From the Department of Hematology and Oncology and
the § Department of Bioregulatory Medicine and Rheumatology,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyoku,
Tokyo 113, Japan
Received for publication, May 29, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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The CrkL adaptor protein is involved in
signaling from the receptor for erythropoietin (Epo) as well as
interleukin (IL)-3 and activates The growth and differentiation of hematopoietic progenitor cells
are regulated by hematopoietic cytokines, including erythropoietin (Epo)1 and interleukin
(IL)-3, as well as through direct interaction with the bone marrow
microenvironment, composed of stromal cells and extracellular matrix
components, such as fibronectin. The hematopoietic cytokine receptors
mainly couple with Jak2, a member of the JAK family of tyrosine
kinases, to activate various signal transduction pathways, including
those involving the Ras/Erk activation cascade, Stats,
phosphatidylinositol 3'-kinase, and phospholipase C (PLC)- CrkL is a member of the Crk family of adaptor proteins originally
identified as homologues of the product of the v-crk
oncogene and is most abundantly expressed in hematopoietic cells (6). CrkL has the domain structure Src homology (SH)2-SH3-SH3 and has been
shown to bind through its N-terminal SH3 domain with various signaling
molecules including Sos1 and C3G, two guanine nucleotide exchange
factors (GEFs) for the Ras family of small GTP-binding proteins (7, 8).
We have demonstrated previously that CrkL is involved in hematopoietic
cytokine receptor signaling because CrkL becomes
tyrosine-phosphorylated in hematopoietic cells in response to
stimulation with Epo or IL-3 and forms complexes with several
tyrosine-phosphorylated signaling molecules, such as Cbl, Shc, and
SHP-2 (9). Furthermore, we showed that CrkL is involved through its
interaction with C3G in activation of the Ras/Erk signaling pathway
leading to the induction of c-fos gene expression in
response to Epo or IL-3 (10). Intriguingly, hematopoietic cells
overexpressing CrkL or C3G showed an enhanced adhesion through Rap1 is a small GTPase of the Ras family which was first identified as
a homologue of Ras (14) and was also identified independently by its
ability to induce a flat revertant phenotype in K-Ras-transformed cells
(15). Two highly homologous forms of Rap1, Rap1A and Rap1B, have been
identified (16), but their functional difference has been unclear.
Recent studies have shown that Rap1 is activated in fibroblasts after
stimulation with a variety of growth factors, including
platelet-derived growth factor, epidermal growth factor, and
lysophosphatidic acid (17, 18). Rap1 is also activated after
stimulation of the T cell or B cell antigen receptor in lymphocytes
(19, 20) and by stimulation with thrombin in platelets (21). Depending
on cell types, different types of intracellular second messengers,
including calcium, diacylglycerol, and cyclic AMP, have been implicated
in Rap1 activation (17, 18). Recently identified, two GEFs for Rap1,
Epac (22), which is directly activated by cyclic AMP, and CalDAG-GEFI
(23), which is sensitive to both calcium and diacylglycerol, have been
supposed to mediate Rap1 activation in response to these second
messengers. C3G, which forms a complex with CrkL, also efficiently
activates Rap1 in vitro as well as when expressed in COS
cells (24). Furthermore, C3G as well as CrkL has recently been
implicated in Rap1 activation induced by stimulation with nerve growth
factor (25), interferon- Previously, M'Rabet et al. (28) reported that GM-CSF, a
hematopoietic cytokine that shares a receptor chain with IL-3,
stimulates Rap1 in mature neutrophils. Rap1 is also activated in
neutrophils by other stimuli that are involved in neutrophil
activation, including fMet-Leu-Phe, platelet-activating factor, and
IgG-coated particles (28). However, the signaling mechanisms for
GM-CSF-induced Rap1 activation in neutrophils and its physiological
significance remain to be understood. Furthermore, although Rap1 has
been shown to be activated in highly differentiated and specialized
hematopoietic cells, such as mature neutrophils (28) and platelets
(21), it has remained to be determined whether Rap1 is involved in
cytokine receptor signaling in hematopoietic progenitor cells.
In the present study, we have examined the possible involvement of Rap1
in cytokine receptor signaling and reveal that Epo as well as IL-3
activates Rap1 in hematopoietic 32D cells, most likely through the
signaling pathways involving the CrkL-C3G complex and PLC- Cells and Reagents--
A clone of IL-3-dependent
32D cells expressing the wild-type murine Epo receptor (EpoR),
32D/EpoR-Wt, was described previously (29) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 1 unit/ml human
recombinant Epo. 32DE/TA cells, which inducibly express the
tetracycline transactivator (tTA) when withdrawn from tetracycline, and
32DE/Tet-CrkL or 32DE/Tet-C3G cells, which inducibly overexpress CrkL
or C3G, respectively, when withdrawn from tetracycline, were also
described previously (11). A human leukemic cell line expressing the
endogenous EpoR, UT-7 (30), was kindly provided by Dr. Norio Komatsu
(Jichi Medical School, Tochigi, Japan). COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.
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). Phorbol 12-myristate 13-acetate (PMA),
ionomycin, and forskolin were purchased from Sigma Chemicals (St.
Louis, MO). U73122 and U73343 were from Calbiochem (La Jolla, CA).
Antibodies against Rap1A, CrkL, C3G, and EpoR were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphotyrosine
(4G10), Erk1/2 (Erk1/2-CT), and PLC- Plasmids--
Expression plasmids for human CrkL, pSG-CrkL (31),
and dominant negative R-Ras, pcDNA-R-Ras-43N (32), were kindly
provided by Dr. John Groffen (Childrens Hospital Los Angeles, Los
Angeles, CA) and Dr. Erkki Ruoslahti (La Jolla Cancer Research Center, La Jolla, CA), respectively. An expression plasmid for C3G
(pcDNA-C3G) as well as those for its activated and dominant
negative mutants, pcDNA-C3G-dN and pcDNA-C3G-dSS, respectively,
was described previously (11). Expression plasmids for dominant
negative and constitutively activated mutants of Rap1/K-Rev1 (33)
tagged with the T7 epitope, pSR Transfection--
Transfection for stable expression was carried
out essentially as described previously (29). In brief, 32DE/TA cells
were transfected with 10 µg of pTet-Rap1A-63E along with 1 µg of
pMAM2-BSD, purchased from Funakoshi (Tokyo, Japan), by electroporation
at 960 microfarads and 300 V, followed by selection in medium
containing blasticidin-S (Funakoshi) and 500 ng/ml tetracycline. Six
clones transfected with pTet-Rap1A-63E were isolated by limiting
dilution and examined for the induction of Rap1A-63E expression by
anti-T7 immunoblotting of cell lysates prepared after withdrawal from tetracycline for 24 h. The clone inducibly expressing the highest level of Rap1A-63E, 32DE/TA-Rap1A-63E, was selected for the subsequent studies.
