Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Author for correspondence (e-mail:
peles{at}weizmann.ac.il)
Accepted 2 December 2002
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Summary |
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Key words: PDZ domain, Tyrosine phosphatase, RPTPß, Tight junctions, Adherens junctions, MAGI
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Introduction |
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Proteins containing PDZ (PSD-95/discs large/zona occludens 1) domains have
been identified as key components in the organization of protein complexes at
the plasma membrane (Fanning and Anderson,
1999). These proteins have been implicated in several functions,
including localization of proteins to specialized cell junctions
(Gonzalez-Mariscal et al.,
2000
), clustering of transmembrane receptors
(Sheng and Pak, 2000
) and the
recruitment of cytosolic proteins to generate multi-signaling complexes
(Scott and Zuker, 1998
). Many
PDZ-domain-containing proteins are found in a multidomain arrangement that
also includes other modules of protein-protein interactions. One particular
class consists of the MAGUKs (membrane-associated-guanylate kinase), a family
of scaffolding proteins containing several PDZ domains, SH3 domains and a
catalytically inactive guanylate kinase domain, all of which mediate
protein-protein interactions (Harris and
Lim, 2001
). The presence of three distinct domains that mediate
protein-protein interaction, along with the ability of these domains to form
higher-order oligomers through intermolecular interactions between the SH3 and
the guanylate kinase domain, enables the MAGUKs to recruit signaling proteins
and to control the assembly of multivalent protein complexes
(McGee et al., 2001
;
Nix et al., 2000
). MAGI
(membrane-associated guanylate kinase with inverted orientation) proteins are
a distinct subgroup of the MAGUKs and include MAGI-1/BAP1
(Dobrosotskaya et al., 1997
;
Shiratsuchi et al., 1998
),
MAGI-2/AIP/ARIP/S-SCAM (Hirao et al.,
1998
; Shoji et al.,
2000
; Wood et al.,
1998
) and MAGI-3 (Wu et al.,
2000b
). In the present study we have identified the rat homologue
of MAGI-3 as a protein that interacts with the C-terminal tail of RPTPß.
Furthermore, we found that MAGI-3 interacts with tyrosine-phosphorylated
proteins in different cell types, suggesting that it may serve as a scaffold
to position substrates for receptor tyrosine phosphatases at the plasma
membrane.
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Materials and Methods |
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cDNA cloning and constructs
For cloning of a full-length MAGI-3, an EcoRI-XhoI
fragment containing the third and fourth PDZ domains, isolated from one of the
clones obtained from the two-hybrid library (pGH#707), was used as a probe to
screen a ZAP-PC12 cDNA library. The sequence of rat Slipr/MAGI-3 cDNA
was determined on both strands by priming with synthetic oligonucleotides and
deposited in GenBank (accession number AF255614). Expression constructs were
made by subcloning the full-length cDNA into pCDNA3 (Invitrogen) to generate
pC3-rMAGI-3. The RPTPß expression construct containing a hemagglutinin
tag has previously been described (Adamsky
et al., 2001).
Fusion proteins and antibodies
GST-fusion proteins containing the intracellular region of RPTPß
(GST-ßD12, amino acids 799-1440), the first phosphatase domain
(GST-ßD1, amino acids 799-1154), the second phosphatase domain including
the C-terminal tail (GST-ßD2, amino acids 1120-1440) and the
intracellular region of RPTPß lacking the last eight amino acids
(GST-ßD12dCT) were made by cloning the corresponding cDNA fragments into
pGEX-6P (Pharmacia-Amersham). A catalytically inactive RPTPß mutant was
constructed by replacing aspartic acid (1026) with alanine residues to
generate GST-ßD12DA. Polyclonal antibodies against MAGI-3 were generated
by immunizing rabbits with a synthetic peptide (RLNRTELPTRSAPQES,
corresponding to amino acid residues 835-850 of MAGI-3) coupled to keyhole
limpet hemocyanin (Sigma). Antibodies were affinity purified on a column of
the peptide antigens covalently coupled to agarose beads (Pierce). A second
antibody to MAGI-3 was generated by immunizing rabbits with a GST-fusion
protein containing the third and fourth PDZ domains of rat MAGI-3 (Ab#M23).
