From the University of Nebraska Medical Center, College of Dentistry and Eppley Cancer Center, Omaha, Nebraska 68198
Received for publication, November 10, 2002, and in revised form, February 24, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cadherins are calcium-dependent
glycoproteins that function as cell-cell adhesion molecules and are
linked to the actin cytoskeleton via catenins. Newly synthesized
cadherins contain a prosequence that must be proteolytically removed to
generate a functional adhesion molecule. The goal of this study was to
examine the proteolytic processing of N-cadherin and the assembly of
the cadherin-catenin complex in cells that express endogenous
N-cadherin. A monoclonal antibody specific for the proregion of human
N-cadherin was generated and used to examine N-cadherin processing. Our
data show that newly synthesized proN-cadherin is phosphorylated and
proteolytically processed prior to transport to the plasma membrane. In
addition, we show that Cadherins comprise a family of
calcium-dependent cell-cell adhesion proteins that play
important roles in the embryonic development and maintenance of normal
tissue architecture. As the transmembrane component of cellular
junctions, the cadherins are composed of three segments,
i.e. an extracellular domain comprised of five homologous
repeats that mediates adhesion, a single pass transmembrane domain, and
a conserved cytoplasmic domain that interacts with catenins to link
cadherins to the actin cytoskeleton (1-5). The catenins were first
identified as proteins that co-immunoprecipitated with cadherins and
were termed In addition to catenins, p120ctn, which was originally
identified as a Src substrate, binds to the cytoplasmic domain of
cadherins and has been suggested to play a role in regulating the
adhesive activity of cadherins (10-12). p120ctn binds to
the juxtamembrane domain of cadherins, a domain that has been
implicated in cadherin clustering and cell motility (13-16). It is
thought that p120ctn influences the strength of
cadherin-mediated adhesion, perhaps by influencing the organization of
the actin cytoskeleton (17-19). The goal of the present study was to
further understand the sequence of events that leads to the formation
of a functional cadherin-catenin-p120ctn complex.
Cadherins are synthesized as precursor proteins that must be
proteolytically cleaved to generate functional, mature proteins (20,
21). All of the classical cadherins have similar proteolytic cleavage
sites within the proregion, suggesting that each is processed by
proteases with similar specificities. Ozawa and Kemler (21) showed that
mutant forms of E-cadherin missing the proteolytic cleavage sites were
transported to the cell surface when transfected into cadherin-negative
cells but were not active in cell-cell adhesion. These precursor forms
could be converted to active molecules by exogenous cleavage of the
proregion at the cell surface. An emerging idea in the cadherin field
is that cadherin family members promote cell type-specific phenotypes.
For example, we have presented evidence suggesting that N-cadherin
expression can promote motility in epithelial cells, whereas E-cadherin
suppresses motility in the same cells (22-24). Thus, it is important
to examine the activity and processing of cadherins in cells that
endogenously express these proteins. To facilitate these studies, we
developed monoclonal antibodies against the proregion of N-cadherin
that would allow us to define the sequence of events that occur during
the synthesis, processing, and transport to the cell surface of an
endogenous N-cadherin-catenin-p120ctn complex.
Cell Culture--
HeLa, VA13, and HT1080 cells were obtained
from ATCC (Manassas, VA) and maintained in Dulbecco's modified
Eagle's medium (DMEM; Sigma) supplemented with 10% fetal bovine serum
(Hyclone Laboratories, Logan, UT). A431D cells have been described
(25).
Detergent Extraction of Cells--
Confluent monolayers were
rinsed three times with phosphate-buffered saline and extracted
in TNE extraction buffer (10 mM Tris acetate, pH 8.0, 0.5%
Nonidet P-40, 1 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, and 2 mM sodium
orthovanadate). The cells were placed on ice, scraped, and triturated
vigorously for 10 min. Insoluble material was pelleted by
centrifugation at 14,000 × g for 15 min at 4 °C,
and the supernatant was used immediately for immunoprecipitations or
stored at Detergent-free Extraction of Cells--
Confluent monolayers of
cells were rinsed three times with phosphate-buffered saline and
scraped from the flask in TE buffer (10 mM Tris acetate, pH
8.0, 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride). The cells were Dounce homogenized on ice by using five strokes of the tight pestle. The membrane fraction was removed by
centrifugation at 14,000 × g for 15 min at 4 °C,
and the resulting supernatant was used immediately for
immunoprecipitations or stored at Antibodies--
We produced antibodies specific for human
proN-cadherin by immunizing mice with a maltose-binding protein
(MBP)1 fusion. The upstream
primer 5'-GCG AAT TCG CCA TGG TCT CTG TAG AGG CTT CTG G-3' and the
downstream primer 5'-GCC TCG AGT TAA CCT CTC TTC TGC CTT TGT AG-3' were
used to PCR amplify nucleotides 269-682 of human N-cadherin
(GenBankTM accession NM_001792) encoding amino acids
22-159. The PCR product was digested using EcoRI and
XhoI and ligated to pMAL-C2 (New England Biolabs, Beverly,
MA) prepared by digestion with EcoRI and SalI.
