From the Istituto di Ricerche Farmacologiche Mario
Negri, 20157 Milan, Italy, ¶ Microscopy and Image Analysis,
, Scientific Institute San Raffaele-Dibit, 20132 Milan,
Università di Milano-Bicocca, Milan, Italy, and
Università degli Studi dell'Insubria, 21100 Varese, Italy
Received for publication, August 3, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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We report here that junctional adhesion molecule
(JAM) interacts with calcium/calmodulin-dependent serine protein kinase
(CASK), a protein related to membrane-associated guanylate
kinases. In Caco-2 cells, JAM and CASK were coprecipitated and found to
colocalize at intercellular contacts along the lateral surface of the
plasma membrane. Association of JAM with CASK requires the
PSD95/dlg/ZO-1 (PDZ) domain of CASK and the putative PDZ-binding motif
Phe-Leu-ValCOOH in the cytoplasmic tail of JAM.
Temporal dissociation in the junctional localization of the two
proteins suggests that the association with CASK is not required for
recruiting JAM to intercellular junctions. Compared with mature
intercellular contacts, junction assembly was characterized by both
enhanced solubility of CASK in Triton X-100 and reduced amounts of
Triton-insoluble JAM-CASK complexes. We propose that JAM association
with CASK is modulated during junction assembly, when CASK is partially
released from its cytoskeletal associations.
Junctional adhesion molecule
(JAM)1 is an integral
membrane protein that belongs to the family of immunoglobulin-like cell adhesion molecules (1) and mediates homophilic adhesive interactions (2). It is expressed at the intercellular cleft of epithelial and
endothelial cells and colocalizes with the tight junction components
occludin, cingulin, and ZO-1 (1). Several tight junction proteins may
interact with each other and with cytoskeletal and signaling molecules
(3). We recently found that JAM interacts with ZO-1 and cingulin and
that such associations may favor the junctional recruitment of occludin
(4). Also, JAM interacts with the junctional molecule all-1 fused gene
on chromosome 6 (AF-6) (5).
While searching for potential molecular partners of JAM, we noticed
that a sequence at the cytoplasmic carboxyl terminus of JAM fits a
consensus motif for binding type II PSD95/dlg/ZO-1 (PDZ) domains (6).
PDZ domains are protein-binding modules involved in the assembly of
transmembrane molecules and signaling complexes at specialized domains
of the plasma membrane (7). Membrane-associated guanylate kinases, a
subfamily of modular PDZ domain-containing proteins, are found at
intercellular junctions, neuronal synapses, and polarized membrane
domains (8).
Based on a peptide library study that defined the binding specificity
of several PDZ domains (6), we hypothesized that calcium/calmodulin-dependent serine protein kinase (CASK) might be a
ligand for the carboxyl terminus of JAM. The membrane-associated guanylate kinase protein CASK is composed of a
Ca2+-calmodulin kinase, a PDZ, an SH3, and a guanylate
kinase domain. Although predominantly expressed at the neuronal
presynaptic membrane (where it binds neurexins), CASK is also expressed
in epithelial cells (9). In nematodes, the CASK homolog LIN-2 is an
essential component of a multiprotein complex that targets the Let-23
receptor tyrosine kinase to the basolateral membrane of vulval
precursors (10). In vertebrates, CASK localizes at the lateral surface of epithelial cells and binds syndecan-2 (11). In the present study, we
have examined the putative interaction of JAM and CASK in human
epithelial cells.
Antibodies and Cells--
Mouse anti-human JAM mAb BV16, rat
anti-murine JAM mAb BV12, and rabbit anti-JAM serum were produced as
described previously (1, 4). Anti-CASK mAb C63120 and anti-AF-6 mAb
A60520 were purchased from Transduction Laboratories, anti-CASK mAb
5230 was purchased from Chemicon International, and anti-ZO-1 serum was
purchased from Zymed Laboratories Inc. mAb 6H (which
recognizes the
Human intestinal epithelial Caco-2 cells were cultured in DMEM plus
10% fetal calf serum and split 1:3 every week. For experiments with
Caco-2 transfectants, the mJAM and mJAM Immunoprecipitation and Western Blot Analysis--
Confluent
Caco-2 cells were lysed with lysis buffer (0.5% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, and protease
inhibitors (pH 7.5)). Lysates were precleared with protein A-Sepharose
and immunoprecipitated with antibodies coupled to protein A-Sepharose. Immunocomplexes were separated by SDS-polyacrylamide gel
electrophoresis and finally analyzed by Western blot, as described
previously (4). To study protein partitioning in the Triton
X-100-soluble and -insoluble fractions, in some experiments lysates
were centrifuged (14,000 × g for 10 min) to separate
the supernatant (soluble fraction) from the pellet. The pellet was
incubated with 0.02% SDS in lysis buffer, resuspended by gentle
pipetting, and centrifuged, and the supernatant was collected
(insoluble fraction). In control experiments, Western blot analysis of
the remaining pellet (i.e. material insoluble in 0.02% SDS)
did not reveal the presence of JAM, whereas a minor fraction of CASK
(between 5% and 10% of total CASK) was still detectable in the
pellet, both in cells with mature junctions and during junction assembly.
