1 Division of Biochemistry and Cellular Biology, National Institute of
Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo
187-8502, Japan
2 Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka
University, Suita, Osaka 565-0871, Japan
* Present address: Department of Cell Biology, University of Virginia,
Charlottesville, VA 22908, USA
Present address: Division of Cellular Proteomics, Institute of Medical
Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan
Author for correspondence (e-mail:
as6by{at}virginia.edu)
Accepted 3 October 2002
![]() |
Summary |
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Key words: Cas, Cell adhesion, Chat, Guanine nucleotide exchange factor, Rap1, Small GTPase
![]() |
Introduction |
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It has been well characterized that Src family tyrosine kinases and Cas
family docking proteins play a pivotal role in the integrin-mediated signaling
pathways (Hanks and Polte,
1997; O'Neill et al.,
2000
; Bouton et al.,
2001
). Cas is a multivalent docking protein that transduces the
adhesion-dependent tyrosine-phosphorylation signal. At the submembranous
region of integrin adhesion sites, activated focal adhesion kinase (FAK) forms
a complex with Cas and Src family kinases, which induces
tyrosine-phosphorylation of the Cas substrate domain
(Schlaepfer et al., 1997
;
Ruest et al., 2001
). The Src
homology 2 (SH2) domain of Crk binds to tyrosine-phosphorylated residues of
the Cas substrate domain (Sakai et al.,
1994
), recruiting downstream Crk SH3-domain-binding effectors
toward Cas (Tanaka et al.,
1994
; Hasegawa et al.,
1996
).
It has also been shown that various small GTPases function in
integrin-mediated signaling pathways. Rap1 regulates inside-out modulation of
integrin function (Katagiri et al.,
2000; Reedquist et al.,
2000
; Caron et al.,
2000
; Ohba et al.,
2001
; Bos et al.,
2001
). R-Ras is also involved in the inside-out activation of
integrins (Zhang et al.,
1996
). Ras activates the extracellular signal-regulated kinase
(ERK) downstream of integrin adhesion
(Clark and Hynes, 1996
). Two
major effectors of Crk are C3G and DOCK 180, which, respectively, activate the
small GTPases Rap1 and Rac1 (Gotoh et al.,
1995
; Kiyokawa et al.,
1998
). Rac1 activates the c-Jun N-terminal kinase (JNK) cascade
that transmits the integrin-mediated mitotic signal to the nucleus
(Dolfi et al., 1998
) and
promotes cell migration through actin filament reorganization
(Klemke et al., 1998
).
Recently, we have identified a novel signaling molecule, Chat
(Cas/HEF1-associated signal
transducer), as a binding partner of the Cas family proteins, Cas
and HEF1 (Sakakibara and Hattori,
2000). Chat contains an SH2 domain and is phosphorylated by
mitogen-activated protein (MAP) kinase, suggesting that Chat integrates
signals from tyrosine kinases and MAP kinase. We also showed that Chat and Cas
were associated with each other through their C-terminal domains. Chat is a
member of a structurally related protein family that consists of NSP1
(Lu et al., 1999
), AND-34
(Cai et al., 1999
)/BCAR3
(van Agthoven et al.,
1998
)/NSP2 and Chat/SHEP1
(Dodelet et al., 1999
)/NSP3.
All of the family proteins are reported to be associated with Cas. Besides the
Cas-binding activity, the C-terminal domain of Chat family proteins shares
weak amino-acid sequence similarity with the catalytic domain of guanine
nucleotide exchange factors (GEFs) (reviewed in
Overbeck et al., 1995
) for Ras
family small GTPases (Dodelet et al.,
1999
; Gotoh et al.,
2000
). Overexpression of AND-34 was reported to activate small
GTPases, RalA, Rap1 and R-Ras, which was inhibited by co-expression of Cas and
AND-34 (Gotoh et al., 2000
).