Similarly, a clone of 32D/EpoR-Wt, 32DE/CrkL, which constitutively
overexpresses CrkL, was obtained by transfection of pSG-CrkL.
Transfection of expression plasmids into COS-7 cells was carried out
using the LipofectAMINE reagent (Life Technologies, Inc.), as described
previously (35). 2 days after transfection, cells were harvested for analysis.
Immunoprecipitation and Rap1 Activation Assay--
For
immunoprecipitation experiments, cells were lysed in a lysis buffer
containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each aprotinin and leupeptin. Cell lysates were subjected to
immunoprecipitation as described previously (36).
Rap1 activation in cells was determined by using an activation-specific
probe, essentially as described by Zwartkruis et al. (37).
In brief, cells were lysed in a lysis buffer containing 1% Nonidet
P-40, 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 2.5 mM MgCl2, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each aprotinin and leupeptin. Cell lysates were incubated with the GST-RalGDS-RBD fusion
protein, prepared as described previously and precoupled to
glutathione-agarose beads. After incubation for 45 min at 4 °C,
beads were washed three times in lysis buffer. Rap1-GTP bound to beads
was eluted by heating at 100 °C for 5 min in 1 × Laemmli's sample buffer and detected by immunoblotting with anti-Rap1 antibody using the enhanced chemiluminescence Western blotting detection system
(Amersham Pharmacia Biotech, Buckinghamshire, U. K.). For immunoblot
analysis of total cell lysates, samples were prepared by mixing an
aliquot of cell lysates with an equal volume of 2 × Laemmli's
sample buffer and heating at 100 °C for 5 min. All Rap1 activation
assays were performed at least three times with reproducible results.
Quantification of blots was performed with NIH Image software.
Cell Adhesion Assays--
Adhesion assays of transiently
transfected cells were performed essentially as described previously
(11). In brief, 32D/EpoR-Wt and UT-7 cells were electroporated with the
indicated amounts of relevant plasmids and 1 µg of a control
luciferase plasmid, pGL-cont. The total amount of expression plasmids
for each transfection was adjusted to become equal by adding an empty
plasmid. After a recovery period of 1 day in medium containing 1 unit/ml Epo, cells were plated in triplicate on wells coated with 5 µg/ml fibronectin and incubated at 37 °C for 30 min. After wells
were washed three times to remove unbound cells, cells remaining
attached to the wells were measured by the luciferase assay. After
subtraction of background cell binding to bovine serum albumin-coated
wells, the percentage of adherent cells was determined by dividing the luciferase activity of the adherent cells by that of the initial cell
input. For adhesion assays of stably transfected clones, cells were
labeled with 5 µM
2'7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein acetoxymethyl
ester (Dojindo, Kumamoto, Japan) at 37 °C for 1 h. After being
washed three times, fluorescently labeled cells were subjected to cell
adhesion assay as described above. Cells were measured by Cytofluor II
fluorescent plate reader (PerSeptive Biosystems, Foster City, CA).
Alternatively, unlabeled cells were subjected to cell adhesion assays
and measured by using the sodium XTT colorimetric assay kit (Boehringer
Mannheim, Indianapolis, IN), as described previously (11). All cell
adhesion assays under "Results" were carried out using
fibronectin-coated plates unless described otherwise and repeated at
least three times, and the results were reproducible.
IL-3 or Epo Stimulation Induces Rap1 Activation in Hematopoietic
Cells--
Previously, we have shown that CrkL is involved in
hematopoietic cytokine receptor signaling and plays a role in
activation of integrin-mediated hematopoietic cell adhesion through its
interaction with C3G (9, 11), a GEF that most efficiently activates
Rap1 (24). To address the possibility that Rap1 may be involved in the
CrkL-mediated cytokine receptor signaling pathway regulating hematopoietic cell adhesion, we first examined whether IL-3 or Epo
induces Rap1 activation in hematopoietic cells. As shown in Fig.
1A, the affinity purification
assay using the GST-RalGDS-RBD, as described under "Experimental
Procedures," demonstrated that IL-3 as well as Epo stimulation
induces activation of Rap1 in 32D/EpoR-Wt cells, a clone of murine
IL-3-dependent 32D cells expressing the EpoR (29). The Rap1
activation induced by IL-3 or Epo was observed as early as 1 min after
stimulation, increased to plateau at 5-20 min, and was sustained as
long as 60 min. As shown in Fig. 1B, Rap1 was significantly
activated when cells were stimulated with Epo or IL-3 at a
concentration as low as 1 unit/ml or 1 ng/ml, respectively. When
stimulated with a higher concentration of Epo (10 units/ml) or IL-3 (10 ng/ml), a further increase in Rap1 activity was observed. Anti-Rap1
blotting of total cell lysates showed that the total amount of Rap1 in
the cells did not change significantly after stimulation with IL-3 or
Epo (Fig. 1, lower panels). These results thus demonstrate that IL-3 and Epo induce time- and dose-dependent
activation of Rap1 in hematopoietic cells.
Involvement of CrkL and C3G in IL-3- or Epo-induced Activation of
Rap1--
To examine whether CrkL is involved in cytokine-induced
activation of Rap1 in hematopoietic cells, we next examined the
cytokine-induced activation of Rap1 in a clone of 32D/EpoR-Wt cells,
32DE/Tet-CrkL, which inducibly overexpresses CrkL when withdrawn from
tetracycline (11). As shown in Fig.
2A and B, the Rap1
activation induced by IL-3 as well as the basal level of Rap1 activity
in 32DE/Tet-CrkL was augmented when cells were depleted overnight from
tetracycline. Anti-Rap1 and anti-CrkL blotting of total cell lysates
confirmed that the depletion of tetracycline increased the expression
level of CrkL but not that of Rap1 in 32DE/Tet-CrkL cells. Similarly, Epo-induced activation of Rap1 was augmented when 32DE/Tet-CrkL cells
were induced to overexpress CrkL (data not shown). These results
strongly suggest that CrkL is involved, most likely through its
interaction with C3G, in IL-3- and Epo-induced activation of Rap1.
