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INTRODUCTION |
Protein-tyrosine-phosphatases
(PTPs)1 are intracellular
signaling molecules in superior eukaryotic organisms that have been demonstrated to be involved in a large set of cellular events such as
migration, proliferation, differentiation, and transformation by
regulating the level of tyrosine phosphorylation in cellular proteins
issued from the action of protein-tyrosine kinases (PTKs) (1). Because
many oncogenes encode PTKs, it was thought that PTPs might represent
tumor suppressors, although it remains to be conclusively demonstrated.
It has been shown that hemizygous deletions of the gene for PTP
mapped to chromosome 3p21 have been detected in tumor cells of renal
and pulmonary origin (2), and some of PTPs can suppress transformation
in cultured cells (3-9). Overexpression of LAR in cultured cells
reduced the extent of both phosphorylation and stability of
p130CAS and induced apoptosis (10).
PTPs identified to date can be classified into receptor-type and
nonreceptor type PTP subfamilies. Leukocyte common antigen-related (LAR) protein-tyrosine-phosphatase is a prototypic member of the receptor-type PTP (RPTP) subfamily that is a transmembrane molecule composed of a 1234-amino acid extracellular receptor-like region and a
623-amino acid cytoplasmic region containing two tandemly repeated PTP
domains (11). The extracellular region is composed of three
immunoglobulin (Ig)-like domains and eight repeats of fibronectin type
III domains and resembles neural-cell adhesion molecules (11).
Full-length constitutively spliced LAR transcripts are expressed in
breast and other tissues, whereas alternatively spliced isoforms are
preferentially expressed in the nervous system (12). The findings that
LAR is expressed in mammalian neurons and that its expression is
regulated during neural development by nerve growth factor (13-15)
suggested that LAR might modulate mammalian neuronal survival and/or
neurite outgrowth. This hypothesis was supported by studies showing the
reduced size of basal forebrain cholinergic neurons and loss of
cholinergic innervations of the dentate gyrus in
LAR-deficient transgenic mice (16) and aberrant motor neuron
pathfinding in Drosophila LAR loss-of-function mutants (17).
LAR-deficient transgenic mice have also been found to impair
outgrowth of sensory fibers during sciatic nerve regeneration (18).
The RET gene codes for a transmembrane tyrosine kinase that
displays a cadherin-like domain and a cysteine-rich motif in the extracellular region (19, 20), and it is expressed in cells of neural
crest origin such as enteric neurons and in the developing excretory
system (19, 21, 22). It is also expressed in tumors originating from
neural crest cells, such as neuroblastoma, pheochromocytoma, and
medullary thyroid carcinoma (23-25).
The gene for LAR is localized to human chromosome 1p32, a
region frequently deleted in neuroblastoma, pheochromocytoma, and medullary thyroid carcinoma (26, 27), and RET germ-line
mutations are responsible for multiple endocrine neoplasia (MEN) 2A and 2B that develop medullary thyroid carcinoma and pheochromocytoma (28).
These findings led us to postulate that LAR may modulate the function
of the RET-MEN2 proteins in human tumors. In this study, to explore the
potential role of LAR for RET tyrosine kinase activities, we
cotransfected LAR and RET with a MEN2A
or MEN2B mutation into the NIH 3T3 cells. As a result, we
found that LAR reduced the levels of phosphorylation, tyrosine kinase
activity, and oncogenic activity of RET-MEN2A but not RET-MEN2B.
