From the Department of Immunology and Cell Biology,
Graduate School of Medicine and § Graduate School of
Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, and the
¶ Department of Cell Pharmacology, Graduate School of
Medicine, Nagoya University, Nagoya, Aichi 466-8550, Japan
Received for publication, November 21, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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In the present study, we showed that SPA-1, a
Rap1 GTPase-activating protein (GAP), was bound to a
cytoskeleton-anchoring protein AF-6. SPA-1 and AF-6 were
co-immunoprecipitated in the 293T cells transfected with both cDNAs
as well as in normal thymocytes. In vitro binding studies
using truncated fragments and their mutants suggested that SPA-1 was
bound to the PDZ domain of AF-6 via probable internal PDZ ligand motif
within the GAP-related domain. The motif was conserved among Rap1 GAPs,
and it was shown that rapGAP I was bound to AF-6 comparably with SPA-1.
RapV12 was also bound to AF-6 via the N-terminal domain, and SPA-1 and
RapV12 were co-immunoprecipitated only in the presence of AF-6,
indicating that they could be brought into close proximity via AF-6 in
cells. Immunostaining analysis revealed that SPA-1 and RapV12 were
co-localized with AF-6 at the cell attachment sites. In HeLa cells
expressing SPA-1 in a tetracycline-regulatory manner, expression of
AF-6 inhibited endogenous Rap1GTP and Rap1 is a member of Ras family GTPases and is suggested to play
roles in the regulation of cell proliferation, differentiation, and
cell adhesion (reviewed in Ref. 1). Rap1 is activated by a wide variety
of extracellular stimuli through different kinds of specific guanine
nucleotide exchange factors, which are coupled with various receptors
or the second messengers via distinct interaction motifs (1). The
amounts and duration of intracellular Rap1GTP, on the other hand, are
controlled by specific GTPase-activating proteins
(GAPs).1 So far, two families
of Rap1 GAPs are identified including rapGAPs (I and II) and SPA-1
family proteins (SPA-1, E6TP1/SPAR/SPA-Ls), which may have different
tissue distribution profiles (2-7). Whereas all the Rap1 GAPs share a
highly conserved domain, called GAP-related domain (GRD), responsible
for GAP catalytic activity, they additionally bear unique functional
domains. For instance, rapGAP II has a G We reported previously (9-11) that activated Rap1 induced cell
adhesion mediated by Cells and Antibodies--
Thymocytes, splenic T and B
cells were obtained from normal BALB/c mice. 293T, HeLa, and Caki-2
(human kidney cancer) cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. HeLa cells
stably transfected with SPA-1 cDNA (HeLa/Tet-SPA-1) in a pTRE
vector (Clontech) were reported before (9) and
maintained in complete Dulbecco's modified Eagle's medium containing
10 ng/ml doxycycline (Dox) (Sigma), unless indicated specifically.
Antibodies used in the present study included anti-SPA-1 (9), anti-AF-6
(19), anti-Rap1, anti-Rap1 GAP (Santa Cruz Biotechnology), and anti-T7
antibodies (Novagen). Biotinylated anti-SPA-1 antibody was prepared
using EZ-link sulfo-NHS-LC biotin (Pierce). Anti-VLA-4 and anti-VLA-5
antibodies were provided by Dr. T. Kinashi, Kyoto University, Kyoto, Japan.
Plasmid Construction and cDNA Transfection--
cDNA of
AF-6 lacking Ras/Rap1-binding domain ( In Vitro Binding of SPA-1 and AF-6--
cDNAs of truncated
SPA-1 fragments, fragment 1 (residues 1-211), 2 (residues 212-532), 3 (residues 538-680), 4 (residues 681-1038), and 5 (residues
748-1038), were amplified by PCR and subcloned into
BamHI/XhoI sites of a pSP73 vector. PCR-amplified
cDNAs of subfragments of SPA-1 GRD (fragment 2), G1 (residues
338-532), G2 (398-459), and G3 (residues 435-489) as well as the
mutants of G2 fragment, M1 (V432A) and M2 (F433A) generated by a
site-directed mutagenesis kit (Stratagene Quick), were subcloned into
BglII/EcoRI sites of a pSP73 vector. A cDNA
fragment of rapGAP I (residues 263-322) corresponding to the G2
fragment of SPA-1 was amplified by PCR and also subcloned into a pSP73
vector. cDNAs of a series of truncated AF-6 fragments, fragment 1 (residues 36-494), 2 (residues 495-909), 3 (residues 914-1129), and
4 (residues 1130-1612), as well as AF-6 and Immunoprecipitation and Immunoblotting--
Cells were lysed
with lysis buffer (0.5% Triton X-100, 10 mM Tris-HCl, pH
7.6, 150 mM NaCl, protease inhibitor mixture), incubated with specific antibodies overnight at 4 °C with gentle rotation, and
then precipitated with protein A-Sepharose beads for 30 min at 4 °C.
