* Division of Signal Transduction, Nara Institute of Science and Technology, Nara 630-01, Japan; Department of Molecular Cell
Biology, Weizmann Institute of Science, Rehovot 76100, Israel; § Department of Virology II, National Institute of Infectious
Diseases, Tokyo 162, Japan; and
Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
RAS (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing,
guanine nucleotide-binding protein for various membrane receptors including tyrosine kinase receptors. Ras participates in the regulation of cell proliferation,
differentiation, and morphology (for reviews see Satoh et al.,
1992 The dynamic rearrangement of cell-cell contacts is a critical step in various cellular processes including the establishment of epithelial cell polarity and developmental patterning (for review see Gumbiner, 1996 In light of these observations, we investigated roles of
Ras and its target AF-6 in the regulation of cell-cell contacts. We found that AF-6 accumulated at cell-cell contact
sites of MDCKII epithelial cells and had a distribution
similar to that of ZO-1. AF-6 interacted with ZO-1, and
this interaction was inhibited by activated Ras.
Materials and Chemicals
MDCKII cell, mouse anti-ZO-1 antibody and rat anti- Cell Culture
MDCKII and Rat1 cells were grown in DME containing 10% FCS, penicillin, and streptomycin in an air-5% CO2 atmosphere at constant humidity. Rat1 RasVal A1 cells were grown in DME containing 10% FCS, 1 mg/ml
Geneticin, 0.4 mg/ml Hygromycin B, penicillin, and streptomycin in an
air-5% CO2 atmosphere at constant humidity. PC12 cells were grown in
DME containing 10% FCS, 5% horse serum, penicillin, and streptomycin
in an air-5% CO2 atmosphere at constant humidity. For Ca2+ switch assay,
subconfluent MDCKII cells were grown in normal growth media and
transferred to low Ca2+ medium (growth media containing 4 mM EGTA)
for 6 h and were then transferred back to the normal Ca2+ medium. To examine the distribution of AF-6 in Rat1 RasVal A1 cells, Rat1 RasVal A1
cells plated on 13-mm-round glass coverslips were incubated either with
or without 5 mM isopropyl- Immunofluorescence and Laser Scanning
Confocal Microscopy
MDCKII, PC12, Rat1, and Rat1 RasVal A1 cells plated on 13-mm-round
glass coverslips were fixed in 4% paraformaldehyde in PBS for 10 min and
permeabilized with 0.2% Triton X-100 in PBS for 10 min. The fixed cells
were incubated with primary antibodies for 1 h at room temperature and
washed three times for 10 min with PBS. After the first labeling, the cells
were incubated for 1 h with secondary antibodies and washed three times
for 10 min with PBS. The distributions of AF-6, ZO-1, and Immunoelectron Microscopy
Immunoelectron microscopy was carried out as described previously
(Burry et al., 1992 Immunocytochemistry of Mouse Intestinal Epithelium
Mice were anesthetized with halothane and fixed by transcardiac perfusion with PBS containing 2% paraformaldehyde, 1 mM CaCl2 and 8% sucrose. The intestine was removed, immersed in the same fixative, and cryoprotected through a range of increasing sucrose concentrations (10, 15, 20, and 25%) in 50 mM Tris-buffered saline. The intestine was embedded in
OCT compound, quick-frozen, and cut into 8-µm-thick sections in a cryostat. The frozen sections, mounted on slides, were then washed with PBS,
fixed in 4% paraformaldehyde in PBS for 10 min, and permeabilized with
0.2% Triton X-100 in PBS for 10 min. The samples were subjected to immunostaining and examined as described above.
Affinity Column Chromatography
GST fusion proteins (each 6 nmol) were immobilized on 200 µl of glutathione Sepharose 4B packed into columns. Bovine membrane fraction
or crude Sf-9 cell lysate expressing HA-AF-6 was loaded onto the affinity
columns. The columns were washed with 2 ml (10 vol) of buffer B (20 mM
Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2, 10 µM
[p-amidino-phenyl] methanesulfonyl fluoride, 10 µg/ml leupeptin), followed by washing with 2 ml (10 vol) buffer B containing 50 mM NaCl. The
proteins bound to the affinity columns were eluted three times by the addition of 660 µl (3.3 vol) of buffer B containing 10 mM reduced glutathione. The eluates were subjected to SDS-PAGE and transferred to
PVDF membranes. The eluted native AF-6 and recombinant HA-AF-6
were immunodetected with the anti-AF-6 antibody.
In Vitro Binding Assay (In Vitro-Translated AF-6)
The in vitro translation of pRSET-AF-6 (36-494 amino acids), AF-6 (495-
909 amino acids), AF-6 (914-1,129 amino acids), and AF-6 (1,130-1,612
amino acids) were performed using the TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI) under the conditions described in the
manufacturer's instruction manual. GST fusion proteins were immobilized onto 31 µl of glutathione Sepharose 4B beads and washed with 310 µl
(10 vol) of buffer B. The immobilized beads were added to 40 µl of the in
vitro-translated products labeled with [35S]methionine containing 1 mg/ml
BSA and incubated for 1 h at 4°C with gentle mixing. The beads were
washed three times with 102 µl (3.3 vol) of buffer B, and the bound proteins were eluted with GST fusion proteins three times by the addition of
102 µl (3.3 vol) of buffer B containing 10 mM reduced glutathione. The
eluates were subjected to SDS-PAGE and vacuum dried. The 35S-labeled
bands corresponding to in vitro-translated AF-6 were visualized with an
image analyzer (BAS-2000; Fuji, Tokyo, Japan).
In Vitro Binding Assay (MBP-AF-6)
The expression of maltose-binding protein (MBP)-AF-6 (36-206 amino
acids) in E. coli was performed as described previously (Kuriyama et al.,
1996 Dissociation of AF-6 from ZO-1 by Activated Ras
GST-Ha-Ras and GST-Rac were produced in E. coli DH5 Other Procedures
SDS-PAGE was performed as described (Laemmli, 1970 Colocalization of AF-6 with ZO-1 But Not
with To clarify the functions of AF-6, we examined its intracellular distribution in MDCKII epithelial cells. First, immunoblot analysis was performed on cell lysates from MDCKII
cells, using a polyclonal anti-AF-6 antibody. The anti-AF-6
antibody recognized two isoforms of AF-6 with molecular
masses of ~180 and 195 kD in the sample obtained from
MDCKII cells (Fig. 1) as described for bovine brain extract (Kuriyama et al., 1996
We then examined the AF-6 localization in confluent
MDCKII cells, which show characteristics of polarized epithelial cells and form the junctional complex, including the
tight junctions and adherens junctions, at cell-cell contact
sites (Gonzalez-Mariscal et al., 1985
Immunoelectron Microscopic Localization of AF-6 and
ZO-1 in Confluent MDCKII Cells
The ultrastructural localization of AF-6 and that of ZO-1
in MDCKII cells was further analyzed by the pre-embedding procedure. The gold signals immunoreactive for AF-6
were identified on the cytoplasmic surface of the plasma
membranes in the junctional complex region (Fig. 3 a). The
area immunoreactive for AF-6 was 400-500 nm long in
cross-section and usually started from the apical-most point
where the apical-adjacent plasma membranes converged (Fig. 3 a). A few gold particles were noted along the lateral plasma membranes outside the junctional complex region.