The PCR product was sequenced and found to encode the correct amino
acid sequence. Hybridomas secreting antibodies specific for the
proregion of N-cadherin were generated as described previously (26,
27). Epitope mapping of the anti-proN-cadherin antibodies was performed
by testing their ability to bind to deleted fusion proteins. The PCR
product was truncated at either the endogenous HindIII site
or the endogenous PstI site to create two MPB fusion proteins. The former included amino acids 22-92 and the latter amino
acids 22-77 of N-cadherin. Recombinant fusion proteins were resolved
by SDS-PAGE, and anti-proN-cadherin antibodies were tested by
immunoblot analysis.
Anti- Immunoprecipitation and Immunoblot--
All polypropylene tubes
were rinsed with 0.1% Nonidet P-40 and dried prior to use in
immunoprecipitations. The indicated volume of cell extract was added to
300 µl of hybridoma-conditioned medium and gently mixed at 4 °C
for 30 min. 50 µl of packed anti-mouse IgG affinity gel (ICN
Pharmaceuticals, Costa Mesa, CA) was added, and mixing continued for 30 min. Immune complexes were washed five times with TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween
20, and 2 mM sodium orthovanadate). After the final wash, the packed beads were resuspended in 50 µl of 2× Laemmli sample buffer (29), boiled for 5 min, and the proteins were resolved by
SDS-PAGE. Proteins were electrophoretically transferred overnight to
nitrocellulose membranes and blocked in 5% nonfat dry milk in TBST for
45 min. Blocking solution was removed by washing for 15 min followed by
2× 5 min in TBST. Hybridoma conditioned media was used at 1:100
dilution in TBST for 1 h. Membranes were washed 15 min followed by
2× 5 min in TBST. Membranes were incubated with horseradish
peroxidase-conjugated anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories) at 1:10,000 for 1 h. Secondary antibody was removed by washing 15 min followed by 4× 5 min in TBST.
Immunoreactive bands were detected using Super Signal Pico substrate (Pierce).
Immunofluorescence--
Cells were grown on glass coverslips to
80% confluence and fixed in 1% paraformaldehyde for 30 min and
permeabilized in methanol at Metabolic Labeling--
For pulse-chase experiments, HeLa cells
grown in a 25-cm2 flask were cultured in DMEM lacking
methionine and cysteine and supplemented with 1% dialyzed fetal bovine
serum (Hyclone) for 4 h. Cells were pulsed with media
containing 250 µCi of Tran35S-label (ICN Pharmaceuticals)
for 15 min and chased for the indicated times with DMEM supplemented
with 10% fetal bovine serum.
For 33P labeling, HeLa cells were cultured overnight in
DMEM lacking phosphate and supplemented with 10% dialyzed fetal bovine serum. Fresh media containing 1 mCi of
H333PO4 was then added for 2 h. Following labeling, Nonidet P-40 cell extracts were prepared and
immunoprecipitated with the appropriate antibody. Immunoprecipitates
were resolved by SDS-PAGE and transferred to nitrocellulose.
Nitrocellulose sheets were exposed to Biomax MR1 film (Kodak) at
Generation of Monoclonal Antibodies Specific for the Proregion of
Human N-cadherin--
To facilitate the examination of the processing
that is required to generate mature N-cadherin from proN-cadherin, we
developed monoclonal antibodies specific for the proregion of human
N-cadherin. The proregion (Fig.
1A) of human N-cadherin was
fused to MBP, expressed in bacteria, affinity purified, and hybridoma
cell-lines secreting antibodies specific for the proregion of
N-cadherin were produced (Fig. 1B). Epitope mapping using
truncated fusion protein showed that the epitope recognized by
monoclonal antibodies 10A10 and 19D8 resides in the carboxyl-terminal
portion of the proregion. The 10A10 and 19D8 antibodies each recognized
a fusion protein containing amino acids 22-159 but did not recognize a fusion protein containing amino acids 22-77 or amino acids 22-92. Immunoblot analysis of HeLa cell lysates using anti N-cadherin proregion antibodies revealed two distinct bands that migrated slower
than mature N-cadherin on SDS-PAGE (compare lanes 1 and 2 in Fig. 1C with lane 3). Lanes
1 and 2 contain ~3 times as much protein as
lane 3. The 13A9 antibody recognized both the processed and
unprocessed forms of N-cadherin, and a faint band corresponding to the
unprocessed form can be seen in lane 3. When we loaded sufficient protein to clearly identify proN-cadherin using the 13A9
antibody, the lane was overloaded and the bands smeared together, making it difficult to separate them from one another. The results with
13A9 show that the vast majority of the N-cadherin in HeLa cells at
steady state lacks the proregion. Immunoblot analysis of HT1080 and
VA13, two other cell-lines expressing endogenous N-cadherin, produced
similar results (data not shown).
Immunolocalization of proN-cadherin in HeLa
Cells--
Immunofluorescence analysis of HeLa cells using the
anti-proN-cadherin antibody produced a staining pattern that closely
resembled that seen for the endoplasmic reticulum protein calnexin
(Fig. 2, D-F) and
the Golgi marker, mannosidase II (Fig. 2, G-I). No proN-cadherin staining was observed at cell borders (Fig. 2,
A, D, and G). This suggests that the
majority of proN-cadherin is localized to the endoplasmic reticulum and
Golgi complex and that proN-cadherin is not transported to the plasma
membrane. In contrast, HeLa cells stained with an antibody directed
against the cytoplasmic domain of N-cadherin (13A9) that recognizes
both mature N-cadherin and proN-cadherin stained mainly cell-cell
borders with a small amount of staining in the endoplasmic
reticulum/Golgi area (Fig. 2B).
