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INTRODUCTION |
Members of the cadherin family of cell-cell adhesion molecules are
key players in morphogenetic processes, and regulation of cadherin
function, as opposed to transcription and translation, is thought to be
responsible for many of the rapid changes that occur during
development. Classic cadherins are characterized by a highly conserved
intracellular domain that interacts with the actin-containing
cytoskeleton, an interaction essential for function. This interaction
is mediated by
- and
-catenin (1-4);
-catenin associates
directly with a 20-amino acid domain near the carboxyl terminus of
cadherin (5, 6) and with
-catenin, which, in turn, interacts with
actin, either directly (7) or indirectly, through
-actinin (8).
-catenin not only performs a bridging role between cadherin and
actin, but free
-catenin can be translocated to the nucleus where it
regulates transcription of cadherin and other gene products (9, 10).
Thus, the regulation of free
-catenin is of critical importance,
and, consequently, the interaction of
-catenin with cadherin has
multiple ramifications on cellular function (11, 12).
Regulation of the interaction of
-catenin with N-cadherin is
mediated by the phosphorylation of tyrosine residues on
-catenin (13, 14). In embryonic chick neural retina cells, hyperphosphorylation of
-catenin is correlated with loss of its association with
N-cadherin and loss of cadherin function (13, 14). Enhanced
phosphorylation of
-catenin has also been correlated with loss of
E-cadherin function (15-19). These data suggest that tyrosine kinases
and/or phosphatases must play a critical role in maintaining
-catenin association with cadherin and/or its ability to mediate the
cytoskeletal linkage. We have reported that the nonreceptor
protein-tyrosine phosphatase PTP1B binds to the cytoplasmic domain of
N-cadherin and regulates its function by dephosphorylating
-catenin
(13, 14). Furthermore, transfection of mouse L-cells constitutively expressing N-cadherin with a catalytically inactive PTP1B (substitution of cysteine 215 for serine) abolishes the ability of these cells to
form N-cadherin-mediated adhesions. The mutant PTP1B associates with
N-cadherin displacing endogenous PTP1B, resulting in dissociation of
the cadherin-actin connection and accumulation of cadherin-free tyrosine-phosphorylated
-catenin (14).
PTP1B is targeted to many distinct cellular locations based on specific
residues or domains in the molecule. The largest single pool is
localized to the cytoplasmic face of the endoplasmic reticulum through
a carboxyl-terminal domain (20). PTP1B also interacts with the
insulin receptor and the EGF receptor and is phosphorylated on tyrosine
residues in response to receptor stimulation (21-23). We have also
reported that PTP1B is physically and functionally associated with
focal adhesion complexes (24). This association may depend on binding
to p130cas through a proline-rich site
(25). Binding of PTP1B to N-cadherin requires that PTP1B itself be
phosphorylated on tyrosine residues (13, 14). In this study we show
that the in vitro and in situ interaction between
PTP1B and N-cadherin depends on phosphorylation of tyrosine residue 152.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Monoclonal mouse anti-PTP1B antibody was
purchased from Calbiochem. Anti-N-cadherin antibodies were
NCD-2, a rat monoclonal specific to chick N-cadherin (grown in our
laboratory from a culture provided by M. Takeichi, Kyoto University,
Kyoto, Japan), and polyclonal anti-pan-cadherin (Sigma).
Monoclonal rabbit anti-phosphotyrosine antibody (PY20) was from
Transduction Laboratories (Lexington, KY). Anti-HA antibody was from
Babco, Richmond, CA).
HRP1-conjugated anti-mouse
and anti-rat secondary antibodies were from Organon Teknika Co.
(Durham, NC). Goat-HRP anti-rabbit antibody and fluorescein
isothiocyanate-conjugated anti-rat IgG were from Jackson Immunoresearch
Laboratories, Inc. (West Grove, PA). Antibodies conjugated to magnetic
beads, used in immunoprecipitations, were from PerSeptive Biosystems
(Farmingham, MA).
