Department of Molecular Biology, Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany
Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology
with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTP.
PTP
biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex.
Both LAR and PTP
underwent further proteolytical processing upon treatment of cells with either calcium
ionophore A23187 or phorbol ester TPA. Induction of
LAR processing by TPA in 293 cells did require overexpression of PKC
. Induced proteolysis resulted in
shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTP
cleavage site between amino acids Pro821
and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of
plakoglobin. Consistent with this observation, we found
direct association of plakoglobin and
-catenin with the
intracellular domain of LAR in vitro. Taken together,
these data suggested an involvement of LAR and PTP
in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After
processing and shedding of the extracellular domain,
the catalytically active intracellular portions of both
PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism
that regulates the activity and target specificity of these
PTPases. Calcium withdrawal, which led to cell contact
disruption, also resulted in internalization but was not
associated with prior proteolytic cleavage and shedding
of the extracellular domain. We conclude that the subcellular localization of LAR and PTP
is regulated by
at least two independent mechanisms, one of which requires the presence of their extracellular domains and
one of which involves the presence of intact cell-cell
contacts.
A key element in the regulation of cell-cell and cell-
matrix contacts is the tyrosine phosphorylation of
proteins that are localized in focal adhesions and
at intercellular junctions (for reviews see Kemler, 1993;
Clark and Brugge, 1995). While much is known about the
protein tyrosine kinases involved in the phosphorylation of cell adhesion components, very little information exists
about the identity of protein tyrosine phosphatases (PTPases),1 which are responsible for the dephosphorylation
and thereby regulation of these structural complexes.
Probable candidates are those receptor-like PTPases that
contain cell adhesion molecule-like extracellular domains
and could therefore regulate their intrinsic phosphatase activity in response to cell contact. Recent reports suggest
that some PTPases do, in fact, possess properties that resemble those of classical cell adhesion molecules (for review see Brady-Kalnay and Tonks, 1995). A direct involvement in cell-cell contact has so far been demonstrated
for PTPµ (Brady-Kalnay et al., 1993; Gebbink et al., 1993)
and PTP PTPµ (Gebbink et al., 1991), PTP We report here that PTP Cell Lines and Culture Media
A431 (CRL 1555; American Type Culture Collection, Rockville, MD)
and HeLa (CCL 2; American type Culture Collection) cells were grown in
Dulbeco's minimal essential medium (DMEM) containing 4.5 mg/ml glucose and supplemented with 10% FCS. For growth of 293 cells (CRL
1573; American Type Culture Collection), DMEM containing 1.0 mg/ml
glucose and 10% FCS was used. All growth media were supplemented
with 2 mM L-glutamine before use. For starvation experiments, A431 and
HeLa cells were grown for 48 h and 293 cells for 24 h in their respective
growth media, which were diluted 1:40 with identical serum-free medium.
All media and supplements were purchased from GIBCO BRL (Eggenstein, Germany).
cDNA Constructs
For transient expression experiments, the human LAR cDNA was cloned
into the cytomegalovirus early promoter-based (Eaton et al., 1986) expression plasmid pRK5. For subcloning purposes, pSP65-LAR was kindly
provided by H. Saito (Harvard Medical School, Boston, MA). pSP65-
LAR was cut with restriction enzymes EcoRI and NruI, and the two resulting fragments of 4,448 (EcoRI/EcoRI) and 2,004 bp (EcoRI/NruI)
containing the complete coding region of human LAR were inserted in
the pRK5 plasmid, which had been linearized with restriction enzymes
EcoRI and EcoRV. The pRK5 expression plasmid containing the cDNA
of rat PTP The plasmid coding for the GST-hPTP LARi fusion protein was constructed by amplification of the cDNA sequence between amino acids
1,259 to 1,881 of human LAR using PCR with oligonucleotides 5 Antibodies
Rabbit antisera Transient Expression in 293 Cells and Stimulation
of Cells
293 cells were seeded in 20% confluency and were transfected 24 h later
using the calcium phosphate precipitation technique described by Chen
and Okayama (1987). 16 h after transfection, cells were washed once with
starvation medium (DMEM with 0.25% FCS) and grown for an additional
24 h in the same medium. Alternatively for metabolic labeling with
[35S]methionine, cells were washed and grown in methionine-free minimal essential medium with 0.25% dialyzed FCS. 50 µCi/ml [35S]methionine
(1,000 Ci/mmol, Amersham Intl., Amersham, UK) were added 16 h before lysis. Before lysis, cells were stimulated with 10 Immunoprecipitation and Immunoblotting
Cells were washed once with ice-cold PBS and lysed in Triton X-100 lysis
buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 10% glycerol, 1% Triton
X-100, 100 mM NaF, 10 mM Na4P2O7, 2 mM Na3V04, 5 mM EGTA, 1 mM
PMSF, 1 µg/ml each leupeptin, pepstatin, antipain, and chymostatin). Lysates were centrifuged for 20 min at 12,500 g to obtain the supernatant
fraction, and protein concentration was determined by using the method
described by Bradford (1976). Equal amounts of proteins were used in
each experiment. For immunoprecipitation, protein A-Sepharose (Pharmacia Biotech) was preincubated with specific antisera, washed twice with
HNTG (50 mM Hepes, pH 7.2, 150 mM NaCl, 10% glycerol, 0.1% Triton
X-100, 1 mM Na3V04, 1 mM PMSF), and added to the lysates. For binding
to WGA-Sepharose (Sigma Chemical Co.), lysates were diluted 1:5 in
HNTG and tissue culture supernatants centrifuged twice at 1,000 g for 15 min before adding WGA-Sepharose. Glutathione-S-transferase (GST) fusion proteins were expressed in Escherichia coli and purified as described
(Smith and Johnson, 1988). 3 µg of GST-hPTP LARi fusion protein and a threefold molar excess of GST were incubated with equal amounts of cell
lysates and immobilized by adding glutathione-Sepharose (Sigma Chemical Co.). All immobilization steps were performed for 4-16 h, and the resulting complexes were washed three times with HNTG. Samples were
boiled in SDS sample buffer for 10 min followed by separation in SDS-PAGE. For immunoblotting analysis, the enhanced chemiluminescence
system (Amersham Intl.) was used in conjunction with goat anti-rabbit
antibodies (Bio Rad Labs). For reprobing purposes, blots were stripped in
62.5 mM Tris/HCl, pH 6.8, 2% SDS, and 100 mM NH2-terminal Sequencing
PTP Immunofluorescence Microscopy
For immunofluorescence studies, A431 cells (CRL 1555; American Type
Culture Collection) were grown for 48 h on uncoated glass coverslips to
different degrees of confluency. Control cells or cells incubated with either TPA, EGTA, or ionophore for the respective time intervals (see Figs.
7-10, legends) were fixed with 2% formaldehyde freshly prepared from
paraformaldehyde in PBS (pH 7.4, 0.12 M sucrose). Autofluorescence was
quenched with PBS glycine (100 mM), and the cells were permeabilized
with 0.5% saponin in PBS (5 min). Unspecific antibody binding was
blocked for 1 h with phosphate buffered gelatine (PBG: PBS, 0.5% bovine
serum albumin, 0.045% cold-water fish gelatine). Primary antibody incubation was done at room temperature for 2 h after dilution in PBG, 1:50 for rabbit antisera
Scanning Electron Microscopy
For scanning electron microscopy, cells were fixed with 2% formaldehyde/
1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, 0.12 M sucrose on
ice), postfixed with 0.02 M osmium tetroxide for 30 min, flushed with 30%
ethanol, and dehydrated through graded ethanol and 100% dried acetone.
After critical point drying with CO2 for 60 min (Polaron, Watford, UK) the
coverslips were attached to scanning electron microscopy stubs with conductive carbon paint and left overnight. Before examination on a scanning
electron microscope (35; Jeol, Tokyo, Japan) they were gold sputtered to a thickness of 20 nm (SCD 020 Coating Unit; Balzers, Liechtenstein).