To further demonstrate that proN-cadherin is restricted to the cytosol,
we compared the staining pattern of permeabilized and non-permeabilized
HeLa cells using an anti-proN-cadherin antibody or an antibody that
recognizes all forms of N-cadherin. Non-permeabilized HeLa cells showed
no immunostaining using the anti-proN-cadherin antibody 10A10 (Fig.
3A). An antibody specific for
the cytoplasmic domain of N-cadherin (13A9) was also negative on
non-permeabilized cells (Fig. 3D). The fact that cells were
present in panels A and D is shown by the
corresponding DAPI staining in panels B and E,
respectively. Staining with an anti N-cadherin antibody directed
against the extracellular domain (8C11) showed that mature N-cadherin
was present on the cell surface (Fig. 3G). HeLa cells permeabilized by methanol treatment and immunostained using 10A10 revealed cytoplasmic staining localized to ER and Golgi compartments (Fig. 3C), whereas 13A9 showed typical cell border staining
(Fig. 3F).
ProN-cadherin Is Removed by a Single Proteolytic
Event--
Because proN-cadherin must be cleaved to generate a
functional cadherin, we sought to detect the cleaved proregion of
N-cadherin using immunoblots of HeLa cell extracts. The cleaved
proregion of N-cadherin was undetectable on straight immunoblots,
perhaps because it is not very abundant (data not shown). However, when anti-N-cadherin proregion antibodies were used to immunoprecipitate HeLa cell extracts, and the immunoprecipitated proteins were
subsequently immunoblotted using the same antibody, the proregion was
detected as a single band at ~15kDa (Fig.
4). Anti-proN-cadherin antibodies (10A10
and 19D8) each immunoprecipitated a peptide of ~15kDa, which is
consistent with the proregion of N-cadherin being ~135-140 amino
acids. The 10A10 and 19D8 antibodies themselves contain multiple
immunoglobulin chains that migrate between 25 and 68 kDa, which were
identified by resolving the antibody in the absence of HeLa cell
extract (Fig. 4, lanes 1 and 2).
Catenins Co-immunoprecipitate with
proN-cadherin--
To estimate the stoichiometric ratio of cadherin to catenin, we
immunoprecipitated HeLa cell extracts with either anti-proN-cadherin or
anti-N-cadherin antibodies such that the resulting immunoblot signals
for
It has been suggested that Catenins Co-localize with Unprocessed Cadherin--
The quantity
of unprocessed N-cadherin in HeLa cells is very small. We can detect it
in immunofluorescence using anti-proN-cadherin, because proN-cadherin
is discretely localized within the endoplasmic reticulum and the Golgi
complex and because the antibody detects only the unprocessed
N-cadherin. It was not possible to use HeLa cells to examine the
co-localization of unprocessed N-cadherin and catenins, because the
catenin antibodies recognize all the catenins, including those
complexed with mature N-cadherin. Because most of the N-cadherin in
these cells is mature (Fig. 1C), the majority of the
immunofluorescence signal corresponding to catenins is at the cell
surface, which tends to obscure any signal that may be in the cytosol.
During the course of a separate project, we prepared a series of
N-cadherin-E-cadherin chimeras (23) and introduced these constructs
into the cadherin-negative cell line A431D. The protein encoded by one
construct called N/E5a-myc (23) was highly expressed by A431D cells but
was not processed and not transported to the plasma membrane.
Presumably, the failure of N/E5a-myc to leave the endoplasmic reticulum
was due to inappropriate protein folding. A431D cells do not stably
express catenins until they are transfected with a cadherin, and the
majority of the stable catenin they express after transfection is
associated with the transfected cadherin (25). Thus, in A431D cells
expressing the N/E5a-myc chimera, the technical complication of cell
surface catenin is not a problem for immunofluorescence experiments.
A431D cells expressing the N/E5a-myc chimera were lysed, and the
extracts were used for immunoblot analysis. Antibodies against the
N-cadherin proregion (10A10 and 19D8), the N-cadherin extracellular domain (8C11), or the C-terminal Myc tag (9E10) were used to identify the chimera. In contrast to the results with HeLa cells (Fig. 1C), each antibody identified one band with the same
mobility (Fig. 6A). Thus, the
majority of the N/E5a-myc cadherin contained the proregion and was
unprocessed. Immunofluorescence staining of the transfected A431D cells
revealed that the N/E-chimeric cadherin was present in a reticular
staining pattern consistent with a protein that is trapped in the
endoplasmic reticulum. Cells expressing the N/E5a-myc chimera also
stably expressed The Cytoplasmic Domain of N-cadherin Is Phosphorylated Prior to the
Removal of the Proregion--
Anti-proN-cadherin antibodies recognized
two major bands in immunoblots of HeLa cell extract (Fig. 1). Thus, we
performed pulse-chase experiments to examine the synthesis of
proN-cadherin and determine which band was the earlier precursor form
(Fig. 7A). The faster
migrating form of proN-cadherin was labeled during a 15-min pulse with
radioactive amino acids (lane 1). After a 10-min chase, the
slower migrating form began to appear (lane 2), and by 30 min the majority of the protein was in this form (lane 4).
By 60 min nearly all the signal had been chased to the slower migrating
form of proN-cadherin, and the overall signal had begun to diminish due
to removal of the proregion by protease cleavage (lane
5).