Site-directed Mutagenesis--
All mutant forms of PTP1B were
generated using recombinant PCR. For bacterial expression in pGEX-KG
(Amersham Pharmacia Biotech), we added a SmaI and an
XhoI restriction site at the 5' and 3' ends, respectively.
The oligonucleotide primers were as follows: forward primer,
5'-TCCCCCGGGGGACATGGAGATCGAGAAGGAGTTCC-3'; reverse primer,
5'-CCGCTCGAGCGGCCATCAATGAAAACATACCCTG-3'. The
underlined bases indicate the start and stop codon. For
expression in eukaryotic cells, the forward primer included a
KpnI restriction site and an HA tag at the 5' end, and the
reverse primer contained an XhoI restriction site at the 3'
end to facilitate cloning into the pcDNA3.1(+)zeo mammalian
expression vector (Invitrogen, Carlsbad, CA). The oligonucleotide
primers used were as follows: 5' primer with a KpnI
restriction site,
5'-GGGGTACCGCCACCATGGCATACCCATACGATGTTCCAGATTACGCTGAGATCGAGAAGGAGTTCCA-3'; 3' primer with an XhoI restriction site,
5'-CCGCTCGAGCGGCCATCAATGAAAACATACCCTG-3'. The
underlined bases indicate the start and stop codon.
The oligonucleotide primers designed to introduce the C215S point
mutation were as follows (the underlined bases
indicate the changes from the naturally occurring nucleotides): forward C215S,
5'-GAGTATGGACCTGTTGTGGTGCACTCCAGTGCAGGAATTGGAAGATCAGG-3'; reverse C215S,
5'-CCTGATCTTCCAATTCCTGCACTGGAGTGCACCACAACAGGTCCATACTC-3'. In addition, three tyrosine residues (Tyr-66, Tyr-152, and Tyr-153) were replaced with phenylalanine in different combinations. The oligonucleotide primers used were as follows: forward Y66F,
5'-GGTGACAATGACTTTATCAATGC-3'; reverse Y66F,
5'-GCATTGATAAAGTCATTGTCACC-3'; forward Y152F,
5'-GATATAAAATCATTTTACACAGTACG-3'; reverse Y152F,
5'-CGTACTGTGTAAAATGATTTTATATC-3'; forward Y153F, 5'-GATATAAAATCATATTTCACAGTACG-3'; reverse Y153F,
5'-CGTACTGTGAAATATGATTTTATATC-3'; forward
Y152F/Y153F,
5'-GATATAAAATCATTTTTTCACAGTACG-3';
reverse Y152F/Y153F,
5'-CGTACTGTGAAAAATGATTTTATATC-3'.
To achieve high fidelity PCR products, Elongase (Life
Technologies, Inc., Grand Island, NY) was used for recombinant PCR. All
PCR products were subcloned into pGEM-T TA cloning vector (Promega,
Madison, WI) and confirmed by DNA sequencing.
Preparation of GST Fusion Proteins--
PTP1B cDNA
constructs were subcloned in pGEX-KG as SmaI/XhoI
fragments. The resulting plasmids were transformed into Epicurian coli TKB1 cells (Stratagene, La Jolla, CA) that constitutively express a tyrosine kinase. Cultures were induced with 0.4 mM isopropyl-1-thio-
-D-galactopyranoside and
allowed to express GST-PTP1B fusion proteins for 3 h. Induced cultures were harvested by centrifugation at 3,000 × g
for 10 min, and the bacterial pellets were stored at
70 °C until
ready for use. The frozen bacterial pellets were resuspended in B-PER bacterial protein extraction reagent (Pierce) containing 1% protease inhibitor mixture (Sigma) and 1 mM sodium orthovanadate
(Sigma). The suspended cultures were incubated for 15 min at room
temperature with gentle shaking. Soluble proteins were separated from
insoluble residue by centrifugation at 27,000 × g for
15 min and stored at
70 °C for future use. Expression of GST-PTP1B
was confirmed by SDS-PAGE and Western blot.