Binding Assay--
To produce the soluble PDZ domain of CASK,
Caco-2 cell RNA was reverse-transcribed according to standard
procedures. Human cDNA encoding the CASK PDZ domain was amplified
by polymerase chain reaction using the sense oligonucleotide
5'-GCGGATCCAGAGTTCGGCTGGTACAGTTTC-3' (which introduces the
underlined BamHI site upstream of nucleotide 1459) as forward primer and antisense oligonucleotide
5'-GGAAGCTTTCAGCGGTAACTTGGCACAA TCTTG-3' (which introduces a
stop codon and the underlined HindIII site downstream of
nucleotide 1716) as reverse primer. The
BamHI-HindIII fragment was inserted in the pQE30
vector (Qiagen), which adds an amino-terminal hexahistidine sequence,
and used to transform competent M15 Escherichia coli. The
protein (6HisCASK-PDZ) was purified on a Ni2+-nitriloacetic
acid resin. For the binding assay, 6HisCASK-PDZ was immobilized
on Ni2+-NTA-Sepharose beads. After blocking with 1 mg/ml
bovine serum albumin and 0.1% Tween 20, beads (20 µl) were diluted
in binding buffer and incubated with either GST-JAM or GST as
fluid-phase ligands (60 min at 4 °C, with rotation). Beads were
washed five times, resuspended with 20 µl of sample buffer, and
boiled for 5 min. Proteins were analyzed by Western blot using an
anti-GST antibody (Biotech).
Immunofluorescence Microscopy--
Cells were grown on glass
coverslips and fixed with methanol. Mowiol 488-mounted coverslips were
analyzed by confocal microscopy (with Kalman filtering), using an MRC
1024 Bio-Rad microscope equipped with a krypton/argon laser (13).
CASK Associates with the Cytoplasmic Tail of JAM--
To study the
association of JAM with CASK, JAM was first immunoprecipitated from
Caco-2 lysates using the anti-JAM mAb BV16. Immunoprecipitates were
then resolved by SDS-polyacrylamide gel electrophoresis and analyzed by
Western blot with the anti-CASK mAb C63120. mAb BV16 coprecipitated a
protein with an apparent relative mass of ~110 kDa that was
recognized in Western blot by the anti-CASK antibody (Fig.
1A, lane 1). The
coprecipitated protein displayed the same electrophoretic mobility of
authentic CASK when the latter was analyzed in parallel by Western blot in lysates from either Caco-2 cells (lane 2) or rat brain
(lane 3).
To define the molecular determinants of the JAM-CASK association, the
cytoplasmic domain of JAM (from Gly256 to the
carboxyl-terminal residue Val300) was expressed as a fusion
protein with GST (GST-JAM) and immobilized onto glutathione-Sepharose
beads. GST-JAM (but not GST) bound CASK in Caco-2 lysates as assessed
by Western blot analysis with the anti-CASK antibody, indicating that
the association of JAM with CASK is mediated the cytoplasmic tail of
JAM (Fig. 1B).
JAM Association with CASK Is PDZ-dependent--
As
mentioned above, the cytoplasmic domain of JAM contains a putative
PDZ-binding motif. To test its involvement in the association with
CASK, we produced a deletion mutant of murine JAM lacking the
carboxyl-terminal residues
Phe298-Leu299-Val300 (mJAM
We then evaluated whether the association of JAM with CASK is mediated
by the PDZ domain of CASK. To this end, the PDZ domain (from
Arg487 to Arg572) was produced as a
polyhistidine-tagged soluble protein (6HisCASK-PDZ), immobilized onto
Ni2+-NTA-Sepharose beads, and used in a binding assay as
solid-phase ligand. Either GST-JAM or GST alone was used as fluid-phase
ligand. Bound GST proteins were then eluted and analyzed by
SDS-polyacrylamide gel electrophoresis and Western blot using an
anti-GST antibody. As shown in Fig. 1D, GST-JAM was
specifically bound by immobilized 6HisCASK-PDZ (lane 4),
whereas no binding of either GST to 6HisCASK-PDZ-coated beads
(lane 2) or GST-JAM to beads alone (lane 3) was detectable.