Affinity purification was performed by first removing the antibodies against
GST using Sepharose-GST (Pierce) and then running it on a column of
GST-MAGI-3#707. Antibodies to HA-tag were purchased from Roche Molecular
Biochemicals and antibodies to ZO-1 and phosphotyrosine (PY20) from
Transduction Laboratories. Antibodies against ß-catenin, desmoplakin and
cingulin were generously provided by Benny Geiger (The Weizmann
Institute).
Northern blots and RT PCR
A 1 kb DNA fragment corresponding to positions 1404-2427 of rat MAGI-3 cDNA
was used as a probe for northern blot analysis. The DNA fragment was isolated,
labeled by random priming, and purified on a Sepharose G-50 column
(Amersham-Pharmacia). Hybridization to multiple tissue northern blots (MTN
Blots, Clontech) was carried out as described previously
(Poliak et al., 1999). PCR
analysis was done with various cDNA sources using a set of primers
(5-GGCAAAGTCATAAATAAAG-3, 5-CTCTGCAAGAAAGCC-3) specific to MAGI-3, which did
not recognize rat MAGI-1 or MAGI-2.
Immunoprecipitation and peptide pulldown experiments
Immunoprecipitation and immunoblotting analyses from different cell lines
or rat brain membrane lysates were done essentially as described previously
(Adamsky et al., 2001).
Co-immunoprecipitation from HEK-293T cells transfected with RPTPß,
MAGI-3, or both proteins, was done using a solubilization buffer containing 1%
Triton X-100 as previously described
(Peles et al., 1995
). For
GST-pulldown experiments, SF763T cells were solubilized in TNTG buffer (20 mM
Tris 7.5, 0.1% Triton X-100, 150 mM NaCl, 10% glycerol and protease
inhibitors), and the lysates were incubated with various GST-fusion proteins
coupled to glutathione-Sepharose (Amersham-Pharmacia). For peptide-pulldown
experiments, rat brains were solubilized in RIPA buffer (10 mM sodium
phosphate pH 7, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 4 µg/ml
aprotinin, 4 µg/ml leupeptin, 1 mM PMSF), the lysates were diluted 1:1 with
TNTG, and incubated with 20 µg of biotinylated peptides coupled to
Neutravidin beads (Pierce). Bound proteins were washed three times with TNTG,
separated on SDS gels and immunoblotted with an anti-MAGI-3 antibody. Peptide
used for pulldown experiments include ßCT-NIAESLESLV,
ßCTS-SISENEVLAL, Caspr2CT-NFTETIDESKKEWLI and Caspr2dCT-NFTETIDESKKE
(Poliak et al., 1999
). For
co-immunoprecipitation of phosphorylated proteins, cells were treated with 1
mM Na3 VO4 for 30 minutes at 37°C and solubilized
using RIPA buffer containing 1mM Na3 VO4. Lysates were
immunoprecipitated with anti-MAGI-3 antibodies (BM#4112; Sigma), and were
western blotted with Py-20 anti-phosphotyrosine antibodies (Transduction
Laboratories).
Dephosphorylation experiments
Phosphorylated p130 was co-precipitated with an anti MAGI-3 antibody from
vanadate-treated SF763T cells. The immunocomplexes where divided equally
between several tubes and incubated with GST-RPTPß fusion protein coupled
to beads in 50 µl of buffer containing 25 mM MES, pH 5.5, 5 mM DTT or with
5U shrimp alkaline phosphatase (Roach) in 50 mM Tris (pH 8.0), 5 mM DTT. All
reactions were carried out for 2 hours at 23°C with gentle shaking.
Dephosphorylation of p130 was then monitored by immunoblotting using anti-PY
antibodies (PY20). Phosphatase activity of GST-RPTPß constructs was
measured as p-phosphate hydrolysis in 0.5 ml of 25 mM MES, pH 5.5,
containing 5 mM DTT and 10 mM pNPP incubated at 23°C. The
reaction was stopped by the addition of 100 µl of 1 N NaOH, and the
absorbance was measured at 405 nm.
Immunofluorescence
Caco2 cells or cultures of primary rat astrocytes
(Adamsky et al., 2001) were
grown on glass coverslips precoated with 10 µg/ml polylysine. Slides were
fixed/permeabilized with 0.2% Triton X-100/4% paraformaldehyde for 3 minutes
and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 15
minutes. Primary antibodies were diluted in 0.1 M PBS, pH 7.4, containing 0.1%
Triton X-100 (PBST) and incubated for 1 hour. After three washes with PBS,
slides were incubated with Alexa-488-conjugated anti-mouse and Cy3-conjugated
anti-rabbit antibodies in DAPI-containing PBTS. Slides were subsequently
washed with PBS and mounted with Elvanol for observation. Immunofluorescence
slides were viewed and analyzed using a BioRad confocal microscope or a Zeiss
Axioplan microscope equipped with a SPOT-II (Diagnostic Instruments) cooled
CCD camera.