The cDNA fragment corresponding to the cytoplasmic domain of
N-cadherin (cyt-N-cad) was generated by PCR and subcloned as a
SmaI/XbaI fragment into pGEX-KG. The
oligonucleotide primers used were as follows (the underlined
bases are nucleotides corresponding to the 5' or 3' end of the
cytoplasmic sequence of N-cadherin cDNA): forward,
5'-TCCCCCGGGGGACTTCGTAGTATGGATGAAGCG-3'; reverse, 5'-GCTCTAGAGCGTCAGTCACTCAGTCATCACCTCCACC-3'. The GST
fusion protein of cyt-N-cad was prepared as described above and
purified using glutathione-Sepharose 4B according to the
manufacturer's instructions (Amersham Pharmacia Biotech). The purified
GST fusion protein was confirmed by SDS-PAGE and Western blot.
In Vitro Binding Assay--
Purified GST-cyt-N-cad was
biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce), and
biotinylation was confirmed by immunoblot using streptavidin-HRP.
Biotinylated cyt-N-cad (30 µg/well in PBS) was applied to a
streptavidin-coated 96-well plate (Roche Molecular Biochemicals). The
plate was incubated for 1 h at room temperature and washed three
times with PBS, blocked with 2% BSA (Sigma) in PBS for 1 h at
room temperature, and washed again with PBS. Aliquots of GST-PTP1B
mutants (50 µg/well in PBS) were added to the wells, and the plate
was incubated for 1 h at room temperature. After several washes in
PBS, anti-PTP1B antibody (in 0.5% BSA, PBS) was added to the wells,
followed by a 1-h incubation at room temperature and three washes with
TBST (50 mM Tris, 150 mM NaCl, 0.2% Tween 20).
Polyclonal anti-mouse HRP antibody (in 0.5% BSA, TBST) was then added,
and the plate was incubated for 1 h at room temperature and washed
three times with TBST. O-Phenylenediamine dihydrochloride
(Sigma) was used as substrate, and absorbance was measured at 492 nm.
Stable Transfection of PTP1B Mutants into Cells Constitutively
Expressing N-cadherin--
Mouse fibroblast cells constitutively
expressing N-cadherin (LN-cells) were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) containing 5% fetal bovine
serum (Life Technologies, Inc.), 1% penicillin-streptomycin (Life
Technologies, Inc.), and 100 µg/ml Geneticin (G418; Life
Technologies, Inc.). 24 h prior to transfection, cells were seeded
in a 6-well plate at 1 × 105 cells per well and
allowed to reach 80% confluence. Cells were transfected in OptiMEM
(Life Technologies, Inc.) using LipofectAMINE (Life Technologies, Inc.)
according to manufacturer's directions. Stable colonies were selected
with 1 mg/ml Zeocin (Invitrogen). 6 to 12 stable colonies were selected
for each transfection and used within 2 weeks.
Immunoprecipitation and Immunoblotting--
Cells were washed
with ice-cold PBS and incubated for 30 min on ice with lysis buffer
(1% Nonidet P-40 and protease inhibitor mixture (Sigma) in PBS). Cells
were harvested by scraping, and the cell lysate was centrifuged at
15,000 × g for 10 min. Aliquots containing equivalent
amounts of protein were incubated overnight at 4 °C with 1 µl of
rabbit anti-HA tag antibody (1 mg/ml). 10 µl of goat anti-rabbit IgG
conjugated to magnetic beads were then added to the supernatant, and
the mixture was incubated for 1 h at 4 °C with mixing. The
magnetic beads were collected using a magnetic stand, washed one time
with lysis buffer and three times with PBS, dissolved in SDS sample
buffer, separated by SDS-PAGE, and transferred to PVDF membranes. The
membranes were immunoblotted with anti-PTP1B, anti-HA, and
anti-N-cadherin antibodies as described (14).