Subcellular Distribution of JAM and CASK--
The distribution of
JAM and CASK in Caco-2 cells was analyzed by confocal laser microscopy.
As shown in Fig. 2, both CASK (Fig.
2A) and JAM (Fig. 2B) distributed and colocalized
(Fig. 2C) at intercellular contacts. In vertical sections,
CASK and JAM colocalized at the lateral region of the plasma membrane
with minimal overlap at the apical-most portion of the latter (Fig. 2,
a-c). Localization of either molecule at the apical surface was never observed. To define JAM distribution in greater detail, JAM
was costained with either the basolateral marker
Na+,K+-ATPase or the tight junction component
cingulin. ATPase (Fig. 2, D and d) and JAM (Fig.
2, E and e) codistributed at intercellular contacts (Fig. 2F) and colocalized along the lateral surface
(Fig. 2f), confirming the localization of JAM at the lateral
domain of the plasma membrane. At variance, a subpopulation of
intercellular JAM (Fig. 2G) localized to the apical-most
region of the lateral membrane (Fig. 2g) and colocalized
(Fig. 2, I and i) with cingulin, which
exclusively decorated tight junctions (Fig. 2, H and
h).
Association with CASK Is Not Required for Recruiting JAM to the
Junctions--
Because several cytoplasmic PDZ-proteins determine the
subcellular localization of transmembrane partners (7), we tested whether association with CASK was required for recruitment of JAM to intercellular contacts. To this end, confluent monolayers of Caco-2 cells were incubated in the low calcium medium minimum essential medium for suspension cultures (S-MEM) to disrupt
junctions and then switched back to DMEM, which contains physiological
calcium concentrations, to induce junction reassembly. Junctional
staining of both JAM and CASK was evaluated at different time points
after calcium addition. We found that JAM was recruited to the
junctions as early as 10 min after calcium addition, whereas
CASK recruitment was only observed at 6 h (Fig.
3A). In additional
experiments, junctional staining of CASK was not yet detectable at
2 h after calcium addition (data not shown), indicating
that junctional recruitment of CASK occurs between 2 and 6 h under
these experimental conditions. At variance with CASK, junctional
staining of cingulin, ZO-1, and AF-6 was already detectable at 30 min
after calcium addition (Fig. 3B). Temporal
dissociation in the junctional recruitment of JAM and CASK suggests
that JAM localizes to intercellular junctions independently of its
association with CASK. The suggestion is further supported by the
observation that the deletion mutant mJAM Modulation of the Interaction between JAM and CASK during Junction
Assembly--
To test the hypothesis that the association of JAM with
CASK may be modulated by the state of maturation of intercellular adhesive complexes, confluent Caco-2 monolayers were first incubated (for 18 h) in low calcium medium to disrupt junctions and then incubated for additional 2 h in medium containing physiological calcium concentrations and finally lysed with 0.5% Triton X-100 (Fig.
4, JUNCTION ASSEMBLY). As a
control, Caco-2 cells were maintained in the presence of physiological
calcium levels throughout the assay (Fig. 4, MATURE
JUNCTIONS). Upon lysis, both Triton-soluble and -insoluble
fractions were analyzed for the presence of JAM and CASK, either alone
(by Western blot with the respective antibodies) or in complex (by
immunoprecipitation with anti-JAM and Western blot with anti-CASK
antibodies). Compared with mature junctions, JAM distribution in the
two fractions was unchanged during junction assembly, as assessed by
Western blot analysis, with JAM being much more abundant in the soluble
fraction. Notably, detection of biotin-labeled JAM suggests that JAM
was expressed at the cell surface in both fractions (Fig.