To confirm that C3G is also involved in cytokine-induced Rap1
activation, we examined 32DE/Tet-C3G cells, which overexpress C3G when
withdrawn from tetracycline (11). As shown in Fig. 2C, the
Rap1 activation induced by Epo or IL-3 was augmented significantly when
C3G was overexpressed. Anti-phospho-Erk blotting of total cell lysates
demonstrated that the Epo- or IL-3-induced activation of Erk is also
enhanced when C3G was overexpressed, which is in agreement with our
previous observation that C3G is also involved in cytokine-induced
activation of the Ras/Erk signaling pathway. Together, these results
strongly suggest that the CrkL-C3G complex is involved in Epo- and
IL-3-induced activation of Rap1.
Rap1 Is Involved in CrkL-induced Activation of Integrin-mediated
Adhesion of Hematopoietic Cells--
To explore the possibility that
CrkL-mediated activation of hematopoietic cell adhesion is mediated by
Rap1, we first employed Rap1A-17N, a putative dominant negative mutant
of Rap1 (25, 38). However, because the dominant negative effect of
Rap1A-17N has been disputed by recent in vitro studies (39),
we first examined whether Rap1A-17N inhibits Rap1 activation mediated
by C3G in COS cells. As shown in Fig.
3A, when transiently
overexpressed in COS cells, the activated form of wild-type Rap1, but
not that of Rap1A-17N, was detected by affinity purification assays
with GST-RalGDS-RBD. In accordance with a previously report (24), when
C3G was coexpressed, the activated form of wild-type Rap1 was increased
drastically. However, the C3G-induced activation of wild-type Rap1 was
abrogated by coexpression of Rap1A-17N (Fig. 3A, upper
panel), which indicates that Rap1A-17N inhibits the activation of
Rap1 by C3G in a dominant negative manner. Anti-T7 blotting of total
cell lysates demonstrated that the expression level of wild-type Rap1
is significantly higher than that of Rap1A-17N in repeated experiments
(Fig. 3A, middle panel, and data not shown). In
addition, when coexpressed with and activated by C3G, the expression level of Rap1 was found to be significantly increased in repeated experiments (Fig. 3A, middle panel, and data not
shown). It is thus possible that the activated form of Rap1 may be more
stable than the inactive form, although further studies are required to
address this possibility. Irrespective of this, these results have
demonstrated unequivocally that Rap1A-17N inhibits C3G-mediated Rap1
activation in cells.
Having established the dominant negative effect of Rap1A-17N in cells,
we examined the effect of this mutant on CrkL-enhanced cell adhesion to
fibronectin. As shown in Fig. 3B, transient overexpression of CrkL enhanced 32D/EpoR-Wt cell adhesion. In accordance with our
previous report (11), coexpression of a dominant negative R-Ras mutant,
R-Ras-43N, partially inhibited the CrkL-enhanced adhesion. Similar to
R-Ras-43N, Rap1A-17N also exhibited an inhibitory effect on the
CrkL-enhanced adhesion. Furthermore, when coexpressed with R-Ras-43N,
Rap1A-17N showed an additive inhibitory effect and nearly abrogated the
CrkL-enhanced cell adhesion (Fig. 3B). These results thus
suggest that Rap1 as well as R-Ras may be involved in CrkL-mediated
activation of hematopoietic cell adhesion.
To explore further the possible involvement of Rap1 in integrin
activation of hematopoietic cells, we next employed a constitutively activated mutant of Rap1, Rap1A-63E. As shown in Fig. 3C,
transient expression of Rap1A-63E in 32D/EpoR-Wt cells induced a
dose-dependent activation of cell adhesion to fibronectin.
We also established a 32D/EpoR-Wt clone, 32DE/TA-Rap1A-63E, which
inducibly expresses Rap1A-63E when withdrawn from tetracycline, and
examined the effect of Rap1A-63E on cell adhesion. As shown in Fig.
3D, when 32DE/TA-Rap1A-63E cells were cultured overnight in
medium containing various concentrations of tetracycline, the
expression of Rap1-63E was induced significantly at tetracycline
concentrations equal to or lower than 10 ng/ml. At these
concentrations, 32DE/TA-Rap1A-63E cells exhibited significantly increased adhesion to wells coated with 5 µg/ml fibronectin (Fig. 3D). Fig. 3E further demonstrates that
32D/TA-Rap1A-63E cells exhibited significantly enhanced adhesion to
wells coated with various concentrations of fibronectin when the
expression of Rap1A-63E was induced. As shown in Fig. 3F,
the Rap1A-63E-induced as well as basal adhesion of 32DE/TA-Rap1A-63E
cells was abrogated by incubation with anti- Rap1 Activation in Hematopoietic Cells Is Also Mediated by the
Second Messengers of PLC-
Next, the effects of these chemical reagents on cell adhesion were
examined. Fig. 4E demonstrates that PMA treatment
dramatically increases the adhesion of 32D/EpoR-Wt cells to
fibronectin. Treatment with ionomycin also increased, though modestly,
the cell adhesion in repeated experiments (Fig. 4E and data
not shown). In contrast, U73122 drastically inhibited the adhesion of
32DE/Tet-CrkL cells in the presence of tetracycline (Fig.
4F). U73122, however, showed only a modest inhibitory effect
on the 32DE/Tet-CrkL cell adhesion which was enhanced remarkably by
overexpression of CrkL in the absence of tetracycline (Fig.
4F). The inhibitory effects of U73122 on activation of Rap1
and cell adhesion were examined further in 32D/EpoR-Wt as well as in
32DE/CrkL, a clone of 32D/EpoR-Wt which constitutively overexpresses
CrkL. As shown in Fig. 4G, the Epo-induced Rap1 activation
as well as cell adhesion was inhibited significantly by U73122 at a
concentration as low as 0.01 µM in 32D/EpoR-Wt cells and
was decreased at 1 µM to about one-fifth of that observed
in the absence of U73122. On the other hand, the Epo-induced Rap1
activation as well as cell adhesion of 32DE/CrkL cells was inhibited
significantly by U73122 only at a concentration as high as 1 µM (Fig. 4H). Therefore, the effects of these
pharmacological reagents on the cell adhesion correlated with their
effects on Rap1 activation in 32D/EpoR-Wt clones, which supports the
idea that Rap1 plays an important role in activation of hematopoietic cell adhesion. In addition, it was suggested that CrkL mediates the
Epo-induced Rap1 activation independently of PLC-
We demonstrated previously that 32DE/Tet-CrkL cells overexpressing
CrkL, but not 32DE/TA cells, significantly adhere to fibronectin-coated wells even when starved from cytokines (11). In the absence of any
cytokines, 32DE/TA-Rap1A-63E cells inducibly expressing Rap1A-63E also
showed a significant adhesion (Fig. 4I). Importantly, U73122
did not show any significant inhibitory effect on the adhesion of
32DE/TA-Rap1A-63E cells in the absence of cytokines, although the
Epo-augmented adhesion of Rap1E-expressing cells was partly inhibited
by the PLC inhibitor (Fig. 4I). These results thus agree
with our hypothesis that Rap1 is activated downstream of PLC- Involvement of CrkL and Rap1 in Signaling from the Endogenous
EpoR--
Because the 32D/EpoR-Wt cells heterologously express the
EpoR, we next examined the human leukemic UT-7 cell line to confirm the
involvement of CrkL and Rap1 in signaling from the endogenously expressed EpoR. As shown in Fig.