Activation levels of signaling molecules potentially downstream of RET
kinase were also down-regulated to variable degrees. These results thus suggested that LAR could play a role in the regulation of the function
of RET-MEN2A proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
Anti-LAR, anti-PLC
, and anti-AKT/PKB
monoclonal antibodies were purchased from Transduction Laboratories
(Lexington, KY). Anti-RET rabbit polyclonal antibody was developed
against the C-terminal 19 amino acids of the RET long isoform as
described previously (29). Anti-phospho-RET (tyrosine 1062)-specific
polyclonal antibody was developed against with a synthetic peptide
(IENKLpYGMSDP) corresponding to the residues around tyrosine 1062 of
human RET. Anti-phosphotyrosine monoclonal antibody was purchased from
Upstate Biotechnology Inc. (Lake placid, NY). Anti-phospho-ERK1/2
(Thr-202/Tyr-204) and anti-phospho-AKT (serine 473) polyclonal
antibodies were purchased from New England BioLabs (Beverly, MA), and
anti-ERK 2 polyclonal antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Cell Culture and Transfection--
The construction of the
Aptag-1 vector containing a full-length RET cDNA with a
MEN2A (Cys-634
Tyr) or MEN2B (Met-918
Thr) mutation was described previously (30). pMT LAR plasmid containing
a full-length LAR cDNA was provided kindly by Dr. H. Saito (Harvard Medical School, Boston, MA). As a control, a truncated mutant LAR cDNA (LAR-
TP) was generated by
ligating a 2638-bp EcoRI/NheI human
LAR cDNA fragment, resulting in the generation of a
protein of 756 amino acids (Fig. 1a). NIH 3T3 cells (5 × 105 in a 6-cm-diameter dish) were cotransfected with
0.2-0.5 µg of RET-MEN2A or
RET-MEN2B plasmid DNA and 1 µg of
LAR or LAR-
TP plasmid DNA, and stable clones
resistant to 250 µg/ml hygromycin B were selected and maintained in a
humidified atmosphere of 5% CO2 and 95% air at 37 °C
in Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical,
Tokyo, Japan) supplemented with 8% bovine calf serum (Hyclone
Laboratories, Logan, UT).
Immunoprecipitation and Western Blot Analysis--
Cells were
grown subconfluently in 60-mm dishes and serum-starved for 6 h.
Then they were washed once with ice-cold phosphate-buffered saline and
lysed in radioimmune precipitation buffer (RIPA) (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1%
Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride
and 1 mM sodium orthovanadate. The lysates were clarified
by centrifugation (15,000 × g) for 1 h and
incubated with 1-2 µg of primary antibodies for 2 h, and the
immunocomplexes were precipitated with protein A- or G-Sepharose beads
(Amersham Pharmacia Biotech) for 1 h at 4 °C. After
immunoprecipitation, beads were washed four times with RIPA buffer,
suspended in sample buffer (2.0% (w/v) SDS, 20 mM Tris-HCl
(pH 6.8), 2 mM EDTA, 10% (w/v) sucrose, and 20 µg/ml bromphenol blue) in the absence or presence of 80 mM
dithiothreitol and boiled for 3 min. The lysates were subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), separated on 5-10%
gradient gels and transferred onto polyvinylidene difluoride membranes
(Nihon Millipore Kogyo KK, Tokyo, Japan) overnight at 100 mA. The
transferred proteins on the membrane were treated with 0.5% (v/v)
Tween-phosphate-buffered saline containing 3% (w/v) ovoalbumin for
1 h. Then, the membrane was incubated overnight at 4 °C with
different primary antibodies. Secondary antibodies used for Western
blotting were peroxidase-conjugated anti-mouse (1:1000) or anti-rabbit
(1:1000) immunoglobulins (Dako, Glostrup, Denmark). The filters were
washed four times with Tween-phosphate-buffered saline and examined by
using a chemiluminescence ECL detection kit (Amersham Pharmacia
Biotech, Tokyo). Before reprobing with different primary antibodies,
the blots were stripped by incubation in stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol) for 45 min at 55 °C. Protein concentrations
were determined using the Bio-Rad protein assay kit.
In Vitro Dephosphorylation of RET Proteins by LAR--
RET
proteins were purified from 500 µg of cellular proteins by
immunoprecipitation with anti-RET polyclonal antibody. The immunoprecipitated RET proteins were washed three times with the RIPA
buffer and twice with the LAR protein-tyrosine-phosphatase (PTP) buffer
(25 mM Tris-HCl, pH 7.0, 50 mM NaCl, 2 mM Na2EDTA, 2 mM dithiothreitol,
0.01% Brij-35) and divided into two portions. Then they were suspended
in 30 µl of LAR PTP buffer containing 1 unit of purified LAR and
incubated for 30 min at 30 °C. The reactions were terminated by
adding an equal volume of 2 × SDS sample buffer, boiled for 5 min, and subjected to immunoblotting as described above.