Biosynthesis and Processing of LAR and PTP LAR and PTP
To compare the biosynthesis of human LAR and rat
PTP The proteins immunoprecipitated with the same antiserum from [35S]methionine-labeled cells were identical
(Fig. 1 C). As previously demonstrated with LAR (Streuli
et al., 1992; Yu et al., 1992), the noncovalent linkage between the E and P subunit was stable under standard cell
lysis conditions and during immunoprecipitation. The
LAR E subunit of 150 kD could thus be coimmunoprecipitated with the antiserum directed against the P subunit
(Fig. 1 C). For PTP When 293 cells that overexpressed the PTPases were
treated with the calcium ionophore A23187 before lysis,
additional proteins of 70 (LAR) and 72 kD (PTP The antiserum specific for the COOH terminus of the
PTPases recognized an additional protein of 76 kD in 293 cells that expressed PTP Involvement of PKC Proteolytic processing of transmembrane proteins could
be shown to depend on the activation of PKC
As shown in Fig. 2, induced processing of overexpressed
LAR in 293 cells occurred only when PKC In TPA-treated cells the P subunits of LAR, whether
processed or unprocessed, showed a shift to a higher molecular weight in comparison to nontreated cells or to cells
in which processing was induced by calcium ionophore
(Fig. 2). In contrast to the TPA-induced proteolytic processing of LAR, this shift to a higher molecular weight of
the noncleaved P subunit occured even in the absence of
overexpressed PKC Processing of LAR and PTP In human platelets, cytosolic PTP1B was proteolytically
processed after treatment with A23187 and TPA. This
processing occurred in a calcium-dependent manner and
was mediated by the intracellular protease calpain (Frangioni et al., 1993). Therefore, we studied the calcium dependence as well as the influence of a specific calpain inhibitor, calpeptin (Tsujinaka et al., 1988), on the proteolytic
processing of LAR and PTP
In the cell lines, we examined the expression of PTP
In agreement with the results obtained with 293 cells
(Fig. 2), the treatment of cells with TPA resulted in proteolytic processing and differed from the one induced by
ionophore treatment, since it caused a shift of the P subunits as well as the processing products to a higher molecular weight. As opposed to overexpressed LAR in 293 cells (Fig. 2), the inducible processing of endogenous LAR
by TPA treatment in the same cells as well as in HeLa and A431 cells occurred without transfection of PKC Time Course of LAR and PTP We performed a time course study to compare calcium
ionophore and TPA-induced processing of LAR and
PTP
The TPA-induced processing was comparable in both
cell lines. Proteolytic processing of both PTPases was maximal after ~40 min and could not be increased further. In
A431 cells especially, the lower molecular weight P subunit was affected by TPA treatment. Since the data shown
in Fig. 2 already indicated a greater susceptibility of PTP It is noteworthy that TPA as well as ionophore treatment for up to 2 h caused the appearance of the same processing products observed after short time treatment. The
absence of any additional degradation products after long-term incubation supported the conclusion that proteolytic
processing of LAR and PTP Inducible Shedding of LAR and PTP For further characterization, we determined the position
of the site where the inducible proteolytic processing takes
place. The calculated molecular weight of the intracellular
domains and the transmembrane regions of both PTPases
was 74 kD. Because the molecular weight of the processing products of the P subunits was 72 and 70 kD (Fig. 1),
cleavage most likely occurred within or near the transmembrane region. To determine whether cleavage occurred in the extra- or intracellular part of the P subunits we performed the following experiments. A431 cells were
treated with vehicle, A23187, or TPA before lysis. Cell lysates were immunoprecipitated with antiserum 320, and
glycosylated proteins were bound to WGA-Sepharose. In
parallel, glycosylated proteins from the supernatants of
the same cells were enriched by binding to WGA-
Sepharose. The presence of LAR and PTP Shedding of the E subunits of the PTPases could not be
observed in overexpressing 293 cells, even though cleavage of the P subunits resulted in loss of the linkage between E and P subunits (Fig. 1, B and C) comparable to
other cell lines (Fig. 3 and 5). This fact is most likely due to
inhibition of solubilization of the E subunits by the calcium precipitation technique used for transfection of the
cells.
Proteolytic Cleavage Site of PTP Because the inducible processing of LAR and PTP
Intracellular Localization of LAR and PTP To study whether the two transmembrane PTPases are associated with any specific subcellular site, we analyzed
their localization in A431 cells by immunolabeling and fluorescence laser confocal microscopy. LAR- and PTP Treatment of the cells with EGTA alone (Fig. 7 E) or
subsequent incubation with ionophore (Fig. 7 F), which
did not lead to inducible processing of the PTPases (Fig.
3), induced internalization of the intracellular domains of
the PTPases. Here fluorescent label was detected as a ring-shaped structure in the perinuclear cytosol, whereas prefixation treatment with ionophore resulted in an internalization of the fluorescence and an even cytosolic staining
of the intracellular domains of the PTPases (Fig. 7 G). Significant labeling with antibodies directed against the extracellular domains of the PTPases could no longer be detected
in ionophore-treated cells (data not shown). Incubation
with TPA was associated with only a partial internalization of the P subunits, while a significant fluorescence remained localized in the plasma membrane (Fig. 7 H). Concentration of this label at sites of cell-cell contact is
reduced in comparison to nontreated cells (Fig. 7 D).
The extent of proteolytic processing after treatment of
A431 cells with ionophore or TPA (Figs. 3-5) was paralleled by an internalization of the intracellular domains of
the PTPases, whereas the extracellular domains, as expected, could no longer be detected at the cell surface.
EGTA-induced internalization was not paralleled by proteolytic processing of the PTPases and was therefore distinct from ionophore and TPA-induced internalization.
Surface Morphology of Intercellular Junctions
The localization of LAR and PTP Colocalization of LAR and PTP We used antibodies directed against plakoglobin ( These data indicated that LAR and PTP Localization of LAR and PTP The previous experiments demonstrated that the TPA-
induced shedding of the E subunit and the internalization
of the P subunit of LAR and PTP In contrast to ionophore and TPA treatment, the disruption of intercellular junctions by EGTA did not induce
shedding of the E subunits but instead led to an internalization of the intracellular as well as the extracellular part
of LAR and PTP In Vitro Association of LAR with Proteins of
the Cell Adhesion Complexes in Adherens Junctions
and Desmosomes
To determine whether the PTPases not only localize to adherens junctions and desmosomes but can also associate
with known proteins of the cell adhesion complexes at
these sites, we performed in vitro association experiments.