The gold signals for ZO-1 were also identified to be almost
exclusively concentrated on the cytoplasmic surface of the
plasma membranes in the junctional complex region (Fig.
3 b). The area immunoreactive for ZO-1 was 300-500 nm
long and always started from the apical-most point of the
junctional complex region. When this junctional complex region was double labeled with anti-AF-6 polyclonal antibody and anti-ZO-1 monoclonal antibody, AF-6 immunoreactivity and that of ZO-1 was colocalized in the main
part of the junctional complex region (Fig. 3, c and d). In
Fig. 3 d, a cross-section of a junctional complex is seen in
the center, and an oblique section of another junctional
complex is seen in the right side of the photograph. Taken
together, these results indicate that AF-6 is colocalized with ZO-1 in the junctional complex.
Ca2+-dependent Distribution of AF-6 and ZO-1 at
Cell-Cell Contact Sites of MDCKII Cells
We examined the localization of AF-6 in MDCKII cells during the formation and disappearance of cell-cell contacts.
The formation of cell-cell contacts and the junctional
complex can be reversed by transferring cells between low
Ca2+ and normal Ca2+ media (Ca2+ switch experiment).
When the MDCKII cells were cultured in low Ca2+ media
for 6 h, cell-cell contacts were disrupted and the cells became rounded up, as described previously (Fig. 4 d; Gonzalez-Mariscal et al., 1985
Localization of AF-6 in Mouse Intestinal
Epithelial Cells
Because intestinal epithelial cells have tight junctions at
the apical cell borders, we examined the distribution of AF-6 in intestinal epithelial cells. Cryosections of mouse intestine were double labeled with AF-6 and ZO-1 antibodies
and examined by laser scanning confocal microscopy. AF-6
and ZO-1 were colocalized at the apical cell borders (Fig.
5). These results indicate that AF-6 is colocalized with
ZO-1 at tight junctions of intestinal epithelial cells.
Localization of AF-6 in Cells Lacking Tight Junctions
In cells lacking tight junctions such as nonepithelial cells,
ZO-1 accumulates at cell-cell contact sites with cadherin
(Howarth et al., 1992
Interaction of Bovine AF-6 with ZO-1 and Occludin
To examine the complex formation of AF-6 with tight junction components including ZO-1 and occludin, bovine brain
membrane fraction was loaded onto the affinity columns
immobilized with GST, GST-ZO-1, GST-occludin, GST-
E-cadherin, GST-
Interaction of Recombinant AF-6 with ZO-1
To determine whether recombinant AF-6 interacts with
ZO-1 and occludin, crude lysates of Sf-9 cells infected with
baculovirus carrying the cDNA of HA-AF-6 (36-1,608
amino acids) were loaded onto the GST, GST-ZO-1, GST-occludin, and GST-CD44 affinity columns. The proteins
bound to the affinity columns were eluted with GST fusion
proteins by the addition of glutathione, and the eluted HA-AF-6 was detected with the anti-AF-6 antibody. HA-AF-6 was detected in the eluate of the GST-ZO-1 affinity
column and weakly in that of GST-occludin affinity column, but not in those of the GST or GST-CD44 affinity
columns (Fig. 8). These results indicate that recombinant
AF-6 interacts with ZO-1. The reason for the weak interaction of recombinant AF-6 and occludin is not clear at
present. The recombinant AF-6 produced from Sf-9 cells
may have a lower affinity for occludin than that of native
AF-6, or the interaction of AF-6 and occludin is indirect
and this interaction may be mediated by ZO-1. When the
same samples were examined with HA antibody, smaller
size bands were detected in addition to full size AF-6 in
the eluate of the GST-ZO-1 affinity column (data not
shown). Since the AF-6 used in this experiment was tagged with HA in its NH2 terminus, these smaller bands are
thought to represent the products degraded from the
COOH terminus. Since the size of the major smaller band
was ~50 kD, the interacting region of AF-6 with ZO-1
may contain the Ras-binding domain, which is identified
as 36-206 amino acids (Kuriyama et al., 1996
We further examined which domain of AF-6 interacts
with ZO-1, using in vitro-translated AF-6 such as AF-6
(36-494 amino acids), AF-6 (495-909 amino acids), AF-6
(914-1,129 amino acids), and AF-6 (1,130-1,612 amino acids) (Fig. 9 a). Affinity beads immobilized with GST-ZO-1
were mixed with the in vitro-translated AF-6 (36-494 amino acids), AF-6 (495-909 amino acids), AF-6 (914-1,129
amino acids), and AF-6 (1,130-1,612 amino acids), and the
interacting proteins were then eluted with GST-ZO-1 by
the addition of glutathione. As shown in Fig. 9, b-e, AF-6
(36-494 amino acids) bound to GST-ZO-1 and weakly to
GST-occludin, but not to GST or GST-CD44, whereas the
other domains slightly bound to GST-ZO-1 and not to GST,
GST-occludin, or GST-CD44. These results together with
the above observations indicate that mainly the NH2-terminal domain of AF-6 is responsible for the binding of AF-6
to ZO-1.
Dissociation of AF-6 from ZO-1 by Activated Ras
Since the Ras-binding domain and ZO-1-binding domain
are very close as noted above, we speculated that activated
Ras inhibited the interaction of AF-6 and ZO-1. To test
this possibility we first examined whether ZO-1 could bind
to MBP-AF-6 (36-206 amino acids), which binds to activated Ras as previously described (Kuriyama et al., 1996
Localization of AF-6 in Activated Ras-expressing Rat1
Fibroblast Cells
As previously described, some ras-transformed fibroblast
cells display anchorage-independent growth and reduced
cell-cell contacts. We examined the localization of AF-6 in
activated Ras-expressing Rat1 fibroblast cells. For this purpose, we used a Rat1 cell line that contains the activated
ras (rasV12) gene under Lac repressor control, designated
as Rat1 RasVal A1 cells. The addition of IPTG efficiently
induced the expression of RasV12 (Fig. 11 A). The distributions of AF-6 and ZO-1 were examined as described above.