In Vitro RET Receptor Tyrosine Kinase Assay--
Two hundred
micrograms of total proteins was immunoprecipitated with 1 µg of
anti-RET polyclonal antibody and incubated with protein A-Sepharose
beads. The beads were washed twice with RIPA buffer (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1%
Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride
and 1 mM sodium orthovanadate and three times with kinase
buffer (10 mM Tris-HCl, pH 7.4, 5 mM
MgCl2) and suspended in the kinase buffer with 2.0 µg of
myelin basic protein (MBP) (Sigma Chemical Co., St. Louis, MO) as an
exogenous substrate, and 370 kBq of [
-32P]ATP
(PerkinElmer Life Sciences, Wilmington, DE). They were incubated for 20 min at 30 °C. The reactions were terminated by adding an equal
volume of 2 × SDS sample buffer and loaded onto SDS-10% polyacrylamide gels. The gels were incubated three times for 30 min in
a fixing solution (20% methanol/10% acetic acid), dried, and
visualized by autoradiography.
GST Fusion Protein Binding Assay--
A cDNA sequence
corresponding to the LAR PTP domain (amino acids 1275-1881) was
inserted into the BamHI site of the pGEX-2T plasmid (11, 31,
32). The LAR-CS (Cys-1552
Ser) mutant has been described
previously (32). Escherichia coli NM522 was transformed with
the expression plasmids and grown at 24 °C, and protein synthesis
was induced by the addition of 0.1 mM
isopropyl-
-D-thiogalactoside. The bacterial pellet was
suspended in ice-cold NETN buffer (1% Nonidet P-40, 20 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl) and
sonicated six times (each for 15 s). Bacterial debris was removed
by centrifugation, and fusion proteins in the supernatant were bound
to glutathione-Sepharose beads (Amersham Pharmacia Biotech), washed
three times with 10 volumes of buffer (1% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, 250 mM NaCl), and
resuspended in NETN buffer. The resulting cell lysates containing 400 µg of total cellular proteins were incubated for 2 h at 4 °C
with GST fusion proteins. The GST protein complexes were washed four
times with RIPA buffer, and the beads were resuspended in 30 µl of
Laemmli buffer (62.5 mM Tris-HCl (pH 6.8), 20% glycerol,
2% SDS, 1.4 mM
-mercaptoethanol, 20 µg/ml bromphenol
blue), boiled for 5 min, and then fractionated on SDS-10%
polyacrylamide gels. Immunoblotting with anti-RET antibody was
performed as described above.
Soft Agar Assay and Tumor Growth in Scid Mice--
Soft agar
assays were carried out in 6-cm dishes as described by Clark et
al. (33). Briefly, 5 × 104 cells were suspended
in 0.3% agar in DMEM containing 10% CS and 10 mM HEPES
(pH 7.4) and added on a bottom layer of 0.6% agar. The plates were
incubated at 37 °C in a humidified 7.5% CO2 incubator for 2 weeks. The colonies were also stained with 500 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide], Sigma,
Deisenhofen) for 2-3 h and counted with a microscope. All assays were
done in quadruplicates.
For tumorigenesis studies, 1 × 107 cells were washed
twice, suspended in 0.2 ml of DMEM, and then inoculated subcutaneously into 10-week-old male scid mice (five mice/group). Local tumor growth
was measured daily, and mice were sacrificed 16 days after injection to
weigh the tumors.
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RESULTS |
Expression of LAR, RET-MEN2A, and RET-MEN2B in NIH 3T3
Cells--
LAR is initially synthesized as a ~200-kDa proprotein
that is processed at a pentabasic site by an endogenous protease (a subtilisin-like protease), leading to a complex of two noncovalently associated subunits; the extracellular- or E-subunit (150 kDa) contains
the cell adhesion molecule-like domain and the phosphatase or P-subunit
(85 kDa) contains an 82-amino acid extracellular region and the
transmembrane and cytoplasmic domains (Fig.