E-cadherin,
Association of The observed, direct in vitro association between the intracellular domain of LAR with plakoglobin and We compared structural and functional characteristics of
the highly related PTPases LAR and PTP Solubilization of extracellular domains of transmembrane proteins by proteolytic processing has been observed for several transmembrane proteins (for review see
Ehlers and Riordan, 1991). The exact cleavage sites, however, are in most cases not characterized. The transmembrane molecules that have been most thoroughly investigated are those growth factors that are released as soluble
proteins by proteolytic processing from a transmembrane
proprotein. The cleavage sites of these different growth
factors also show no significant sequence homology but
display clusters of small nonpolar amino acids (for review
see Massague and Pandiella, 1993). The same enrichment
of nonpolar amino acids was found in the cleavage position of PTP The observation that the characteristics as well as the
size of the resulting cleavage proteins were comparable in
all the cell lines we studied indicated that the processing of
LAR and PTP To explore putative biological targets for the cleaved
PTPases, we studied how the inducible processing would
affect the subcellular localization of LAR and PTP When we analyzed the consequences of proteolytic processing on the subcellular localization of LAR and PTP Based upon these observations, we conclude that at
least two independent mechanisms for the internalization
of LAR and PTP The proteins of the cadherin/catenin complexes at focal
cell-cell contacts underwent a very similar internalization
and, later, degradation when cells were treated with
EGTA (Kartenbeck et al., 1991) or TPA (Fabre and De
Herreros, 1993). However, the time course of internalization for plakoglobin and the PTPases differed in our experiments and were dependent on the agent used. Whereas short-time treatment with TPA left the integrity of intercellular junctions and the localization of plakoglobin unaffected, it already induced a significant yet incomplete internalization of the PTPases. At longer periods of incubation,
intercellular junctions became more and more disrupted,
and plakoglobin was also redistributed from the plasma
membrane to the cytosol, but not in parallel and at a much
slower rate than the intracellular phosphatase domains. In
contrast to the effect observed with TPA, ionophore and
EGTA, two agents that disrupted cell-cell contacts, induced a rapid and simultaneous internalization of both
plakoglobin and the intracellular PTPase domains. We
conclude from these data that the agents that we have
shown to disrupt intercellular junctions simultaneously induce the internalization of LAR and PTP LAR and PTP (Sap et al., 1994), for which a homophilic interaction between their extracellular domains was found. The
localization of PTPµ (Brady-Kalnay et al., 1995; Gebbink et al., 1995), PTP
(Fuchs et al., 1996), and PCP-2 (Wang et
al., 1996) was restricted to sites of cell-cell contact and surface expression of PTPµ (Gebbink et al., 1995), and PTP
(Fuchs et al., 1996) was increased in a cell density-dependent manner. Moreover, a direct association of PTP
(Fuchs et al., 1996) and PTPµ (Brady-Kalnay et al., 1995)
with members of the cadherin/catenin family suggests that
proteins of the cell adhesion complex represent physiological substrates for these PTPases. A possible regulatory
function in cell-matrix adhesion has been proposed for
LAR, another receptor-like PTPase, which associated with
focal cell-substratum adhesions via the newly identified
LAR interacting protein 1, LIP-1 (Serra-Pages et al., 1995).
(Jiang et al., 1993;
Fuchs et al., 1996), PTP
(Krueger et al., 1990; Mizuno et
al., 1993, Pulido et al., 1995a), PCP-2 (Wang et al., 1996),
and LAR (Streuli et al., 1988, Pot et al., 1991) are members of the so-called type II receptor-like PTPases. The extracellular domains of these PTPases contain a variable
number of Ig-like and fibronectin type III-like (FNIII) domains (for review see Charbonneau and Tonks, 1992). With the exception of PCP-2 (Wang et al., 1996), these PTPases also share characteristics in their biosynthesis. They
all underwent proteolytic processing by a furin-like endoprotease and were expressed at the cell surface in two
subunits which were not covalently linked (Streuli et al.,
1992; Yu et al., 1992; Jiang et al., 1993; Brady-Kalnay and
Tonks, 1994; Gebbink et al., 1995; Pulido et al., 1995a;
Fuchs et al., 1996). It was shown for LAR that the E subunit, which contains the cell adhesion molecule-like extracellular domain, was shed from the cell surface when cells
were grown to a high density (Streuli et al., 1992). This
shedding of the E subunit of LAR was the result of an additional proteolytic processing step that could also be induced by treatment of the cells with the phorbol ester
TPA (Serra-Pages et al., 1995). An accumulation of E subunits in the supernatant of cells was also observed for PTPµ (Gebbink et al., 1995) and PTP
(Pulido et al.,
1995a), and this suggests a common mechanism in the regulation of type II PTPases. However, the effect of proteolytic processing on either the catalytic activity, the substrate specificity, or the cellular localization of these
PTPases has not yet been determined.
, a recently identified new
member of the family of receptor-like type II PTPases
(Pan et al., 1993; Walton et al., 1993; Yan et al., 1993;
Ogata et al., 1994; Zhang et al., 1994), underwent biosynthesis and proteolytic processing in a manner that resembled that of the most closely related PTPase LAR. Moreover, further proteolytic processing of PTP
as well as of
LAR could be induced by treatment of the cells with TPA or the calcium ionophore A23187. Transient expression
studies indicated that TPA-induced processing of LAR,
but not PTP
, was dependent on the coexpression of
PKC
. Inducible processing of both PTPases took place in
the extracellular segment of the P subunit in a juxtamembrane position and led to the shedding of the E subunit.
Both LAR and PTP
were predominantly localized in regions of cell-cell contact and accumulated in dot-like
structures that could be identified as adherens junctions
and desmosomes by colocalization with plakoglobin
(Cowin et al., 1986). Moreover, plakoglobin and
-catenin,
another component of E-cadherin-containing cell adhesion complexes in adherens junctions, associated directly with the intracellular domain of LAR in vitro. The inducible shedding of the E subunit of LAR and PTP
was followed by a redistribution of the PTPases within the cell
membrane and by an internalization of the cleaved P subunits. It therefore represents a mechanism through which
the phosphatase activity of these PTPases could be regulated in response to cell-cell contact. The cell adhesion molecule-like character of LAR and PTP
was further
supported by the fact that the internalization of LAR and
PTP
occurred independently of the proteolytic processing if cells were grown in calcium-depleted growth medium. The analogies in specific localization as well as internalization behavior of PTP
and LAR, with molecules of
the cadherin/catenin family, strongly suggest a direct involvement of PTP
and LAR in the formation or maintenance of intercellular contacts.
Materials and Methods
(Yan et al., 1993) was kindly provided by Y. Schlessinger
(New York University Medical Center, New York).
-CATGGATCCAAAAAGGAAAAGGACCCAC-3
and 5
-GATCAGATCTTCACGTTGCATAGTGGTCAAAGC-3
. The PCR product was cut
with restriction enzymes BamHI and BglII and was inserted in the appropriate pGEX vector (Pharmacia Biotech, Uppsala, Sweden). Human
-catenin and plakogobin (these sequence data are available from GenBank/EMBL/DDBJ under accession number Z19054 and M23410, respectively) were amplified from cDNA generated from MCF7 cells using the
PCR method and were cloned in pRK5 expression plasmid. The integrity
of subcloned PCR products was confirmed by sequence analysis. The
CMV-driven expression plasmid for PKC
and rabbit antiserum 105 directed against PKC
were described elsewhere (Seedorf et al., 1995).
LAREN and
LAREC were generated against synthetic
peptides corresponding to NH2 (amino acids 5-18) and COOH-terminal
(amino acids 1,129-1,142) regions of the LAR E subunit, respectively.
Rabbit antisera 320 and 322 were kindly provided by Y. Schlessinger
(New York University Medical Center, New York). Antiserum 320 is directed against a peptide corresponding to the COOH-terminus of LAR
(amino acids 1868-1881) and PTP
(amino acids 1465-1478), whereas antiserum 322 is directed against a peptide corresponding to the NH2 terminus of PTP
(amino acids 5-18). Anti-plakoglobin (
-catenin) and anti-
-catenin antibodies were purchased from Transduction Laboratories
(Lexington, KY).
5 M calcium ionophore A23187 (Sigma Chemical Co., Taufkirchen, Germany), 1 µM phorbol ester TPA (Sigma Chemical Co.), 5 mM EGTA, or 30 µM calpeptin
(Calbiochem, Bad Soden, Germany). Pervanadate was freshly prepared
from sodiumorthovanadate and H2O2 and was used in a final concentration of 0.1 mM Na3V04 and 3 × 10
7 M H2O2. Time intervals of incubation
are given in the figure legends.
-mercaptoethanol at
50°C for 1 h.
was expressed transiently in 293 cells, and the cells were incubated
for 1 h with 10
5 M A23187 before lysis in Triton X-100 lysis buffer described above, without the phosphatase inhibitors NaF, Na4P2O7, and
Na3V04. Lysates of four 15-cm tissue culture plates were immunoprecipitated with antiserum 320, immunprecipitates separated in 8% SDS-PAGE, and transferred to ProBlotTM-membrane (Applied Biosystems).