In the Rat1 RasVal A1 cells in the absence of IPTG, AF-6
accumulated at cell-cell contact sites and was colocalized with ZO-1, as seen in the wild-type strain of Rat1 cells (Fig. 11, B, c and d). When Rat1 RasVal A1 cells were treated
with IPTG, their morphology changed dynamically and
the cell-cell contacts were perturbed. In these cells, the
colocalization of AF-6 and ZO-1 was decreased at the surface of the cells dissociated from neighbor cells (Fig. 11, B,
e and f). These results suggest that AF-6 may contribute to
the perturbation of cell-cell contacts by activated Ras, or
that the altered cell-cell contacts by activated Ras may
change the cellular distribution of AF-6.
AF-6 Is a Peripheral Component of Junctional Complex
Our immunofluorescent and immunoelectron microscopic
analysis showed that AF-6 is concentrated at tight junctions in confluent MDCKII cells, and that its distribution
closely overlaps with that of ZO-1. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 is thought to
directly interact with occludin and to serve as an essential
peripheral component of tight junctions (Furuse et al.,
1994 A recent study showed that ZO-1 interacts with catenins
during the early stages of the assembly of tight junctions
(Rajasekaran et al., 1996 ZO-1-binding Domain of AF-6
Since it has been reported that some PDZ domains can
bind to other PDZ domains (Brenman et al., 1996 The GST-ZO-1 used in this study contains three PDZ
domains, an SH3 domain, and a guanylate kinase domain.
It is possible that the SH3 domain of ZO-1 interacts with
the proline-rich domain of AF-6. Indeed, our in vitro binding assay showed that AF-6 (1,130-1,612 amino acids) interacts with ZO-1, but this interaction is weaker than that
of AF-6 (36-494 amino acids). The AF-6-binding domain of ZO-1 remains to be identified. The interaction of recombinant AF-6 with occludin is very weak. It remains to
be clarified whether AF-6 directly interacts with occludin
in vivo.
Possible Roles of AF-6 in the Regulation of
Cell-Cell Contacts
AF-6 is composed of several domains including Ras-binding, ZO-1-binding, myosin V-like, PDZ, and the COOH-terminal domains. Although the roles of AF-6 in the regulation of cell-cell contacts remain to be clarified, AF-6 may
be a scaffolding protein as a peripheral component at the
cell-cell contact sites. AF-6 shows the strong sequence homology with Drosophila Canoe and shares a common domain organization with Canoe (Kuriyama et al., 1996 We have shown here that AF-6 can interact with ZO-1
in vitro and is colocalized with ZO-1 at cell-cell contact
sites including tight junctions. This suggests that AF-6 can
modulate the function of ZO-1. ZO-1 is thought to be a
linkage molecule between occludin and actin cytoskeleton
and to play important roles in the rearrangement of cell-
cell contacts and cytoskeletons (Furuse et al., 1994 We have recently found that AF-6 interacts with filamentous actin by a cosedimentation assay (data not shown).
Actin cytoskeleton is known to be linked to adhesion molecules through certain peripheral components and to support the cell-cell adhesions. Although the mode of interaction between AF-6 and filamentous actin is not known at
present, the actin-binding activity of AF-6 may be necessary for the regulation of the cell-cell adhesions through
AF-6. The COOH-terminal domain of AF-6 has the proline-rich region, which is thought to interact with certain
proteins containing the SH3 or WW domain (Sudol, 1996 Regulation of the Interaction of AF-6 and ZO-1 by
Activated Ras
As described above, it is likely that the Ras-binding domain and ZO-1-binding domain of AF-6 are quite close.
This led us to examine whether Ras inhibits the interaction of AF-6 with ZO-1. Indeed, activated Ras can dissociate MBP-AF-6 (36-206 amino acids) from ZO-1. This suggests that activated Ras inhibits the interaction of AF-6
and ZO-1 at cell-cell adhesions. Our preliminary experiments, however, showed that activated Ras dissociates full
length recombinant AF-6 from ZO-1 less efficiently than
MBP-AF-6 (36-206 amino acids) from ZO-1 (data not
shown). The reason for the less efficient dissociation of full
length AF-6 from ZO-1 is not known. Another domain of
AF-6 in addition to the NH2-terminal domain containing the Ras-binding domain may strengthen the interaction of
AF-6 and ZO-1. Alternatively, activated Ras may change
the state of the AF-6-ZO-1 complex without dissociating
AF-6 from ZO-1. In addition, since we used the recombinant Ras produced from bacteria, which was not modified
with lipid at the COOH terminus, it is possible that lipid-modified, activated Ras can efficiently dissociate full length
AF-6 from ZO-1.
Regulation of Cell-Cell Contacts by Ras
Ras is thought to regulate the dynamics of cell-cell contacts. Some ras-transformed epithelial cells display a fibroblastic morphology and reduced cell-cell contacts, as previously described (Basolo et al., 1991; McCormick, 1994
). Activated ras oncogenes have been
identified in various forms of human cancers including epithelial carcinomas of the lung, colon, and pancreas (Barbacid, 1987
). These cancer cells, as well as those that have
been experimentally transformed by the activated ras
gene, exhibit morphological changes and alterations of cell
adhesions. Ras is thought to affect cell adhesions and cytoskeletons via alterations of the signaling pathways downstream of Ras. Ras has GDP-bound inactive and GTP-bound active forms, the latter of which interact with targets
(Marshall, 1995b
). The Raf kinase family, consisting of
c-Raf-1 (for reviews see Blenis, 1993
; Daum et al., 1994
),
A-Raf (Vojtek et al., 1993
), and B-Raf (Catling et al., 1994
;
Jaiswal et al., 1994
; Moodie et al., 1994
; Yamamori et al.,
1995
), is one of the direct targets for Ras. The activated
Raf phosphorylates mitogen-activated protein (MAP)1 kinase kinase and activates it. Consequently the activated
MAP kinase kinase activates MAP kinase, leading to the
expression of certain genes such as c-fos (for reviews see
Cano and Mahadevan, 1995
; Marshall, 1995a
). Several
molecules interacting with activated Ras in addition to Raf
have been identified as Ras targets in mammals. These include phosphatidylinositol-3-OH kinase (Rodriguez-Viciana et al., 1994
), Ral guanine nucleotide dissociation stimulator (Kikuchi et al., 1994
; Spaargaren and Bischoff, 1994
),
and Rin1 (Han and Colicelli, 1995
). We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6)
as a novel Ras target (Kuriyama et al., 1996
). AF-6 was identified as the fusion partner of the acute lymphoblastic leukemia-1 (ALL-1) protein (Prasad et al., 1993
). The ALL-1/
AF-6 chimeric protein is the critical product of the t(6;11)
abnormality associated with some human leukemia. AF-6
has a PDZ (GLGF/DHR) domain that is found in a number of other proteins, including postsynaptic density protein 95 (PSD-95) (Cho et al., 1992
), Drosophila discs-large
tumor suppressor gene product (Dlg) (Woods and Bryant,
1991
), and a tight junction-associated protein, ZO-1 (Itoh
et al., 1993
; Willott et al., 1993
). The PDZ domain is thought
to localize these proteins at the specialized sites of cell-cell
contact by forming a complex with specific proteins such as
the NMDA receptor and the K+ channel (Kim et al., 1995
;
Kornau et al., 1995
).