After extensive washing, the beads were eluted with SDS sample buffer,
boiled, and electrophoresed in SDS-PAGE followed by immunoblotting and
ECL detection (Amersham Biosciences). To detect intracellular Rap1GTP,
cell lysates (0.8-1 mg of proteins) were incubated with a GST fusion
protein of RalGDS-RBD coupled with glutathione-Sepharose 4B (Amersham
Biosciences) on ice for 1 h, washed, and eluted with SDS sample
buffer followed by immunoblotting with anti-Rap1 antibody.
Immunostaining--
HeLa/Tet-SPA-1 cells were transfected with
pEF-BOS AF-6 and selected with puromycin (Sigma) to establish a stable
cell line (HeLa/Tet-SPA-1/AF-6). The latter cells were transfected
transiently with T7-tagged RapV12 cDNA in a pSR Cell Adhesion Assay--
96-Well flat-bottom plates were coated
with 5 µg/ml fibronectin (FN) (Sigma) overnight at 4 °C followed
by blocking with 3% bovine serum albumin/phosphate-buffered saline for
1 h at 37 °C. 293T or HeLa/Tet-SPA-1 cells were treated with
trypsin/EDTA, washed, resuspended in serum-free Dulbecco's modified
Eagle's medium containing 0.02% bovine serum albumin and 10 mM Hepes, rotated in suspension for 2 h, and plated in
triplicate onto FN-coated wells at 0.5 × 105 cells
per well. After incubation for 30 min at 37 °C, nonadherent cells
were removed gently by aspiration, and the remaining adherent cells
were fixed with 3.7% paraformaldehyde followed by staining with 0.5%
crystal violet in 20% methanol. After extensive washing with distilled
water, dye was extracted with extract solution (50% ethanol in 50 mM sodium citrate, pH 4.5) and measured using an
enzyme-linked immunosorbent assay reader (Molecular Devices). Absorbance in uncoated wells was subtracted from that in the FN-coated wells to show specific adhesion.
Statistics Analysis--
Statistics analysis was done by
Student's t test.
Association of SPA-1 and AF-6 in Normal Cells--
By yeast
two-hybrid screening of a mouse spleen cell cDNA library using a
full-length SPA-1 as bait, AF-6 was identified as a potential
SPA-1-binding protein (data not shown). We therefore examined the
association of SPA-1 and AF-6 by transient gene expression in 293T
cells, which expressed only marginal SPA-1 and AF-6 if any. In the 293T
cells co-transfected with SPA-1 and AF-6 cDNAs, SPA-1 (130 kDa) was
co-immunoprecipitated by anti-AF-6, and reciprocally AF-6 (~200 kDa)
was co-immunoprecipitated by anti-SPA-1 antibody, although AF-6 tended
to be degraded in overexpression system (Fig. 1A). Neither protein was
immunoprecipitated by control preimmune IgG. We then investigated
whether the association of SPA-1 and AF-6 occurred physiologically in
normal cells. As shown in Fig. 1B, thymocytes abundantly
expressed both SPA-1 and AF-6 among normal lymphoid cells, and the
endogenous SPA-1 and AF-6 were co-immunoprecipitated with anti-AF-6 and
anti-SPA-1 antibodies, respectively, indicating that SPA-1 and AF-6
were associated physiologically in normal cells. It was estimated that
around 40% of SPA-1 and 15% of AF-6 were associated with AF-6 and
SPA-1, respectively, in normal thymocytes.
Involvement of GRD of SPA-1 and PDZ Domain of AF-6 for
Binding--
To identify the domains involved in the association of
SPA-1 to AF-6, binding of truncated SPA-1 fragments to AF-6 was
examined in vitro. Among the fragments, a GRD fragment of
SPA-1 (fragment 2, residues 212-532) was bound to the AF-6-coated
beads, whereas none of the other fragments were bound (Fig.
2A). Although not shown,
fragment 2 was not bound to the control beads. Similar analysis was
performed using truncated fragments of AF-6. As also indicated in Fig.