By contrast, Dodelet et al. reported that SHEP1, an alternatively transcribed
isoform of Chat, binds to R-Ras and Rap1 without activating them
(Dodelet et al., 1999
). Thus,
the function of Chat family members in the regulation of Ras family proteins
still remains unclear.
Therefore, to further investigate the Chat function, we examined the effect of Chat expression on Ras family GTPases. The expression of Chat induced a significant activation of the small GTPase Rap1, but not H-Ras, R-Ras or RalA, in 293T cells. The membrane-targeted form of Chat (Myr-Chat) showed higher activity in Rap1 activation. Interestingly, co-expression of Cas enhanced the activation of Rap1, and dominant-negative mutants of Cas, Crk or C3G counteracted the effect of Chat. These results suggest that the Chat-dependent Rap1 activation was not mediated by the direct effect of Chat on Rap1 but was rather dependent on the Chat-Cas interaction and downstream Crk-C3G pathway. We also found that expression of Myr-Chat in NIH3T3 cells induced cell protrusions at the cell periphery, which was frequently accompanied by branched cell morphology. This cell-shape-converting activity also required the Cas-Crk-C3G signaling pathway and Rap1 function. Furthermore, Myr-Chat expressing 293T cells showed an increased adhesion to the fibronectin matrix. These results suggest that the Chat-Cas complex plays a role in coupling certain tyrosine-kinase-derived signals to modulate cell adhesion via Rap1 activity.
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Materials and Methods |
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Pull-down assay for GTP-bound small GTPases
The relative amount of GTP-bound small GTPases was determined by Bos's
pull-down method with the following modifications
(Franke et al., 1997;
Ohba et al., 2000a
). The
following binding domains for Ras family GTPases (RBDs) fused to glutathione
S-transferase (GST) were used for GTP-bound GTPase pull-down; Raf-RBD
for H-Ras and R-Ras, RalGDS-RBD for Rap1 or RalGDS-RBD for RalA.
Semi-confluent 293T cells on poly-L-lysine-coated dishes were transiently
transfected with expression plasmids for FLAG-tagged small GTPases by using
Lipofectamine 2000 reagent (Life Tech.). 30 hours after transfection, cells
were lysed with ice-cold PD buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM
MgCl2, 1% Triton X-100, 1 mM Na3VO4, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin),
and samples were kept on ice for 10 minutes. After clarification by
centrifugation, GTP-bound small GTPases were pulled down with GST-RBD
fusion-protein-adsorbed glutathione-Sepharose beads (Amersham Pharmacia
Biotech.) for 30 minutes on ice. The complex were washed three times with PDW
buffer (PD buffer containing 0.5% Triton X-100) and then eluted with sample
buffer for SDS-polyacrylamide gel electrophoresis. Immunoblotting of the
samples was carried out as previously described
(Sakakibara and Hattori,
2000
).
Co-immunoprecipitation assay
293T cells were transiently transfected with the indicated expression
constructs using a calcium phosphate method. 36 hours after transfection,
cells were lysed in ice-cold IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 50
mM NaF, 1% Triton X-100, 1 mM Na3VO4, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin),
followed by centrifugal clarification. Samples were immunoprecipitated with
anti-Cas or anti-Crk 3A8 mouse monoclonal antibodies by using protein A
Sepharose (Amersham Pharmacia Biotech.). Co-immunoprecipitated Chat was
detected by anti-Chat immunoblotting.
Immunofluorescence microscopy
Indirect immunofluorescence microscopy was carried out as described above
(Sakakibara and Hattori,
2000). NIH3T3 cells on glass cover slips were transfected with
pCAX-Chat or pCAX-Myr-Chat using Lipofectamine Plus reagent (Life Tech.)
followed by 30 hours of incubation. After fixation of the cells, anti-FLAG was
used as a primary antibody for detection of Chat or Myr-Chat. Fluorescein
(FITC)-conjugated goat anti-mouse IgG (Jackson Lab.) or Texas-Red-conjugated
phalloidin (Molecular Probes) was, respectively, used for visualizing the
anti-Chat immune complex or the actin filament.