). Cell-cell junctions
are categorized into at least three groups: tight, adherens,
and gap junctions (for reviews see Farquhar and Palade,
1963
; Tsukita et al., 1993
). Tight junctions, the most apical
components of the junctional complex, form a diffusion
barrier that regulates the flux of ions and hydrophilic molecules through the paracellular pathway (for reviews see
Diamond, 1977
; Gumbiner, 1987
). Tight junctions are mediated by molecules such as occludin and its associated
ZO-1 and -2 (Anderson et al., 1988
; Furuse et al., 1994
; for
review see Anderson, 1996
). Adherens junctions are characterized by a well-developed plaque structure in which
actin filaments are densely associated. Adherens junctions
are mediated by adhesion molecules such as cadherin and
its associated catenins (for reviews see Takeichi, 1990
; Hülsken et al., 1994
). These junctions undergo dynamic remodeling when cells move or enter the mitotic phase. Although these junctions are suspected to be regulated by
certain intracellular signaling pathways, little is known at
present about how they are actually regulated. Accumulating evidence suggests that small GTPases Ras and Rho
family members including Rho, Rac, and Cdc42 participate in the regulation of cell adhesions (for review see
Hall, 1994
).
Materials and Methods
-catenin antibody
were kindly provided by Dr. S. Tsukita (Kyoto University, Kyoto, Japan;
Itoh et al., 1991
). Rat1 and Rat1 RasVal A1 cells were kindly provided by
Dr. Y. Kaziro (Tokyo Institute of Technology, Yokohama, Japan). Rabbit
polyclonal antibody against AF-6 (1,130-1,612 amino acids) was generated as described previously (Harlow and Lane, 1988
). FITC-conjugated
anti-rabbit IgG, Texas red-conjugated anti-mouse IgG, and [35S]methionine were purchased from Amersham Intl. (Arlington Heights, IL). The expression plasmids of glutathione-S-transferase (GST)-mouse ZO-1 (1-862
amino acids), GST-mouse ZO-2 (1-400 amino acids), GST-mouse occludin (264-521 amino acids), GST-E-cadherin cytoplasmic domain, GST-mouse
-catenin and GST-
-catenin were kindly provided by Dr. S. Tsukita
(Kyoto University). GST fusion proteins were expressed in Escherichia
coli BL21(DE3) and purified according to the manufacturer's instructions.
Bovine brain membrane fraction was prepared as described previously
(Kuriyama et al., 1996
) and preabsorbed to remove the native GST with
glutathione Sepharose 4B (Pharmacia Biotech Inc., Grand Island, NY).
To obtain recombinant HA-AF-6 (36-1,608 amino acids), the baculovirus
expression plasmid pAcYM1-HA-AF-6 (36-1,608 amino acids) was constructed as follows. The cDNA fragment encoding AF-6 (36-1,608 amino
acids) was amplified by polymerase chain reaction from the full length
AF-6 cDNA in pBluescript and was subcloned into the KpnI site of
pAcYM1-HA (Matsuura et al., 1987
). Sf9 cells infected with baculovirus
carrying the cDNA of HA-AF-6 (36-1,608 amino acids) were suspended
in buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, 5 mM
MgCl2, 10% sucrose, 10 µM [p-amidino-phenyl] methanesulfonyl fluoride, 10 µg/ml leupeptin). The suspension was sonicated and centrifuged at
100,000 g for 60 min at 4°C. The supernatant was used for affinity column
chromatography. Other materials and chemicals were obtained from commercial sources.
-D-thiogalactoside (IPTG) for 24 h.
-catenin were examined with a laser scanning confocal microscope (Carl Zeiss, Inc.,
Thornwood, NY) equipped with an argon laser and a helium-neon laser
for double fluorescence at 488 and 543 nm (emission filter; BP510-525 and
LP590). 20 horizontal confocal sections were obtained for MDCKII cells
and used to generate three-dimensional images.
; Jongens et al., 1994
; Uchida et al., 1996
). MDCKII cells
were cultured on tissue culture-treated polycarbonate filters (Transwell;
Coaster Corp., Cambridge, MA) with a pore size of 0.4 µm and a diameter
of 6.5 mm, and maintained for 2 wk after being confluent. The samples
were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, rinsed with PBS for 10 min, and permeabilized with PBS containing
5% BSA and 0.02% saponin for 10 min. The fixed cells were incubated
with rabbit anti-AF-6 (1:100) in PBS containing 0.005% saponin or a hybridoma culture medium containing mouse anti-ZO-1 antibody for 2 h,
followed by three washes with PBS containing 0.005% saponin for 20 min
in each step. The cells were incubated with an anti-rabbit antibody or an
anti-mouse antibody coupled with 1.4-nm gold particles (Nanoprobes, Inc.,
Stony Brook, NY) for 2 h, followed by three washes with PBS containing
0.005% saponin. For the double staining study, the samples were incubated with a hybridoma culture medium containing mouse anti-ZO-1 antibody and 0.005% saponin for 2 h and washed three times with PBS
containing 0.005% saponin. The cells were then incubated with HRP-conjugated anti-mouse antibody (1:1,000; Jackson ImmunoResearch Lab.,
Inc., West Grove, PA) followed by three washes with PBS containing
0.005% saponin. The samples were incubated twice with 0.1 M PB (0.1 M
sodium phosphate buffer at pH 7.4) for 5 min, fixed with 0.1% glutaraldehyde in 0.1 M PB for 10 min, and rinsed with 0.1 M PB for 5 min. The samples were then incubated three times with 50 mM Hepes, at pH 5.8, for 10 min. The signals were silver enhanced with the use of HQ silver (Nanoprobes) for 8 min at 18°C in the dark, followed by three washes with 0.1 M
PB. The samples were incubated with a DAB solution (50 mM Hepes at
pH 7.4, 0.05% 3,3
-diaminobenzidine tetrahydrochloride) for 30 min and
then incubated with the DAB solution containing 0.01% H2O2 for 20 min.