1a) (34). After cotransfection
of NIH 3T3 cells with RET-MEN2A or RET-MEN2B and
LAR or LAR-
TP, stable clones resistant to 250 µg/ml hygromycin B were selected and maintained in DMEM supplemented with 8% bovine calf serum. Immunoblotting of the lysates from cells
transfected with the LAR gene demonstrated about 5-fold increase in the abundance of the LAR protein compared with that of the
endogenous LAR protein in untransfected cells (Fig. 1b). The
migration of the transfected LAR protein species, a ~200-kDa precursor and a ~150-kDa proteolytically cleaved extracellular domain
subunit, was detected by anti-LAR monoclonal antibody directed against
the LAR N terminus, whereas cells transfected with the truncated
LAR cDNA expressed a protein with an apparent molecular mass of ~98 kDa (Fig. 1b). However, when the same filter
was stripped and reprobed with anti-RET polyclonal antibody, there was
no significant difference in the expression levels of the RET proteins
in all cell lines examined (Fig. 1b).

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Fig. 1.
A, schematic illustration of wild-type
LAR and LAR- TP. The full-length wild-type LAR and the truncated LAR
mutant LAR- TP, which generates a protein of 756 amino acids, are
shown. The wild-type LAR protein is cleaved into two subunits that
remain associated by a noncovalent linkage. B, expression of
LAR and LAR- TP in NIH 3T3 cells expressing RET-MEN2A or RET-MEN2B.
LAR or LAR- TP was cotransfected with
RET-MEN2A (Cys-634 Tyr) or RET-MEN2B (Met-918
Thr) into NIH 3T3 cells. Total cell lysates (25 µg of proteins)
were prepared from the designated cell lines, subjected to SDS-PAGE
under reducing conditions, and analyzed by immunoblotting with anti-LAR
monoclonal antibody. Two protein bands of LAR, a ~200-kDa LAR
precursor and a 150-kDa extracellular subunit of LAR were detected in
RET-MEN2A/LAR and RET-MEN2B/LAR cells, whereas cells transfected with
LAR- TP expressed a protein with an apparent molecular
mass of ~98 kDa (upper panel). The same filter was
stripped and reprobed with anti-RET antibody to show equal expression
of the RET proteins in each cell line (lower panel). 175- and 155-kDa RET proteins are shown.
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LAR Effectively Inhibits Autophosphorylation and Kinase Activity of
RET-MEN2A but Not RET-MEN2B--
We first measured the levels of
tyrosine phosphorylation and kinase activity of RET in the established
cell lines. Equal protein amounts of their lysates (25 µg) were
subjected to Western blotting with anti-phosphotyrosine monoclonal
antibody, 4G10. As shown in Fig.
2a, the level of tyrosine
phosphorylation of the 175-kDa RET in RET-MEN2A/LAR cells was
significantly lower than that in RET-MEN2A and RET-MEN2A/LAR-
TP
cells, whereas there was no difference in the phosphorylation levels of
RET among RET-MEN2B, RET-MEN2B/LAR, and RET-MEN2B/LAR-
TP cells.
Because the decrease of tyrosine phosphorylation of the RET protein was
observed in three independent RET-MEN2A/LAR clones (date not shown), we
pooled them for the following experiments. In addition, this result was
confined by immunoprecipitation of the lysates with anti-RET antibody,
followed by immunoblotting with anti-phosphotyrosine antibody (Fig.
2b).

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Fig. 2.
Effects of LAR on tyrosine phosphorylation of
RET. a, total cell lysates (25 µg of proteins) were
prepared from the designated cell lines and resolved by SDS-PAGE,
followed by immunoblotting with anti-phosphotyrosine antibody
(upper panel). The same blot was stripped and immunoblotted
with anti-RET antibody (lower panel). b, equal
proteins (300 µg) from the designated cell lines were
immunoprecipitated with anti-RET antibody, followed by immunoblotting
with anti-phosphotyrosine (upper panel) or anti-RET antibody
(lower panel). 175- and 155-kDa RET proteins are
shown.
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We next measured the RET tyrosine kinase activity in each cell line.