To characterize the difference between the two proN-cadherin bands, we
metabolically labeled HeLa cell cultures using 33P and
immunoprecipitated proN-cadherin. Immunoprecipitates were resolved by
SDS-PAGE and transferred to nitrocellulose; the 33P labeled
bands were identified by autoradiography of the nitrocellulose membrane
prior to immunoblotting with anti-proN-cadherin antibody (Fig.
7B, lane 2). From this experiment we determined
that the slower migrating band was phosphorylated, whereas the faster
migrating band was not phosphorylated. These data indicate that
N-cadherin is phosphorylated while in the endoplasmic reticulum or
Golgi complex prior to removal of the proregion.
It has been suggested that phosphorylation of the cytoplasmic domain of
cadherin regulates catenin binding. Thus, we used The goal of the present study was to investigate endogenous
N-cadherin processing and cadherin-catenin complex formation. We chose
to use HeLa cells that endogenously express N-cadherin and catenins and
form N-cadherin-containing cell-cell junctions. To monitor the
processing of N-cadherin, we generated monoclonal antibodies specific
for the proregion of N-cadherin. Immunoblot analysis of HeLa cell
extracts with the proN-cadherin antibodies revealed two major bands.
Immunoblots of extracts prepared from other cells expressing endogenous
N-cadherin (HT1080 and VA13) gave similar results (data not shown).
Metabolic labeling of HeLa cell cultures with 33P
identified the faster migrating form of proN-cadherin as a
non-phosphorylated form and the slower migrating form as a
phosphorylated form. In pulse-chase experiments, the earliest
(non-phosphorylated) form of proN-cadherin that we could detect rapidly
chased into the slower migrating (phosphorylated) form. It was this
slower migrating form that co-immunoprecipitated with -catenin and plakoglobin associate only with
phosphorylated proN-cadherin, whereas p120ctn can
associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at
the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and
is transported to the plasma membrane where linkage to the actin
cytoskeleton can be established.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin,
-catenin, and
-catenin (plakoglobin)
according to their mobility on SDS-PAGE. Either
-catenin or
plakoglobin binds directly to the cadherin and to
-catenin, whereas
-catenin associates directly and indirectly with actin filaments
(6-9). The ability of cadherins to simultaneously self-associate and
link to the actin cytoskeleton allows strong cell-cell adhesion.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
70 °C.
-catenin (5H10), anti
-catenin (1G5), anti-plakoglobin
(11E4), anti-N-cadherin cytoplasmic domain (13A9), anti-c-Myc tag
(9E10), and anti-N-cadherin extracellular domain (8C11) have been
described (23, 26-28). Rabbit anti-
-catenin was purchased from
Sigma. Anti-p120ctn was purchased from BD Transduction
Laboratories (Lexington, KY). Rabbit anti calnexin was purchased from
Stressgen (Victoria, British Columbia, Canada) and rabbit anti
mannosidase II was purchased from Chemicon (Temecula, CA).
20 °C for 5 min. For experiments
comparing permeabilized cells to non-permeabilized cells, the
coverslips were fixed in 1% paraformaldehyde for 10 min with or
without methanol treatment. After three 5 min washes in serum-free
culture medium, the coverslips were blocked in 10% goat serum in
culture medium for 30 min. Coverslips were incubated with primary
antibodies for 20 min, washed 3× 5 min with culture medium, and
incubated in fluorescein isothiocyanate or rhodamine-conjugated
secondary antibody for 20 min. Coverslips were washed 3× 5 min in
culture medium and briefly rinsed in distilled water prior to mounting
in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images
were collected on a Zeiss Axiovert 200 M equipped with an
ORCA-ER (Hamamatsu) digital camera. Images were collected and processed
using OpenLab software from Improvision Inc. (Boston, MA)
70 °C using an intensifying screen (Transcreen LE, Kodak)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Production of antibodies against the
proregion of human N-cadherin. A, schematic diagram of
human N-cadherin, including the proregion (pro), the 5 extracellular repeats (EC1-5), the transmembrane domain
(TM), and the cytoplasmic domain. Two previously
characterized antibodies were used in this study, namely 13A9, which
binds to the cytoplasmic domain of N-cadherin, and 8C11, which binds to
the extracellular domain between EC3 and EC4. B, MBP fused
to the proregion of human N-cadherin was used as an antigen to make
monoclonal antibodies. MBP-fusion proteins containing amino acids
22-159 of N-cadherin (lane 1), amino acids 22-77 of
N-cadherin (lane 2), and amino acids 22-92 of N-cadherin
(lane 3) were resolved by SDS-PAGE, transferred to
nitrocellulose, and blotted with anti-maltose-binding protein, 10A10,
or 19D8. Monoclonal antibodies 10A10 and 19D8 each bound to a region
included in amino acids 92-159. 19D8 also cross-reacts with a
bacterial protein that co-migrates with the fusion protein
corresponding to amino acids 22-77 of N-cadherin. C, HeLa
cell extract was separated by SDS-PAGE and transferred to
nitrocellulose, and the membrane was blotted with antibodies specific
for proN-cadherin, 10A10 (lane 1), and 19D8 (lane
2) or the cytoplasmic domain of N-cadherin, 13A9 (lane
3).
View larger version (46K):
[in a new window]
Fig. 2.