The samples were postfixed with 0.5% OsO4 in 0.1 M PB for 60 min at
4°C, washed twice with 0.1 M PB for 5 min, dehydrated by passage through
a graded series of ethanol (50, 60, 70, 80, 90, 95, and 100%) and propylene
oxide, and then embedded in epoxy resin (Polysciences, Inc., Warrington,
PA). Ultrathin sections were cut, stained with lead citrate, and observed with an immunoelectron microscope (JEM-1200; JEOL, Tokyo, Japan).
). GST-ZO-1 (0.1 nmol) was immobilized on 100 µl of glutathione
Sepharose 4B packed into columns. E. coli BL21(DE3) producing MBP-AF-6 (36-206 amino acids) was suspended in buffer A. The suspension
was sonicated and centrifuged at 100,000 g for 60 min at 4°C. The supernatants containing various concentrations of MBP-AF-6 (36-206 amino acids) were then loaded onto the GST-ZO-1 columns. The columns were
washed with 1 ml (10 vol) of buffer B, followed by washing with 1 ml (10 vol) buffer B containing 50 mM NaCl. The proteins bound to GST-ZO-1
columns were eluted with GST-ZO-1 three times by the addition of 330 µl
(3.3 vol) of buffer B containing 10 mM reduced glutathione. The eluted
MBP-AF-6 was immunodetected with the anti-MBP antibody. The immunodetected MBP-AF-6 was visualized and estimated with a densitograph
(ATTO, Tokyo, Japan).
and cleaved
with thrombin according to the manufacturer's instructions. The guanine
nucleotide-bound forms of Ha-Ras and Rac were made by incubating
Ha-Ras or Rac (5 nmol) for 1 h at 30°C with 50 µM GDP or guanosine 5
-
(3-O-thio)-triphosphate (GTP
S) in 1 ml of a reaction mixture (20 mM
Tris/HCl at pH 7.5, 10 mM EDTA, 1 mM DTT, 5 mM MgCl2). The binding of MBP-AF-6 (36-206 amino acids) to GST-ZO-1 columns was carried
out as described above. The proteins bound to GST-ZO-1 columns were
eluted three times by the addition of 330 µl (3.3 vol) of 2 µM GDP/Ha-Ras, GTP
S/Ha-Ras, or GTP
S/Rac. The eluted MBP-AF-6 was immunodetected with the anti-MBP antibody.
). Protein concentrations were determined with BSA as the reference protein as described (Bradford, 1976
). Immunoblot analysis was carried out as described (Harlow and Lane, 1988
).
Results
-Catenin
). Preincubation of the antibody with the recombinant AF-6 abolished the immunoreactivity, indicating the expression of AF-6 in MDCKII
cells.
Fig. 1.
Immunoblot analysis of MDCKII, Rat1, and PC12 cell
lysates with anti-AF-6 antibody. Lane 1, MDCKII cell lysate with
preimmuneserum; lane 2, MDCKII cell lysate with anti-AF-6 antibody preincubated with recombinant AF-6; lane 3, MDCKII cell
lysate with anti-AF-6 antibody; lane 4, Rat1 cell lysate with anti-
AF-6 antibody; lane 5, PC12 cell lysate with anti-AF-6 antibody.
The results shown are representative of three independent experiments. The arrowheads denote the position of AF-6.
[View Larger Version of this Image (21K GIF file)]
). The immunoreactivity
of AF-6 was specifically localized at sites where a cell contacted a neighbor cell, but not at the free ends, and showed
a belt-like pattern of staining along the cell-cell contact
sites (Fig. 2, a and d). The cytosol exhibited a relatively
low level of the anti-AF-6 staining. To examine whether AF-6 exists in the tight junctions or adherens junctions,
the cellular distribution of AF-6 was compared with those
of ZO-1 and
-catenin, which are marker proteins of tight
junctions and adherens junctions, respectively, by laser
scanning confocal microscopy. Three-dimensional images
of confluent MDCKII cells, doubly stained with antibodies
against AF-6 and ZO-1 or
-catenin, were generated from
serial optical sections obtained by laser scanning confocal microscopy. En face views showed that the localization of
AF-6 overlapped with that of ZO-1 (Fig. 2, a-c), but was
somewhat different from that of
-catenin (Fig. 2, d-f). To
confirm that AF-6 is localized at apical lateral membranes,
five serial optical sections (one section every 0.8 µm) were
shown for each staining (Fig. 2, g-j). These images showed
that AF-6 and ZO-1 were concentrated at the apical sections, whereas
-catenin was found at more basal sections.
When the three-dimensional images were rotated latitudinally by 90°, the rotated images showed that AF-6 was
colocalized with ZO-1 at the apical cell borders but not
with
-catenin (Fig. 2, k and l). Thus, it is likely that AF-6
is colocalized with ZO-1 at the tight junctions rather than
adherens junctions of the confluent MDCKII cells.
Fig. 2.
Confocal microscope images of confluent MDCKII cells
showing the distributions of AF-6, ZO-1, and -catenin. Confluent MDCKII cells were doubly stained with a rabbit polyclonal
antibody against AF-6 and a mouse monoclonal antibody against
ZO-1 (a-c, h, and k) or a rat monoclonal antibody against
-catenin (d-f, j, and l), followed by FITC-conjugated anti-rabbit IgG
and Texas red-conjugated anti-mouse IgG or Texas red-conjugated anti-rat IgG antibodies. 20 serial optical sections were obtained at 0.8-µm intervals, and three-dimensional images were
generated. In images a, c, d, f, k, and l, AF-6 is shown in green,
and ZO-1 (b, c, and k) or
-catenin (e, f, and l) is shown in red.