Equal amounts (250 µg) of lysates from the cell lines were
immunoprecipitated with the anti-RET antibody and incubated in the
presence of [
-32P]ATP and MBP. Phosphorylated products
were resolved by SDS-PAGE and detected by autoradiography (Fig.
3a). The relative levels of
catalytic activities were calculated by densitometric analyses of each
band, and the results were plotted as percent decrease of
phosphorylation levels relative to those in
RET-MEN2A/LAR-
TP- expressing cells (Fig. 3, b and
c). We found the RET autophosphorylation and phosphorylation
of MBP that was used as an exogenous substrate were reduced by 80 and
70%, respectively, in RET-MEN2A/LAR cells compared with those in
RET-MEN2A/LAR-
TP cells. Consistent with the previous results (35,
36), the autophosphorylation of RET-MEN2B proteins from RET-MEN2B/LAR
and RET-MEN2B/LAR-
TP cells was much lower than that of RET-MEN2A
proteins. However, the inhibitory effects of LAR on RET-MEN2B
autophosphorylation and kinase activity were not observed in
RET-MEN2B/LAR cells (Fig. 3). These results confirmed that
overexpression of LAR inhibited the tyrosine kinase activity of only
RET-MEN2A.

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Fig. 3.
In vitro kinase assay of RET
proteins. A, equal amounts of proteins (400 µg) from
the designated cell lines were immunoprecipitated with anti-RET
antibody, and the immunoprecipitated RET proteins were incubated in the
presence of [ -32P]ATP and myelin basic protein
(MBP) and subjected to SDS-PAGE on a 15% gel.
Autoradiography of the gel is shown (upper panel). Bands
representing autophosphorylated RET proteins and phosphorylated MBP are
indicated. Aliquots of the immunoprecipitated RET proteins from each
cell line were subjected to immunoblotting with anti-RET antibody to
show the presence of equal amounts of RET proteins in the samples
(lower panel). B and C, phosphorylated
RET and MBP bands were quantitated by the densitometric analysis.
Results are plotted as percent decrease in phosphorylation of RET and
MBP relative to that in the sample from RET-MEN2A/LAR- TP-expressing
cells.
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LAR Effectively Dephosphorylates RET-MEN2A and RET-MEN2B in
Vitro--
The above results suggest that RET may be a direct
substrate of LAR. To test this possibility, phosphorylated RET was
immunoprecipitated and incubated with purified LAR in vitro.
As shown in Fig. 4, RET was efficiently
and completely dephosphorylated, suggesting that RET is a preferred and
efficient substrate of LAR tyrosine phosphatase in
vitro.

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Fig. 4.
In vitro dephosphorylation of RET
by LAR. Phosphorylated RET was immunoprecipitated from the
designed cell lines, mixed with LAR protein-tyrosine-phosphatase in the
phosphatase reaction buffer and incubated for 30 min. After
dephosphorylation, the proteins were resolved by SDS-PAGE on a 10% gel
and immunoblotted with anti-phosphotyrosine antibody (upper
panel), or anti-RET antibody (lower panel).
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LAR Binds to RET-MEN2A and RET-MEN2B in Vitro--
To investigate
the mechanism of how LAR inhibits the RET-MEN2A kinase activity and
reduces the level of its autophosphorylation, we examined whether LAR
interacts with the RET proteins. Initially, cell extracts were
incubated with anti-RET polyclonal antibody in the presence of PTP
inhibitor (sodium vanadate). Proteins that bound to RET were
precipitated with protein A-Sepharose beads and analyzed by
immunoblotting with the anti-LAR antibody. However, no LAR was detected
by this approach, presumably because active LAR may rapidly
dephosphorylate and dissociate the substrate in vivo despite
the presence of phosphatase inhibitor (data not shown). To overcome
this problem, we used mutant LAR in which Cys-1552 was changed to Ser
(C1552S, abbreviated as CS). Because Cys-1552 is located in the
catalytic center of LAR, the CS mutant has no phosphatase activity
(31), whereas it should retain its affinity for the substrates. A
physical association was observed between an analogous CS mutant of the
3CH134PTP and its putative substrate; p42 MAPK (37). Equal protein
amounts (250 µg) of the lysates prepared from each transfectant were
incubated with GST, GST-LAR, and GST-LAR-CS immobilized on
glutathione-Sepharose beads and then separated by SDS-PAGE under
reducing conditions. Immunoblot analysis with anti-RET antibody
indicated that GST-LAR-CS but not GST-LAR bound to both RET-MEN2A and
RET-MEN2B, suggesting that association of wild-type LAR with mutant RET
proteins may be transient (Fig. 5).