Proteins were stained with Coomassie blue R-250, and the processing
product of the PTP
P subunit was isolated. Microsequencing was performed by using a sequencer (model 494; Applied Biosystems) using standard reagents and programs as suggested by the manufacturer.
LAREN, 320, and 322, and 1:200 for monoclonal anti-plakoglobin antibody. After three washes in PBG, primary antibody binding
was detected with isotype-specific secondary antibody, FITC(DTAF)-
conjugated donkey-anti-rabbit IgG (1:200), or Cy3-conjugated goat-anti-
mouse IgG (1:300; Jackson ImmunoResearch Laboratories, West Grove,
PA). For double labeling experiments, antibody decoration was done consecutively. Controls were incubated with either species-specific nonimmune serum or with secondary antibody alone. Coverslips were mounted
under glycerol-2.4% Dabco (1,4 Diazabicyclo [2.2.2*octane]) and were
viewed with appropriate band pass filters on a laser confocal microscope
(LSM 410; Carl Zeiss, Oberkochen, Germany) using a 40× oil immersion
objective of aperture 1.3. Images were recorded with a voxel size of 0.082 mm and smoothed for printouts by subdividing the pixels and linear interpolation. Controls were recorded at identical settings. To visualize the localization of antibody binding together with the cellular morphology, a
gray scale transmission image (pseudo-phase contrast) and the two confocal fluoresence images (FITC and Cy3) were superimposed in AVS (Advances Visual Systems, Waltham, MA).
Fig. 7.
Localization of LAR and PTP. A431 cells were grown
on glass coverslips, fixed, and labeled for laser confocal microscopy, as indicated in Materials and Methods. A-D are representative of unstimulated control cells. E-H were treated as follows:
(E) 5 mM EGTA for 45 min; (F) EGTA (5 mM) for 45 min plus
A23187 (10
5 M) for the last 30 min; (G) A23187 (10
5 M) for 30 min; (H) TPA 1 µM for 40 min. Antibodies were (A)
LAREN
(NH2 terminus of LAR), (B) 322 (NH2 terminus of PTP
), (C- H) 320 (COOH terminus of LAR and PTP
). Note the concentration of the label along the cell-cell contact sites in A, B, and D and the punctate staining along the circumference of solitary cells
(C). EGTA induced a ring-shaped core of internalized fluorescent label (E and F). Ionophore treatment (G) resulted in an
even cytosolic distribution of antibody labeling. After TPA treatment, some dot-like fluorescence was detected in the cytosol, but
predominant labeling remained at intercellular junctions. Bar, 25 µm.
[View Larger Version of this Image (88K GIF file)]
Fig. 8.
Surface morphology of intercellular adhesions. A431 cells were fixed
and prepared for scanning
electron microscopy as described in Materials and
Methods. Prefixation treatment was as follows: (A) control; (B) vehicle (DMSO);
(C) EGTA (5 mM) for 45 min; (D) EGTA (5 mM) for
45 min plus A23187 (105 M)
for the last 30 min; (E)
A23187 (10
5 M) for 30 min;
(F) TPA (1 µM) for 40 min.
Note intact cell-cell contacts
in A, B, and F and largely disrupted intercellular junctions in C, D, and E. Bar, 10 µm.
[View Larger Version of this Image (182K GIF file)]
Fig. 9.
Colocalization of
LAR and PTP with plakoglobin after ionophore and TPA
treatment. A431 cells were
fixed and labeled for plakoglobin (A, C, E, and G) or double
labeled with anti-plakoglobin
and antiserum 320 (COOH
terminus of LAR and PTP
;
B, D, F, and H) as described in
Materials and Methods. Laser
confocal fluorescence images
were superimposed on a transmission image to show protein
localization together with cellular and junctional morphology. Red pseudocolor indicates
monoclonal anti-plakoglobin
antibody; green, polyclonal 320 antiserum; and yellow, regions of colocalization of both. Prefixation treatment was as
follows: (A and B) controls; (C
and D) A23187 (10
5 M) for
30 min; (E and F) TPA (1 µM)
for 40 min; (G and H) TPA (1 µM) for 240 min. Note rapid
internalization of label and
disruption of cell-cell contacts
in C and D. While 40 min of
TPA neither disrupted intercellular junctions nor altered
plakoglobin localization (E),
antibody 320 label was already
found in the cytosol (F). After
4 h of TPA treatment, significant plakoglobin label (G) and
even more antibody 320 label
(H) were found internalized in comparison to the label remaining at the plasma membrane or
at cell-cell adhesions. Bar, 25 µm.
[View Larger Version of this Image (76K GIF file)]
Fig. 10.
Colocalization of
LAR and PTP with plakoglobin after EGTA treatment.
Cells were prepared, labeled,
and imaged as indicated in Fig.
9, legend. Prefixation treatment was EGTA (5 mM) for
45 min (A, C, E, and G) and
EGTA (5 mM) for 240 min (B,
D, F, and H). Cells were labeled with anti-plakoglobin
antibody (A and B), anti-plakoglobin antibody plus antiserum 320 (C and D), antiserum
LAREN (NH2 terminus of
LAR; E),
LAREN plus anti-plakoglobin antibody (G), antiserum 322 (NH2 terminus of
PTP
; F), and 322 plus anti-plakoglobin antibody (H).
Note that EGTA treatment induces rapid disruption of intercellular junction and internalization of plakoglobin (A and
B) together with the intracellular phosphatase domain (C
and D) from the plasma membrane. EGTA also induces internalization of the extracellular E subunits of the PTPases
(E and F), which can be colocalized with plakoglobin (G
and H). Bar, 25 µm.
[View Larger Version of this Image (82K GIF file)]
Results
are highly related PTPases whose rat homologues display a sequence identity of 79% in their proximal membrane PTPase domain, 90% in their COOH-terminal PTPase domain, and 57% in their extracellular
domain (Zhang et al., 1994). LAR contains three Ig- and
eight FNIII-like domains in the extracellular domain and
two intracellular PTPase domains. Three splice variants of PTP
are known so far. The rat protein we analyzed (Pan
et al., 1993; Walton et al., 1993; Yan et al., 1993) differs
from LAR in so far as it lacks the FNIII-like domains four
through seven. While it was shown that LAR was expressed in two subunits (Streuli et al., 1992; Yu et al.,
1992), the biosynthesis of PTP
has not yet been studied.
We hypothesized that PTP
would be processed in a manner that is analogous or similar to the processing of LAR
because a polyclonal antiserum directed against one of its FNIII-like domains recognized a protein of ~100 kD instead of the 168 kD that would have been predicted from
the full length sequence of PTP
(Yan et al., 1993; Rotin
et al., 1994). Fig. 1 A shows the schematic structure of
LAR and PTP
, the proposed biosynthesis of PTP
, and
the recognition sites of subunit-specific antibodies used in
this study.
Fig. 1.
Physiological- and calcium ionophore-induced processing of LAR and PTP after transfection into 293 cells. (A) Schematic
representation of the biosynthesis of LAR and PTP
. Dashed lines indicate a gap introduced for alignment purposes. Antibody 320 specifically recognizes the P subunit, while
LAREC and 322 recognize the E subunits of LAR and PTP
, respectively. (B) LAR or PTP
were transiently expressed in 293 cells and before lysis cells were treated for 1 h with or without 10
5 M A23187, as indicated. Control
cells were transfected with the expression plasmid pRK5. Lysates were either separated by 8% SDS-PAGE (TRITON) or after binding
to WGA-sepharose beads. Proteins were transferred to nitrocellulose and analyzed by immunoblotting of the membrane with antisera
specific for the COOH terminus of LAR and PTP
(320), the NH2 terminus of PTP
(322), or the COOH terminus of the LAR E subunit (
LAREC). Arrows on the left indicate molecular weight standards, and arrows on the right indicate the position of the LAR and
PTP
subunits. (C) 293 cells transfected with LAR, PTP
, or control plasmid were labeled with [35S]methionine (16 h) and incubated
with or without A23187, as described above. Lysates were immunoprecipitated with antiserum specific for LAR and PTP
(320) or with
nonimmune serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, and the dried gel was exposed to X-ray film for 24 h.