2A, only a fragment containing the PDZ domain of AF-6
(fragment 3, residues 914-1129) was bound specifically to the
SPA-1-coated beads. To confirm these results, we generated a
GRD-deletion mutant of SPA-1 ( Binding of SPA-1 to PDZ Domain of AF-6 via Probable Internal
PDZ Ligand Motif, a Common Property of Rap1 GAPs--
We next
intended to investigate whether the binding of SPA-1 and AF-6 occurred
by PDZ-mediated protein interaction. By further truncation of a SPA-1
GRD fragment (fragment 2), it was shown that smaller fragments, G1
(residues 338-532) and G2 (residues 398-459), were bound to
AF-6-coated beads, whereas another overlapping fragment G3 (residues
435-489) was not (Fig. 3B),
suggesting the binding site was in the region between the residues 398 and 434. By using peptide libraries, it was reported that the AF-6 PDZ preferred a class 2 ligand motif ( Co-localization of SPA-1 and Rap1 with AF-6 at Cell Attachment
Sites--
We next examined the intracellular localization of SPA-1 in
relation to AF-6 by using HeLa/Tet-SPA-1 cells and those stably transfected with AF-6 (HeLa/Tet-SPA-1/AF-6), in which SPA-1 expression was repressed at the undetectable level in the presence of 1.0 ng/ml
Dox while induced strongly in the presence of 0.1 ng/ml Dox within
24 h. In HeLa/Tet-SPA-1 cells cultured with 0.1 ng/ml Dox, SPA-1
was expressed diffusely at the cortical area as well as in the cytosol
with little expression of AF-6 (Fig.
4A). On the other hand, AF-6
was localized predominantly at the cell attachment sites with
undetectable SPA-1 expression in the HeLa/Tet-SPA-1/AF-6 cells cultured
with 1.0 ng/ml Dox (Fig. 4B). In HeLa/Tet-SPA-1/AF-6 cells
additionally transfected with T7-tagged RapV12 and cultured with 1.0 ng/ml Dox, a significant proportion of RapV12 was co-localized with
AF-6 (Fig. 4C). Strong nuclear staining by anti-T7 antibody was nonspecific, because it was detected in untransfected cells as
well. When both SPA-1 and AF-6 were induced to express in
HeLa/Tet-SPA-1/AF-6 cells in the presence of 0.1 ng/ml Dox, on the
other hand, SPA-1 was co-localized with AF-6 at the cell attachment
regions (Fig. 4D). It was noted that those cells expressing
both SPA-1 and AF-6 tended to become slender, which was followed by the
cell detachment from a dish in 2 days, much earlier than HeLa/Tet-SPA-1
cells (see below). As shown in Fig. 4E, SPA-1 was
co-localized also with endogenous Rap1 under this condition. These
results strongly suggested that both SPA-1 and Rap1GTP were recruited
to the cell attachment sites by AF-6.
AF-6 Induces the Association of SPA-1 and Rap1GTP and
Enhances Rap1 Inactivation--
A possibility that AF-6 recruited both
SPA-1 and its substrate Rap1GTP was investigated more directly. 293T
cells were transfected with SPA-1 and T7-tagged RapV12 with or without
AF-6 cDNA, and the lysate was immunoprecipitated by anti-T7
antibody. In the absence of AF-6, SPA-1 was co-immunoprecipitated
barely with RapV12 (Fig. 5A),
conforming to a general consensus that the interaction of GAPs with the
substrate was only transient (21). In the presence of AF-6, however, a
significant proportion of SPA-1 was co-immunoprecipitated with
RapV12 along with AF-6 (Fig. 4A). On the other hand,
expression of AF-6 Enhances the Inhibitory Effect of SPA-1 on
In the present study, we demonstrated that a Rap1
GTPase-activating protein SPA-1 was bound to AF-6. SPA-1 was
co-immunoprecipitated specifically with AF-6 and vice versa not only in
the 293T cells co-transfected with SPA-1 and AF-6 cDNAs but also in
normal thymocytes, indicating that the association was physiological.