To analyze the precipitation of endogenous PTP1B with N-cadherin,
anti-N-cadherin antibody NCD-2 was covalently linked to protein
G-agarose beads (Pierce) and incubated with neutral detergent extracts
of cells prepared as described above. Bound protein was eluted,
fractionated by SDS-PAGE, transferred to PVDF membranes, and
immunoblotted with the appropriate antibodies and developed as described.
Adhesion Assays--
96-well plates coated with protein L
(Pierce) were incubated with anti-N-cadherin antibody NCD-2 (20 µg/ml
in PBS; 50 µl/well) overnight at 4 °C. The wells were washed three
times with PBS and blocked with 1% BSA for 1 h at room
temperature. Cells in semiconfluent monolayers were washed in
serum-free medium and incubated overnight in methionine-free
Dulbecco's modified Eagle's medium containing 1 µCi/ml
3H-methionine (PerkinElmer Life Sciences). The cells
were then washed twice in HBSGKCa (20 mM HEPES, 150 mM NaCl, 3 mM KCl, 2 mM glucose, 1 mM CaCl2), released from the plate with a
0.002% trypsin solution prepared in the same buffer, washed, and
resuspended in the same buffer containing 0.1% BSA, 10 µg/ml DNase,
and 0.4 mM AESBF (Calbiochem). Approximately 4 × 104 cells were added to each well. The plate was
incubated for 45 min at 37 °C and washed 4 times with HBSGKCa. The
cells remaining on the wells were solubilized in 0.5% SDS, and
radioactivity was determined by liquid scintillation.
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RESULTS |
Tyrosine Residues 66, 152, and 153 in PTP1B Are Targets for
Phosphorylation--
The amino acid sequence of chick PTP1B has eleven
tyrosine residues; however, only three of those fit the consensus
substrate site for most protein-tyrosine kinases (26). To determine the residues essential for interaction between N-cadherin and PTP1B we used
the catalytically inactive C215S PTP1B mutant to create point mutations
substituting phenylalanine for tyrosine residues 66, 152, and 153. This
substitution is the most conservative, maintaining the structure and
size of the amino acid, but eliminating the phosphorylation site. A
diagram of all the constructs is shown in Fig.
1. The mutated PTP1B cDNAs were
subcloned into pGEX-KG and expressed as GST fusion proteins in the
bacterial strain TKB, which expresses a tyrosine kinase with broad
specificity, able to phosphorylate a variety of proteins. The GST
fusion proteins were analyzed for reactivity with anti-PTP1B and
anti-phosphotyrosine antibodies (Fig.
2A). All PTP1B fusion proteins
migrate as multiple bands on SDS-PAGE, with apparent molecular masses
between ~60 and 76 kDa (Fig. 2A), reflecting the added
masses of GST (~26 kDa) and PTP1B (~50 kDa). The multiple bands do
not appear to reflect differential phosphorylation, as immunoblotting
with an anti-phosphotyrosine antibody reveals only two major bands. The triple mutant, Y66F/Y152F/Y153F, does not show any reactivity with
anti-phosphotyrosine antibody, demonstrating that these tyrosine residues are indeed the only substrate sites for Src-like tyrosine kinases. The wild-type enzyme also shows minimal tyrosine
phosphorylation as compared with the C215S mutants because of its
phosphotyrosine phosphatase activity.

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Fig. 1.
Diagrammatic representation of PTP1B showing
all the mutations analyzed in these studies, the relative position of
the catalytic domain, and the targeted tyrosine residues.
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Fig. 2.