4B). In contrast, partitioning of CASK between the two
fractions was severely affected because much lower amounts of CASK were
detected in the insoluble fraction during junction assembly as compared
with mature junctions. Similarly, the amount of CASK coprecipitated
with JAM in the insoluble fraction was reduced during the assembly, as
evaluated by immunoprecipitation and Western blot (Fig. 4B;
see also Fig. 4C for a quantitative analysis).
The major findings of this study are: (i) JAM association with
CASK requires the PDZ domain of CASK and the putative PDZ-binding motif
F-L-V of JAM, (ii) the two proteins colocalize at the lateral surface
in epithelial cells, (iii) association with CASK is not required for
recruitment of JAM to the junctions, and (iv) junction assembly
is accompanied by decreased amounts of JAM-CASK complexes in the Triton
X-100-insoluble fraction of cell lysates. These data suggest that
binding of JAM with CASK is dynamically regulated during junction assembly.
PDZ-dependent Association of JAM with CASK--
The
molecular association was determined by independent and complementary
assays. First, CASK was coimmunoprecipitated with JAM. Second, CASK
coeluted with the GST-JAM fusion protein. Third, the 6HisCASK-PDZ
protein interacted with GST-JAM in a direct binding assay. Hence, the
association requires the cytoplasmic tail of JAM (in particular, the
carboxyl-terminal residues Phe-Leu-ValCOOH, as demonstrated
by the inability of mJAM Subcellular Distribution of JAM and CASK--
Molecular
interaction of JAM and CASK is mirrored by their colocalization at the
lateral surface of the plasma membrane. Whereas the lateral
distribution of CASK (11) and human JAM (16) has been reported
previously, murine JAM was shown to codistribute with tight
junction-specific molecules in cell lines and to concentrate to the
tight junction-containing segment of the intercellular cleft in tissue
sections (1). Conceivably, differences in animal species, antibodies,
cell type, and maturation state of cell junctions are likely
determinants of such discrepancy. Data reported here indicate the
existence of two distinct subpopulations of JAM in Caco-2 cells. The
former decorates the lateral cell surface and colocalizes with the
basolateral membrane marker Na+,K+-ATPase. The
latter concentrates more apically and codistributes with the tight
junction marker cingulin. Interestingly, the transmembrane protein
occludin is also distributed both at basolateral membrane and at tight
junctions, perhaps reflecting differences in the maturation of
intercellular contacts (17). In our experimental system, CASK
colocalized with both lateral JAM and (albeit to a much lesser extent)
junctional JAM. Whereas a putative localization of CASK at epithelial
tight junctions requires further analysis, it is noteworthy that CASK
is a molecular component of the neuronal presynaptic plaque, another
junctional specialization of the plasma membrane (18).
Association with CASK and Junctional Localization of
JAM--
Several PDZ proteins are instrumental in recruiting
transmembrane ligands to specific domains of the plasma membrane (7). Among the immunoglobulin-like cell adhesion molecules known to interact
with PDZ proteins, fasciclin II and nectin are recruited to
neuromuscular and adherens junctions by the PDZ molecules discs-large and afadin, respectively (19, 20). However, a role for CASK in
recruiting JAM is questioned by the observations that JAM localizes to
the junctions earlier than CASK and that deletion of the
CASK-interacting sequence Phe-Leu-ValCOOH does not abolish
JAM recruitment to the junctions. CASK-independent targeting of JAM to
intercellular junctions might be attributable to other intercellular
molecules (such as ZO-1, cingulin, and AF-6) that, in contrast to CASK, localized to the junctions at early time points of junction assembly.
Modulation of JAM-CASK Complexes during Junction Assembly--
A
calcium switch protocol that induces disassembly and subsequent
reassembly of cadherin-based intercellular junctions (21, 22) was used
to evaluate whether junction assembly modulates the interaction of JAM
with CASK. Junction assembly strikingly influenced partitioning of CASK
(either alone or in association with JAM) between soluble and insoluble
fractions of Triton X-100 lysates, with enrichment of CASK in the
soluble fraction and reduction of JAM-CASK complexes in the insoluble
fraction. Because CASK interacts with protein 4.1 (11), it is
conceivable that the enhanced solubility reflects a transient loss of
CASK interactions with the cortical F-actin cytoskeleton, even if the
molecular interaction(s) primarily responsible for the observed effect
remains unknown. Regardless of the precise molecular mechanism, we
propose a dynamic model for the regulation of the JAM-CASK interaction during junction assembly (Fig. 5). In
mature junctions, CASK is found in association with JAM and can be
coprecipitated in both Triton X-100-soluble and -insoluble fractions of
cell lysates. Triton-insoluble JAM-CASK complexes are likely to be
associated with the actin cytoskeleton, possibly via protein 4.1 (11). At variance, upon junction assembly, CASK is released from its cytoskeletal associations, and JAM-CASK complexes are almost
exclusively detectable in the Triton-soluble fraction outside of the
junctions, whereas another subpopulation of JAM likely associates at
the junctions with other proteins, such as AF-6 (5), ZO-1, and cingulin
(4). We speculate that the interaction with JAM might be instrumental
in preventing CASK from being prematurely assembled into nascent
junctional complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 subunit of Na+,K+-ATPase)
and anti-cingulin serum were generous gifts of Drs. G. Pietrini
(University of Milan, Milan, Italy) and S. Citi (University of Padova,
Padova, Italy).