In vitro binding studies revealed that the binding was
mediated by the interaction between GRD of SPA-1 and the PDZ domain of
AF-6. Most known PDZ-mediated protein interactions occur through
recognition of short C-terminal PDZ ligand motifs of partner proteins
(22, 23). However, exceptional interactions of PDZ domain with internal
motifs of partner proteins have been also reported (24-27). It was
reported that neuronal nitric-oxide synthase was bound to a PDZ domain
of syntrophin via the internal region, in which a PDZ ligand motif in a
sharp In polarized epithelial cells, it was reported that AF-6 was localized
at the cadherin-based cell-cell adhesion sites such as tight and
adherens junctions, in which association with a tight junction protein
ZO-1 might play a role (19). The present results indicated that AF-6
expressed in non-polarized HeLa cells was located focally at the cell
attachment sites to the matrix. On the other hand, SPA-1 expressed in
HeLa/Tet-SPA-1 cells, which barely expressed endogenous AF-6, was
detected diffusely at the cortical area as well as in the cytosol. In
the HeLa/Tet-SPA-1 cells transfected with AF-6, however, SPA-1 was
co-localized significantly with AF-6 at the cell adhesion sites. It was
shown also that RapV12 was co-localized with AF-6 irrespective of the
presence of SPA-1, conforming to the previous report (16) that RapV12
was bound to AF-6. The present results further indicated that SPA-1 and Rap1V12 could be co-immunoprecipitated significantly in the presence of
AF-6 but not of It was reported that Ras GAP activity of a catalytic fragment of
p120RasGAP was inhibited in vitro by an RBD
fragment of AF-6 due to competitive binding of the two fragments to
overlapping sites of RasGTP (15). Independent binding of SPA-1 and
Rap1GTP to the distinct domains of AF-6 may prevent such a possible
interference with the GAP catalytic activity of SPA-1 by AF-6. Rather,
it was shown that the GAP activity of SPA-1 in vivo was
enhanced significantly in the presence of AF-6, indicating that AF-6
recruited SPA-1 and its substrate Rap1GTP into close proximity in the
cells and facilitated the efficient catalytic interaction between them.
We reported previously (9-11) that Rap1GTP activated
Integrin-mediated cell adhesions are regulated dynamically during
various cellular functions. In the immune system, for instance, leukocyte movement and migration involve a spatio-temporally organized regulation of cell adhesion (28, 29). Also, T cell activation in immune
responses depends on the integrin-mediated intimate cell-cell
interactions between T cells and antigen-presenting cells called
immunological synapse, the extent and duration of which profoundly
affect the modes and intensity of immune responses (30). We reported
previously (12) that Rap1 activation and its regulation by SPA-1 played
crucial roles in the immunological synapse formation through
LFA-1/ICAM-1 interaction. The present results indicated that AF-6 was
expressed abundantly in the normal thymocytes partly in association
with SPA-1, and our results3
using SPA-1 transgenic mice showed that Rap1 activation was essential for the differentiation and expansion of thymocytes in vivo.
It thus seems possible that AF-6 plays a significant role in the fine
control of integrin-mediated cellular functions via regulation of Rap1
activation in the immune as well as other systems.
1
integrin-mediated cell adhesion to fibronectin in SPA-1-induced
conditions, whereas it affected neither of them in SPA-1-repressed
conditions. These results suggested that AF-6 could control
integrin-mediated cell adhesion by regulating Rap1 activation through
the recruitment of both SPA-1 and Rap1GTP via distinct domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i-binding domain
and is recruited to plasma membrane, inducing Rap1 inactivation there
and concomitant increase in the basal Ras/extracellular
signal-regulated kinase-signaling in certain cells (7). SPAR bearing
actin-binding domains is located at the dendritic spines of neurons in
association with actin-cytoskeleton and controls the spine morphology
(5). E6TP1 is shown to bind human papilloma virus E6 oncoprotein via
the C-terminal region and is targeted for protein degradation (8).
1 as well as
2
integrins, and SPA-1 could negatively regulate the integrin-mediated
cell adhesion via Rap1 GAP activity. Thus, overexpression of SPA-1 in T
cells almost completely suppressed the immunological synapse formation with the specific antigen-presenting cells by inhibiting T
cell-receptor-induced activation of LFA-1 (12). In the present study,
we have attempted to identify the molecules that are associated with
SPA-1 in order to understand how the intracellular localization and the
functions of SPA-1 are regulated. Here we report that SPA-1 is bound to AF-6, which has been isolated originally as a fusion partner of ALL-1
in human acute myeloid leukemia (13). AF-6 (also called afadin) is an
actin-binding multidomain protein and is reported to bind a number of
proteins such as Ras family GTPases including Rap1 (14-16), a
tight-junction protein ZO-1 (17), actin-regulatory profilin (16), and
subsets of Eph-related receptor protein tyrosine kinases (18). Although
these features imply that AF-6 may function as a molecular scaffold
integrating the signals related to cell adhesion and cytoskeletal
reorganization, its exact functions remain to be seen. We provide
evidence that AF-6 binds both SPA-1 and its specific substrate Rap1GTP
via distinct domains and can control integrin-mediated cell adhesion by
regulating Rap1 activation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RBD AF-6) was obtained by
deleting an N-terminal region between NotI and
AflII sites (1-128 residues) from a full-length AF-6
cDNA. cDNA of AF-6 lacking PDZ domain (
PDZ AF-6) was
constructed by deleting a fragment between BamHI and
SalI sites (910-1612 residues) from a full-length AF-6
cDNA, to which PCR-amplified fragments (910-990 and 1078-1612 residues) were ligated back consecutively. SPA-1 cDNA lacking GRD
(
GRD SPA-1) and C3G cDNA bearing a CAAX box
sequence at the C terminus (C3G-F) were reported before (9). These
plasmids were transfected into 293T or HeLa cells using a
CaPO4 precipitation method or Effectene Transfection
Reagent (Qiagen).