Immunoblots of PTP1B and N-cadherin fusion
proteins. A, Western transfers of SDS-PAGE of wild-type
PTP1B (WT), catalytically inactive PTP1B (CS),
and catalytically inactive PTP1B containing single, double (indicated
by residue numbers), and triple (Tp) mutations at
tyrosine residues were blotted with anti-PTP1B (top) and
anti-phosphotyrosine (bottom). GST indicates
fusion produced from vector lacking an insert. B, Western
transfers of SDS-PAGE of biotinylated N-cadherin fusion protein
(bio) blotted with a pan-cadherin antibody (left)
and with HRP-avidin (right).
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Tyr-152 Is the Crucial Residue for PTP1B Binding to the Cytoplasmic
Domain of N-cadherin in Vitro--
To determine the tyrosine
residue(s) critical for the interaction of PTP1B with N-cadherin, we
analyzed the ability of the various GST-PTP1B mutants to bind to the
cytoplasmic domain of N-cadherin in vitro. cyt-N-cad was
prepared as a GST fusion protein, purified on glutathione-conjugated
Sepharose 4B, and covalently labeled with biotin on lysine residues
(Fig. 2B, bottom). The labeled cyt-N-cad was
further purified to eliminate free biotin and bound to
neutravidin-coated 96-well plates. The amount of bound biotin-cyt-N-cad
was determined by enzyme-linked immunosorbent assay using an antibody
to the carboxyl terminus of N-cadherin. Wells coated with saturating
amounts of N-cadherin or BSA were then incubated with the various
GST-PTP1B fusions, as well as with GST only, as a control. After
washing and blocking the wells with BSA, the amount of PTP1B bound was
determined using anti-PTP1B antibody, which recognizes all the PTP1B
mutants equally well (see Fig. 2A), followed by an
HRP-conjugated secondary antibody. Optimal binding of PTP1B to
immobilized N-cadherin depends on phosphorylated tyrosine residues.
Fusion proteins lacking phosphorylated tyrosine residues, the C215S
triple mutant (Y66F/Y152F/Y153F), and the wild-type bind minimally,
showing only about 25% that of the C215S mutant with no substituted
tyrosine residues (Fig. 3A).
Among the C215S mutants bearing one Tyr
Phe substitution, only the Y152F shows a significant reduction in binding, suggesting that residue 152 is the most critical determinant of PTP1B binding to
N-cadherin in vitro. In agreement with this, the C215S
double mutants containing a 152 mutation (Y66F/Y152F and Y152F/Y153F) also show reduced binding, whereas the C215S Y66F/Y153F double mutant
binds as well as the unsubstituted C215S (Fig. 3). These results are
true over a wide concentration range (Fig. 3B);
concentrations of C215S PTP1B that show saturation binding still fail
to show binding of the Y152F mutant. It is interesting to note that the Y66F mutant actually facilitates binding.

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Fig. 3.
Binding of wild-type (WT)
and catalytically inactive PTP1B containing each of the single, double,
and triple mutants (indicated by residue numbers) to
N-cadherin fusion protein. A, 50 µg/ml of PTP1B fusion
protein was added to wells containing the immobilized N-cadherin
cytoplasmic domain. Asterisks indicate binding groups,
within which there is no statistical difference (p < 0.01). The difference between binding of 66 and catalytically inactive
(CS) is not statistically significant (p < 0.05). B, binding of increasing concentrations of wild-type
(WT), catalytically inactive (CS), or PTP1B
bearing mutations at all three tyrosines Tp,
(Y66F/Y152F/Y153F) to the immobilized N-cadherin cytoplasmic domain.
Data are graphed as a percentage of control (CS at 50 µg/ml).