The yellow area indicates the colocalization of AF-6 and ZO-1 (c
and k) or
-catenin (f and l). Images a-f are unrotated en face
view. In images k and l, images c and f are rotated latitudinally by
90°, respectively. Images g-j show five confocal sections for each
staining. Images g and i show the distribution of AF-6, and images h and j show that of ZO-1 and of
-catenin, respectively.
Subsequent sections from apical to basal were shown from left to
right. The results shown are representative of three independent
experiments. Bars, 10 µm.
[View Larger Versions of these Images (99 + 48K GIF file)]
Fig. 3.
The ultrastructural localization of AF-6 and that of
ZO-1 in confluent MDCKII cells. Immunoelectron micrographs
of the junctional complex region in MDCKII cells stained with a
rabbit polyclonal antibody against AF-6 (a), with a mouse monoclonal antibody against ZO-1 (b), or doubly stained with both
anti-AF-6 polyclonal antibody (gold particles) and anti-ZO-1
monoclonal antibody (HRP reaction products; c and d). (a) The
gold particles for AF-6 are accumulated on the cytoplasmic surface of the plasma membranes in the junctional complex region.
(b) ZO-1 labeling is exclusively concentrated in the junctional
complex region. (c and d) AF-6 immunoreactivity (gold particles)
and ZO-1 immunoreactivity (HRP reaction products) are intermixed. The results shown are representative of three independent experiments. Bar, 500 nm.
[View Larger Version of this Image (115K GIF file)]
). Under such conditions, AF-6 and
ZO-1 showed cytoplasmic staining without specific localization at the cell surface (Fig. 4, e and f). When the cells
were transferred to normal media, they formed cell-cell
contacts (Fig. 4 g), and AF-6 and ZO-1 had similar distributions at the cell-cell contact sites (Fig. 4, h and i), suggesting that AF-6 and ZO-1 show similar dynamic behavior during the formation and disappearance of cell-cell
contacts.
Fig. 4.
Immunofluorescence localization of AF-6 and that of
ZO-1 in Ca2+ switch experiments with MDCKII cells. Subconfluent MDCKII cells were grown in normal growth media (a-c) and
transferred to low Ca2+ media (growth media containing 4 mM
EGTA) for 6 h (d-f) and then transferred back to the normal
Ca2+ medium for 2 h (g-i). The cells were doubly stained with a
rabbit polyclonal antibody against AF-6 (b, e, and h) and a mouse
monoclonal antibody against ZO-1 (c, f, and i), followed by
FITC-conjugated anti-rabbit IgG and Texas red-conjugated anti-
mouse IgG antibodies, and examined using laser scanning confocal microscopy. Images a, d, and g are phase contrast images. The
results shown are representative of three independent experiments. Bar, 10 µm.
[View Larger Version of this Image (147K GIF file)]
Fig. 5.
Immunofluorescence localization of AF-6 and that of
ZO-1 in the frozen mouse intestinal epithelium. Cryosections of
mouse intestine were doubly stained with a rabbit polyclonal antibody against AF-6 (a) and a mouse monoclonal antibody against
ZO-1 (b), followed by FITC-conjugated anti-rabbit IgG and Texas
red-conjugated anti-mouse IgG antibodies, and examined using
laser scanning confocal microscopy. The results shown are representative of three independent experiments. Bar, 5 µm.
[View Larger Version of this Image (48K GIF file)]
; Itoh et al., 1993
). A recent study
showed that ZO-1 interacts with catenins during the early
stages of the assembly of tight junctions (Rajasekaran et al.,
1996
). We examined whether AF-6 accumulates at cell-
cell contact sites in cells lacking tight junctions such as
Rat1 fibroblasts and PC12 rat pheochromocytoma cells.
First, immunoblot analysis was performed on cell lysates from Rat1 and PC12 cells. The anti-AF-6 antibody recognized two bands in the samples from Rat1 and PC12 cells
as described for MDCKII cells (Fig. 1). We next examined
the distribution of AF-6 in Rat1 and PC12 cells. AF-6 accumulated at the cell-cell contact sites and was colocalized
with ZO-1 in both the Rat1 fibroblasts and the PC12 rat
pheochromocytoma cells (Fig. 6). In the Rat1 fibroblasts,
the accumulation of AF-6 was discontinuous rather than a
belt-like accumulation as observed in the MDCKII epithelial cells. These results indicate that AF-6 is colocalized
with ZO-1 in cell-cell contact sites of cells lacking tight
junctions.
Fig. 6.
Colocalization of AF-6 with ZO-1 in Rat1 and PC12
cells. Subconfluent Rat1 (a-c) and PC12 (d-f) cells were doubly
stained with a rabbit polyclonal antibody against AF-6 (a, c, d,
and f) and a mouse monoclonal antibody against ZO-1 (b, c, e,
and f), followed by FITC-conjugated anti-rabbit IgG and Texas
red-conjugated anti-mouse IgG antibodies, and examined using
laser scanning confocal microscopy. AF-6 is shown in green (a, c,
d, and f) and ZO-1 (b, c, e, and f) is shown in red. In images c and
f, the yellow area indicates the colocalization of AF-6 and ZO-1.
The results shown are representative of three independent experiments. Bars: (a-c) 10 µm; (d-f) 5 µm.
[View Larger Version of this Image (55K GIF file)]
-catenin, GST-
-catenin, and GST-CD44.
The proteins bound to the affinity columns were eluted
with the GST fusion proteins by the addition of glutathione, and the eluted AF-6 was detected with an anti-AF-6
antibody. AF-6 was detected in the eluate of the GST-ZO-1
affinity column and weakly in that of the GST-occludin affinity column, but not in those of the GST, GST-E-cadherin,
GST-
-catenin, GST-
-catenin, or GST-CD44 affinity columns (Fig. 7). These results indicate that native AF-6 directly or indirectly interacts with ZO-1 and occludin.
Fig. 7.
Interaction of bovine AF-6 with ZO-1. Bovine brain
membrane fraction was loaded onto affinity columns immobilized
with GST (lane 1), GST-ZO-1 (lane 2), GST-occludin (lane 3),
GST-E-cadherin (lane 4), GST--catenin (lane 5), GST-
-catenin (lane 6), and GST-CD44 (lane 7). The interacting proteins
were eluted with GST proteins by the addition of glutathione.
The eluates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-AF-6 antibody. The arrowheads denote
the position of bovine AF-6. The results shown are representative
of three independent experiments.
[View Larger Version of this Image (36K GIF file)]
).