Cell area estimation
NIH3T3 cells plated on 0.5 µg/ml fibronectin-coated 35 mm glass base
dish (Iwaki) were transiently transfected with pIRES-EGFP vector-based
expression constructs using Lipofectamine 2000 reagent. 30 hours after
transfection, fluorescent images of EGFP-expressing cells were recorded using
a Zeiss Axiovert microscope (Carl Zeiss) with a cooled CCD camera (Roper
Scientific), controlled by MetaMorph2 software (Universal Image). The cell
area of each GFP-positive cell was estimated by measuring the
EGFP-fluorescence-emitting region using MetaMorph2 software. For
co-transfection experiments, cells were transfected with a five-fold amount of
a mutant protein expressing plasmid together with pIRES-EGFP-Myr-Chat.
Cell adhesion assay
In this study, we have developed a novel system for cell adhesion assays
using transiently transfected 293T cells. An expression plasmid for EGFP was
always cotransfected, and the adhesion of transfected cells was quantified by
measuring the EGFP-derived fluorescence. Briefly, 293T cells were
co-transfected with 1.5 µg pCAX-EGFP and 3.5 µg plasmid to be examined
as described in the small GTPase pull-down assay section. 24 hours after
transfection, cells were serum starved for 6 hours at 37°C in an adhesion
medium (DMEM supplemented with 0.5% bovine serum albumin). Microwell culture
plates (96 well) were coated with PBS containing 0.2 µg/ml fibronectin
(Sigma) for 2 hours at 37°C, blocked for 1 hour with DMEM supplemented
with 3% bovine serum albumin followed by three washes with the adhesion
medium. The cells were dispersed in PBS containing 0.01% trypsin, followed by
adding 10 µg/ml trypsin inhibitor and washing with PBS. After re-suspension
in the adhesion medium, 2x105 cells were added into each well
and allowed to adhere for 30 minutes at 37°C. EGFP-derived fluorescence
was measured as the total loaded cell-derived fluorescence (Ft) using a
Fluorimager (Molecular Dynamics). To remove non-adherent cells, the wells were
washed four times with the adhesion medium. Remaining fluorescence was
measured as the adherent cell-derived fluorescence (Fa). The percentage of
adherent cells was calculated from the ratio of Fa/Ft.
![]() |
Results |
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First, we found that the expression of Chat caused a slight increase in
GTP-bound Rap1 in 293T cells (Fig.
1B) when the active Rap1 was analyzed using a modified pull-down
assay originally developed by Bos' group
(Franke et al., 1997;
Ohba et al., 2000a
) (see
Materials and Methods). By measuring the intensity of the corresponding bands,
a statistically significant increase in GTP-bound Rap1 (2.4±1.2 times,
n=5, P<0.05) was reproducibly observed in Chat
overexpressing cells compared with control pCAX-transfected cells. An
N-terminally myristoylated Chat variant (Myr-Chat) showed higher activity in
Rap1 activation (3.7±1.4 times, n=5, P<0.01). To
elucidate whether the Chat C-terminal Cas/HEF1 association (GEF-like) domain
was required for the activation of Rap1, we examined the effect of
C-terminally truncated mutants, Chat-
CT and Myr-Chat-
CT, on Rap1
activation. Both mutants did not show statistically significant activation of
Rap1 (1.2±0.4 times, n=3, P<0.5 and 1.3±0.2
times, n=3, P<0.1, respectively), indicating that the
function of the Chat C-terminal domain was essential for the Rap1
activation.