ProN-cadherin co-localizes with endoplasmic
reticulum and Golgi apparatus proteins. HeLa cells were
fixed in 1% paraformaldehyde, permeabilized in methanol at 20 °C,
and stained with antibodies specific for proN-cadherin,
10A10 (panel A), and 19D8 (panel D and
G), mature N-cadherin, 13A9 (panel B), the
endoplasmic reticulum marker calnexin (panel E), or the
Golgi marker mannosidase II (panel H). Panel C is
a phase contrast image of the cells in panel B. Panel
F is a merged image of panels D and E. Panel I is a merged image of panels G and
H. The scale bar in panel A
is 1 µm.
View larger version (70K):
[in a new window]
Fig. 3.
ProN-cadherin is not present on the surface
of HeLa cells. HeLa cells grown on glass coverslips were fixed in
1% paraformaldehyde and stained with anti-proN-cadherin (panel
A), anti N-cadherin cytoplasmic domain (panel D), or
anti N-cadherin extracellular domain (panel G).
Alternatively, cells were permeabilized after fixation and stained with
anti-proN-cadherin (panel C) or anti N-cadherin cytoplasmic
domain (panel F). Panels B and E are
DAPI staining of panels A and D respectively to
show the location of the cells. The scale bar in panel
G is 1 µm.
View larger version (29K):
[in a new window]
Fig. 4.
Immunoprecipitation of the proregion of
N-cadherin. Anti-proN-cadherin antibodies were used to
immunoprecipitate buffer alone (lanes 1 and 2) or
HeLa cell extract (lanes 3 and 4). The
immunoprecipitates were resolved by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with anti-proN-cadherin
(10A10). Note that 10A10 and 19D8 each immunoprecipitated a
protein that migrated at 15 kDa, consistent with the entire proregion
of human N-cadherin.
-catenin, plakoglobin, and
p120ctn each directly bind to the cytoplasmic domain of
N-cadherin (11, 28).
-catenin is localized to the cadherin-catenin
complex via binding
-catenin or plakoglobin. We sought to determine
at which point the catenins assemble on N-cadherin by examining the
pool of proN-cadherin in HeLa cells. ProN-cadherin was
immunoprecipitated from HeLa cell extract, and the immunoprecipitate
was resolved by SDS-PAGE. Immunoblot analysis using antibodies specific
for
-catenin,
-catenin, p120ctn (Fig.
5A), or plakoglobin (data not
shown) demonstrated that each catenin co-immunoprecipitated with
proregion-containing N-cadherin, suggesting that all four catenins can
assemble on N-cadherin before the proregion is cleaved. HeLa cells
express two isoforms of p120ctn, and both isoforms were
associated with proN-cadherin (Fig. 5A).
View larger version (27K):
[in a new window]
Fig. 5.
Catenins associate with proN-cadherin.
A, HeLa cell extract was immunoprecipitated (IP)
with anti-proN-cadherin monoclonal antibody (Pro-region aby)
10A10. The immunoprecipitate (left lane) and HeLa cell
extract (right lane) were resolved by SDS-PAGE, transferred
to nitrocellulose, and immunoblotted with antibodies specific for
-catenin,
-catenin, or p120ctn. B, 900 µl of HeLa cell extract was immunoprecipitated with
anti-proN-cadherin (19D8; left lane), and 150 µl of HeLa
cell extract was immunoprecipitated with anti-mature N-cadherin (13A9;
right lane). Immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with anti
-catenin
(1G5) and anti N-cadherin (13A9). C, cytosolic HeLa extract
prepared in the absence of Nonidet P-40 was immunoprecipitated with
anti
-catenin (
-catenin aby; 1G5) or no antibody
(no aby). The immunoprecipitation was resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with antibodies
specific for
-catenin or N-cadherin. Cytosolic and membrane extracts
were also immunoblotted to show the absence of N-cadherin in the
cytosolic extract.
-catenin were roughly equal. To accomplish this, different
amounts of cell extract were used for each immunoprecipitation. The
nitrocellulose membrane was immunoblotted simultaneously for
-catenin and N-cadherin using the 13A9 antibody that recognizes all
forms of N-cadherin (Fig. 5B). When roughly equal amounts of
-catenin were present in each immunoprecipitation, the N-cadherin signals were approximately equal. This result indicates that
proN-cadherin-catenin complexes are stoichiometrically similar to
mature cadherin-catenin complexes, suggesting that the majority of the
proregion containing cadherin in HeLa cells is complexed with
-catenin. This result clearly demonstrates that assembly of the
complex does not require removal of the proregion.
-catenin binds to E-cadherin
co-translationally, but
-catenin binds at the time of proteolytic cleavage or after the complex has been transported to the plasma membrane (30, 31). The data presented in Fig. 5B suggest
that both
-catenin and
-catenin can associate with unprocessed
N-cadherin. One possibility is that
-catenin and
-catenin form a
complex in the cytosol before either one associates with N-cadherin. To determine whether
-catenin and
-catenin could associate with one
another in the absence of cadherin, we Dounce homogenized HeLa cells in
the absence of detergent to generate cadherin-free cytosolic extract.
We then analyzed the cytosolic extract for complexes of
-catenin and
-catenin by co-immunoprecipitation experiments. Fig. 5C
shows that
-catenin co-immunoprecipitated with
-catenin in these
extracts. Reciprocal experiments showed that
-catenin
co-immunoprecipitated with
-catenin (not shown), and immunoblots
confirmed that there was no cadherin in the cytosolic extract (Fig.