5,A and B, Epo
induced the time- and dose-dependent tyrosine
phosphorylation of CrkL in UT-7 cells. The UT-7 cell adhesion to
fibronectin-coated wells in the presence of Epo was enhanced
significantly by transient overexpression of CrkL or an activated
mutant of C3G, C3G-dN, and was inhibited by that of a dominant negative
C3G mutant, C3G-dSS (Fig. 5C). U73122 also inhibited the
UT-7 cell adhesion in the presence of Epo (Fig. 5D). These
results indicate that CrkL is involved in signaling from the endogenous
EpoR and, along with C3G, plays a role in activation of adhesion of
UT-7 cells. In addition, the inhibitory effect of U73122 indicates that
PLC-
It was also confirmed that Epo activates Rap1 in UT-7 cells (Fig.
5,E and F). The Epo-induced activation of Rap1,
conspicuously observed when cells were stimulated with as low as 0.1 unit/ml Epo, peaked at 5-15 min after Epo stimulation and gradually
declined thereafter. We also examined the effect of Epo on the adhesion of UT-7 cells starved overnight from Epo. As shown in Fig.
5G, Epo modestly increased the adhesion of cytokine-starved
UT-7 cells even when added immediately before cells were allowed to
adhere to fibronectin-coated wells for 30 min. The effect of Epo on
cell adhesion peaked at the preincubation period of 30 min and declined slightly at 60 min. We also examined the dose effect of Epo on UT-7
cell adhesion. As shown in Fig. 5H, the cell adhesion was stimulated significantly by Epo at a concentration as low as 0.1 unit/ml. Because Rap1 as well as cell adhesion was activated
significantly by Epo at a concentration as low as 0.1 unit/ml and
within 30 min of stimulation, these observations in UT-7 cells are in
accordance with our hypothesis that Epo stimulates cell adhesion
through activation of Rap1.
The present studies have demonstrated that IL-3 and Epo induce
Rap1 activation in a hematopoietic cell line, 32D/EpoR-Wt. We have
found previously that CrkL forms a stable complex with C3G in
hematopoietic cells and is recruited to the activated cytokine receptor
complex through interaction between the CrkL SH2 domain and
tyrosine-phosphorylated signaling molecules, including Cbl, Shc, and
SHP-2 (9). Our previous studies have demonstrated further that the
CrkL-C3G complex is involved in cytokine-induced activation of Ras
(10). Because C3G activates Rap1 more efficiently than Ras, it is
plausible to hypothesize that the recruitment of the CrkL-C3G complex
to the cytoplasmic membrane is involved in cytokine-induced activation
of Rap1 as well as that of Ras. In accordance with this hypothesis, the
cytokine-stimulated Rap1 activation was enhanced by the induced
overexpression of CrkL or C3G in 32DE/Tet-CrkL or 32DE/Tet-C3G cells,
respectively (Fig. 2). Recently, the CrkL-C3G complex has also been
implicated in Rap1 activation in cells stimulated with other growth
factors and cytokines as described in the Introduction.
Rap1 was also activated in 32D/EpoR-Wt cells treated with PMA or a
calcium ionophore, ionomycin, but the cytokine-induced Rap1 activation
was inhibited by a PLC inhibitor, U73122. It is thus hypothesized that
PLC- Tsukamoto et al. (46) reported previously that, when
cultured in G-CSF, the Rap1 activity increased gradually over several days in a subclone of 32D cells which undergoes granulocytic
differentiation with concomitant increases in expression of various
adhesion molecules, such as VLA-4, LFA-1, and CD44, and in cell
adhesiveness under the same condition. Tsukamoto et al. (46)
further demonstrated that overexpression of SPA-1, a Rap1-specific
GTPase-activating protein, drastically inhibited the activation of Rap1
and adhesion of 32D cells without affecting the G-CSF-induced
morphological differentiation and augmentation in expression of
adhesion molecules. It was thus speculated that Rap1-GTP may be
required for the adhesion of 32D cells cultured in G-CSF. However, the
adhesion molecules involved in adhesion of differentiated 32D cells
induced by G-CSF were not examined. Furthermore, because the
G-CSF-induced increase in Rap1 took place very gradually over a very
long period of time (46), it has remained to be known whether Rap1 is
involved directly in signaling mediated through the G-CSF receptor or
in inside-out signaling mediating G-CSF-induced integrin activation.
Several observations in the present studies, on the other hand,
indicate that Rap1 plays a critical role in cytokine-induced activation
of hematopoietic cell adhesion mediated through While this manuscript was in preparation, Katagiri et al.
(49) reported that an activated Rap1 mutant, Rap1-12V, stimulates adhesion of a murine pro-B-cell line, BA/F3, through heterologously expressed human LFA-1, a 1 integrin-mediated
hematopoietic cell adhesion through its interaction with C3G, a guanine
nucleotide exchange factor for Rap1. We demonstrate here that Epo as
well as IL-3 activates Rap1 in an IL-3-dependent
hematopoietic cell line, 32D, expressing the Epo receptor. The
cytokine-induced activation of Rap1 was augmented in cells that
inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of
cell adhesion was inhibited by expression of a dominant negative mutant
of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E,
activated
1 integrin-dependent adhesion of
hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol
12-myristate 13-acetate and ionomycin, which also enhanced cell
adhesion to fibronectin, whereas U73122, an inhibitor of phospholipase
C, inhibited both cytokine-induced activation of Rap1 and cell
adhesion. It was also demonstrated that Rap1 as well as CrkL is
involved in signaling from the EpoR endogenously expressed in a human
leukemic cell line, UT-7. These results suggest that Epo and IL-3
activate Rap1 at least partly through the CrkL-C3G complex as well as
through additional pathways most likely involving phospholipase C
and strongly implicate Rap1 in regulation of
1
integrin-mediated hematopoietic cell adhesion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1). Most of the hematopoietic cytokines, such as stem cell factor,
IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF),
granulocyte colony-stimulating factor (G-CSF), thrombopoietin, and Epo,
also activate adhesion of hematopoietic cells to extracellular matrix
components through integrins of the
1 subfamily, mainly
VLA-4 (
4
1) and VLA-5
(
5
1) (2-5). However, the mechanisms by
which receptors for these cytokines transduce signals to activate
integrins (inside-out signaling) have remained elusive.
