inhibitory
effect of R-Ras43N on the CrkL-enhanced cell adhesion was only modest,
it is speculated that other signaling molecules, most likely other
small GTPases, may play more significant roles in integrin activation
by CrkL and C3G.
| |
DISCUSSION |
In this study, we have showed that overexpression of CrkL activates the
adhesion of hematopoietic cells to fibronectin. The enhancement of cell
adhesion was observed without changes in expression levels of VLA-4 and
VLA-5 but was specifically blocked by antibodies against these
integrins, thus indicating that overexpression of CrkL activates VLA-4
and VLA-5 to increase the cell adhesion to fibronectin. Studies using
CrkL mutants have showed that the N-terminal SH3 domain of CrkL,
required for binding C3G, plays a crucial role for integrin activation,
because a mutant defective in this domain decreased the cell adhesion
to fibronectin. In accordance with this, overexpression of C3G also
increased the cell adhesion to fibronectin, whereas a C3G mutant
defective in the guanine nucleotide exchange domain significantly
inhibited the basal and CrkL-enhanced adhesion. These data indicate
that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic
cells through the guanine nucleotide exchange activity of C3G.
During the preparation of this report, Senechal et al44
reported that overexpression of CrkL in hematopoietic cells increased adhesion to fibronectin. Senechal et al44 further showed
that individual mutations or deletions of each SH2 and SH3 domain of CrkL abrogated the increase in adhesion. This is at variance with our
structure function studies, which showed that the SH2 or C-terminal SH3
domain of CrkL is partially or totally, respectively, dispensable for
the increase in cell adhesion and that the N-terminal SH3 domain-defective mutant exerted a dominant negative effect on adhesion.
The basis for these discrepancies are unknown but may reflect
differences in the structures of CrkL mutants or other experimental
conditions. Although Senechal et al44 suggested the
involvement of integrins in CrkL-induced cell adhesion by showing an
inhibitory effect of RGD peptides, the integrins involved in adhesion
were not identified. The present study, thus, complements and extends
that of Senechal et al44 by showing that VLA-4 and VLA-5,
without changes in their expression levels, mediate the CrkL-induced
cell adhesion to fibronectin and that CrkL transduces the signal
leading to integrin activation through the guanine nucleotide exchange
activity of C3G, which is complexed through the N-terminal SH3 domain
of CrkL.
Although it has remained to be known how the CrkL-C3G complex activates
the signaling pathway leading to integrin activation through the
guanine nucleotide exchange activity of C3G, it is reasonable to
speculate that CrkL functions as an adapter protein to recruit C3G to
its substrate involved in regulation of the integrin function. In this
regard, it is noteworthy that the SH2 domain of CrkL was also required,
although partially, for the enhancement of cell adhesion, because
previous studies have shown that CrkL binds through its SH2 domain to
tyrosine-phosphorylated adhesion-associated proteins, such as
paxillin,45 CAS,46 HEF-1,47,48 and
Cbl29,31-33,49,50 in hematopoietic cells expressing BCR/Abl or stimulated with cytokine. In addition, overexpression of CrkL in
fibroblasts has been shown to activate many of the same signal transduction pathways as BCR/Abl51 and to induce tyrosine
phosphorylation of paxillin and its association with
CrkL.44 In accordance with these observations, Cbl was
constitutively tyrosine phosphorylated and associated with CrkL in
32DE/Tet-CrkL cells withdrawn from tetracycline and starved from
cytokine (Y.N., A.A., O.M., unpublished observation, July
1998). Thus, it is possible that overexpression of CrkL induces
recruitment of C3G to the vicinity of its substrates involved in
regulation of integrin function through interaction between the CrkL
SH2 domain and these tyrosine-phosphorylated proteins.
Previously, R-Ras was shown to upregulate the binding affinity of
integrins, including VLA-4 and VLA-5 in 32D cells.38 In contrast, H-Ras or its downstream kinase Raf-1, inhibited the activation of chimeric integrins with multiple and subunit cytoplasmic domains expressed in CHO cells.43 Although C3G
most efficiently activates Rap1/K-Rev1, it also activates R-Ras
moderately and H-Ras rather weakly.52 Therefore, the
possible involvement of R-Ras or H-Ras in CrkL-induced activation of
cell adhesion was examined in the present study. In accordance with the
report by Hughes et al,43 a dominant negative mutant of
H-Ras or that of Raf-1 expressed alone or in combination with CrkL
significantly increased the 32D cell adhesion to fibronectin (Fig 5C).