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Fig. 5.
In vitro binding of RET protein to
LAR. The lysates from the designed cells were precipitated with 5 µg of purified GST, GST-LAR, or GST-LAR-CS fusion protein. The GST
protein complexes were washed four times with RIPA buffer, and
subjected to immunoblotting with anti-RET antibody.
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Down-regulation of RET-MEN2A-induced Signal Transduction by
LAR--
The effects of RET tyrosine kinase are mediated by the
concerted activation of several signaling pathways, including SHC, phospholipase C
, phosphatidylinositol 3-kinase, and MAPK (38). Thus,
we investigated whether all kinase-dependent events are affected to a similar degree by expression of LAR. To demonstrate these, we have analyzed some occurrences of RET-dependent
intracellular signaling in the transfectants. First, equal amounts of
the cell lysates (250 µg) were immunoprecipitated with anti-PLC
,
followed by immunoblotting with anti-phosphotyrosine antibody.
Overexpression of LAR in RET-MEN2A cells caused an ~80% reduction in
the tyrosine phosphorylation of PLC
, compared with that in
RET-MEN2A/LAR-
TP cells (Fig.
6a). However, the tyrosine
phosphorylation levels of PLC
were approximately equal in
RET-MEN2B/LAR and RET-MEN2B/LAR-
TP cells. After stripping this
membrane, it was reprobed with anti-PLC
antibody to verify equal
protein loading (Fig. 6a, lower panel).

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Fig. 6.
LAR expression impairs activation of
PLC , ERK1/2, and AKT/PKB by RET-MEN2A.
A, PLC proteins were immunoprecipitated from the lysates
(250 µg) of the designated cell lines with anti-PLC antibody and
analyzed by immunoblotting with anti-phosphotyrosine antibody
(upper panel). The blots were reprobed with anti-PLC
antibody (lower panel). B, the cell lysates (25 µg) were analyzed by immunoblotting with anti-phospho-ERK1/2
(Thr-202/Tyr-204) antibody (upper panel). The same filter
was stripped and reprobed with anti-ERK2 (lower panel).
C, the lysates (25 µg) were immunoblotted with
anti-phospho-AKT (serine 473) antibody (upper panel). The
same filter was reprobed with anti-AKT/PKB antibody (lower
panel). D, the lysates (25 µg) from the RET-MEN2A and
RET-MEN2A (Y1062F) cells were immunoblotted with anti-phospho-RET
(tyrosine 1062)-specific antibody (upper panel) or
anti-phosphotyrosine antibody (middle panel). The membrane
was reprobed with anti-RET antibody (lower panel).
E, the lysates (25 µg) of the designed cells were
immunoblotted with anti-phospho-RET (tyrosine 1062)-specific antibody
(upper panel). The membrane was reprobed with anti-RET
antibody (lower panel).
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We also tested the effect of LAR on ERK1/2 and AKT/PKB activation. Cell
lysates (25 µg) from each cell line were subjected to SDS-PAGE and
analyzed by immunoblotting with anti-phospho-ERK1/2 (Thr-202/Tyr-204)
or anti-phospho-AKT (Ser-473). As shown in Fig. 6 (b and
c, upper panels), the phosphorylation levels of
both proteins significantly decreased in RET-MEN2A/LAR cells compare with those in RET-MEN2A/LAR-
TP cells. In contrast, no marked difference was observed between the RET-MEN2B/LAR-
TP and
RET-MEN2B/LAR cells. Equal protein loading was verified by reprobing
the membrane with anti-ERK 2 and anti-AKT antibodies (Fig. 6,
b and c, lower panels).