FLV constructs were cloned
in the PINCO retroviral vector to transfect the Phoenix packaging cell
line (gifts of Drs. P. G. Pelicci (European Institute of Oncology,
Milan, Italy) and G. P. Nolan (Stanford University, Stanford,
CA) and then to infect Caco-2 cells, as described previously (12).
Production of mJAM and mJAM
FLV has been described previously (4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PDZ-dependent association of JAM
with CASK. A, anti-JAM mAb BV16 coprecipitates CASK.
Caco-2 cell lysates were immunoprecipitated with mAb BV16 and analyzed
by Western blot with anti-CASK mAb C63120 (lane 1). Lysates
from Caco-2 cells (lane 2) and rat brain (lane 3)
were used as positive controls. B, the cytoplasmic tail of
JAM mediates the association with CASK. GST (lane 1) or
GST-JAM (lane 2) was immobilized onto glutathione-Sepharose
beads and incubated with Caco-2 lysates. C, deletion of the
putative PDZ-binding motif FLV in the JAM tail abolishes the
coprecipitation of CASK with JAM. Lysates from Caco-2 cells expressing
murine JAM (either full-length murine JAM (mJAM) or a
deletion mutant (mJAM FLV)) were
immunoprecipitated with either mAb BV12 or BV16, which recognizes
transfected murine (lanes 1 and 3) and endogenous
human (lanes 2 and 4) JAM, respectively.
D, the cytoplasmic tail of JAM directly binds the PDZ domain
of CASK. Fluid-phase GST (lanes 1 and 2) or
GST-JAM (lanes 3 and 4) was incubated with either
6HisCASK-PDZ immobilized on Ni2+-NTA beads (lanes
2 and 4) or beads alone (lanes 1 and
3). Proteins were eluted, separated by 7.5%
SDS-polyacrylamide gel electrophoresis under reducing conditions, and
analyzed by Western blot with either anti-CASK mAb C63120
(A-C) or anti-GST antibody (D). Molecular size
standards (kDa) are indicated at the left of each
panel.
FLV).
Caco-2 cells were then transfected with either mJAM
FLV or
full-length murine JAM (mJAM). As evaluated by fluorescence-activated cell-sorting analysis, mJAM and mJAM
FLV were expressed at
comparable levels at the cell surface (26.4% and 26.9% positive
cells, respectively). Endogenous human JAM and transfected murine JAM
(both mJAM and mJAM
FLV) were first immunoprecipitated using
anti-human BV16 and anti-murine BV12 mAbs, respectively. Then, the
association with CASK was tested by Western blot using the anti-CASK
mAb C63120 (Fig. 1C). As expected, mAb BV16 coprecipitated
CASK together with endogenous human JAM in both mJAM (lane
2) and mJAM
FLV transfectants (lane 4). At variance,
mAb BV12 only coprecipitated CASK in mJAM (lane 1) and not
in mJAM
FLV transfectants (lane 3). Preliminary
experiments had shown that the FLV deletion does not impair the ability
of mAb BV12 to immunoprecipitate mJAM
FLV (4).
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Fig. 2.
Immunofluorescence analysis of JAM and CASK
distribution. Caco-2 cells were stained with anti-CASK mAb 5230 (A), anti-JAM serum (B and E)
and mAb BV16 (G),
anti-Na+,K+-ATPase mAb 6H
(D), and anti-cingulin serum (H). Confocal laser
scanning micrographs of x, y (A-I) and x,
z (a-i) optical sections (first and
second columns), and merging of the two staining patterns
(third column) are shown.