Immunoelectron microscopy
For immunogold electron microscopy, Caco2 cells were fixed with a freshly
prepared solution of 4% paraformaldehyde, 2% acrolein and 0.1 M sucrose in 0.1
M cacodylate buffer containing 5 mM CaCl2 for 1 hour at 24°C.
The cells were scraped from the culture dish; the pellets were incubated in
10% gelatin at 37°C for 30 minutes and postfixed in the same fixative at
40°C for 24 hours. Fixed cell pellets were cryoprotected overnight with
2.3 M sucrose in cacodylate buffer and frozen by injection into liquid
nitrogen. Ultrathin (75 nm) sections were cut with a diamond knife (Drukker)
at -115°C using a Reichert Ultracat-S ultramicrotome. The sections were
transferred to 200-mesh nickel grids coated with formvar. The sections were
treated with PBS-containing 0.5% BSA, 3% normal goat serum (Sigma), 0.1%
glycine and 1% Tween 20 for 5 minutes to block non-specific binding, followed
by 2 hours of incubation with M-23 antibodies. After extensive washing in
PBS-0.1% glycine, the primary antibody was detected with goat anti-rabbit 10
nm colloidal gold conjugate (1:20; Zymed). The grids were then washed in
PBS-glycine, stained with neutral uranyl acetate oxalate for 5 minutes,
quickly washed and then stained with 2% uranyl acetate in H2O for
10 minutes. Embedding was done in 2% methyl cellulose/uranyl acetate.
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Results |
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Expression of MAGI-3 mRNA and protein
Analysis of the expression of MAGI-3 mRNA in various rat tissues by
northern blots revealed the presence of a single 6.4 kb transcript in the
brain, heart, lung, liver and at a lower level in the kidney
(Fig. 2A). No MAGI-3 transcript
was detected in the spleen, skeletal muscle and testis. The size of the
transcript, as well as its expression in various tissues, differed from those
of MAGI-1 (Dobrosotskaya et al.,
1997; Shiratsuchi et al.,
1998
) and MAGI-2 (Hirao et
al., 1998
; Shoji et al.,
2000
; Wood et al.,
1998
), indicating that the probe we used specifically detected
MAGI-3 and not the related MAGIs. RT-PCR analysis using a set of primers
specific to MAGI-3 detected its expression in rat brain, retina,
astrocytes and in neuroendocrine GH3 cell lines but not in neural stem cells
or testis (Fig. 2B). We next
examined the expression of MAGI-3 protein in various cell lines, rat brain
lysates and primary astrocytes using antibodies that were raised against a
peptide that is found in MAGI-3 but not in MAGI-1 and MAGI-2. As shown in
Fig. 2C,D, this antibody, but
not the pre-immune serum, recognized a major band at 140 kDa and two minor
bands at 170 kDa and 130 kDa in GH3 cells, primary cultured astrocytes and in
rat brain. In C6 and PC12 cells, two major forms at 140 kDa and 130 kDa, as
well as a minor band at 170 kDa, were detected. Similar results were obtained
using a monoclonal antibody specific to MAGI-3 (data not shown), suggesting
that MAGI-3 has multiple isoforms, as do MAGI-1 and MAGI-2, which are
generated by alternative splicing
(Dobrosotskaya et al., 1997
;
Hirao et al., 2000
).
Nevertheless, although several splice isoforms of MAGI-3 do exist in the
databases (data not shown), we found no indication that the minor 170 kDa band
results from alternative splicing.