PDZ AF-6 cDNAs were
subcloned into KpnI and SalI sites of a pSP73
vector. In vitro transcription and translation (IVTT) of
each cDNA was performed using TNTTM
T7/SP6-coupled wheat germ extract system (Promega) in the presence of
[35S]methionine. In vitro binding assay was
performed as follows. Cell lysate of the 293T cells transfected with
pEF-BOS-AF-6 or pSR
-SPA-1 was immunoprecipitated with anti-AF-6,
anti-SPA-1, or preimmune IgG as a control followed by the precipitation
with protein A-Sepharose beads (Amersham Biosciences). The labeled IVTT
products above were incubated with such conjugated beads for 1 h
at 4 °C with gentle rotation. The beads were extensively washed,
eluted with SDS sample buffer, and electrophoresed in regular SDS-PAGE
or Tricine-buffered SDS-PAGE for smaller molecular mass IVTT products
followed by autoradiography.
vector by using
Effectene Transfection Reagent (Qiagen). These cells were cultured on
coverslips in the presence of Dox at an either inductive (0.1 ng/ml) or
non-inductive (1 ng/ml) dose for 24 h. The cells were rinsed with
Tris-buffered saline, fixed with 3% paraformaldehyde, permeated with
0.5% Triton X-100/Tris-buffered saline, blocked with 2% bovine serum
albumin/Tris-buffered saline, and incubated with anti-AF-6 or anti-Rap1
antibody followed by AlexaFluor 546 anti-rabbit IgG (Molecular Probes).
After washing, the cells were incubated further with biotinylated
anti-SPA-1 or anti-T7 antibody followed by AlexaFluor 488 streptavidin
or anti-mouse IgG. Normal rabbit or mouse IgG was used as a control for
the corresponding primary antibody. The stained cells were analyzed
using a confocal microscopy (Olympus).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Intracellular association of SPA-1 and AF-6.
A, 293T cells were transfected with SPA-1, AF-6, or both
cDNAs (1 µg each), and 2 days later aliquots of cell lysate of
each group were immunoprecipitated (IP) with anti-SPA-1,
anti-AF-6, or preimmune IgG followed by immunoblotting. SPA-1 was
detected as a 130-kDa band and AF-6 as an ~200-kDa band with a ladder
of smaller bands likely representing protein degradation. The
experiments were repeated three times with similar results.
B, left, freshly isolated mouse thymocytes and
splenic T and B cells were lysed and immunoblotted with indicated
antibodies. Right, thymocytes lysate was immunoprecipitated
with anti-SPA-1, anti-AF-6, or preimmune IgG and immunoblotted with the
indicated antibodies. Relative intensities of the immunoprecipitated
bands of heterologous antibody combinations to those of homologous
antibody combinations are indicated.
GRD SPA-1) and a PDZ-deletion mutant
of AF-6 (
PDZ AF-6) by IVTT. As shown in Fig. 2B,
GRD SPA-1 and
PDZ AF-6 failed to be bound to the beads coated with AF-6
and SPA-1 respectively, whereas full-length proteins were bound to the
partner proteins. These results strongly suggested that SPA-1 and AF-6
were associated via interaction between GRD of SPA-1 and PDZ domain of
AF-6.
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Fig. 2.
Requirement of GRD of SPA-1 and PDZ domain of
AF-6 for the binding. A, a series of truncated fragments of
SPA-1 (left) or AF-6 (right) were generated by
IVTT in the presence of [35S]methionine and
analyzed with SDS-PAGE followed by autoradiography (left
panel of each). Lysate of 293T cells transfected with AF-6 or
SPA-1 cDNA was immunoprecipitated with specific antibody and
protein A-Sepharose beads. Each IVTT fragment was incubated with the
AF-6- or SPA-1-coated Sepharose beads, and the specific binding was
determined by a pull-down assay followed by autoradiography
(right panel of each). Although not shown, none of the
fragments were precipitated by the control beads incubated with the
cell lysates and preimmune IgG. The experiments were repeated twice
with identical results. B, left, wild type
(wt) and GRD SPA-1 proteins generated by IVTT (left
panel) were incubated with AF-6-coated beads, and the washed beads
were eluted with SDS sample buffer followed by autoradiography
(right panel). Right, wild type and
PDZ AF-6
proteins generated by IVTT (left panel) were incubated with
SPA-1-coated beads, and the washed beads were eluted with SDS sample
buffer followed by autoradiography (right panel).