5C). Although these data do not prove that
-catenin and
-catenin form dimers before they associate with N-cadherin, when
considered in conjunction with the knowledge that
-catenin co-immunoprecipitates with proN-cadherin, they are consistent with the
hypothesis that
-catenin and
-catenin could load onto the
cadherin as a dimer.
-catenin, which co-localized with the cadherin
(Fig. 6, E-G) and with the endoplasmic reticulum marker,
calnexin (Fig. 6, B-D). In addition,
-catenin (Fig. 6,
H-J) and p120ctn (Fig. 6, K-M) were
stably expressed in the transfected cells, and these cytoplasmic
proteins were also found in a reticular staining pattern that
co-localized with
-catenin and, thus, with the N/E5a-myc chimeric
cadherin. These data confirm that the catenins and other cytoplasmic
proteins of the adherens junction can assemble with cadherins prior to
cadherin localization at the plasma membrane.
View larger version (31K):
[in a new window]
Fig. 6.
Catenins co-localize with proN-cadherin.
A, cell extracts prepared from A431D cells transfected with
the N/E5a-myc chimeric cadherin were separated by SDS-PAGE, transferred
to nitrocellulose, and immunoblotted with antibodies against the
N-cadherin proregion (10A10 and 19D8), the N-cadherin extracellular
domain (8C11), and the C-terminal Myc tag (9E10). B-L,
transfected cells were fixed in 1% paraformaldehyde, permeabilized in
methanol at 20 °C, and co-stained with mouse anti
-catenin
(panel B) and rabbit anti calnexin (panel C);
mouse anti N-cadherin (panel E) and rabbit anti
-catenin
(panel F); mouse anti
-catenin (panel H) and
rabbit anti
-catenin (panel I); and mouse anti
p120ctn (panel K) and rabbit anti
-catenin
(panel L). Panels D, G, J,
and M are corresponding merged images. The scale bar in
panel B is 1 µm. Note that not all the cells in this
experiment were transfected with the chimeric cadherin. Thus,
panel C includes cadherin-negative and cadherin-positive
cells, which are all positive with the calnexin antibody. Likewise,
panel L includes cadherin-negative and cadherin-positive
cells, which are all positive for p120ctn.
View larger version (37K):
[in a new window]
Fig. 7.
Characterization of two proN-cadherin
species. A, HeLa cells were starved for 4 h in
methionine- and cysteine-deficient media and then pulsed for 15 min in
deficient media supplemented with 35S-labeled methionine
and cysteine. Cultures were chased with complete media for the
indicated times, and cells were extracted in TNE buffer.
Anti-proN-cadherin (10A10) was used to immunoprecipitate proN-cadherin
from labeled extracts. The immunoprecipitates were resolved by SDS-PAGE
and transferred to nitrocellulose. The nitrocellulose was exposed to
autoradiographic film at 70 °C. B, HeLa cells were
cultured in phosphate-deficient media overnight and then labeled with
33PO4-containing media for 2 h. Cells were
extracted in TNE buffer and immunoprecipitated with 10A10. The
immunoprecipitate was resolved by SDS-PAGE, transferred to
nitrocellulose, and exposed to film (autorad.; lane
2) prior to immunoblotting with anti N-cadherin (anti-N-cad
blot; 13A9; lane 1). C, HeLa cell extract
was immunoprecipitated with anti-
-catenin, anti-plakoglobin, and
anti-p120ctn. Immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted with
anti-proN-cadherin (10A10).
-catenin,
plakoglobin, and p120ctn antibodies to co-immunoprecipitate
proN-cadherin and anti-proN-cadherin antibodies to detect which
proN-cadherin species are co-immunoprecipitated with each catenin (Fig.
7C). All three catenins were associated with the slower
migrating phosphorylated form of proN-cadherin, whereas only
p120ctn was associated with the faster non-phosphorylated
form. Thus, p120ctn appears to bind N-cadherin in its most
immature state, although
-catenin and plakoglobin bind after the
cadherin has been phosphorylated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin and
plakoglobin. In addition to the experiments with HeLa cells, the
experiments with the N/E5a-myc chimeric cadherin showed that catenins
could associate with cadherins that are in the endoplasmic reticulum. Taken together, these data suggest that the phosphorylation of proN-cadherin and the subsequent assembly of the cadherin-catenin complex occurs while the cadherin is in the endoplasmic reticulum and
Golgi complex. Fig. 8 presents a model
depicting the sequence of events leading to N-cadherin-catenin complex
localization at the plasma membrane.
View larger version (36K):
[in a new window]
Fig. 8.
Model of N-cadherin processing and complex
formation. N-cadherin is synthesized in the endoplasmic reticulum
where p120ctn binds to the cytoplasmic juxtamembrane
domain. The cytoplasmic domain is then phosphorylated by casein kinase
II (CKII), which leads to the binding of - and
-catenin. The proregion is removed by furin protease, and the
complex is transported to the plasma membrane.
The proregion of cadherins must be removed in order to generate functional adhesion molecules (21). Recently, Ozawa (32) used L cells to show that the proregion of E-cadherin not only prevented cell aggregation (which requires the formation of adhesion dimers) but also prevented the formation of lateral dimers. The fact that we found catenins complexed with proN-cadherin suggests that the catenins can load onto the monomeric form of this cadherin. Because our pulse-chase experiments showed that the earliest detectible form of proN-cadherin chases into a slower migrating form that co-immunoprecipitates all the catenins, it is likely that the proN-cadherin-catenin complex forms in the endoplasmic reticulum.