1 integrins VLA-4 and VLA-5 in response to cytokines,
whereas cells expressing a dominant negative mutant of CrkL or C3G
showed an impairment in adhesion (11). The CrkL-mediated enhancement of
hematopoietic cell adhesion, also reported by others (12, 13), was
observed without changes in expression levels of these integrins, thus
suggesting that it is through the increase in ligand binding activity
of integrins (11). These observations, thus, indicate that CrkL as well
as C3G is involved in cytokine signaling leading to the activation of
hematopoietic cell adhesion through
1 integrins.
However, the CrkL-mediated activation of hematopoietic cell adhesion
was independent of Ras activation (11). Thus, it has remained unknown
how the CrkL-C3G complex activates the downstream signaling pathways
leading to integrin activation.
(26), and hepatocyte growth factor (27) or
by stimulation of the T cell antigen receptor (19). With an effector
domain that is nearly identical to Ras and binds to many of the same effectors, Rap1 has been hypothesized to have antagonistic effects on
the downstream signaling events of Ras. However, roles for Rap1 in
physiological signaling pathways largely remain elusive (17, 18).
.
Furthermore, the present study indicates that Rap1 is involved in the
regulation of hematopoietic cell adhesion through
1 integrins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (05-163) were purchased from
Upstate Biotechnology (Lake Placid, NY). Anti-phospho-Erk
(Thr-202/Tyr-204) and anti-phospho-Stat5 (Tyr-694) were purchased from
New England Biolabs (Beverly, MA). A monoclonal antibody against the T7
epitope was purchased from Novagen (Madison, WI). Monoclonal antibodies
against murine
1 integrin (HA2/5, hamster IgM,
) and
murine
5 integrin (5H10-27, Rat IgG2a,
) were
purchased from Pharmingen (San Diego, CA). A monoclonal antibody
against murine
4 integrin (428, Rat IgG2a,
) and
control murine monoclonal antibodies (IgM,
, and IgG2a,
) were
purchased from Seikagaku Corp. (Tokyo, Japan) and Sigma Chemicals, respectively.
-T7-Rap1A-17N and
pSR
-T7-Rap1A-63E, respectively, were kindly provided by Drs. Makoto
Noda and Masakazu Hattori (Kyoto University, Kyoto, Japan). For
construction of pTet-Rap1A-63E, a tetracycline-responsive expression
plasmid for Rap1A-63E, the BamHI/EcoRV fragment
of pSR
-T7-Rap1A-63E coding for T7-tagged Rap1A-63E, was excised and
subcloned between the BamHI and HindIII sites in
pcDNA3 (Invitrogen, San Diego, CA) to give pcDNA-Rap1A-63E. The
region coding for T7-tagged Rap1A-63E was then excised from
pcDNA3-Rap1A-63E by digestion with both HindIII and
XbaI and subcloned between the HindIII and
SpeI sites of pTet-Splice to give pTet-Rap1A-63E. The
pGEX-RalGDS-RBD plasmid encoding a glutathione S-transferase (GST) fusion protein containing the 97-amino acid Rap1 binding domain
(RBD) of the RalGDS protein (34) was kindly provided by Dr. Johannes L. Bos (Utrecht University, Utrecht, The Netherlands). A control
luciferase plasmid, pGL-cont, was purchased from Promega (Madison, WI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Epo as well as IL-3 activates Rap1 in
32D/EpoR-Wt cells. 32D/EpoR-Wt cells, a clone of the
IL-3-dependent 32D cell line expressing the EpoR, were
starved from Epo overnight in serum-free medium and stimulated with 10 units/ml Epo or 1 ng/ml IL-3 for various times as indicated in
A or stimulated with the indicated concentrations of Epo
(units/ml) or IL-3 (ng/ml) for 15 min in B. Activated Rap1
(Rap1-GTP) was affinity purified from cell lysates using a
GST-RalGDS-RBD fusion protein, as described under "Experimental
Procedures." Eluates from the precipitates (upper panels)
or total cell lysates (lower panels) were subsequently
subjected to immunoblotting with anti-Rap1 antibody. Relative Rap1
activities, determined by densitometric analysis, are shown between the
two blots.
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Fig. 2.
Cytokine-induced activation of Rap1 is
augmented in cells inducibly overexpressing CrkL or C3G.
A and B, a clone of 32D/EpoR-Wt cells,
32DE/Tet-CrkL, which overexpresses CrkL when withdrawn from
tetracycline, was starved from Epo overnight in serum-free medium
containing 100 ng/ml tetracycline (Tet+) or without
tetracycline (Tet ), as indicated. Cells were then
stimulated with 1 ng/ml IL-3 for the indicated times in A or
with the indicated concentrations (ng/ml) of IL-3 for 15 min in
B and subjected to the Rap1 activation assays. Eluates from
the GST-RalGDS-RBD precipitates (top panels) or total cell
lysates (middle and bottom panels)
were subsequently subjected to immunoblotting with anti-Rap1 or
anti-CrkL antibody, as indicated. Relative Rap1 activities are shown
between the two anti-Rap1 blots. C, 32DE/Tet-C3G cells,
which overexpress C3G when withdrawn from tetracycline, were starved
from Epo overnight in serum-free medium containing 100 ng/ml
tetracycline (Tet+) or without tetracycline
(Tet
), as indicated. Cells were then stimulated with 10 units/ml Epo (E) or 1 ng/ml IL-3 (I), as
indicated, for 15 min and subjected to the Rap1 activation assays.
Eluates from the GST-RalGDS-RBD precipitates (top panel) or
total cell lysates (middle and bottom panels)
were subsequently subjected to immunoblotting with anti-Rap1,
anti-phospho-Erk, or anti-C3G antibody, as indicated.
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Fig. 3.