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Tyr-152 Is Essential for PTP1B Interaction with N-cadherin--
To
determine the interaction of the PTP1B mutants with N-cadherin in
cells, the several PTP1B cDNA constructs were subcloned into the
pcDNA3.1(+)zeo vector and transfected into LN-cells (14). A
9-amino acid sequence coding for the hemagglutinin sequence was added
to the amino terminus of the PTP1B sequence to facilitate detection of
the transfected enzyme. Stable cell clones were established by
culturing in the presence of Zeocin and Geneticin (for stable N-cadherin expression). Cells were grown to near confluency, lysed with
nonionic detergent in the presence of tyrosine phosphatase inhibitors,
and immunoprecipitated with anti-HA antibody. Immunoprecipitated material was fractionated by SDS-PAGE and transferred to PVDF membranes, and the membranes were probed with anti-N-cadherin antibody
(NCD-2) and anti-PTP1B antibody (Fig.
4A). In agreement with what we
observed in the in vitro binding assays, the Y152F mutation
alone is enough to eliminate binding to N-cadherin (Fig. 4A). Furthermore, all combinations of mutant tyrosine
residues that include Tyr-152 behave identically (not shown), whereas
mutation at tyrosine residues 66 and 153 alone (Fig. 4A) or
in combination (not shown) have no effect on binding of PTP1B to
N-cadherin.

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Fig. 4.
In situ interaction of N-cadherin with
PTP1B. Neutral detergent extracts of LN-cells transfected with
HA-tagged PTP1B mutants were immunoprecipitated with anti-HA antibody
(A) or anti-N-cadherin antibody (B), separated by
SDS-PAGE, transferred to PVDF, and blotted with the indicated
antibodies. CS, cells expressing the C215S mutant;
66, 152, and 153, cells expressing the
C215S mutant in conjunction with mutations at each of the indicated
tyrosine residues; Vec, cells transfected with empty
vector.
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As in embryonic chick retina cells (13), endogenous PTP1B is associated
with N-cadherin in control LN-cells (transfected with vector alone) and
is phosphorylated on tyrosine residues (Fig. 4B). Expression
of the dominant-negative C215S mutant PTP1B in LN-cells prevents the
association of endogenous PTP1B with N-cadherin (see Fig. 4B
and Ref. 14). In contrast, expression of PTP1B carrying both the C215S
and the Y152F mutations does not alter the association of endogenous
PTP1B with N-cadherin. Thus tyrosine 152 is critical for in
situ binding and displacement of endogenous PTP1B from cadherin.
The Y152F Mutation Reverses the C215S Dominant-Negative Effect on
N-cadherin-mediated Adhesion--
The catalytically inactive C215S
PTP1B mutant acts as a dominant-negative when introduced into LN-cells,
inhibiting N-cadherin-mediated cell interaction (14). By introducing a
mutation that eliminates binding to N-cadherin in the C215S PTP1B, the
dominant-negative effect should be abolished; this is indeed the
case (Fig. 5). N-cadherin-mediated
cell adhesion is abolished in the C215S mutants but restored in the
C215S mutants that also have a Y152F mutation. In comparison, mutations
in tyrosine residues 66 and 153 alone or in combination have no effect
(Fig. 5). This effect on N-cadherin-mediated adhesion is reflected in
the cells phenotype; LN-cells grow in clusters of tightly adherent
cells because of expression of N-cadherin (Fig.
6A; see also Ref. 14). In the
dominant-negative C215S mutant this phenotype is lost because of
inactivation of N-cadherin (compare Fig. 6, A and
B) but recovered in the C215S mutant bearing the Y152F
mutation (Fig. 6C). In contrast, mutation of either tyrosine
66 or 153 has little or no effect on the dominant-negative phenotype.

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Fig. 5.
Adhesion of LN-cells expressing each of the
PTP1B constructs to N-cadherin. The data are expressed as the
percentage of input cells adhering to the substrate. WT,
wild-type PTP1B; CS, catalytically inactive PTP1B;
numbers indicate mutations at the indicated tyrosine
residues; +NCD indicates adhesion in the presence of the
function blocking antibody NCD2; IP, immunoprecipitate; Vec, vector;
Tp, Y66F/Y152F/Y153F.
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Fig. 6.