Fig. 8.
Interaction of recombinant AF-6 with ZO-1.
Crude lysates of Sf-9 cells infected with baculovirus carrying HA-AF-6 cDNA were loaded onto affinity columns
immobilized with GST (lane
1), GST-ZO-1 (lane 2), GST-occludin (lane 3), and GST-CD44
(lane 4). The interacting proteins were eluted with GST fusion
proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with
anti-AF-6 antibody. The arrowheads denote the positions of
HA-AF-6. The results shown are representative of three independent experiments.
[View Larger Version of this Image (35K GIF file)]
Fig. 9.
Interaction of in vitro-translated AF-6 (36-494 amino
acids) with ZO-1. (a) Domain diagram of AF-6 and the recombinant fragments used for the in vitro binding assay. RB, Ras-binding domain; Myosin V, Myosin V-like domain; PDZ, PDZ domain. Bold lines show the recombinant fragments used for the in
vitro binding assay. (b-e) In vitro-translated AF-6 (36-494 amino
acids; b), AF-6 (495-909 amino acids; c), AF-6 (914-1,129 amino
acids; d), and AF-6 (1,130-1,612 amino acids; e) were mixed with
GST (lane 1), GST-ZO-1 (lane 2), GST-occludin (lane 3), and
GST-CD44 (lane 4) immobilized to glutathione Sepharose 4B
beads. The interacting proteins were eluted with GST fusion proteins by the addition of glutathione. The eluates were subjected
to SDS-PAGE and vacuum dried. The in vitro-translated AF-6
fragments were visualized with an image analyzer. The arrowheads denote the position of in vitro-translated AF-6 fragments. The results shown are representative of three independent experiments.
[View Larger Version of this Image (19K GIF file)]
).
E. coli lysate containing MBP-AF-6 (36-206 amino acids)
was loaded onto affinity columns immobilized with GST, GST-ZO-1, GDP/GST-Ha-Ras, GTP
S/GST-Ha-Ras, and
GST-CD44. The proteins bound to the affinity columns
were then eluted with the GST fusion proteins by the addition of glutathione, and the eluted MBP-AF-6 (36-206 amino
acids) was detected with an anti-MBP antibody. MBP-AF-6
(36-206 amino acids) was detected in the eluates of the GST-ZO-1 or GTP
S/GST-Ha-Ras affinity columns, but
only slightly in those of the GST, GDP/GST-Ha-Ras, or
GST-CD44 affinity columns (Fig. 10 a). The weak binding
of MBP-AF-6 (36-206 amino acids) to GST, GDP/GST-Ha-Ras, or GST-CD44 affinity columns appears to be background. Similar observations were obtained when MBP-AF-6 (36-494 amino acids) was used instead of MBP-AF-6
(36-206 amino acids; data not shown). To assure the specificity of the binding, we carried out the kinetic study on
the binding of AF-6 to ZO-1. As shown in Fig. 10 b, MBP-AF-6 (36-206 amino acids) bound to GST-ZO-1 in a dose-dependent manner, and this binding was saturable when the amounts of MBP-AF-6 (36-206 amino acids) were increased. The apparent Kd value for the binding of MBP-AF-6 (36-206 amino acids) to GST-ZO-1 was estimated
to be ~260 nM under the conditions. Next, to examine
whether activated Ras dissociates AF-6 from ZO-1, we applied GTP
S/Ras onto a GST-ZO-1 column, which retained
MBP-AF-6 (36-206 amino acids). As shown in Fig. 10 c,
the bound MBP-AF-6 (36-206 amino acids) was eluted
with GTP
S/Ras, but was weakly eluted with GDP alone,
GTP
S alone, GDP/Ras, or GTP
S/Rac. It may be noted
that two bands corresponding to MBP-AF-6 were eluted from the GST-ZO-1 or GTP
S/Ras affinity columns by
the addition of glutathione, whereas the upper band was
eluted more efficiently than the lower band by the addition
of GTP
S/Ras. The smaller size of MBP-AF-6 was probably the degradation product. We can not give the precise
reasons for this complexity, but it is probable that the
smaller size of MBP-AF-6 binds strongly to ZO-1 and
hardly dissociates from ZO-1 by the addition of GTP
S/
Ras. Taken together, these results indicate that ZO-1 interacts with the Ras-binding domain of AF-6 (36-206
amino acids) and that this interaction may be inhibited
specifically by activated Ras.
Fig. 10.
Dissociation of the Ras-interacting domain of AF-6
from ZO-1 by activated Ras. (a) Interaction of MBP-AF-6 (36-
206 amino acids) with ZO-1. Crude lysates of E. coli expressing
MBP-AF-6 (36-206 amino acids) were loaded onto affinity columns immobilized with GST (lane 1), GST-ZO-1 (lane 2), GDP/
GST-Ha-Ras (lane 3), GTPS/GST-Ha-Ras (lane 4), and GST-CD44 (lane 5). The interacting proteins were eluted with GST fusion
proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with
anti-MBP antibody. The arrowheads denote the positions of MBP-AF-6 (36-206 amino acids). (b) The kinetic study on the binding
of MBP-AF-6 (36-206 amino acids) to GST-ZO-1. E. coli lysates
containing various concentrations of MBP-AF-6 (36-206 amino
acids) were loaded onto the GST-ZO-1 and GST-CD44 affinity
columns (0.1 nmol). The proteins bound to GST-ZO-1 columns
were eluted with GST-ZO-1 by the addition of glutathione. The
eluates were subjected to SDS-PAGE and followed by immunoblot analysis with anti-MBP antibody. The immunodetected MBP-AF-6 were visualized and estimated with a densitograph.
The values shown are means ± SEM of triplicate experiments.
,
with GST-ZO-1;
, with GST-CD44. (c) Dissociation of MBP-AF-6 (36-206 amino acids) from ZO-1 by activated Ras. Crude lysates of E. coli expressing MBP-AF-6 (36-206 amino acids) were
loaded onto affinity columns immobilized with GST-ZO-1. The
proteins bound to the GST-ZO-1 columns were eluted by the addition of buffer containing GDP alone (lane 1), GTP
S alone
(lane 2), GDP/Ras (lane 3), GTP
S/Ras (lane 4), and GTP
S/Rac
(lane 5). The eluates were subjected to SDS-PAGE and followed
by immunoblot analysis with anti-MBP antibody. The arrowhead
denotes the position of MBP-AF-6 (36-206 amino acids). The results shown are representative of three independent experiments.