Arrows on the left indicate molecular weight and on the right the position of the P subunits of LAR and PTP
, respectively.
[View Larger Versions of these Images (35 + 47 + 48K GIF file)]
, both proteins were transiently expressed in human
embryonic kidney 293 cells (Fig. 1, B and C). As shown in
Fig. 1 B, antiserum (320) directed against the identical
COOH terminus of human LAR and rat PTP
recognized
proteins of 205 and 84 kD in cell lysates of LAR expressing 293 cells and proteins of 158 and 80 kD in PTP
- expressing 293 cells. These protein bands represented the
precursor and the proteolytically processed P subunits of
both PTPases, respectively. The reduced size of the PTP
precursor and E subunit of 40 kD was in good agreement
with the presence of only four FNIII-like domains instead
of eight such domains in LAR. The molecular mass of the
P subunit of PTP
, on the other hand, varied only by 4 kD.
The large amount of unprocessed precursor protein that was detected is most likely the result of overexpression in
the 293 cell system.
, a coimmunoprecipitated 97-kD protein (Fig. 1 C) was identified as its processed extracellular
domain by immunoblot analysis with an antiserum raised
against an NH2-terminal peptide (Fig. 1 B). Taken together, these data show that the biosynthesis of PTP
is indeed comparable to that of LAR in every aspect (Fig. 1 A).
) could
be immunoprecipitated with the COOH terminus-specific
consensus antiserum 320. In addition, the amount of immunoprecipitated P and E subunits was considerably reduced, whereas the amount of immunoprecipitated precursor of both PTPases was not or much less affected (Fig.
1 C). Immunoblot studies using specific antisera directed
against the E and P subunit indicated that the 70- and 72-kD proteins were derived from the P subunits of LAR and
PTP
, respectively, by proteolytic processing at the NH2
terminus (Fig. 1 B). Since coimmunoprecipitation of the E
subunits with the shortened P subunits was not detected (Fig. 1 C), proteolytic processing induced by calcium ionophore treatment resulted in separation of the E and the P
subunit. This lack of association between the E subunit
and the shortened P subunit could also be demonstrated
by analyzing the binding of subunits of LAR and PTP
to
WGA (Fig. 1 B). In untreated cells, the P subunits of LAR
and PTP
were found enriched in the fraction of WGA-bound proteins. However, after processing was induced, the 70- and 72-kD protein bands were no longer detected
in the WGA-bound protein fraction (Fig. 1 B). This indicates that the P subunits would have to be linked to their
respective E subunits to be detected in the WGA-bound
protein fraction.
. The relative amount of this protein varied from experiment to experiment and was not affected by calcium ionophore treatment of the cells (Fig. 1,
B and C). It therefore most likely represents a degradation
product of PTP
, although a different type of processing cannot be excluded. An equivalent protein product, however, could not be detected in 293 cells that expressed LAR.
in the Proteolytic Processing of
LAR and PTP
in several
instances (for review see Ehlers and Riordan, 1991). We
therefore investigated whether treatment of LAR and
PTP
overexpressing 293 cells with the PKC
activator TPA
(12-O-tetradecanoylphorbol-13-acetate) could induce proteolytic processing of these PTPases. In addition we analysed if this processing is dependent on the coexpression of
PKC
in these cells. 293 cells were transfected with vectors encoding LAR or PTP
alone or together with a PKC
expression plasmid. The effect of TPA treatment of these
cells on the proteolytic processing of the PTPases was determined by immunoblotting of cell lysates with the COOH
terminus-specific antiserum 320 (Fig. 2, top gel). The reprobing of the same membrane with an antiserum specific for
PKC
confirmed comparable expression levels of PKC
(Fig. 2, bottom gel). For immunoblotting we used smaller
quantities of cell lysate from cells that expressed only the PTPases, because coexpression of PKC
consistently reduced
the amount of LAR and PTP
expression in these cells.
Fig. 2.
Effect of TPA treatment and coexpression of PKC on
the processing of LAR and PTP
in 293 cells. LAR or PTP
were
transiently expressed in 293 cells either alone or together with
PKC
. As controls, pRK5 plasmid or PKC
expressing cells were
used. Before lysis, cells were treated for 1 h with or without 1 µM
TPA or 10
5 M A23187 as indicated. Lysates were separated by
7% SDS-PAGE (TRITON), proteins were transferred to nitrocellulose and subsequently analyzed by immunoblotting of the
same membrane with antisera specific for the COOH terminus of
LAR and PTP
(320) and PKC
(105). The amount of cell lysates analyzed is one third in LAR/pRK5-transfected cells and
one half in PTP
/pRK5 transfected cells in comparison to lysates
in the other lanes. Sizes of molecular weight standards in kD are
indicated on the left.
[View Larger Version of this Image (51K GIF file)]
was coexpressed. This indicated a critical involvement of this enzyme
in the TPA-induced processing of LAR. PTP
processing in
response to TPA, on the other hand, was independent of
PKC
overexpression in 293 cells. We cannot exclude that
this effect was mediated by endogenous PKC
and may be
due to a higher susceptibility of PTP
towards TPA-induced processing in comparison to LAR.
and was most likely mediated by endogenous PKC
. We assume that the increase in molecular weight was due to a modification of the LAR P subunits by serine/threonine phosphorylation because we
observed TPA-dependent [32P]orthophosphate incorporation in PKC
coexpressing 293 cells (data not shown). A
shift to higher molecular weight for the P subunits of
PTP
, however, was not observed (Fig. 2) although TPA-induced [32P]orthophosphate incorporation in the P subunits of PTP
was comparable to LAR (data not shown).
in HeLa, A431, and
293 Cells
(Fig. 3). Accordingly, A431,
HeLa cells, and nontransfected 293 cells were treated with the calcium ionophore A23187 or with TPA. Cells that
were treated with A23187 were preincubated either with
calpeptin or with an excess of EGTA to deplete the medium of calcium. Lysates of the cells were immunoprecipitated with antiserum 320 and immunoprecipitates analyzed in immunoblots with antiserum 320 and antiserum
LAREC (Fig. 3).
Fig. 3.
Characteristics of inducible processing of LAR and
PTP in different cell lines. HeLa, A431, and 293 were incubated
with or without A23187 (10
5 M, 1 h) or TPA (1 µM, 1 h) or were
pretreated with EGTA (5 mM, 1h) or calpeptin (30 µM, 1 h). Cell
lysates (1.2 mg protein) were immunoprecipitated with either antiserum specific for LAR and PTP
(320) or nonimmune serum
(NI). Immunoprecipitates were separated by 8% SDS-PAGE,
transferred to nitrocellulose, and analyzed by immunoblotting of
the membrane with antiserum specific for the COOH terminus of
LAR and PTP
(320) or the COOH terminus of the LAR E subunit (
LAREC).
[View Larger Version of this Image (44K GIF file)]
with E subunit-specific (data not shown in Fig. 3; see Fig.
5) as well as P subunit-specific antisera could be clearly detected only in A431 cells, whereas all three cell lines express LAR (Fig. 3). Ionophore-induced proteolytic processing led in all cell lines to protein products of the same
molecular weight. Pretreatment with EGTA eliminated
the appearance of the proteolytic products, suggesting that
proteolysis was indeed calcium dependent. However, pretreatment with the calpain inhibitor calpeptin had no effect, thereby indicating that calpain was not required for
this proteolytic effect. A control experiment using PTP1B
transiently expressed in 293 cells showed that the same
concentration of calpeptin could inhibit PTP1B processing
almost completely (data not shown). This confirmed that,
although the ionophore-induced processing of LAR and
PTP
was calcium dependent, it was not mediated by calpain.