|
Association of RPTPß with MAGI-3 is mediated by its C-terminal
tail
To further corroborate the two-hybrid data, we examined whether RPTPß
interacts with MAGI-3 in transfected cells. As shown in
Fig. 3A, RPTPß could be
immunoprecipitated using an antibody against MAGI-3 from HEK-293T cells
expressing both proteins but not from cells expressing MAGI-3 or RPTPß
alone, or from untransfected cells. Similarly, MAGI-3 was
co-immunoprecipitated using an HA-tag antibody that recognized RPTPß from
cells expressing both proteins. In order to map the region in RPTPß that
mediates its binding to MAGI-3, we used GST-fusion proteins containing the
entire cytoplasmic domain of RPTPß (ßD12), the first phosphatase
domain (ßD1), the second phosphatase domain (ßD2) or a mutant form
lacking the C-terminus (ßD12dCT) of this phosphatase in pulldown
experiments. Cell lysates of HEK-293T cells expressing MAGI-3 were mixed with
the various GST-fusion proteins immobilized on beads, and bound proteins were
detected by immunoblotting with an antibody to MAGI-3. As shown in
Fig. 3B, MAGI-3 was
precipitated with ßD12 and ßD2, but not with ßD1 or
ßD12dCT. Furthermore, MAGI-3 was precipitated from rat brain membrane
lysates using a peptide containing the last 12 amino acids of RPTPß
(ßCT) immobilized on beads but not by a peptide containing a scrambled
sequence (ßCTS), nor by a Caspr2 peptide, which contains a type II
PDZ-binding sequence (Spiegel et al.,
2002) (Fig. 3C).
Yeast two-hybrid analysis using the different RPTPß constructs and the
original MAGI-3 fragment obtained from the screen, which contained PDZ3 and
PDZ4, yielded similar results (Fig.
3D). Taken together, these results show that MAGI-3 and RPTPß
are found in a protein complex when expressed in the same cells and that this
association is mediated by binding of the C-terminal sequence of RPTPß to
the third or fourth PDZ domain of MAGI-3.
|
Cell-type-specific localization of MAGI-3 at cell junctions, focal
adhesions and the nucleus
Proteins containing PDZ domains have been suggested to play a role in the
organization of protein complexes at the plasma membrane
(Fanning and Anderson, 1999).
Accordingly, many of these proteins were found in specialized cell-cell
contacts such as adherens and tight junctions
(Gonzalez-Mariscal et al.,
2000
; Nagafuchi,
2001
). In order to determine the subcellular localization of
MAGI-3, we examined its distribution in epithelial cells and astrocytes.
Double labeling of Caco2 colon carcinoma cells with a MAGI-3-specific antibody
and antibodies to either ZO-1 or cingulin revealed that MAGI-3 colocalized
with these proteins at tight junctions
(Fig. 4). In contrast, MAGI-3
only partially overlapped with ß-catenin and desmoplakin, two proteins
that reside in adherens junctions and desmosoms, respectively. A similar
localization of MAGI-3 was detected in Caco2 cells using a different
monoclonal antibody (Fig. 4M).
Furthermore, MAGI-3 was concentrated at cell-cell contacts in transfected MDCK
cells, which lack endogenous MAGI-3 (Fig.
4N,O). In addition, strong punctuate staining of MAGI-3 was found
in the nucleus, whereas most of the cell cytoplasm remained unstained or only
very weakly labeled. Membrane and nuclear localization of MAGI-3 was also
found by cell fractionation (data not shown). Localization of MAGI-3 to tight
junctions and the nucleus in Caco2 cells was also confirmed by immunoelectron
microscopy (Fig. 5). In
addition, gold particles were frequently detected in the cell microvilli,
demonstrating that in addition to tight junctions, MAGI-3 is also found at the
apical membrane.
|
|
In primary rat astrocytes, a cell type that does not show any tight junction structures in culture, MAGI-3 was found in the nucleus and was localized with ß-catenin in cell-cell contacts (Fig. 6A-C). Double labeling of MAGI-3 and anti-phosphotyrosine antibodies showed that MAGI-3 was concentrated along with tyrosine-phosphorylated proteins at sites of cell contacts but was absent in phosphotyrosine-positive focal adhesion sites at the cell's periphery (Fig. 6D-F). At cell-cell contacts, MAGI-3 appeared as a thin discontinuous line at the cell's junction or as a rod-like shape, labeling extended protrusions between neighboring cells. Surprisingly, in older cultures of astrocytes, MAGI-3 was also found in focal adhesions, where it colocalized with vinculin at the edge of actin filaments (Fig. 6G-L). Localization of MAGI-3 to focal adhesion sites was more prominent in dispersed rather than crowded cultures (data not shown). Thus, we concluded that MAGI-3 is found in the cell nucleus and in specific structures along the plasma membrane. Depending on the cell type and culture conditions, it could be found in tight and adherens junctions, cellular protrusions and focal adhesion sites.