FLV did localize to
intercellular junctions in transfected Caco-2 cells (data not
shown).
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Fig. 3.
Temporal dissociation in the junctional
recruitment of JAM and CASK. Caco-2 cells were grown to confluence
on glass coverslips in DMEM, incubated for 18 h with S-MEM
to disrupt intercellular junctions, and finally switched back to DMEM.
At the indicated time points, cells were fixed for staining with
antibodies against JAM and CASK (A) as well as the other
junctional molecules cingulin, ZO-1, and AF-6 (B).
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Fig. 4.
Effect of junction assembly on the solubility
of JAM, CASK, and JAM-CASK complexes in Triton X-100.
A, schematic diagram of the experimental procedure. High and
low calcium refer to DMEM (plus 10% fetal calf serum) and S-MEM
(plus 0.1% bovine serum albumin), respectively. B, Triton
X-100 lysates from Caco-2 cells were divided into Triton X-100-soluble
(S) and -insoluble (I) fractions and analyzed by
immunoprecipitation with a JAM antiserum followed by Western blot with
anti-CASK mAb C63120 (CASK JAM-bound). Lysates were also
analyzed by Western blot with either mAb C63120 (CASK total)
or anti-JAM mAb BV16 (JAM total). Immunoprecipitation of
cell surface JAM is shown (JAM biotin-labeled).
C, densitometric analysis of the results from experiments
similar to the one shown in B. Optical density values
(mean ± S.D.) for JAM, CASK, and JAM-CASK complexes were
determined in individual samples from five, four, and three independent
experiments, respectively. **, p < 0.01, Student's t test, unpaired samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FLV to coprecipitate CASK) and the PDZ
domain of CASK. Crystal structure (14) and peptide binding studies (6)
have established that CASK is a type II PDZ protein. Members of this
class of PDZ domains bind transmembrane proteins bearing the
carboxyl-terminal consensus sequence Phe/Tyr-X-
(X, any residue;
, hydrophobic). The preferred binding
motif of CASK was predicted to be Phe-Phe-Val(Phe/Ala), and ligands
known to interact with its PDZ domain, i.e. syndecan-2 (11)
and neurexin (9), terminate in Phe-Tyr-Ala and Tyr-Tyr-Val, respectively. The data reported here confirm the prediction that JAM is
a ligand for the PDZ domain of CASK and that binding requires the
Phe-Leu-ValCOOH residues, which are conserved in all the
sequences determined thus far, such as human, bovine (15), murine, and rat JAM (1), and which mediate JAM binding to the PDZ proteins ZO-1 (4)
and AF-6 (5).
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Fig. 5.
Regulation of the interaction between JAM and
CASK during junction assembly. Schematic model of the association
between JAM and CASK in mature junctions (right) and upon
junction assembly (left). See "Discussion" for a
detailed description of the model.
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FOOTNOTES |
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* This study was supported by grants from Human Frontiers Science Program (RG 0006/1997-M), EU (BIO4 CT 980337, BMH4 CT 983380, QLG1-CT-1999-01036, and QLK3-CT-1999-00020), Consiglio Nazionale delle Ricerche (97.01299.PF49), Ministero Università e Ricerca Scientifica e Tecnologica (99063171 57.003), and Associazione Italiana per la Ricerca sul Cancro.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.
§ A recipient of a Fellowship from International Center for Genetic Engineering and Biotechnology (Trieste, Italy).
** To whom correspondence should be addressed: Laboratory of Vascular Biology, Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20157 Milan, Italy. Tel.: 39-02-39014439; Fax: 39-02-3546277; E-mail: bazzoni@marionegri.it.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M006991200
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ABBREVIATIONS |
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The abbreviations used are: JAM, junctional adhesion molecule; GST, glutathione S-transferase; PDZ, PSD95/dlg/ZO-1; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Martin-Padura, I.,
Lostaglio, S.,
Schneemann, M.,
Williams, L.,
Romano, M.,
Fruscella, P.,
Panzeri, C.,
Stoppacciaro, A.,
Ruco, L.,
Villa, A.,
Simmons, D.,
and Dejana, E.
(1998)
J. Cell Biol.
142,
117-127 |
2. |
Bazzoni, G.,
Martinez-Estrada, O. M.,
Mueller, F.,
Nelboeck, P.,
Schmid, G.,
Bartfai, T.,
Dejana, E.,
and Brockhaus, M.
