receptor protein tyrosine phosphatase ptp1b with n- cadherin

ESSENTIAL TYROSINE RESIDUES FOR INTERACTION OF THE NON-

RECEPTOR PROTEIN TYROSINE PHOSPHATASE PTP1B WITH N-

CADHERIN

by

Jinseol Rhee, Jack Lilien and Janne Balsamo

Department of Biological Sciences

The University of Iowa

Iowa City, IA 52242-1342

Running Title: PTP1B Binding to N-cadherin

Key Words: Tyrosine Phosphatase, PTP1B, N-cadherin, Adhesion, ß-catenin

Supported by a grant from the NIH to JL and JB (EY12132)

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 5, 2000 as Manuscript M007656200 by guest on February 15, 2018

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SUMMARY

Expression of a dominant-negative, catalytically inactive form of the non-receptor protein

tyrosine phosphatase PTP1B in L-cells constitutively expressing N-cadherin (LN-cells) results in

loss of N-cadherin-mediated cell-cell adhesion. PTP1B interacts directly with the cytoplasmic

domain of N-cadherin and this association is regulated by phosphorylation of tyrosine residues in

PTP1B. Three tyrosine residues in PTP1B are potential substrates for tyrosine kinases: Y66,

Y152 and Y153. To determine the tyrosine residue(s) that are crucial for the cadherin-PTP1B

interaction we used site-directed mutagenesis to create catalytically inactive PTP1B constructs

bearing additional single, double or triple mutations in which tyrosine was substituted by

phenylalanine. Mutation Y152F eliminates binding to N-cadherin in vitro, while mutations

Y66F and Y153F do not. Overexpression of the catalytically inactive PTP1B with the Y152F

mutation in LN-cells has no effect on N-cadherin-mediated adhesion, and immunoprecipitation

reveals that the mutant Y152F PTP1B does not associate with N-cadherin in situ. Furthermore,

among cells overexpressing the Y152F mutant endogenous PTP1B associates with N-cadherin

and is tyrosine phosphorylated.

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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 α- and ß-catenin (1, 2, 3, 4); ß-catenin associates directly with a 20

amino acid domain near the carboxy-terminus of cadherin (5, 6) and with α-catenin which, in

turn, interacts with actin, either directly (7) or indirectly, through α-actinin (8). ß-catenin not

only performs a bridging role between cadherin and actin, but free ß-catenin can be translocated

to the nucleus where it regulates transcription of cadherin and other gene products (9, 10). Thus,

the regulation of free ß-catenin is of critical importance and, consequently, the interaction of ß-

catenin with cadherin has multiple ramifications on cellular function (11, 12).

Regulation of the interaction of ß-catenin with N-cadherin is mediated by the phosphorylation of

tyrosine residues on ß-catenin (13, 14). In embryonic chick neural retina cells,

hyperphosphorylation of ß-catenin is correlated with loss of its association with N-cadherin and

loss of cadherin function (13, 14). Enhanced phosphorylation of ß-catenin has also been

correlated with loss of E-cadherin function (15, 16, 17, 18, 19). These data suggest that tyrosine

kinases and/or phosphatases must play a critical role in maintaining ß-catenin association with

cadherin and/or its ability to mediate the cytoskeletal linkage. We have reported that the non-

receptor protein tyrosine phosphatase PTP1B binds to the cytoplasmic domain of N-cadherin and


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regulates its function by dephosphorylating ß-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 ß-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 carboxy-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, 22, 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 (La

Jolla, CA). 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,

Japan) and polyclonal anti-Pan Cadherin from Sigma (St. Louis, MO). Monoclonal rabbit anti-

phosphotyrosine antibody (PY20) was from Transduction Laboratories (Lexington, KY). Anti-

HA antibody was from Babco, Richmond, CA). HRP-conjugated anti-mouse and anti-rat

secondary antibodies were from Organon Teknika Co (Durham, MC). Goat-HRP-anti-rabbit

antibody and FITC-conjugated anti-rat IgG were from Jackson Immunoresearch

Laboraboratories, 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 (Pharmacia Biotech, Piscataway, NJ), we added a