We next analyzed the specificity of Chat activity toward several Ras family small GTPases (Fig. 2). As shown in Fig. 2A, overexpression of Chat or Myr-Chat again induced Rap1 activation, although the activation level was rather low compared with that caused by C3G. Chat did not significantly activate other Ras family small GTPases, H-Ras, R-Ras and RalA (Fig. 2B-D). Taken together, the results from several independent experiments established that Chat reproducibly activated Rap1 but not other GTPases (Fig. 1B, Fig. 2; data not shown). We also observed a slight decrease (35% decrease, n=3, P<0.005; Fig. 2D; data not shown) of GTP-bound RalA in Chat- but not Myr-Chat-expressing cells, although the meaning is unclear.
|
Functional Cas-Crk-C3G signaling pathway is required for the
Chat-induced Rap1 activation
Interestingly, we further found that the overexpression of Cas also
activates Rap1 and that co-expression of Chat and Cas caused enhanced
activation of Rap1 (Fig. 3A).
By contrast, co-expression of the Cas C-terminal portion (Cas-C-half;
amino-acid residues 425-874), which lacked both SH3 and substrate domains of
Cas, inhibited the Chat-mediated Rap1 activation. Overexpression of Crk also
robustly activated Rap1 with higher efficiency
(Fig. 3B). These results raised
the possibility that the Chat-induced Rap1 activation was mediated by
Chat-Cas-interaction inducing the upregulation of the downstream Crk-C3G
pathway rather than by intrinsic GEF activity of Chat. Since Crk activates
Rap1 rather strongly, triple expression of Crk, Cas and Chat did not further
increase the activation level (Fig.
3B).
|
Therefore, we next analyzed the effect of several Cas mutants on Rap1
activation. Cas is a multivalent docking protein that consists of an SH3
domain, substrate domain (SD), Src-binding domain and Chat association domain
(Fig. 4A) (see
Nakamoto et al., 1997;
Sakakibara and Hattori, 2000
;
Bouton et al., 2001
). The Cas
SH3 domain interacts with a number of signaling molecules such as, FAK, Pyk2,
PTP-1B, PTP-PEST and C3G. Cas SD contains multiple tyrosine phosphorylation
sites that serve as docking sites for Crk SH2 domain. As shown in
Fig. 4B, deletion of Cas SD
(Cas
SD) almost completely impaired activation of Rap1 (1.0 fold,
n=4). As expected, Cas
SD was not tyrosine phosphorylated at
all and did not bind Crk (Fig.
4C), which may be the reason for its inability to activate Rap1.
Surprisingly, Cas
SCB, lacking the region essential for Src binding and
Chat association, still retained substantial tyrosine phosphorylation level,
Crk association and Rap1-activating activity (2.0 fold, n=4). These
results revealed a crucial role of the Cas substrate domain and its
interaction with Crk in the Cas-induced Rap1 activation. Therefore, we used
Cas
SD mutant in the following experiments as an interfering mutant for
Chat-mediated Rap1 activation.
|
To address whether the Cas-Crk-C3G signaling pathway indeed mediates the
Chat-induced Rap1 activation, we expressed dominant-negative mutants of Cas,
Crk or C3G together with Myr-Chat and evaluated the effect of these mutants on
the activation of Rap1 (Fig.
5). As expected, co-expression of Cas SD (Myr+
SD)
significantly suppressed the Myr-Chat-induced Rap1 activation. Furthermore,
Crk W169L (Myr+W169L), a loss of function mutant of Crk SH3 domain
(Tanaka et al., 1993
), or C3G
dCD (Myr+dCD), C3G devoid of its catalytic domain
(Tanaka et al., 1997
), also
interfered with the Rap1 activation induced by Myr-Chat. A similar inhibitory
effect of these mutants on Rap1 activation was observed when Chat instead of
Myr-Chat was used (data not shown). Thus, the functional Cas-Crk-C3G signaling
pathway is required for Chat-mediated upregulation of Rap1.