[View Larger Version of this Image (20K GIF file)]
Fig. 11.
Localization of AF-6 in Ras-transformed Rat1 cells.
(A) Immunoblot analysis of Rat1 RasVal A1 cells. The expression of RasV12 was induced in Rat1 RasVal A1 cells (lanes 3) by
the addition of IPTG, and the cell lysates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-Ras antibody
(Rask4). Lane 1, Wild-type Rat1 cells; lane 2, Rat1 RasVal A1
cells in the absence of IPTG; lane 3, Rat1 RasVal A1 cells treated
with IPTG for 24 h. (B) Localization of AF-6 in Ras-transformed
Rat1 cells. Wild-type Rat1 cells (a and b), Rat1 RasVal A1 cells in
the absence of IPTG (c and d), and Rat1 RasVal A1 cells treated
with 5 mM IPTG for 24 h (e and f) were doubly stained with a
rabbit polyclonal antibody against AF-6 (a, c, and e) and a mouse
monoclonal antibody against ZO-1 (b, d, and f), followed by
FITC-conjugated anti-rabbit IgG and Texas red-conjugated anti-
mouse IgG antibodies. The results shown are representative of
three independent experiments. Bar, 10 µm.
[View Larger Version of this Image (56K GIF file)]
Discussion
). Thus, these results indicate that AF-6 is a peripheral component of tight junctions. Although the roles of
AF-6 in tight junctions are not clear, it is possible that AF-6
modulates the functions of ZO-1 downstream of Ras. We
have also found that AF-6 from bovine brain interacts
with the GST-occludin affinity beads, whereas the purified
AF-6 from insect cells, which lack ZO-1, only weakly interacts with the same beads. This raises the possibility that
the binding of AF-6 from bovine brain may be mainly mediated by ZO-1.
). In cells lacking tight junctions
such as fibroblasts and astrocytes, ZO-1 is localized at
cell-cell contact sites with cadherin (Howarth et al., 1992
;
Itoh et al., 1993
). We found here that AF-6 is localized with
ZO-1 at cell-cell contact sites in Rat1 fibroblasts and PC12
rat pheochromocytoma cells lacking tight junctions. These
results suggest that AF-6 is recruited with ZO-1 at cell-cell
contact sites, where cadherin is localized, and serves as a
peripheral component of cell-cell adhesions in cells lacking tight junctions.
), we first
thought that the PDZ domain of AF-6 may interact with
the PDZ domain of ZO-1. The present results, however,
indicate that the NH2-terminal domain (36-206 amino acids) of AF-6 is mainly responsible for the interaction with
ZO-1. A previous study showed that the NH2-terminal domain (36-206 amino acids) binds to activated Ras (Kuriyama et al., 1996
). The present findings indicate that the
Ras-binding domain and ZO-1-binding domain of AF-6
are very close or overlapped, though the ZO-1-binding
domain of AF-6 is not necessarily coincident with the Ras-binding domain.
). Canoe is implicated in the formation of cone cells in the
developing compound eye in Drosophila (Matsuo et al.,
1997
). The fates of cone cells are thought to be determined
by cell-cell contacts. The phenotypic effect of canoe mutations on the cone cells depends on the state of Ras (Matsuo et al., 1997
), and Canoe is shown to interact with the
activated Ras in a cell-free system, indicating that Canoe
serves as a target of Ras as described for AF-6 (Kuriyama et al., 1996
). Genetic study of Canoe in Drosophila will
provide us further information on the roles of Canoe/AF-6
in the regulation of cell-cell contacts.
). It is
possible that AF-6 modulates the function of ZO-1 and
subsequently regulates the cell-cell contacts.
).
Searching for interacting proteins with the COOH-terminal domain is under investigation to understand the functions of AF-6.
; Kinch et al., 1995
).
Several molecules interacting with activated Ras in addition to AF-6 have been identified as Ras targets in mammals. These include Raf (Daum et al., 1994
), phosphatidylinositol-3-OH kinase (Rodriguez-Viciana et al., 1994
), Ral guanine nucleotide dissociation stimulator (Kikuchi
et al., 1994
; Spaargaren and Bischoff, 1994
), and Rin1 (Han
and Colicelli, 1995
). One of these Ras targets may account
for the functions of Ras in the regulation of cell-cell contacts. We have shown here that AF-6 specifically accumulates at cell-cell contact sites. None of the above Ras targets except for AF-6 is reported to accumulate at cell-cell
contact sites in an adhesion-dependent fashion. Expression of activated Ras perturbs cell-cell contacts in Rat1 fibroblast cells, followed by a decrease in the accumulation
of AF-6 and ZO-1 at the cell surface. Moreover, we have
shown here that AF-6 interacts with ZO-1, and this interaction is inhibited by activated Ras. Taken together, it is
likely that the Ras-AF-6 pathway plays critical roles in the
regulation of the cell-cell contacts through ZO-1. Alternatively, the altered cell-cell contacts by activated Ras may
change the cellular distribution of AF-6 and ZO-1. The activated Ras-expressing cells change a plethora of characteristics. We do not know exactly what happens in these
cells. Further studies are necessary for better understanding the mechanism of the regulation of cell-cell contacts
by Ras.
Received for publication 13 March 1997 and in revised form 28 August 1997.
Address all corrrespondence to Kozo Kaibuchi, Division of Signal Transduction, Nara Institute of Science and Technology, Nara 630-01, Japan. Tel.: (81) 7437-2-5440. Fax: (81) 7437-2-5449. E-mail: kaibuchi{at}bs.aist-nara.ac.jpWe thank Drs. Masahiko Itoh, Mikio Furuse, and Shoichiro Tsukita (University of Kyoto, Kyoto, Japan) for kindly providing MDCKII cells, anti-ZO-1 antibody, anti--catenin antibody, and expression plasmids of GST-mouse ZO-1, GST-mouse occludin, GST-mouse E-cadherin, GST-mouse
-catenin, and GST-mouse
-catenin; Drs. Hiroshi Itoh and Yoshito Kaziro (Tokyo Institute of Technology, Yokohama, Japan) for kindly providing Rat1 and Rat1 RasVal A1 cells; and A. Takemura for secretarial
assistance.
This study was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture of Japan (1997) and by grants from the Mitsubishi Foundation and Kirin Brewery Company Limited.
GST, glutathione-S-transferase;
IPTG, isopropyl--D-thiogalactoside;
MAP, mitogen-activated protein;
MBP, maltose-binding protein.
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