-X-
, where
is a hydrophobic residue), and a hydrophobic residue was also
preferred at
1 position (20). In the G2 fragment, it was noticed that
a stretch of residues at 432-434, IVF, fitted the predicted PDZ ligand
motif, which was in a probable
-sheet immediately followed by a turn
and a
-sheet as predicted by Chou-Fasman secondary structure
prediction (Fig. 3A). To investigate possible involvement of
this motif, we generated mutant proteins of G2, M1 (V433A), and M2
(F434A), by IVTT, and examined their binding to AF-6-coated beads. As
shown in Fig. 3B, M1 was bound barely to AF-6-coated beads
and M2 with markedly reduced efficiency as compared with G1, suggesting
that both Val-433 and Phe-434 were required for the binding to
AF-6 PDZ domain. Among known Rap1 GAPs, the motif was conserved (IVF for SPA-1 and E6TP1 and VVF for rapGAP), and a fragment of rapGAP I
(residues 263-322) corresponding to G2 of SPA-1 was bound specifically to the AF-6-coated beads comparably (Fig. 3B). Furthermore,
as shown in Fig. 3C, rapGAP I and AF-6 were
co-immunoprecipitated in the 293T cells co-transfected with full-length
rapGAP I and AF-6 cDNAs as well as in a renal cancer cell line
endogenously expressing both rapGAP I and AF-6, suggesting that the
binding to AF-6 was a shared feature of Rap1 GAPs.
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Fig. 3.
Probable involvement of internal PDZ ligand
motifs within GRD in the binding of SPA-1 to AF-6. A,
schematic illustration of G1, G2, and G3 fragments of SPA-1 GRD, and
amino acid sequence of G3 fragment. Aligned sequences of rapGAP and
E6TP1 are also shown. A potential ligand motif for AF-6 PDZ is
underlined, and mutated residues (V for M1
fragment and F for M2 fragment) are indicated in
boldface. A -finger-like structure predicted by
Chou-Fasman secondary structure prediction is also indicated.
B, IVTT products of G1, G2, G3, M1 (V432A), and M2 (F433A)
of SPA-1 and a fragment of rapGAP corresponding to G2 were incubated
with AF-6-coated or control (IgG) beads, and the binding were analyzed
as in Fig. 2A using Tricine-buffered SDS-PAGE. Input of each
IVTT product is shown in the left panel of each group.
C, left, 293T cells were transfected with AF-6
with or without rapGAP I cDNA (1 µg), and the lysates were
immunoprecipitated with anti-AF-6 followed by immunoblotting with
anti-rapGAP. Expression of transfected cDNAs was confirmed by
straight immunoblotting. Right, Caki-2 cells were lysed,
immunoprecipitated with anti-AF-6, anti-rapGAP, or control IgG, and
immunoblotted with anti-AF-6 antibody. These experiments were repeated
twice with reproducible results.
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Fig. 4.
Co-localization of SPA-1 and Rap1 with
AF-6 at the cell adhesion sites. A and B,
HeLa/Tet-SPA-1 (A) and HeLa/Tet-SPA-1/AF-6 cells
(B) were cultured in the presence of inductive (0.1 ng/ml)
and non-inductive (1.0 ng/ml) doses of Dox for 24 h, respectively,
and double-stained with anti-SPA-1 and anti-AF-6 antibodies.
C, HeLa/Tet-SPA-1/AF-6 cells additionally transfected with
T7-tagged RapV12 cDNA were cultured in the presence of 1.0 ng/ml
Dox for 24 h and double-stained with anti-T7 and anti-AF-6
antibodies. Intense nuclear staining with anti-T7 antibody was
nonspecific. D and E, HeLa/Tet-SPA-1/AF-6 cells
were cultured in the presence of 0.1 ng/ml Dox for 24 h and
double-stained with anti-SPA-1 and anti-AF-6 antibodies (D)
or with anti-SPA-1 and anti-Rap1 antibodies (E).
Merge pictures are also shown. Arrows indicate
the localization of proteins at the cell attachment sites.