|
We then tried to identify a signaling complex consisting of Chat, Cas, Crk and C3G by a co-immunoprecipitation assay. As shown in Fig. 6, Chat was recovered in anti-Cas-immunoprecipitates from Chat-expressing 293T cells, and the amount of Chat was greatly increased by co-expression of Cas. In the control experiment, Chat was not detected at all in immunoprecipitates with a control antibody (data not shown). Increased expression of Crk did not affect the amount of Chat in the Cas immune complex. Recovery of Chat in anti-Crk immunoprecipitates of Chat-overexpressed cells was rather low, and its recovery greatly enhanced in Chat-Cas-Crk triple-transfected cells. These results clearly indicate a ternary complex formation consisting of Chat, Cas and Crk and provide substantial support for the Chat-Cas-Crk-C3G signaling pathway inducing Rap1 activation.
|
Overexpression of Cas did not significantly increase the Chat recovery in Crk immunoprecipitates. Assuming that the Chat-Crk interaction is mediated by Cas, excess Cas might inhibit the recovery of Chat in Crk-immunoprecipitates depending on the molar ratios of Chat, Cas and Crk. Finally, we examined a possible interaction between Chat and C3G by using a similar immunoprecipitation method. However, it was hard to detect a significant association to form a quaternary complex containing Chat and C3G (data not shown).
Membrane-targeted Myr-Chat expression induces cell periphery
spreading and cell shape branching
Recent reports from several laboratories implicate Rap1 in the inside-out
modulation of integrin adhesion (Katagiri
et al., 2000; Reedquist et
al., 2000
; Caron et al.,
2000
; Ohba et al.,
2001
), and cell adhesion properties often affect the cell
morphology. Therefore, we studied the effect of the membrane-targeted form of
Chat (Myr-Chat) on cell morphology. Intracellular localization of Chat or
Myr-Chat was visualized by anti-FLAG immunofluorescence together with
phalloidin staining of actin filaments to illustrate the cell shape. Chat was
distributed throughout the cytoplasm, and no apparent cell morphological
alteration was observed compared with non-transfected cells in the same field
(Fig. 7A,B) (data not shown).
Myr-Chat-derived fluorescence signal was mainly from the plasma membrane and
intracellular vesicle-like structures (Fig.
7C,E). Interestingly, Myr-Chat-expressing cells frequently showed
highly spread cell periphery and branched cell shape
(Fig. 7C-E). This morphology
was obviously distinct from a typical pancake-like spreading image often
induced by constitutive active Rac (Nobes
and Hall, 1995
; Kiyokawa et
al., 1998
). Co-expression of Cas with Chat does not alter the
morphology of the cells, even though it activated Rap1 robustly (data not
shown).
|
To quantify the morphological change, we used the pIRES-EGFP-based
expression system that could simultaneously express a gene of interest with
EGFP from a single transcript and visualize the transfected live cells. The
area of each transfected cell was measured as the size of the
fluorescence-emitting region. Under our experimental conditions, most of
control pIRES-EGFP-transfected NIH3T3 cells showed spindle-like cell shape
(Fig. 8). As shown in
Fig. 7, Myr-Chat-expressing
cells frequently showed cell protrusions and branched morphology. The
quantified cell area values of these EGFP-positive cells were represented as
histograms (Fig. 8. right
panels). The cells expressing Myr-Chat showed substantially increased cell
area values compared with control cells. Overexpression of wild-type Chat did
not induce a significant increase. As in the case of Rap1 activation, a
C-terminal deletion of Myr-Chat (Myr-CT) completely inactivated the
cell-area-enlarging activity.
|
We further examined the effect of dominant-negative mutants of Cas, Crk and
Rap1 on Myr-Chat-induced cell area enlargement
(Fig. 9). In good agreement
with the inhibitory effect on Myr-Chat-induced Rap1 activation
(Fig. 5), Cas SD, Crk
W169L, and C3G dCD effectively suppressed the spreading and branching of cell
periphery and cell area enlargement induced by Myr-Chat. N17 Rap1, a
dominant-negative mutant of Rap1, also interfered with the Myr-Chat-induced
cell shape conversion. Under these conditions, the membranous localization of
Myr-Chat was not affected by co-expression of these mutants (data not shown).