Sma I and a Xho I restriction site at the 5’ and 3’ ends, respectively. The oligonucleotide

primers were:

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 a Xho I restriction site at the 3’ end to facilitate cloning into the


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pcDNA3.1(+)zeo mammalian expression vector (Invitrogen, Carlsbad, CA). The

oligonucleotide primers used were as follows:

5’-primer with Kpn I restriction site,

5’-GGGGTACCGCCACCATGGCATACCCATACGATGTTCCA

GATTACGCTGAGATCGAGAAGGAGTTCCA-3’;

3’-primer with Xho I 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’-CCTGATCTTCCAATTCCTGCACTGGAGTGCACCACAACA GGTCCATACTC-3’.

In addition, three tyrosine residues (Y66, Y152 and Y153) 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’-CGTACTGTGTAA AATGATTTTATATC-3’ ;

forward Y153F, 5’-GATATAAAATCATATTTCACAGTA CG-3’;

reverse Y153F 5’-CGTACTGTGAAATATGATTTTATATC-3’;


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forward Y152/153F, 5’-GATATAAAATCATTTTTCACAGTACG-3’,

reverse Y152/153F, 5’-CGTACTGTGAAAAATGATTTTATATC-3’.

To achieve high fidelity PCR products, Elongase (Gibco, 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

Sma I/Xho I 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 0.4M IPTG and allowed to express GST-PTP1B fusion proteins for 3 hours. Induced

cultures were harvested by centrifugation at 3,000g, for 10min and the bacterial pellets stored at

–70oC until ready to use. The frozen bacterial pellets were resuspended in B-PER Bacterial

Protein Extraction Reagent (Pierce, Rockford, IL), containing 1% Protease Inhibitor Cocktail

(Sigma) and 1mM Sodium orthovanadate (Sigma). The suspended cultures were incubated for

15min at room temperature with gentle shaking. Soluble proteins were separated from insoluble

residue by centrifugation at 27,000g for 15min and stored at –70oC 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 Sma I/Xba I 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’;


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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 (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

(Boehringer Mannheim, Germany). The plate was incubated for 1 hour at room temperature and

washed three times with PBS, blocked with 2% BSA (Sigma) in PBS for 1 hour 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 incubated for 1 hour 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 hour incubation at room temperature and three washes with TBST (50 mM Tris, 150 mM

NaCl, 0.2% Tween20). Polyclonal anti-mouse HRP antibody (in 0.5% BSA, TBST) was then

added, the plate incubated for 1 hour 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 - LN

cells, mouse fibroblast cells constitutively expressing N-cadherin, were grown in DMEM

medium (Gibco) containing 5% FBS (Gibco), 1% penicillin-streptomycin (Gibco) and 100

_µg/ml Geneticin (G418, Gibco). 24h prior to transfection, cells were seeded in a six well plate


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at 1x105 cells per well and allowed to reach 80% confluence. Cells were transfected in

OptiMEM (Gibco) using Lipofectamine (Gibco) according to manufacturer’s directions. Stable

colonies were selected with 1 mg/ml Zeocin (Invitrogen). 6 to12 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% NP-40 and protease inhibitor cocktail (Sigma)

in PBS]. Cells were harvested by scraping and the cell lysate centrifuged at 15000g for 10 min.

Aliquots containing equivalent amounts of protein were incubated overnight at 4oC 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 incubated for 1 hour at 4oC 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 PDVF membranes and immunoblotted with the appropriate

antibodies and developed as described.


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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 hour at room temperature. Cells

in semi-confluent monolayers were washed in serum free medium and incubated overnight in

methionine-free DMEM containing 1 µCi/ml 3H-methionine (NEN). The cells were then washed

twice in HBSGKCa (20mM HEPES, 150 mM NaCl, 3mM KCl, 2mM glucose, 1mM 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 DNAase and 0.4 mM AESBF

(Calbiochem). Approximately 4x104 cells were added to each well. The plate was incubated for

45 min at 37oC and washed 4 times with HBSGKCa. The cells remaining on the wells were

solubilized in 0.5% SDS and radioactivity determined by liquid scintilation.