RBD AF-6, which failed to be co-immunoprecipitated
with RapV12, hardly induced the co-immunoprecipitation of SPA-1 with
RapV12 either (Fig. 4A). The results indicated that AF-6
could bind both SPA-1 and Rap1GTP simultaneously via distinct domains,
leading to the efficient association of SPA-1 and Rap1GTP. We then
examined the effect of AF-6 expression on the efficiency of Rap1
inactivation by SPA-1 in the cells. Expression of SPA-1 in 293T cells
suppressed the amount of endogenous Rap1GTP generated by the
transfection of C3G-F cDNA in a dose-dependent manner
as reported previously (9). Additional expression of AF-6 significantly
enhanced the Rap1 inactivation by SPA-1 (Fig. 4B). Thus,
transfection of 0.3 µg of SPA-1 cDNA resulted in almost complete
inhibition of Rap1GTP generation in the presence of AF-6, whereas 40%
of Rap1GTP still remained in the absence of AF-6 (Fig. 4B).
As also shown in Fig. 4B, the enhancing effect of AF-6 on
the Rap1 inactivation by SPA-1 was reduced markedly by the deletion of
the PDZ domain.
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Fig. 5.
Association of SPA-1 with Rap1GTP and
enhanced efficiency of Rap1 inactivation by SPA-1 in the presence of
AF-6. A, 293T cells were transfected with an empty
vector ( ), AF-6 (wt), or
RBD AF-6 cDNA along with SPA-1 and
T7-tagged RapV12 cDNAs (1 µg each). Two days later, the cells
were lysed and immunoprecipitated (IP) with anti-T7 antibody
followed by immunoblotting with anti-AF-6, anti-SPA-1 or anti-Rap1
antibody (right panels). Expression of each protein was
determined by immunoblotting of the straight lysates (left
panels). The experiment was repeated twice with similar results.
B, 293T cells were transfected with C3G-F (1 µg) and
varying doses of SPA-1 cDNA (0, 0.3, and 1.0 µg) with or without
AF-6 or
PDZ AF-6 cDNA (1 µg). Two days later, Rap1GTP in the
cell lysates (1 mg of proteins) was detected by a GST-RalGDS RBD
pull-down assay. Expression of total Rap1, SPA-1, and AF-6 was
determined by immunoblotting of the straight lysates. Relative
intensities of Rap1GTP bands are indicated. The experiment was repeated
twice with reproducible results.
1-Integrin-mediated Cell Adhesion--
We finally
investigated functional effects of the association of SPA-1 and AF-6 on
cell adhesion. 293T cells in suspension adhered to fibronectin
(FN)-coated plates in a manner dependent on VLA4 andVLA5 (Fig.
6A). Although expression of
SPA-1 in 293T cells significantly inhibited the cell adhesion as
reported before (9), that of AF-6,
RBD AF-6, or
PDZ AF-6 alone
hardly affected the cell adhesion (Fig. 6A). However,
co-transfection of AF-6 with SPA-1 cDNA resulted in the greater
inhibition of cell adhesion than that of SPA-1 alone (Fig.
6A). Neither
RBD AF-6 nor
PDZ AF-6 affected the
SPA-1-induced inhibition of cell adhesion, suggesting that the effect
of AF-6 was dependent on the association with SPA-1 and Rap1. Similar
experiments were performed using HeLa/Tet-SPA-1 cells. HeLa/Tet-SPA-1
cells were transfected with a control vector, AF-6, or
PDZ AF-6
cDNA and cultured for 1 day in the presence of Dox at 1.0 or 0.1 ng/ml. At 1.0 ng/ml Dox, SPA-1 expression was almost completely
repressed (Fig. 6B, left). Under this condition, expression of AF-6 or
PDZ AF-6 induced a slight increase in Rap1GTP, and the cell adhesion tended to be enhanced marginally although with
statistical insignificance (Fig. 6B, right). When
the cells were cultured in the presence of 0.1 ng/ml Dox, on the other
hand, SPA-1 expression was induced, and concomitantly Rap1GTP was
reduced, leading to the significant decrease in the cell adhesion (Fig. 6B). Expression of AF-6 under this condition resulted in the
much greater decrease in both cell adhesion and Rap1GTP level, whereas that of
PDZ AF-6 was without effect at all (Fig. 6B).
These results suggested that AF-6 could contribute to the inhibition of
1 integrin-mediated cell adhesion by enhancing the
efficiency of SPA-1-mediated Rap1GTP inactivation.
View larger version (40K):
[in a new window]
Fig. 6.
Enhancement of the inhibitory effect of SPA-1
on 1 integrin-mediated cell adhesion by AF-6.