These results indicate that the morphological change of Myr-Chat-expressing
cells is mediated by the Cas-Crk-C3G signaling pathway, leading to Rap1
activation.
|
Myr-Chat expression modulates integrin-mediated adhesion of 293T
cells
To gain insight into the cellular mechanism underlying the morphological
effect of Myr-Chat, we studied the adhesion of cells to fibronectin as Rap1
activity had been implicated in cell adhesion. We used the dishes coated with
low concentrations of fibronectin (0.2 µg/ml) to make the difference in
cell adhesion stand out. The cells expressing Myr-Chat showed 50% greater cell
adhesion when compared to cells transfected with a control vector pCAX
(Fig. 10). Under the same
conditions, we could not detect a significant increase in cell adhesion when
the wild-type Chat was overexpressed. Myr-Chat-CT also was ineffective,
indicating the essential role of the Cas/HEF1 association domain in modulating
the cell adhesion. These results suggest a function of Chat-Cas complex in
regulation of cell adhesion.
|
![]() |
Discussion |
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The Chat family consists of three closely related members, NSP1
(Lu et al., 1999), AND-34
(Cai et al., 1999
)/BCAR3
(van Agthoven et al.,
1998
)/NSP2 and Chat/SHEP1
(Dodelet et al., 1999
)/NSP3,
all of which have N-terminal SH2 and C-terminal GEF-like Cas/HEF1 association
domains. Identities in amino-acid sequence among these members are around 40%.
Recently, Gotoh et al. reported that AND-34 activated RalA, Rap1 and R-Ras
(Gotoh et al., 2000
). However,
overexpression of Cas inhibited the GEF activity of AND-34 toward RalA, which
was in striking contrast to our results that co-expression of Cas showed a
synergistic effect on Chat-mediated Rap1 activation
(Fig. 3A). These differential
effects of Cas may reflect the dissimilar mechanism of AND-34 and Chat in the
activation of these small GTPases; AND-34 may function as a direct GEF whose
activity is inhibited by Cas binding, whereas Chat indirectly activates Rap1
through the Cas-Crk-C3G pathway. However, the effect of Cas on Rap1 activation
by AND-34 was not studied; detailed molecular events remain obscure. Bos et
al. reported that AND-34 does not activate Rap1 in vitro as an unpublished
result (Bos et al., 2001
). In
another study, Dodelet et al. identified SHEP1 (which is identical to Chat-H,
hematopoietic cell-specific Chat) as a downstream target molecule for
activated Eph receptors (Dodelet et al.,
1999
). They also described the binding of the C-terminal half of
SHEP1 to R-Ras and Rap1. However, the same region of SHEP1 did not show any
GEF activity in vitro (Dodelet et al.,
1999
).
In our previous studies, we showed that C3G, one of the major downstream
effectors of the Cas-Crk pathway, functions as a GEF for Rap1 and R-Ras
(Gotoh et al., 1995;
Gotoh et al., 1997
;
Ohba et al., 2000b
). We also
reported that another Crk effector DOCK 180 promotes the activation of Rac1
(Kiyokawa et al., 1998
).
Therefore, when Myr-Chat up-regulates the Cas-Crk pathway, it should
theoretically activate not only Rap1 but also R-Ras and Rac1. However, we
could detect activation of neither R-Ras nor Rac1 in our system
(Fig. 2C) (A.S., Y.O., K.K.,
M.M. et al., unpublished). Two explanations may reconcile this observation.
First, as shown in Fig. 2, C3G
activation of Rap1 is much higher than that of R-Ras. Second, the small
GTPases were overexpressed as GST-fusion proteins in the previous studies,
whereas the activation of FLAG-tagged GTPases was measured in this study. In
any case, the activation of Rap1 by Chat has been established in this
study.