Fig. 5.
Shedding of LAR and PTP E subunits in A431 cells.
A431 cells were starved for 2 d, washed once with starvation medium, and treated with or without A23187 (10
5 M, 1 h) or TPA
(1 µM, 40 min). Cell lysates (TRITON) were either immunoprecipitated with antiserum specific for LAR and PTP
(IP:320, 0.9 mg protein) or were bound to WGA-sepharose beads (WGA, 0.6 mg protein). Proteins of the tissue culture supernatant of these cells (MEDIUM, corresponding to 1.5 mg protein of cell lysate) were bound to WGA-sepharose beads (WGA). Immunoprecipitates
and WGA-bound proteins were separated by 8% SDS-PAGE,
transferred to nitrocellulose, and analyzed by immunoblotting of
the membrane with antiserum specific for the COOH terminus of
LAR and PTP
(320), the NH2 terminus of PTP
(322), or the
COOH terminus of the LAR E subunit (
LAREC). Arrows at the
right indicate the position of the LAR or PTP
subunits.
[View Larger Version of this Image (48K GIF file)]
and is
therefore most likely mediated by the endogenous enzyme
in these cells. As seen with the ionophore, TPA-induced
processing occurred at the NH2 terminus of the P subunits
and cleaved the linkage between the E and the P subunits
(Fig. 3). Moreover, treatment with A23187 and TPA
induced an identical proteolytic processing in SK-BR-3, BT-20, MIA-PaCa-2, and PC12 cells (data not shown).
This suggested that this inducible proteolytic processing
pattern is a general feature of LAR and PTP
and occurs
independently of cell type.
Processing in A431 and
HeLa Cells
in different cell lines. HeLa and A431 cells were incubated for different time periods with A23187 and TPA
before lysis, and immunoprecipitates with antiserum 320 were analyzed by immunoblotting with the same antiserum. As shown in Fig. 4, in A431 cells the processing was
complete after 10-20 min of ionophore treatment and
even on the longest exposures of the immunoblot, the P
subunits of the PTPases could not be detected (data not
shown). In contrast, in HeLa cells, the processing of LAR
was maximal but incomplete after 40 min of treatment and
could not be increased further by longer incubation times.
Fig. 4.
Time course of inducible processing of LAR and PTP
in HeLa and A431 cells. A431 and HeLa cells were starved for 2 d
and then incubated for different time intervals (indicated in min)
with A23187 (10
5 M) or TPA (1 µM). Control cells were incubated with vehicle, which is indicated as E (ethanol) and D
(DMSO). Cell lysates (0.9 mg protein) were immunoprecipitated
with antiserum specific for LAR and PTP
(320) or nonimmune
serum (NI). Immunoprecipitates were separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with antiserum specific for the COOH terminus of LAR
and PTP
(320). Arrows at the left indicate the position of the
processing products of the P subunit of LAR and PTP
.
[View Larger Version of this Image (41K GIF file)]
to TPA-induced processing, the higher and lower molecular weight bands in A431 cells most likely represented the
LAR and PTP
P subunits, respectively.
was a specific and therefore
functionally significant event.
-E subunits in
A431 Cells
subunits in
the different fractions was analyzed by subsequent immunoblotting with antibodies directed against the different
subunits (Fig. 5). As shown in Fig. 5, the E subunits of
LAR and PTP
were no longer present in the fraction of
WGA-bound proteins after induced proteolytic processing. Instead, they could be detected in the supernatant.
This demonstrated that the cleavage site for induced proteolytic processing was located in the extracellular domain
of the P subunit and that processing caused shedding of the E subunits of LAR and PTP
. As shown earlier, the
lower molecular weight band that was recognized by the
antibody directed against the COOH terminus of the PTPases
was found to be more sensitive to TPA-induced processing
(Fig. 4). Because the corresponding E subunit found in the
supernatant was recognized by an antiserum specific for
PTP
, the lower molecular weight subunit was now identified as the P subunit of PTP
.
was
identical in cell lines that endogenously express the phosphatases and in transfected 293 cells, we used the 293 cell
overexpression system to isolate the processed P subunit
of PTP
and to determine the cleavage site by NH2-terminal sequence analysis. The NH2-terminal sequence (XVDGEEGLI) of the ionophore-induced processing product of PTP
was identical to amino acids 822-830 of PTP
,
with exception of the first amino acid (X) which could not
be identified (Fig. 6). The cleavage occurred extracellulary
between amino acids Pro821 and Ile822, only six amino acids
away from the transmembrane region. In this region the
protein sequence of LAR does not show a significant homology to the sequence of PTP
, and the proteolytic
cleavage site of LAR is therefore not necessarily in an
analogous position to that of PTP
. The data in Fig. 5,
however, indicated that the inducible processing of LAR
also took place in the extracellular part of the P subunit.
Fig. 6.
Schematic representation of the calcium ionophore-induced cleavage site of PTP in comparison to the corresponding LAR
sequence. Shown are the amino acid sequences of PTP
(amino acids 817-836) and LAR (amino acids 1224-1242). The putative transmembrane regions are boxed, and identical amino acids of both PTPases are indicated by asterisks. The determined NH2-terminal sequence of the PTP
processing product is aligned to the homologous PTP
sequence, and the cleavage site is shown by an arrow.
[View Larger Version of this Image (8K GIF file)]
-specific labeling with either antibodies directed against the
intracellular or the extracellular domains of both PTPases
was observed in dot-like structures at the attachment sites
of the cells to the glass surface (data not shown). Consistent with previous findings for LAR (Serra-Pages et al.,
1995), these structures are likely to represent adhesion
plaques. In addition, a punctate label along the contact
sites of neighboring cells was detected by labeling either
the extracellular domain of LAR (Fig. 7 A) and PTP
(Fig. 7 B) or the intracellular domain of both PTPases
(Fig. 7, C and D). When cells were grown at low density before cell-cell contacts were formed, labeling was found
as punctate staining along the cell membrane (Fig. 7 C).
Focal concentration of fluorescence was detected at intercellular junctions as soon as these had formed between
cells at higher density (Fig. 7 D).
at cell-cell contacts
and their partial or complete internalization raised the
question whether and how EGTA, ionophore, and TPA
had affected the structural integrity of these specialized
membrane regions. On scanning electron microscopy, untreated and vehicle (DMSO)-treated A431 cells were
found to have formed multiple adhesion complexes between neighboring cells (Fig. 8, A and B). Incubation with
EGTA (Fig. 8 C), as well as subsequent treatment with
ionophore (Fig. 8 D) or ionophore alone (Fig. 8 E) induced an almost complete disruption of intercellular junctions, the formation of multiple surface protrusions, and a
rounded elevation of the normally flat cell body from the
surface. 40 min of treatment with TPA, which was also associated with internalization of LAR and PTP
on immunolabeling studies (Fig. 7 H), left the junctional morphology completely intact (Fig. 8 F). These observations indicated that only the EGTA- and ionophore-induced internalization was paralleled by a structural disruption of
intercellular adhesions, whereas TPA-induced internalization occurred independently of this effect.
with Plakoglobin
-catenin), a protein localized at the intracellular site of adherens junctions and desmosomes (Cowin et al., 1986), to
study whether LAR and PTP
colocalize to these specialized areas and to investigate whether the internalization of
the phosphatases from the plasma membrane is paralleled
by a dissociation of plakoglobin from this site. Plakoglobin
(Fig. 9 A) was detected along cell-cell contacts of neighboring A431 cells, strongly colocalized with antiserum 320 label (Fig. 9 B), and therefore identified the subcellular site of LAR and PTP
as adherens junctions and desmosomes. Upon ionophore treatment, plakoglobin (Fig. 9 C)
and the phosphatase domains were internalized in parallel
from the rapidly dissociating intercellular junctions (Fig. 9
D). Incubation with TPA for 40 min left the localization of
anti-plakoglobin labeling unaffected (Fig. 9 E) but induced a significant yet incomplete internalization of 320 label (Fig. 9 F). Extended time course experiments with TPA treatment up to 4 h showed that plakoglobin, in agreement with studies by Fabre and DeHerreros (1993), is redistributed from the plasma membrane to the cytosol (Fig.