Because Rap1/K-Rev1, which C3G activates most efficiently, antagonizes the function of Ras in certain cell lines,53-55 it is
formally possible that the CrkL-C3G complex activates integrins by
downregulating the Ras/Raf-1 signaling pathway through activation of
Rap1/K-Rev1. However, this possibility is unlikely because
overexpression of the CrkL-C3G complex in 32D cells activates the
Raf/MAP kinase pathway in 32D cells (A.A., Y.N., O.M.,
unpublished observation, July 1998). A dominant negative
mutant of R-Ras, on the other hand, downregulated the basal as well as
CrkL-induced adhesion of 32D/EpoR-Wt cells to fibronectin (Fig 5B),
which is in agreement with the previous report.38 So, it is
possible that CrkL may activate VLA-4 and VLA-5 partly through
activation of R-Ras. However, the reduction in cell adhesion induced by
the dominant negative R-Ras, which was expressed at high levels, was
only moderate, thus suggesting that biochemical events involving
signaling molecules other than R-Ras should play more important roles
in activation of hematopoietic cell adhesion induced by CrkL and C3G.
Therefore, to elucidate the signaling pathways mediating the
CrkL-induced activation of cell adhesion, further studies are required
to analyze the possible modulation of the activities of these Ras
family GTPases and to examine the possible involvement of other
signaling molecules. The Rho family GTPases, which have also been
implicated in enhancing cell adhesion,56,57 are of
particular interest because v-Crk has recently been reported to
activate the Rho-signaling pathway in PC12 cells,58
although C3G may not directly activate Rho.
CrkL has been implicated in hematopoietic cell signaling from the
receptors for Epo, IL-3, thrombopoietin, and stem cell factor, because
these factors induce the tyrosine phosphorylation of CrkL and its
binding with tyrosine-phosphorylated signaling molecules, including
Cbl.29-33 Intriguingly, these factors also activate the hematopoietic cell adhesion to fibronectin through VLA-4 and
VLA-5.6-9 It is tempting to speculate that CrkL may mediate
the signal from these receptors to activate integrins ("inside
out" signaling), possibly by recruiting C3G to the vicinity of its
effector molecule at the plasma membrane through the binding of CrkL
SH2 domain with tyrosine-phosphorylated signaling molecules, such as
Cbl. Consistent with this hypothesis, overexpression of CrkL enhanced the Epo- or IL-3-induced cell adhesion as shown in Fig 1D. In addition,
it should be noted that, except for the results shown in Fig 1D, all
the other cell adhesion assays in the present study were performed
under the condition in which cells had been cultured in Epo-containing
medium and subsequently allowed to adhere to fibronectin-coated wells
for 30 minutes in the presence of IL-3 as described in Materials and
Methods. Therefore, the effects of mutants of CrkL or other signaling
molecules examined in this study should represent their effects on
cytokine-stimulated adhesion of hematopoietic cells. As shown in Fig
1D, overexpression of CrkL also activated the adhesion of
cytokine-starved cells. However, this could be explained by the
observation that overexpression of CrkL per se induces tyrosine
phosphorylation of adhesion-associated proteins and their association
with CrkL44 (Y.N., A.A., O.M., unpublished
observation, July 1998). In addition to playing a possible role in the
"inside out" signaling, CrkL may also play a role in the
"outside in" signaling, because binding of ligands with integrins
or cross linking of integrins also induces tyrosine phosphorylation of
signaling molecules, including Hefl48,59 and
Cbl48 and their association with CrkL. Noteworthy in this regard is the observation that the CrkL-overexpressing cells not only
showed increased adhesion but also exhibited morphologic changes when
attached to culture plate (Fig 1A). Further studies are in progress in
our laboratory to examine the effects of CrkL overexpression on the
"outside in" signaling pathways as well as on the control of
growth, differentiation, apoptosis, motility, and morphologic changes
of various hematopoietic cell lines, including those derived from
chronic myelogenous leukemia.
| |
ACKNOWLEDGMENT |
We are grateful to Drs John Groffen, Erkki Rouslahti, and Michiyuki
Matsuda for the generous gifts of expression plasmids for CrkL,
R-Ras43N, and C3G, respectively. We also thank Dr Shuji Tohda for
assistance in photography as well as Eiko Nishimura, Kiyomi Kaneki,
Mihoko Suzuki, and Kaori Okada for excellent technical assistance.
| |
FOOTNOTES |
Submitted October 18, 1998; accepted January 25, 1999.
Supported in part by grants from the Ministry of Education, Science and
Culture of Japan.
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.
Address reprint requests to Osamu Miura, MD, First Department of
Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyoku, Tokyo 113, Japan.
| |
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A. Arai, E. Kanda, Y. Nosaka, N. Miyasaka, and O. Miura
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TOP
ABSTRACT
INTRODUCTION