|
Association of MAGI-3 with tyrosine-phosphorylated proteins
The association of MAGI-3 with RPTPß raises the question of whether it
serves as a substrate for this tyrosine phosphatase. To determine whether
MAGI-3 could be phosphorylated on tyrosine residues, we treated SF763T cells
with sodium orthovanadate, a cell-permeable inhibitor of tyrosine phosphatases
that induces a general increase in tyrosine phosphorylation of proteins within
the cell (Gordon, 1991). Such
treatment caused a marked shift in the apparent molecular weight of MAGI-3 in
these cells, as detected by immunoprecipitating MAGI-3 with two different
antibodies (Fig. 7A, upper
panel). However, this shift was not due to phosphorylation on tyrosine
residues, since MAGI-3 was neither detected by anti-phosphotyrosine after
immunoprecipitation with an anti-MAGI-3 antibody nor by anti-MAGI-3
immunoblotting after immunoprecipitation with anti-phosphotyrosine antibodies.
Furthermore, treatment of MAGI-3 immunocomplexes with alkaline phosphatase did
not change its mobility, indicating that the observed shift in MAGI-3 upon
vanadate treatment was not due to its phosphorylation either on tyrosine or
other residues. Similar results were obtained using potato acid phosphatase
(data not shown). Nevertheless, anti-phosphotyrosine immunoblots revealed the
presence of a 130 kDa protein in MAGI-3 immunocomplexes from vanadate-treated
cells (Fig. 7A, lower panel). A
very low level of phosphorylated p130 was occasionally detected in non-treated
cells. Importantly, this protein was detected when two distinct MAGI-3
antibodies were used for immunoprecipitation and disappeared after treatment
of the immunocomplexes with alkaline phosphatase. Furthermore, similar
analysis done with various cell lines demonstrated that MAGI-3 was associated
with different tyrosine-phosphorylated proteins
(Fig. 7B, lower panel).
Although MAGI-3 was associated with a tyrosine-phosphorylated p130 in SF763T
and C6 cells, in Caco2 cells it was associated with a 90 kDa protein
(Fig. 7B). The identity of
these proteins is presently unknown, although it should be noted that it was
not recognized by antibodies to several candidates such as FAK, p130-Cas, Pyk2
and ß-catenin (data not shown).
|
Dephosphorylation of MAGI-3-associated p130 by RPTPß
The results so far suggest that MAGI-3 functions as a scaffolding protein
linking tyrosine phosphatases such as RPTPß with their phosphorylated
protein substrates. In order to check this possibility, we examined whether
phosphorylated p130 could be dephosphorylated by RPTPß. To this end, we
immunoprecipitated MAGI-3 from vanadate-treated SF763T cells and incubated the
immunocomplexes with GST-fusion proteins containing the cytoplasmic region of
RPTPß (ßD12), a catalytically inactive RPTPß mutant
(ßD12DA), a deletion mutant lacking the C-terminal tail of RPTPß
(ßD12dCT) or a GST-fusion containing only the first phosphatase domain
(ßD1) before analyzing them by anti-phosphotyrosine or anti-MAGI-3
antibodies. As depicted in Fig.
8A, p130 was completely dephosphorylated by ßD12 and, as
expected, not by the catalytically inactive ßD12DA. Surprisingly, p130
was not dephosphorylated by ßD1 or ßD12dCT, although both of these
constructs were active phosphatases as determined using nitrophenyl phosphate
as a substrate (Fig. 9C). These
results demonstrated that p130 could serve as a substrate for RPTPß.
Furthermore, dephosphorylation of p130 required the C-terminal tail of the
phosphatase, suggesting that the interaction of this region with MAGI-3
regulates the activity of RPTPß toward its substrates.
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Discussion |
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The dynamic arrangement of cell-cell contacts is important for maintaining
cell polarity and allowing communication between neighboring cells by precise
deposition of receptors and their downstream effectors at the plasma membrane
(Fanning and Anderson, 1999).
Proteins containing PDZ domains have been localized to tight, adherens and
synaptic junctions, where they interact with the cytoskeletal proteins,
thereby providing a direct link between membrane signals and cell morphology
(Gonzalez-Mariscal et al.,
2000
; Nagafuchi,
2001
). We have found that in epithelial cells, MAGI-3 is localized
with ZO-1 and cingulin to tight junctions, whereas in primary astrocytes it
was found with ß-catenin in adherens junctions and cellular protrusions.