Involvement of Rap1 in CrkL-enhanced
hematopoietic cell adhesion through 1
integrins to fibronectin. A, COS-7 cells were
transfected with 0.5 µg of pcDNA-C3G (C3G),
pSR
-T7-Rap1A-17N (Rap1-17N), or pSR
-T7-Rap1A-Wt
(Rap1-Wt), as indicated. The total amount of expression
plasmids for each transfection was adjusted to become equal by the
addition of pcDNA3. 2 days after transfection, cells were lysed and
subjected to an affinity purification assay with GST-RalGDS-RBD.
Eluates (top panel) and total cell lysates
(middle and lower panels) were then subjected to
anti-T7 or anti-C3G immunoblotting, as indicated. B,
32D/EpoR-Wt cells were transfected with 10 µg of pSG-CrkL
(CrkL), 20 µg of pSR
-T7-Rap1A-17N
(Rap1-17N), or 20 µg of pcDNA-R-Ras-43N
(R-Ras-43N), as indicated, along with 1 µg of pGL-cont.
Transiently transfected cells were subjected to the cell adhesion assay
measured by the luciferase assay as described under "Experimental
Procedures." C, 32D/EpoR-Wt cells were transfected with
the indicated amount of pSR
-T7-Rap1A-63E along with 1 µg of
pGL-cont. Transiently transfected cells were subjected to the cell
adhesion assay as measured by the luciferase assay. D,
32DE/TA-Rap1A-63E cells were cultured overnight with the indicated
concentrations of tetracycline, fluorescently labeled, and subjected to
the cell adhesion assays as measured by fluorometry as described under
"Experimental Procedures." Anti-T7 immunoblotting of the
GST-RalGDS-RBD precipitates obtained from lysates of cells cultured
under the same conditions is also shown. E,
32DE/TA-Rap1A-63E cells, cultured overnight with or without
tetracycline as indicated, were allowed to attach to wells coated with
the indicated concentrations of fibronectin for the cell adhesion
assays as measured by the XTT colorimetric method. Anti-T7
immunoblotting of the GST-RalGDS-RBD precipitates obtained from lysates
of cells cultured under the same conditions is also shown.
F, 32DE/TA-Rap1A-63E cells, cultured overnight with or
without tetracycline (Tet.) as indicated, were fluorescently
labeled, preincubated for 15 min at room temperature with
anti-
1 integrin monoclonal antibody
(Anti-
1) or mouse IgM
(IgM) as a
control as indicated, and subjected to the cell adhesion assays as
measured by fluorometry. G, 32DE/TA-Rap1A-63E cells,
cultured overnight without tetracycline, were fluorescently labeled,
preincubated for 15 min at room temperature with anti-
4,
anti-
5, or anti-
1 integrin monoclonal
antibodies, as indicated, or with mouse IgG2a
(IgG) or
IgM
(IgM) as a control as indicated, and subjected to the
cell adhesion assays as measured by fluorometry.
1 integrin
monoclonal antibody, which indicates that Rap1 activates hematopoietic
cell adhesion to fibronectin through
1 integrins. Fig.
3G further demonstrates that the Rap1A-63E-enhanced cell
adhesion was inhibited by anti-
4 and
anti-
5 integrin antibodies in an additive way, thus
suggesting that both VLA-4 (
4
1) and VLA-5
(
5
1) are involved in Rap1-induced cell
adhesion to fibronectin. Taken together with our previous observation
that CrkL-enhanced cell adhesion is mediated by the
1
integrins VLA-4 and VLA-5 (11), these data strongly support our
hypothesis that Rap1 plays a role in CrkL-mediated activation of
hematopoietic cell adhesion.
--
Recent studies have shown that
calcium, diacylglycerol, and cAMP mediate activation of Rap1 by various
stimuli in cell type-specific ways (17, 18). Previous studies have
shown that the cytokine receptors, including the receptors for Epo (40)
and IL-3 (41), activate PLC-
, which transduces signals through
generation of diacylglycerol as well as through an increase in
intracellular calcium. In accordance with this, Epo as well as IL-3
induced tyrosine phosphorylation of PLC-
in 32D/EpoR-Wt cells (Fig.
4A). Epo stimulation also
induced the physical association of PLC-
with the
tyrosine-phosphorylated EpoR (Fig. 4A), which agrees with a
previous report on UT-7 cells (40). Because PLC-
is activated by
tyrosine phosphorylation (42), we next examined the possible
involvement of the second messengers of PLC-
as well as cAMP in
cytokine-induced Rap1 activation in hematopoietic cells. As shown in
Fig. 4B, treatment of 32D/EpoR-Wt cells with a
diacylglycerol analogue, PMA, induced activation of Rap1, which was
detectable as early as 1 min after PMA addition and peaked at 5 min.
Treatment with a calcium ionophore, ionomycin, similarly induced
activation of Rap1, which peaked at 5 min after treatment. On the other
hand, forskolin, which activates adenylyl cyclase to generate cAMP,
showed no stimulatory activity toward Rap1 in 32D/EpoR-Wt cells.
Treatment of cells with a vehicle, dimethyl sulfoxide
(Me2SO), alone showed no effect on Rap1 activation (data not shown). Anti-Rap1 blotting of total cell lysates demonstrated that
treatment of cells with these reagents had no effect on the expression
level of Rap1 (Fig. 4B, lower panel). It was,
however, revealed that treatment of cells with forskolin for 20 min
induced the appearance of a slower migrating form of Rap1 (Fig.
4B, lower panel), which is in agreement with a
previous observation that protein kinase A, activated by cAMP,
phosphorylates Rap1 (43). These results suggest that diacylglycerol and
calcium, but not cAMP, may be involved in Rap1 activation in 32D cells.
Therefore, we next examined the effect of U73122, an inhibitor of PLC, on cytokine-induced activation of Rap1. As shown in Fig. 4C,
U73122 drastically decreased Epo- as well as IL-3-induced activation of
Rap1. On the other hand, U73343, an inactive analogue of U73122, showed
no significant effect on cytokine-induced activation of Rap1 (data not
shown). The specificity of the inhibitory effect of U73122 was
supported further by the observation that U73122 showed no significant
inhibitory effects on Epo-induced activation of Stat5 and Erk (Fig.
4D), which mediate the two major signaling pathways from the
EpoR (1). Furthermore, because CrkL plays an important role in
Epo-induced activation of the Ras/Erk pathway in 32D/EpoR-Wt cells
(10), the absence of inhibitory effect on Erk suggests that U73122
should not affect the CrkL-mediated signaling pathways. In accordance
with this, U73122 showed no effects on Epo-induced tyrosine
phosphorylation of CrkL and its association with various signaling
molecules in 32D/EpoR-Wt cells (data not shown). Together, these
results suggest that PLC-
may play a crucial role, independently of
CrkL, in cytokine-induced activation of Rap1 in hematopoietic
cells.