A, left, 239T cells in suspension were plated
onto FN-coated wells (5 µg/ml) in the absence or presence of
anti-VLA-4, anti-VLA-5, or both antibodies (40 µg/ml each) for 30 min, and adherent cells were quantified. Middle and
right, 293T cells were transfected with wt AF-6,
RBD
AF-6, or
PDZ AF-6 cDNA (2 µg) with (solid columns)
or without (open columns) SPA-1 cDNA (3 µg). Two days
later, cells were trypsinized and plated on FN-coated wells, and cell
adhesion was determined after 30 min. The means of triplicate plates
and S.E. as well as p values by Student's t test
for the indicated combinations are indicated. These experiments were
repeated three times with similar results. B,
left, HeLa/Tet-SPA-1 cells were transfected with an
empty vector, wt AF-6, or
PDZ AF-6 cDNA (2 µg) and cultured in
the presence of 1 ng/ml or 0.1 ng/ml Dox for 1 day. Rap1GTP in the
lysate of each group (800 µg of proteins) was detected by a pull-down
assay using GST-RalGDS. Relative intensities of the signals to that in
the HeLa/Tet-SPA-1 cells at 1 ng/ml Dox are indicated. Expression of
total Rap1 as well as transfected AF-6 and SPA-1 proteins in each group
was determined by straight immunoblotting. Right,
aliquots of above cells were trypsinized, washed, gently rotated for
2 h in suspension, and plated on FN-coated wells (0.5 × 105 cells/well), and cell adhesion was determined after 30 min. The means and S.E. of triplicate wells as well as p
values by Student's t test for the indicated combinations
are provided. Open columns, 1 ng/ml Dox; solid
columns, 0.1 ng/ml Dox.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-finger structure of neuronal nitric-oxide synthase mimicked a
C-terminal ligand motif as a pseudo-peptide (27). It was reported that
a class 2 ligand motif (
-X-
) with hydrophobic residues also at the
1 position was preferred by AF-6 PDZ domain by using peptide libraries (20). SPA-1 GRD contained a stretch of residues IVF
(residues 432-434), which fitted the predicted ligand motif for AF-6
PDZ, and the mutations of Val-433 to Ala or Phe-434 to Ala markedly
reduced the binding of a minimal fragment of SPA-1 (G2) to AF-6
in vitro. Marginally retained binding activity of a F434A
mutant fragment may be due to the residual activity of alanine as an
anchoring residue. Chou-Fasman secondary structure prediction suggested
that the motif was located in a
-finger-like structure. These
results suggested, but did not prove, that the interaction of internal
PDZ ligand motif in SPA-1 GRD with the PDZ domain of AF-6 mediated the
specific binding of SPA-1 to AF-6. The motif was conserved in rapGAP
and E6TP1, and present results indicated that rapGAP was bound to AF-6
comparably with SPA-1, suggesting that the binding to AF-6 was a shared
feature of Rap1 GAPs. On the other hand, mutations of RKR at positions
421-423 to LIG, which attenuated the GAP catalytic activity in
vivo, did not affect the binding of SPA-1 to AF-6 at
all.2
RBD AF-6. Collectively these results strongly suggested that both SPA-1 and Rap1GTP (RapV12) could be recruited to
AF-6 via independent binding sites, a central PDZ domain and a
N-terminal RBD, respectively.
1 and
2 integrins and induced the
integrin-mediated cell-cell or cell-matrix adhesion, whereas SPA-1
could negatively regulate the cell adhesion by inactivating Rap1. The
present results using HeLa/Tet-SPA-1 cells indicated that expression of
AF-6, but not
PDZ AF-6, induced the decrease in Rap1GTP levels and
significant inhibition of
1 integrin-mediated cell
adhesion to FN in an SPA-1-induced condition, whereas AF-6 did not
affect either of them in an SPA-1-repressed condition. These results
have suggested strongly that AF-6 plays a role in the control of
integrin-mediated cell adhesion by enhancing the efficiency of Rap1
inactivation by SPA-1 at the cell adhesion sites.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. K. Katagiri and T. Kinashi for assistance of cell adhesion assay.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan.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: Dept. of
Immunology and Cell Biology, Graduate School of Medicine, Kyoto
University, Sakyo, Kyoto 606-8501, Japan. Tel.: 81-75-753-4659; Fax:
81-75-753-4403; E-mail: minato@imm.med.kyoto-u.ac.jp.
Published, JBC Papers in Press, February 15, 2003, DOI 10.1074/jbc.M211888200
2 L. Su, M. Hattori, and N. Minato, unpublished observations.
3 K. Kometani, M. Hattori, and N. Minato, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: GAPs, GTPase activating proteins; IVTT, in vitro transcription and translation; GRD, GAP-related domain; RBD, Ras/Rap1-binding domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FN, fibronectin; wt, wild type; Dox, doxycycline.
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