(2000)
J. Biol. Chem.
275,
30970-30976 |
3. | Tsukita, S., Furuse, M., and Itoh, M. (1999) Curr. Opin. Cell Biol. 11, 628-633[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Bazzoni, G.,
Martinez-Estrada, O. M.,
Orsenigo, F.,
Cordenonsi, M.,
Citi, S.,
and Dejana, E.
(2000)
J. Biol. Chem.
275,
20520-20526 |
5. |
Ebnet, K.,
Schulz, C. U.,
Meyer-zu-Brickwedde, M. K.,
Pendl, G. G.,
and Vestweber, D.
(2000)
J. Biol. Chem.
275,
27979-27988 |
6. |
Songyang, Z.,
Fanning, A. S.,
Fu, C.,
Xu, J.,
Marfatia, S. M.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
7. | Fanning, A. S., and Anderson, J. M. (1999) Curr. Opin. Cell Biol. 11, 432-439[CrossRef][Medline] [Order article via Infotrieve] |
8. | Stevenson, B. R., and Keon, B. H. (1998) Annu. Rev. Cell Dev. Biol. 14, 89-109[CrossRef][Medline] [Order article via Infotrieve] |
9. | Hata, Y., Butz, S., and Südhof, T. C. (1996) J. Neurosci. 16, 2488-2494[Abstract] |
10. | Kaech, S. M., Whitfield, C. W., and Kim, S. K. (1998) Cell 94, 761-771[Medline] [Order article via Infotrieve] |
11. |
Cohen, A. R.,
Wood, D. F.,
Marfatia, S. M.,
Walther, Z.,
Chishti, A. H.,
and Anderson, J. M.
(1998)
J. Cell Biol.
142,
129-138 |
12. | Grignani, F., Kinsella, T., Mencarelli, A., Valtieri, M., Riganelli, D., Grignani, F., Lanfrancone, L., Peschle, C., Nolan, G. P., and Pelicci, P. G. (1998) Cancer Res. 58, 14-19[Abstract] |
13. |
Perego, C.,
Bulbarelli, A.,
Longhi, R.,
Caimi, M.,
Villa, A.,
Caplan, M. J.,
and Pietrini, G.
(1997)
J. Biol. Chem.
272,
6584-6592 |
14. | Daniels, D. L., Cohen, A. R., Anderson, J. M., and Brunger, A. T. (1998) Nat. Struct. Biol. 5, 317-325[Medline] [Order article via Infotrieve] |
15. |
Ozaki, H.,
Ishii, K.,
Horiuchi, H.,
Arai, H.,
Kawamoto, T.,
Okawa, K.,
Iwamatsu, A.,
and Kita, T.
(1999)
J. Immunol.
163,
553-557 |
16. |
Liu, Y.,
Nusrat, A.,
Schnell, F. J.,
Reaves, T. A.,
Walsh, S.,
Pochet, M.,
and Parkos, C. A.
(2000)
J. Cell Sci.
113,
2363-2374 |
17. |
Sakakibara, A.,
Furuse, M.,
Saitou, M.,
Ando-Akatsuka, Y.,
and Tsukita, S.
(1997)
J. Cell Biol.
137,
1393-1401 |
18. |
Hsueh, Y.-P.,
Yang, F.-C.,
Kharazia, V.,
Naisbitt, S.,
Cohen, A. R.,
Weinberg, R. J.,
and Sheng, M.
(1998)
J. Cell Biol.
142,
139-151 |
19. | Zito, K., Fetter, R. D., Goodman, C. S., and Isacoff, E. Y. (1997) Neuron 19, 1007-1016[Medline] [Order article via Infotrieve] |
20. |
Takahashi, K.,
Nakanishi, H.,
Miyahara, M.,
Mandai, K.,
Satoh, K.,
Satoh, A.,
Nishioka, H.,
Aoki, J.,
Nomoto, A.,
Mizoguchi, A.,
and Takai, Y.
(1999)
J. Cell Biol.
145,
539-549 |
21. | Rajasekaran, A. K., Hojo, M., Huima, T., and Rodriguez-Boulan, E. (1996) J. Cell Biol. 132, 451-463[Abstract] |
22. | Anderson, J. M., Van Itallie, C. M., Peterson, M. D., Stevenson, B. R., Carew, E. A., and Mooseker, M. S. (1989) J. Cell Biol. 109, 1047-1056[Abstract] |