The furin subgroup of subtilisin-like proprotein convertases is thought to be responsible for cadherin processing. E-cadherin has been shown to be a furin protease substrate in a baculovirus system, and the consensus sequence for furin protease in E-cadherin is identical to that of N-cadherin (20). The furin convertases have been shown to be proteolytically active in the trans-Golgi network (33). Interestingly, our immunofluorescence experiments using anti-proN-cadherin antibodies revealed a staining pattern consistent with proN-cadherin localization to the ER and Golgi network. In addition, immunoprecipitation experiments showed that a 15-kDa fragment, corresponding to the cleaved proregion, was present in HeLa cell extracts, suggesting that the proregion of N-cadherin can be removed in one step, most likely by the furin convertases. The detection of the 15-kDa pro-peptide raises the possibility that it could be secreted and may have some yet to be identified function outside the cell. In permeabilization experiments, proN-cadherin was not present at the plasma membrane of HeLa cells, consistent with the proregion of N-cadherin being removed prior to transport to the plasma membrane.
Studies focusing on E-cadherin-catenin complex formation in L cells
suggested that -catenin was added to the cadherin
-catenin complex after the cadherin proregion was removed (30). In addition, studies on the assembly of E-cadherin complexes in Madin-Darby canine
kidney (MDCK) cells suggested that
-catenin was added to the
adherens junction complex after localization at the plasma membrane
(31). Our results demonstrate that this is not the case for N-cadherin
in HeLa cells. Immunoprecipitations showed that
-catenin,
plakoglobin, p120ctn, and
-catenin were all found
in a complex with proN-cadherin. The studies with E-cadherin raise the
possibility that, although we could find the catenins associated with
proN-cadherin, only a small fraction of the total proN-cadherin is
actually complexed with the catenins. However, the experiment shown in
Fig. 5 demonstrates that the association of
-catenin with
proN-cadherin is not a rare event; the
-catenin-proN-cadherin
complex has the same stoichiometry as the
-catenin-mature N-cadherin
complex. The cadherin-
-catenin-
-catenin complex has been
estimated to have molar ratios of 1:1:1 (30), a conclusion that is
supported by recent structural studies (34, 35). It is likely that
proN-cadherin binds the catenins in the same ratios as does mature
N-cadherin.
To date, p120ctn association with unprocessed cadherin has
not been investigated. Our data show that p120ctn, but not
-catenin or plakoglobin, can bind to non-phosphorylated proN-cadherin, which is the earliest form of N-cadherin we can detect.
Taken together, our results suggest a model where, following synthesis,
proN-cadherin associates immediately with p120ctn.
Following phosphorylation of the cadherin, possibly by casein kinase II
(36),
-catenin or plakoglobin then associates with the cadherin
(Fig. 8). Our data also show that
-catenin-
-catenin complexes can
form in the cytosol, raising the possibility that
-catenin and
-catenin may simultaneously load onto proN-cadherin. The complex is
then transported to the plasma membrane where linkage to the actin
cytoskeleton occurs. The mechanism of cadherin-catenin complex
transport to the plasma membrane is unknown, although recent work
suggests a microtubule-dependent mechanism for the formation of N-cadherin cell-cell contacts (37). Understanding the
sequence of events leading to the formation of the N-cadherin-catenin complex and its localization at the plasma membrane will help clarify
the regulation of N-cadherin mediated cell-cell adhesion and identify
new mechanisms for controlling N-cadherin-mediated cell-cell adhesion.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jill Nieset and Jennifer Oiler for expert technical assistance in preparing proN-cadherin fusion proteins and generating the constructs used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM51188 (to M. J. W.) and DE12308 (K. R. J.).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: University of Nebraska
Medical Center, College of Dentistry and Eppley Cancer Center, 987696 Nebraska Medical Center, Omaha, NE 68198-7696. Tel.: 402-559-3893; Fax:
402-559-3739; E-mail: Jwahl@unmc.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M211452200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MBP, maltose-binding protein; DMEM, Dulbecco's modified Eagle's medium; DAPI, 4,6-diamidino-2-phenylindole.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wheelock, M. J., Soler, A. P., and Knudsen, K. A. (2001) J. Mammary Gland Biol. Neoplasia 6, 275-285[CrossRef][Medline] [Order article via Infotrieve] |
2. | Wheelock, M. J., Knudsen, K. A., and Johnson, K. R. (1996) Curr. Top. Membr. 43, 169-185 |
3. |
Gumbiner, B. M.
(2000)
J. Cell Biol.
148,
399-404 |
4. | Nollet, F., Kools, P., and van Roy, F. (2000) J. Mol. Biol. 299, 551-572[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Yagi, T.,
and Takeichi, M.
(2000)
Genes Dev.
14,
1169-1180 |
6. | Stappert, J., and Kemler, R. (1994) Cell Adhes. Commun. 2, 319-327[Medline] [Order article via Infotrieve] |
7. | Knudsen, K. A., Soler, A. P., Johnson, K. R., and Wheelock, M. J. (1995) J. Cell Biol. 130, 67-77[Abstract] |
8. | Rimm, D. L., Koslov, E. R., Kebriaei, P., Cianci, C. D., and Morrow, J. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8813-8817[Abstract] |
9. |
Nieset, J. E.,
Redfield, A. R.,
Jin, F.,
Knudsen, K. A.,
Johnson, K. R.,
and Wheelock, M. J.