9 G), although at a much slower rate and not in parallel
with the intracellular phosphatase domain (Fig. 9 H).
When antibodies directed against the extracellular domains of LAR and PTP
were used for immunolabeling, both ionophore treatment and TPA incubation induced a
complete disappearance of fluorescence label (data not
shown).
colocalized
with plakoglobin to adherens junctions and desmosomes.
The subsequent internalization of the intracellular phosphatase domains occurred rapidly and in parallel with plakoglobin after ionophore stimulation. TPA-induced internalization of plakoglobin occurred at much longer treatment intervals and not in parallel with that observed for the P
subunits of the PTPases.
after EGTA Treatment
could occur in the presence of intact cell-cell adhesions. Whether, however, the
disruption of cell-cell contacts is automatically associated
with internalization of the P subunit remained unknown.
We showed in Fig. 7, that EGTA induced a rapid and almost complete disruption of intercellular junctions, which should lead to the dissociation of protein complexes at
these subcellular sites (Kartenbeck et al., 1991). Plakoglobin label in A431 cells was indeed found in the cytosol under the plasma membrane after 45 min of EGTA treatment (Fig. 10 A) and in the perinuclear cytosol after 4 h
EGTA (Fig. 10 B). Colocalization with the intracellular
domains of LAR and PTP
demonstrated the same route
of redistribution from the membrane to the cytosol (Fig.
10, C and D). Immunofluorescent staining with LAR and
PTP
antibodies directed against the extracellular domain
alone (Fig. 10, E and F) or in colocalization with plakoglobin (Fig. 10, G and H) resulted in exactly the same appearance as observed for the intracellular domains of the PTPases. Shown here is the 45-min treatment for LAR (Fig.
10, E and G) and the 4-h treatment for PTP
(Fig. 10, F
and H). Immunofluorescent labeling at other time points
was identical for both PTPases (data not shown).
. The previously mentioned experiments showed that this redistribution occurred without
prior proteolytic cleavage of LAR and PTP
and could
therefore involve the entire and intact molecule.
- and
-catenin, and plakoglobin were transiently expressed in 293 cells. Cells were incubated with or
without the phosphatase inhibitor pervanadate before lysis to study the influence of tyrosine phosphorylation of
these proteins on the association with LAR. Lysates were
then incubated with glutathione-sepharose-bound GST-fusion protein, GST-hPTP-LARi, containing the entire intracellular domain of LAR, from amino acids 1,259 to
1,881. Bound proteins were analyzed by immunoblotting
with antibodies directed against
-catenin (Fig. 11 A), plakoglobin (Fig. 11 B),
-catenin (data not shown), and E-cadherin (data not shown).
Fig. 11.
In vitro association of LAR with plakoglobin and -catenin.
-catenin and plakoglobin were transiently expressed in 293 cells, and cells were stimulated for 10 min with pervanadate before lysis. Equal amounts of lysates were incubated with the LAR-GST-fusion protein, GST-hPTP LARi, or a threefold molar excess of GST, complexes were immobilized on glutathione-sepharose, and precipitates were separated by SDS-PAGE. Lysates of control plasmid-transfected 293 cells were bound in the same way to GST-hPTP
LARi-glutathione-sepharose. Bound proteins were analyzed by immunoblotting with antibodies specific for
-catenin (A) or plakoglobin (B). Arrows indicate the proteins of interest; molecular size standards in kD are shown on the left.
[View Larger Version of this Image (15K GIF file)]
-catenin (Fig. 11 A) and plakoglobin
(Fig. 11 B) with the GST-fusion protein, GST-hPTP-LARi, was detected in lysates of cells that expressed these
proteins but was absent in controls. The amounts of associated proteins were independent of prior pervanadate
treatment of the cells. In contrast, no specific association
could be observed with lysates from
-catenin- or E-cadherin-expressing cells (data not shown), even though
overexpression of these proteins in 293 cells was confirmed by immunoblotting of the same lysates with the appropiate antibodies (data not shown).
-catenin
suggests that LAR not only colocalized with cell adhesion
molecules at sites of cell-cell contact but that these proteins
also interact in a functional manner. However, the question whether plakoglobin and
-catenin represent physiological targets of LAR could not be answered, because
under our experimental conditions the association was independent of tyrosine phosphorylation of these proteins.
Discussion
in regard to
spontaneous as well as induced proteolytic processing. In
analogy to LAR (Streuli et al., 1992; Yu et al., 1992) and
other members of the type II class of PTPases (Jiang et al.,
1993; Brady-Kalnay and Tonks, 1994; Pulido et al., 1995a;
Fuchs et al., 1996), PTP
was expressed in two subunits
that were derived from a precursor protein by proteolytic
processing. The similarities in the sequence as well as the
structure among the members of this subfamily of PTPases suggest that they may also have closely related functions.
This assumption is further supported by the observation
that both (LAR and PTP
) underwent analogous processing in response to intracellular calcium concentration increase or in response to treatment with TPA. Interestingly,
while TPA-induced processing of overexpressed LAR required co-overexpression of PKC
in 293 cells, this was not necessary for PTP
, suggesting differential effector
sensitivity of the two PTPases. In 293 cells that overexpressed LAR or PTP
as well as in cell lines that expressed these PTPases endogenously, the inducible processing took place at the NH2 terminus of the P subunit
removing a 14-kD fragment from LAR and an 8-kD fragment from PTP
. It also resulted in shedding of the E subunits of both PTPases. By NH2-terminal sequencing we
identified the cleavage site of PTP
and found it to be located between amino acids Pro821 and Ile822, which corresponds to a distance of merely six amino acids from the
transmembrane region. The analogous cleavage position of LAR does not show any sequence homology to that of
PTP
. Because the E subunit of LAR could be detected in
the cellular supernatant after proteolytic processing, the
cleavage of LAR also occurred extracellulary. Serra-Pages
et al. (1994) have already demonstrated by mutational
analysis that the cleavage of LAR in response to TPA occurred at a site that is located COOH-terminally to amino acid 1,222. Despite the absence of any sequence homology
to the cleavage site of PTP
, the proteolytic processing of
LAR therefore occurred in a completely analogous position that is located between amino acids 1,222 and the first
amino acid of the transmembrane region, Met1235.