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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). In order 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 Figure 1. The mutated PTP1B cDNAs were subcloned into pGEX2T and expressed as

GST fusion proteins in the bacterial strain TKB, which express 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 (PY) antibodies (Fig. 2A). All PTP1B

fusion proteins migrate as multiple bands on SDS-PAGE, with apparent molecular masses

between approximately 60kD to 76kD (Fig. 2A), reflecting the added masses of GST (~26KD)

and PTP1B (~50KD). 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, Y66/152/153F does not show any reactivity with anti-PY 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 to the C215S

mutants, due to its phosphotyrosine phosphatase activity.


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Y152 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. The cytoplasmic domain of N-cadherin (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 ELISA using an antibody to the carboxy-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 second antibody. Optimal binding of PTP1B to immobilized N-cadherin

depends on phosphorylated tyrosine residues. Fusion proteins lacking phosphorylated tyrosine

residues, the C215S triple mutant (Y66/152/153F) and the wild type, bind minimally, showing

only about 25% that of the C215S mutant with no substituted tyrosine residues (Figure 3A).

Among the C215S mutants bearing one Y-F 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 (Y66/152F and Y152/153F) also show reduced binding, while the C215S

Y66/153F 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


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saturation binding still fail to show binding of the Y152F mutant. It is interesting to note that the

Y66F mutant actually facilitates binding.

Y152 is essential for PTP1B interaction with N-cadherin - In order to determine the interaction

of the PTP1B mutants with N-cadherin in cells, the several PTP1B cDNA constructs were

subcloned into the pcDNA3.1zeo vector and transfected into L cells constitutively expressing N-

cadherin (LN-cells; 14). A 9 amino acid sequence coding for the hemaglutinin 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 non-ionic

detergent in the presence of tyrosine phosphatase inhibitors and immunoprecipitated with anti-

HA antibody. Immunoprecipitated material was fractionated by SDS-PAGE, transferred to

PVDF membranes and the membranes 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 Y152 behave identically (not shown),

while 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.

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 (Fig. 4B and ref. 14). In contrast expression


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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 due to expression of N-cadherin (Fig.6A; see also

14). In the dominant-negative C215S mutant this phenotype is lost due to inactivation of N-

cadherin (compare Fig.6A 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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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 co-

immunoprecipitate 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 carboxy 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, 28, 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 ~50kD enzyme, suggesting cleavage (13, 14). The N-cadherin-associated PTP1B


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represents a small fraction of the total and co-localizes 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 ~50kD.

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.


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FIGURE LEGENDS

Figure 1. Diagrammatic representation of PTP1B showing all the mutations analyzed in these

studies, the relative position of catalytic domain and the targeted tyrosine residues

Figure 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).

Figure 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 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 CS is not statistically different

(p < 0.05). B: Binding of increasing concentrations of wild-type (WT), catalytically inactive

(CS), or PTP1B bearing mutations at all three tyrosines (66/152/153) to immobilized N-cadherin

cytoplasmic domain. Date are graphed as percent of control (CS at 50 µg/ml).


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Figure 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 PDVF, and blotted

with the indicated antibodies. CS: cells expressing the C215S mutant; 66, 152, 153: cells

expressing the C215S mutant in conjunction with mutations at each of the indicated tyrosine

residues; Vec: cells transfected with empty vector.

Figure 5. Adhesion of LN-cells expressing each of the PTP1B constructs to N-cadherin. The

data are expressed as the percent 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.

Figure 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 Y152 revert to a tightly adherent population with N-cadherin

present at cell-cell boundaries.


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Jinseol Rhee, Jack Lilien and Janne BalsamoPhosphatase PTP1B With N-cadherin

Essential Tyrosine Residues For Interaction Of The Non-receptor Protein Tyrosine

published online December 5, 2000J. Biol. Chem.

10.1074/jbc.M007656200Access the most updated version of this article at doi:

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receptor protein tyrosine phosphatase ptp1b with n- cadherin

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