(1997)
J. Cell Sci.
110,
1013-1022 |
10. | Shibamoto, S., Hayakawa, M., Takeuchi, K., Hori, T., Miyazawa, K., Kitamura, N., Johnson, K. R., Wheelock, M. J., Matsuyoshi, N., Takeichi, M., and Ito, F. (1995) J. Cell Biol. 128, 949-957[Abstract] |
11. | Reynolds, A. B., Daniel, J., McCrea, P. D., Wheelock, M. J., Wu, J., and Zhang, Z. (1994) Mol. Cell. Biol. 14, 8333-8342[Abstract] |
12. | Daniel, J. M., and Reynolds, A. B. (1995) Mol. Cell. Biol. 15, 4819-4824[Abstract] |
13. |
Finnemann, S.,
Mitrik, I.,
Hess, M.,
Otto, G.,
and Wedlich, D.
(1997)
J. Biol. Chem.
272,
11856-11862 |
14. |
Navarro, P.,
Ruco, L.,
and Dejana, E.
(1998)
J. Cell Biol.
140,
1475-1484 |
15. |
Chen, H.,
Paradies, N. E.,
Fedor-Chaiken, M.,
and Brackenbury, R.
(1997)
J. Cell Sci.
110,
345-356 |
16. |
Yap, A. S.,
Niessen, C. M.,
and Gumbiner, B. M.
(1998)
J. Cell Biol.
141,
779-789 |
17. |
Aono, S.,
Nakagawa, S.,
Reynolds, A. B.,
and Takeichi, M.
(1999)
J. Cell Biol.
145,
551-562 |
18. |
Ohkubo, T.,
and Ozawa, M.
(1999)
J. Biol. Chem.
274,
21409-21415 |
19. |
Thoreson, M. A.,
Anastasiadis, P. Z.,
Daniel, J. M.,
Ireton, R. C.,
Wheelock, M. J.,
Johnson, K. R.,
Hummingbird, D. K.,
and Reynolds, A. B.
(2000)
J. Cell Biol.
148,
189-202 |
20. | Posthaus, H., Dubois, C. M., Laprise, M. H., Grondin, F., Suter, M. M., and Muller, E. (1998) FEBS Lett. 438, 306-310[CrossRef][Medline] [Order article via Infotrieve] |
21. | Ozawa, M., and Kemler, R. (1990) J. Cell Biol. 111, 1645-1650[Abstract] |
22. | Islam, S., Carey, T. E., Wolf, G. T., Wheelock, M. J., and Johnson, K. R. (1996) J. Cell Biol. 135, 1643-1654[Abstract] |
23. |
Kim, J. B.,
Islam, S.,
Kim, Y. J.,
Prudoff, R. S.,
Sass, K. M.,
Wheelock, M. J.,
and Johnson, K. R.
(2000)
J. Cell Biol.
151,
1193-1206 |
24. |
Nieman, M. T.,
Prudoff, R. S.,
Johnson, K. R.,
and Wheelock, M. J.
(1999)
J. Cell Biol.
147,
631-644 |
25. |
Lewis, J. E.,
Wahl, J. K., III,
Sass, K. M.,
Jensen, P. J.,
Johnson, K. R.,
and Wheelock, M. J.
(1997)
J. Cell Biol.
136,
919-934 |
26. | Johnson, K. R., Lewis, J. E., Li, D., Wahl, J., Soler, A. P., Knudsen, K. A., and Wheelock, M. J. (1993) Exp. Cell Res. 207, 252-260[CrossRef][Medline] [Order article via Infotrieve] |
27. | Wahl, J. K., III (2002) Hybrid Hybridomics 21, 37-44[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Sacco, P. A.,
McGranahan, T. M.,
Wheelock, M. J.,
and Johnson, K. R.
(1995)
J. Biol. Chem.
270,
20201-20206 |
29. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
30. | Ozawa, M., and Kemler, R. (1992) J. Cell Biol. 116, 989-996[Abstract] |
31. | Hinck, L., Nathke, I. S., Papkoff, J., and Nelson, W. J. (1994) J. Cell Biol. 125, 1327-1340[Abstract] |
32. |
Ozawa, M.
(2002)
J. Biol. Chem.
277,
19600-19608 |
33. | Nakayama, K. (1997) Biochem. J. 327, 625-635[Medline] [Order article via Infotrieve] |
34. | Huber, A. H., and Weis, W. I. (2001) Cell 105, 391-402[Medline] [Order article via Infotrieve] |
35. | Pokutta, S., and Weis, W. I. (2000) Mol. Cell 5, 533-543[Medline] [Order article via Infotrieve] |
36. |
Lickert, H.,
Bauer, A.,
Kemler, R.,
and Stappert, J.
(2000)
J. Biol. Chem.
275,
5090-5095 |
37. |
Mary, S.,
Charrasse, S.,
Meriane, M.,
Comunale, F.,
Travo, P.,
Blangy, A.,
and Gauthier-Rouviere, C.
(2002)
Mol. Biol. Cell
13,
285-301 |