. While proteases responsible for the shedding of extracellular domains of transmembrane proteins
have not been identified, a protease activity involved in
the proprotein processing of TGF
had been characterized. It is a transmembrane protease whose active center
was shown to be located in its extracellular part (Harano
and Mizuno, 1994). Since the cleavage of the TGF
proprotein could be activated by TPA and A23187 (Pandiella
and Massague, 1991), the same agents that we used to induce the processing of LAR and PTP
, the transmembrane character of this protease can serve as a model for
the mechanisms that allow intracellular signals to increase
the extracellular activity of an enzyme. For the proprotein
processing of TGF
, as opposed to the processing of the
PTPases, the presence of a COOH-terminal valin residue in the short intracellular domain was an essential requirement (Bosenberg et al., 1992). While we cannot conclude
from our study that the same or a related protease mediated the inducible processing of LAR and PTP
, we excluded the possibility that the protease calpain, which mediated the processing of the intracellular PTPase 1B (Frangioni
et al., 1993) was involved here. We therefore suggest that
the processing of LAR and PTP
underlies a different
mechanism and is distinct from the processing of PTP1B.
was a specific process and a ubiquitous
event in cell lines of different origins. Moreover, the fact
that a solubilization of the E subunits could also be observed for PTPµ (Gebbink et al., 1995) and PTP
(Pulido
et al., 1995a) suggested that this form of processing could
be a general feature of type II PTPases. The cell density-dependent shedding of the E subunit of LAR has been
proposed to play a role in the contact inhibition of cell growth (Streuli et al., 1992). The latter report and a study
by Serra-Pages et al. (1994) showed that the shedding of
the E subunits of PTPases in response to high cell density
was caused by proteolytic processing. A cell density-
dependent increase in the catalytic activity of PTPases was
observed in contact-inhibited fibroblasts (Pallen and Tong,
1991) as well as in A431 cells (Mansbridge et al., 1992),
while the phosphatase inhibitor vanadate was found to
abolish the contact inhibition of rat kidney cells under certain experimental conditions (Rijksen et al., 1993). The
underlying mechanism, however, through which the processing of transmembrane PTPases participates in cell
contact inhibition is not yet understood. An increase of
phosphatase expression with growing cell density has been
observed for some PTPases (Longo et al., 1993; Östman et
al., 1994; Celler et al., 1995; Fuchs et al., 1996). Cleavage and subsequent loss of the extracellular domains from receptor-type PTPases could also contribute to increased
phosphatase activity. This hypothesis was supported by
the observation that incubation of fibroblasts with trypsin,
a treatment that permitted the cleavage of extracellular domains from transmembrane proteins, increased the phosphatase activity in these cells (Maher, 1993). However, in
our study neither the in vitro activity of the processed P subunits of LAR and PTP
nor their activity towards possible substrates, such as receptor tyrosine kinases, was found
to be altered in cotransfection studies (data not shown).
and
used confocal microscopy in conjunction with cells that endogenously express both proteins. In untreated A431 cells,
LAR and PTP
were found at focal cell substratum adhesions (data not shown; Serra-Pages et al., 1995) as well as
at sites of cell-cell contacts. The colocalization with plakoglobin (Cowin et al., 1986) identified these structures as
adherens junctions and desmosomes. These subcellular
compartments are known to contain cell adhesion molecules of the cadherin/catenin family, which mediate cell-
cell adhesion by a homologous, calcium-dependent interaction of their extracellular domains and by linking the cell contact sites to the cytoskeleton via proteins bound to
their intracellular portion (for reviews see Grunwald,
1993; Koch and Franke, 1994). Increased tyrosine phosphorylation at the sites of cell-cell and cell-substratum adhesions correlated with a number of biological processes
such as cell migration, malignant transformation, and metastatic spread of tumor cells (for reviews see Kemler, 1993;
Clark and Brugge, 1995; Rosales et al., 1995). The close association between cell contact disruption and malignant
behavior actually led to the conclusion that cadherins represent a separate class of tumor suppressors (Birchmeier
et al., 1995). We now show here that LAR and PTP
colocalize with cell adhesion proteins at sites of cell-cell contact, an observation that suggests that LAR and PTP
may
be involved in either the formation or maintenance of intercellular junctions. This is further supported by the direct association of plakogobin and
-catenin with a GST-fusion protein of the intracellular domain of LAR in vitro.
Although this association was found to be independent of
tyrosine phosphorylation of plakogobin and
-catenin, we
cannot rule out that phosphorylation plays a role for the in
vivo interaction of PTPases with these proteins. An association of proteins of the cadherin/catenin complex was not
only reported for PTPases of the LAR family (Kypta et
al., 1996) but also for two other members of class II PTPases, PTP
(Fuchs et al., 1996) and PTPµ (Brady-Kalnay
et al., 1995). These two PTPases (Brady-Kalnay et al.,
1995; Gebbink et al., 1995; Fuchs et al., 1996) and PCP-2
(Wang et al., 1996) also localized to sites of cell-cell contacts. However, the linkage of LAR to focal cell-substratum adhesions via interaction with the protein LIP-1
(Serra-Pages et al., 1995) and the association of PTP
with LIP-1 (Pulido et al., 1995b) make it likely that the supposed regulation of cadherin/catenin complexes is not the
only function of LAR and PTP
.
we found that the treatment with either TPA or A23187
resulted in a loss of labeling for the extracellular domain.
This finding was in complete agreement with the cleavage
and shedding of the E subunits of both PTPases, which we
observed in biochemical experiments. The effect, however, that these agents showed on the localization of the
shortened P subunit of LAR and PTP
was quite different. A23187, while inducing a complete disruption of cell-
cell contacts within minutes, led to an even cytosolic distribution of the P subunits and a loss of labeling at the cell
membrane. A brief treatment with TPA, on the other
hand, left cell-cell contacts morphologically intact but reduced the concentration of P subunits at these cell-cell
contacts by inducing only a partial internalization of label
to the cytosol. After longer treatment intervals with TPA,
a structural disturbance of cell-cell adhesions was finally observed and was paralleled by a greater internalization of
P subunits. Interestingly, a depletion of calcium in the culture medium with EGTA also resulted in an internalization of both PTPases. This effect, however, occurred independent of proteolytic processing, was not associated with
a shedding of E subunits, and involved both subunits of
the presumably intact PTPases.
exist. One of them involves the induction of proteolytic processing, which, as demonstrated in
the TPA experiments, can lead to internalization of P subunits even in the presence of intact cell-cell contact. The
other mechanism involves the disruption of cell-cell contact but, as shown in the EGTA experiments, can lead to
internalization of the intact PTPases without prior proteolytic processing.
and disturb the
integrity of the cadherin/catenin complexes at the same
subcellular sites. With TPA, on the other hand, the internalization of the PTPases occured before the integrity of
cellular junctions was affected, and the loss of the PTPases
at these subcellular sites presumably contributes to the
later disruption of cell-cell contacts.
did not only colocalize with cell adhesion proteins of the cadherin/catenin family and were subjected to a comparable intracellular redistribution under
experimental conditions, but also their extracellular domains were shed in a manner that was also reported for
cadherins (Wheelock et al., 1987; Roark et al., 1992). Interestingly, the soluble extracellular domain of N-cadherin, NCAD90, remained capable of binding N-cadherin
in a homophilic manner and promoted cell adhesion and
neurite growth in chicken embryo retina cells when presented in a substrate-bound form (Paradies and Grunwald,
1993). Moreover, the soluble extracellular domain of
E-cadherin was able to inhibit cell adhesion (Wheelock et
al., 1987). It is therefore possible that the extracellular domains of receptor-type PTPases, which are also cleaved
from the cell surface upon proteolytic processing, could be
equally involved in the formation, maintenance, or restoration of cell adhesions. While the distinct subcellular redistribution of presumably catalytically active P subunits
of LAR and PTP
under normal conditions may regulate
proliferation and tissue integrity by cell-cell contact through targeting the phosphatase activity to specific intracellular sites, the biological function of the solubilized E
subunits remains at present unknown. Under pathophysiological conditions such as malignant cell transformation,
the regulatory mechanism investigated here may represent
a critical target for subversion leading to dysregulated
growth and metastatic spread.
Received for publication 5 December 1996 and in revised form 18 April 1997.
Please address all correspondence to Axel Ullrich, Department of Molecular Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany. Tel.: (49) 89-8578-2513; Fax: (49) 89-857-7866.We thank Y. Schlessinger for providing the expression vector pRK5-PTP
and antisera 320 and 322, H. Saito for providing LAR cDNA as plasmid
pSP65-LAR, J. Murphy and R. Aebrecht for advice and support with confocal microscopy, A. Kharitonenkov for helpful discussions, and K. Martell for critically reading the manuscript.
This work was supported by a grant from SUGEN, INC.
GST, glutathione-S-transferase; PTPase, protein tyrosine phophatase.
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