Morphology and localization of N-cadherin
among LN-cells transfected with catalytically inactive PTP1B mutated at
key tyrosine residues and visualized with anti-N-cadherin
antibody. WT, wild-type; C215S,
catalytically inactive; Y66F, Y152F,
Y153F, and Y6/2/3F (triple mutant), catalytically
inactive forms containing mutations at the indicated tyrosine residues.
Note that among the forms bearing mutations at tyrosine residues, only
cells transfected with forms mutated at Tyr-152 revert to a tightly
adherent population with N-cadherin present at cell-cell
boundaries.
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DISCUSSION |
Our laboratory has demonstrated that PTP1B interacts directly with
N-cadherin and that phosphorylation of PTP1B on tyrosine residues is
necessary for this association (13, 14). We now identify tyrosine
residue 152 in PTP1B as the critical residue for PTP1B-N-cadherin
interaction. PTP1B mutants that have tyrosine 152 replaced by
phenylalanine do not interact with N-cadherin in in vitro
binding assays. Moreover, in L-cells expressing N-cadherin and
HA-tagged PTP1B carrying the Y152F and C215S double mutation, HA-PTP1B
does not coimmunoprecipitate with N-cadherin, indicating a lack of
association between the two molecules in situ. This is also
reflected in the loss of the dominant-negative effect on adhesion
of the C215S mutation on N-cadherin function. Furthermore, in LN-cells expressing the Y152F mutation endogenous PTP1B is associated with N-cadherin, and it is tyrosine- phosphorylated.
The multiple intracellular roles played by PTP1B require interactions
with many different intracellular partners. The needed binding
specificity appears to be achieved by compartmentalization or by
targeting mediated by specific domains. The carboxyl terminus of PTP1B
directs its localization to the cytoplasmic face of the endoplasmic
reticulum, thus restricting the number of potential interactors (20).
In platelets and activated T-cells, proteolytic cleavage in the ER
targeting domain results in translocation of PTP1B to the
cytoskeletal/membrane fraction (27-29). This cleavage is dependent on
integrin engagement, resulting in increased Ca2+ levels
and, consequently, activation of calpain. We also find that PTP1B
associated with N-cadherin in vivo migrates faster on
SDS-PAGE than the intact ~50-kDa enzyme, suggesting cleavage (13,
14). The N-cadherin-associated PTP1B represents a small fraction of the
total and colocalizes with N-cadherin in sites of cell-cell contacts
and at the tips of growing neurites (14, 30). Elimination of the ER
localization signal does not alter the interaction of PTP1B with
N-cadherin, suggesting that targeting of PTP1B to the N-cadherin
complex does not depend on prior targeting to the ER. Furthermore,
targeting to specific plasma membrane locations does not appear to
depend on cleavage of the ER targeting sequence, as the PTP1B
associated with focal adhesion complexes (24) and the insulin receptor
(22) have an apparent molecular mass of ~50 kDa.
Phosphorylation on tyrosine residues is important for targeting of
PTP1B to at least two of its interacting partners. As we demonstrate
here, phosphorylation of tyrosine 152 is critical for binding to
N-cadherin. Additionally, interaction of PTP1B with the insulin
receptor results in phosphorylation of tyrosine residues 66 and
152/153. Phosphorylation of these residues further promotes binding to
the receptor. Tyrosine 66 is the major target for phosphorylation of
PTP1B by the insulin receptor, creating a site essential for downstream
signaling (22). In contrast, tyrosine phosphorylation on PTP1B does not
appear to play a role in the binding of PTP1B to
p130cas (25). This interaction, which probably
mediates targeting of PTP1B to the integrin complex, is mediated by a
proline-rich, SH3-binding domain in PTP1B (25). These differences
highlight the fact that even though PTP1B is a ubiquitous enzyme, it
plays a pivotal role in regulating many cellular functions through
specific protein-protein interactions.