The latter are reminiscent of the dynamic finger-like structures observed when
keratinocytes in culture make contacts
(Vasioukhin et al., 2000
). Our
results are in agreement with studies describing the subcellular localization
of MAGI-1, which was found at tight junctions in MDCK cells
(Dobrosotskaya and James, 2000
;
Ide et al., 1999
), as well as
in cadherin-based adherens junctions in normal kidney NRK cells
(Nishimura et al., 2000
).
However, in contrast to other family members, MAGI-3 was also found in focal
adhesion sites in aged astrocyte cultures, suggesting that its subcellular
localization not only depends on the cell type, but also on the culture
condition. In addition to its localization to specific sites at the plasma
membrane, MAGI-3 was strongly expressed in the nucleus. MAGI-3 contains two
putative nuclear localization signals located at its N-terminus (KKKKH at aa
6) and in its guanylate kinase domain (KRKK at amino acid 368). Importantly,
nuclear localization of MAGI-3 was detected in all cell types examined using
two distinct antibodies and was also confirmed by biochemical fractionation of
the cells (data not shown). In this regard, the nuclear localization of MAGI-3
is similar to several other junctional proteins containing PDZ domains that
could also be found in the nucleus
(Dobrosotskaya et al., 1997
;
Gottardi et al., 1996
;
Hsueh et al., 2000
). It was
suggested that the binding of these proteins to transmembrane proteins
sequester MAGI at the plasma membrane, thereby regulating their nuclear
localization. Furthermore, two of these proteins, ZO-1 and CASK, were recently
shown to activate gene expression by binding to the Tbr-1 and ZONAB
transcription factors, respectively (Balda
and Matter, 2000
; Hsueh et
al., 2000
). Although transcriptional activity of MAGI-3 remains to
be explored, the subcellular localization of MAGI-3 raises the possibility
that it takes part in a signaling pathway that is regulated by cell adhesion
and cell-cell contact.
Cell adhesion and cell-cell contact are controlled by tyrosine
phosphorylation. We showed that treatment of cells with vanadate induced a
marked shift in the apparent molecular mass of MAGI-3. However, the nature of
this modification is presently not clear. Despite the presence of a conserved
tyrosine residue at position 356 in MAGI-3, which is phosphorylated by Src in
MAGI-1 (Nishimura et al.,
2000), we found no indication that MAGI-3 is tyrosine
phosphorylated. Instead, vanadate treatment revealed that MAGI-3 was
associated with few tyrosine-phosphorylated proteins. A 130 kDa
tyrosine-phosphorylated protein that was associated with MAGI-3 in most of the
cell lines examined served as a substrate for RPTPß. Interestingly, we
found that p130 could serve as a substrate for RPTPß. Strikingly,
dephosphorylation of p130 by RPTPß required the C-terminal tail of this
phosphatase. Given that this region mediates the interaction of RPTPß
with MAGI-3, these results suggest that the latter may function as a
scaffolding protein that bridges between the phosphatase and p130
(Fig. 9). Furthermore, the
multi-domain organization of MAGI-3 may allow it to establish higher order
signaling complexes, as suggested for other multi-PDZ-containing proteins
(Harris and Lim, 2001
). In the
case of RPTPß such a signaling complex may consist of a mesh of several
distinct scaffolding proteins including members of the MAGI and PSD95
families. Accordingly, it was shown that the MAGI-3-related S-SCAM/MAGI-2
interacts with PSD95 and ß-catenin, two proteins that are found in a
complex with RPTPß (Dobrosotskaya and
James, 2000
; Kawachi et al.,
1999
). Our results suggest that the association of RPTPß with
MAGI-3 may allow the recruitment of p130 and additional substrates of this
phosphatase. Interestingly, it was recently demonstrated that both
S-SCAM/MAGI-2 and MAGI-3/MAGI-3 are found in a complex with the tumor
suppressor PTEN, and they regulate its activity by recruiting it to the plasma
membrane where its PtdIns(3,4,5)P3 phospholipid substrate is
localized (Wu et al., 2000a
;
Wu et al., 2000b
). Similarly,
PTPµ interacts with RACK (receptor for activated protein C kinase), a
scaffolding protein that is thought to recruit proteins to the plasma membrane
(Mourton et al., 2001
). The
above data, along with the results presented here, suggest that the use of
scaffolding proteins such as MAGI-3 to bring tyrosine-phosphorylated proteins
in close proximity to their phosphatases, may be a general mechanism that
regulates the function of these enzymes.
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Acknowledgments |
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Footnotes |
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