In general, one of the most important roles of SH2 domains is recruiting
proteins to specific subcellular locations. Although all Chat family proteins
have an SH2 domain at the N-terminal portion, their physiological targets are
still unclear except for the information from overexpression systems
(Lu et al., 1999;
Dodelet et al., 1999
). This
makes it difficult to study Chat function under physiological conditions.
Lipidation signals, such as an N-terminal myristoylation signal and C-terminal
CAAX box, have been widely used for membrane targeting of the molecule to
mimic the activated state (Aronheim et al.,
1994
; Gotoh et al.,
1995
; Hasegawa et al.,
1996
; Kohn et al.,
1996
). Therefore, we employed Chat with a myristoylation signal
(Myr-Chat) for activated Chat. Membrane targeting of Chat upregulated Rap1
induced cell periphery spreading and branched morphology in NIH3T3 cells and
enhanced the integrin-mediated adhesion in 293T cells. Although wild-type Chat
also activated Rap1, it affected neither cell morphology nor adhesion. We also
observed similar induction of cell periphery spreading by My-Chat but not by
Chat in other cell lines (COS7, PC12) (data not shown). Chat-Cas co-expression
even robustly activated Rap1 but was not sufficient for this morphological
alteration in these cells (Sakakibara and
Hattori, 2000
) (data not shown). The requirement for membrane
targeting of Chat resembles that of C3G; although both wild-type and
membrane-targeted C3G activate Rap1, only the membrane-targeted but not
wild-type C3G causes flat reversion of v-Ki-ras-transformed cells
(Gotoh et al., 1995
).
Activation of Rap1 in specific subcellular compartments might be necessary for
these cell biological observations.
Recently, a role for Rap1 in controlling integrin-mediated adhesion has
been proposed using leukocyte systems. Katagiri et al. showed that LFA-1
(ß2 integrin)-mediated adhesion to ICAM1 was regulated by Rap1 in BA/F3 B
cells expressing LFA-1 (Katagiri et al.,
2000). Reedquist et al. described the requirement of Rap1 for
CD31-mediated activation of VLA-4 (ß1 integrin) and LFA-1 in Jurkat T
cells (Reedquist et al.,
2000
). Caron et al. showed that phagocytosis in macrophages, which
is mediated by
Mß2 complement receptor 3, is Rap1 dependent
(Caron et al., 2000
). In the
case of adherent cells, C3G-deficient mouse embryonic fibroblasts showed
impaired cell attachment and spreading phenotypes resulted from the
inefficient Rap1 activation (Ohba et al.,
2001
). These lines of evidence indicate that Rap1 controls
integrin function. Taken together with our observation that Myr-Chat
overexpression modulated cell adhesion, a novel function of the Chat-Cas
signaling pathway in regulating integrin adhesion via Rap1 activity is
suggested.
The only known tyrosine phosphorylated protein that binds to the Chat SH2
domain is activated Eph receptors (Dodelet
et al., 1999). Activation of Eph receptors by ephrin ligands
transmits repulsive cell-cell interaction signals, which play pivotal roles in
controlling cell migration, axon guidance and compartmental boundary formation
during the embryogenesis (reviewed in
Holder and Klein, 1999
;
Schmucker and Zipursky, 2001
).
Several studies have demonstrated that the ephrin-Eph system controls the
adhesion property of integrins (Huynh-Do
et al., 1999
; Miao et al.,
2000
), although the mechanism is not known. It is an intriguing
possibility that Chat is involved in this regulation of cell adhesion
downstream of the ephrin-Eph system.
In the present study, we provided evidence that Chat, an adaptor protein for Cas family proteins, indirectly regulates the activity of Rap1. Furthermore, we proposed a potential regulatory system for cell adhesion by a Chat-Cas signaling pathway through controlling Rap1 activity. This pathway may be involved in the modulation of cell adhesion mediated by certain receptor tyrosine kinases, such as Eph receptors, and may play a role in the development of the multicellular organisms.
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Acknowledgments |
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