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Fig. 4.
Effects of various pharmacological reagents
on Rap1 activation and cell adhesion. A, after being
starved overnight from Epo, 32D/EpoR-Wt cells were stimulated with 10 units/ml Epo (E) or 10 ng/ml IL-3 (I), as
indicated, for 5 min. Cells were lysed and subjected to
immunoprecipitation with anti-PLC- . Immunoprecipitates were resolved
by SDS-PAGE and subjected to anti-phosphotyrosine
(
PY) blotting followed by reprobing with
anti-EpoR (
EpoR) and anti-PLC-
(
PLC
), as indicated.
Tyrosine-phosphorylated PLC-
(PLC
-PY) and
EpoR (EpoR-PY) are indicated. B, 32D/EpoR-Wt
cells were starved from Epo overnight in serum-free medium and
incubated with 100 ng/ml PMA, 1 µM ionomycin, or 50 µM forskolin, for various times, as indicated, and
subjected to the Rap1 activation assays. Rap1-GTP eluted from
GST-RalGDS-RBD (upper panel) and total cell lysates
(lower panel) was subjected to immunoblotting with anti-Rap1
antibody. C, after starvation, 32D/EpoR-Wt cells were
pretreated with 1 µM U73122 or 0.01% Me2SO
for 30 min at 37 °C, as indicated. Cells were stimulated with 10 units/ml Epo or 10 ng/ml IL-3 for the indicated times and subjected to
the Rap1 activation assays. D, 32D/EpoR-Wt cells were
starved overnight from Epo, pretreated for 30 min at 37 °C with the
indicated concentrations of U73122, and stimulated with Epo for 15 min.
Cell lysates were subjected to immunoblotting with anti-phospho-Stat5
(
Stat5-PY) followed by reprobing with
anti-phospho-Erk (
Erk-P) and anti-Erk1/2
(
Erk), as indicated. E, 32D/EpoR-Wt
cells, transiently transfected with pGL-cont., were subjected to the
cell adhesion assays in the presence or absence of 100 ng/ml PMA or 1 µM ionomycin, as indicated. F, 32DE/Tet-CrkL
cells were cultured overnight in Epo-containing medium with or without
100 ng/ml tetracycline, as indicated. Cells were fluorescently labeled,
pretreated for 30 min at 37 °C with 0.1% Me2SO or with
1 µM U73122, as indicated, and subjected to the cell
adhesion assays measured by fluorometry as described under
"Experimental Procedures." G and H,
32D/EpoR-Wt (G) or 32DE/CrkL (H) cells were
fluorescently labeled, pretreated for 30 min at 37 °C with the
indicated concentrations of U73122, and subjected to the cell adhesion
assays as measured by fluorometry. The values of cell adhesion in the
presence of U73122 are expressed relative to that in the absence of
U73122. For measurement of Rap1 activities, 32D/EpoR-Wt (G)
or 32DE/CrkL (H) cells were starved overnight from Epo,
pretreated for 30 min at 37 °C with the indicated concentrations of
U73122, stimulated with Epo for 15 min, and subjected to the Rap1
activation assays. Eluates from the GST-RalGDS-RBD precipitates
(upper panels) or total cell lysates (lower
panels) were subsequently subjected to immunoblotting with
anti-Rap1 antibody. Relative Rap1 activities, determined by
densitometric analysis, are shown between the two blots. I,
32DE/TA (TA) or 32DE/TA-Rap1A-63E (Rap1A-63E)
cells were removed from tetracycline overnight in culture medium with
or without 1 unit/ml Epo, as indicated. Cells were fluorescently
labeled, pretreated for 30 min at 37 °C with 0.1% Me2SO
or 1 µM U73122, as indicated, and subjected to the cell
adhesion assays as measured by fluorometry.
because the
inhibitory effect of U73122 on Epo-induced Rap1 activation in cells
overexpressing CrkL was much less significant than that in parental cells.
as
well as by the CrkL/C3G signaling pathway and activates integrin-mediated hematopoietic cell adhesion.
, activated by Epo in UT-7 cells (40), may also play a role in
Epo-induced activation of UT-7 cell adhesion.
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Fig. 5.
Involvement of Rap1 and CrkL in signaling
from the endogenous EpoR. A and B, UT-7 cells
were starved from Epo overnight and stimulated with 10 units/ml Epo for
the indicated times (A) or with the indicated concentrations
of Epo for 15 min (B). Cell lysates were immunoprecipitated
with anti-CrkL and subjected to anti-phosphotyrosine
( PY) immunoblotting followed by reprobing with
anti-CrkL (
CrkL), as indicated. C,
UT-7 cells were transfected with 50 µg of pSG-CrkL (CrkL),
pcDNA-C3G-dN (C3G-dN), or pcDNA-C3G-dSS
(C3G-dSS), as indicated, along with 1 µg of pGL-cont.
Transiently transfected cells were subjected to the cell adhesion
assays measured by the luciferase assay as described under
"Experimental Procedures." D, UT-7 cells cultured in
Epo-containing medium were fluorescently labeled, pretreated for 30 min
at 37 °C with 0.1% Me2SO or with 1 µM
U73122, as indicated, and subjected to the cell adhesion assays as
measured by fluorometry. E and F, UT-7 cells,
starved overnight from Epo in serum-free medium, were stimulated with
10 units/ml Epo for indicated times (E) or with the
indicated concentrations of Epo for 15 min (F), and
subjected to the Rap1 activation assays. Eluates from the
GST-RalGDS-RBD precipitates (upper panels) or total cell
lysates (lower panels) were probed with anti-Rap1. Relative
Rap1 activities are shown between the two anti-Rap1 blots. G
and H, UT-7 cells were starved overnight from Epo and
fluorescently labeled. Cells were left unstimulated as a control
(C) or stimulated with 10 units/ml Epo for indicated times
(G) or with the indicated concentrations of Epo for 30 min
(H) before being allowed to adhere to fibronectin-coated
wells for 30 min for the cell adhesion assays as measured by
fluorometry.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which Epo as well as IL-3 activates in hematopoietic cells
(40, 41), also mediates Rap1 activation through its second messengers,
diacylglycerol and calcium. The strong inhibitory effects of U73122 on
cytokine-induced activation of Rap1 and cell adhesion further imply
that the PLC-mediated pathway may play a more important role than that
involving the CrkL-C3G complex in the Epo- or IL-3-induced activation
of Rap1 and cell adhesion. It should be noted, however, that dominant negative mutants of CrkL and C3G significantly inhibited
cytokine-induced cell adhesion in our previous report (11). In
addition, because U73122 exhibited less profound inhibitory effects on
activation of Rap1 and cell adhesion in cells overexpressing CrkL, it
is speculated that CrkL mediates the activation of Rap1 and cell adhesion independently of PLC-
. Recent studies have also shown that
PLC plays a critical role in Rap1 activation induced by the B cell
receptor stimulation in B lymphocytes and that induced by thrombin in
platelets (20, 21). CalDAG-GEF-I (23), a Rap1 GEF that is highly
expressed in hematopoietic cells and is activated by diacylglycerol or
calcium, has been implicated in PLC-mediated activation of Rap1. A
recent study in platelets (44), however, has demonstrated that protein
kinase C, which is activated by diacylglycerol, may also be involved in
a delayed and sustained second phase of Rap1 activation, which is
observed following a very rapid and transient activation mediated by
calcium in platelets stimulated with thrombin. Because the
cytokine-induced Rap1 activation in hematopoietic cells we observed in
this study is also sustained, it is possible that protein kinase C may
be involved in its activation. It should be also mentioned that an
intracellular calcium chelator, BAPTA-AM, showed no inhibitory effect
on Epo- or IL-3-induced activation of Rap1 in 32D
cells,2which is in contrast
to previous reports on PLC-dependent Rap1 activation
observed rapidly and transiently following thrombin stimulation in
platelets or that observed in lymphocytes stimulated though the B cell
antigen receptor (20, 21). It is thus possible that, although Epo
stimulation induces an increase in intracellular calcium (45), it may
not play a critical role in Rap1 activation. Further studies are
required to elucidate the downstream signaling pathways mediating the
PLC-
-dependent Rap1 activation in cytokine-stimulated hematopoietic cells.
1 integrins. First, the basal as well as cytokine-stimulated Rap1 activity was increased by the induction of CrkL or C3G overexpression in 32D cells (Fig. 2), which also enhanced the basal as well as cytokine-stimulated adhesion of cells through VLA-4 and VLA-5 as
demonstrated previously (11). The Rap1 activity, thus, correlated with
the
1 integrin-mediated cell adhesiveness in these
cells. Second, the correlation was also observed in cells treated with various chemical reagents, including U73122, which drastically inhibited both cytokine-induced Rap1 activation and cell adhesion. (Fig. 4). Last, a dominant negative mutant of Rap1, Rap1A-17N, inhibited CrkL-enhanced adhesion of hematopoietic cells, whereas an
activated Rap1 mutant, Rap1A-63E, enhanced cytokine-induced cell
adhesion or induced adhesion of cells starved from cytokines (Fig. 3).
It should be also noted that the cell adhesion induced by
overexpression of CrkL and that induced by expression of Rap1A-63E are
both mediated through the
1 integrins VLA-4 and VLA-5,
as demonstrated by inhibitory effects of specific antibodies. These observations are in agreement with the idea that the CrkL-C3G complex
mediates cytokine-induced integrin activation, at least partly, by
activating Rap1. R-Ras may also be involved in this process because a
dominant negative mutant of R-Ras, R-Ras-43N, also showed an inhibitory
effect on CrkL-enhanced cell adhesion in an additive way with Rap1A-17N
(Fig. 3B). This is in accordance with previous reports that
R-Ras activates integrin in 32D cells (32). Previous reports have also
implicated PLC-
in inside-out signaling to activate
1
integrins in hematopoietic cells (47, 48), which is consistent with our
observation that U73122 inhibited cytokine-induced cell adhesion (Fig.
4). In contrast, U73122 failed to inhibit cytokine-independent adhesion
of cells expressing Rap1A-63E, which is in agreement with the notion
that Rap1 functions downstream of PLC-
to mediate the
cytokine-induced integrin activation signal. Together, these results
support the hypothesis that the receptors for Epo and IL-3 transduce
inside-out signaling to activate
1 integrins by
stimulating Rap1 through the CrkL/C3G and PLC-
pathways.
2 integrin, to ICAM-1 and
further suggested that Rap1 may be involved in T cell receptor-mediated
activation of LFA-1. Furthermore, Reedquist et al. (50)
reported that Rap1-12V stimulates T lymphocyte adhesion through VLA-4
to VCAM-1 as well as through LFA-1 to ICAM-1. Reedquist et
al. (50) further demonstrated CD31-dependent
activation of Rap1 and its involvement in CD31-dependent cell adhesion. Whereas Reedquist et al. (50) suggested, by
using an activation-specific antibody, that Rap1 may not directly
increase the ligand binding affinity of LFA-1, Katagiri et
al. (49) showed that Rap1 increases the affinity of LFA-1 for
ICAM-1. It is thus possible that Rap1 may activate
2
integrin-mediated cell adhesion through different mechanisms in
different cell types. On the other hand, the mechanism by which Rap1
stimulates hematopoietic cell adhesion through
1
integrins as well as the downstream signaling events mediating this
process has remained to be examined. Irrespective of the mechanisms,
our study and these reports collectively indicate that Rap1 is involved
in inside-out signaling to activate both
1 and
2 integrins in hematopoietic or lymphoid cells in
response to various stimuli.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. John Groffen, Erkki Ruoslahti, Michiyuki Matsuda, Makoto Noda, Masakazu Hattori, Johannes L. Bos, and Norio Komatsu for the generous gifts of experimental materials. We also thank Drs. Masayuki Yoshida and Toshihiko Iizuka for assistance in fluorometric adhesion assays, Drs. Yoji Ikawa and Masakazu Hattori for helpful discussions, and Eiko Nishimura and Kaori Okada for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan and the Ichiro Kanehara Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-3-5803-5952; Fax: 81-3-5803-0131; E-mail: miura.med1@med.tmd.ac.jp.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M004627200
2 A. Arai, Y. Nosaka, E. Kanda, and O. Miura, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: Epo, erythropoietin; EpoR, Epo receptor; IL, interleukin; Stat, signal transducers and activators of transcription; Erk, extracellular signal-regulated kinase; PLC, phospholipase C; GM-CSF, granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; SH, Src homology; GEF, guanine nucleotide exchange factor; Tet, tetracycline; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; RBD, Rap1 binding domain; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; ICAM, intercellular adhesion molecule; VCAM, vascular cellular adhesion molecule; XTT, 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate; LFA-1, lymphocyte function-associated antigen-1.
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