Because ERK1/2 and AKT are known to be activated through phosphorylated
tyrosine 1062 in RET (39), we investigated its phosphorylated state
using anti-phospho-RET (tyrosine 1062)-specific antibody. To define the
specificity of this antibody, we established NIH 3T3 cells expressing
RET-MEN2A proteins in which tyrosine 1062 was replaced with
phenylalanine (designated Y1062F). As shown in Fig.
6d, anti-phospho-RET (tyrosine 1062)-specific antibody detected tyrosine phosphorylation of RET-MEN2A proteins but not RET-MEN2A (Y1062F) proteins (upper panel), although there
was no obvious difference in tyrosine phosphorylation between RET-MEN2A and RET-MEN2A (Y1062F) proteins (Fig. 6d, middle
panel). As expected, LAR expression significantly reduced
tyrosine-1062 phosphorylation in RET-MEN2A but not in RET-MEN2B (Fig.
6e, upper panel). The levels of RET protein
expression were comparable in each cell line (Fig. 6, d and
e, lower panels).
LAR Inhibits Dimerization and Phosphorylation RET-MEN2A
Proteins--
The MEN2A mutations activate RET by inducing its
ligand-independent dimerization, whereas the MEN2B mutations activate
RET without dimerization (30). Thus, we investigated whether LAR expression affects the dimerization of the RET-MEN2A proteins. When the
lysates from each transfectant were analyzed by immunoblotting under
reducing or nonreducing conditions, LAR expression significantly decreased the dimerization and phosphorylation of RET-MEN2A proteins, demonstrating that their decreased kinase activity correlates with
their decreased dimer formation (Fig.
7).

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Fig. 7.
Effect of LAR on dimerization of RET-MEN2A
proteins. Total lysates (25 µg) prepared from the designated
cells were separated on SDS-5% polyacrylamide gels under reducing or
nonreducing conditions and analyzed by immunoblotting with
anti-phosphotyrosine antibody (upper panel). The same blot
was reprobed with anti-RET antibodies (lower panel). 350-kDa
RET dimers and 175- and 155-kDa monomers are shown.
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LAR Inhibits RET-MEN2A-mediated Transformation and Soft Agar
Growth--
We examined whether LAR can reverse cell transformation by
RET-MEN2A. RET-MEN2A/LAR-
TP cells were rounded, refractile, and highly transformed, whereas the RET-MEN2A/LAR cells resumed a more
flattened morphology (Fig. 8,
a-c). Morphological reversion of RET-MEN2B/LAR
cells was not observed (Fig. 8, d and e). These findings demonstrate that transformation by RET-MEN2A, but not by
RET-MEN2B, can be reversed partially by LAR overexpression.

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Fig. 8.
Colony formation of the transfectants in soft
agar. a-e, morphological appearance of transfected NIH
3T3 fibroblast subclones. (a) NIH 3T3 cells, (b)
RET-MEN2A/LAR- TP cells, (c) RET-MEN2A/LAR cells,
(d) RET-MEN2B/LAR- TP cells, (e) RET-MEN2B/LAR cells.
Partial morphological reversion of RET-MEN2A/LAR cells was observed.
f-j, soft agar growth of transfectants. Cells were seed at
a density of 50,000 cells/ml in semi-solid agar, and then photographed
after 2 weeks. (f) NIH 3T3 cells, (g)
RET-MEN2A/LAR- TP cells, (h) RET-MEN2A/LAR cells,
(i) RET-MEN2B/LAR- TP cells, (j) RET-MEN2B/LAR
cells. RET-MEN2A/LAR cells formed small colonies compare with
RET-MEN2A/LAR- TP cells. k-o, colonies in soft agar
stained with MTT. (k) NIH 3T3 cells, (l)
RET-MEN2A/LAR- TP cells, (m) RET-MEN2A/LAR cells,
(n) RET-MEN2B/LAR- TP cells, (o) RET-MEN2B/LAR
cells. Colonies were photographed after 2 weeks. Similar results were
obtained in three independent experiments.
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To test possible biological consequences of LAR expression, we
performed soft agar colony formation assay. RET-MEN2A/LAR-
TP cells
displayed typical large colony formation in soft agar (Fig. 8,
g and l) similar to that of RET-MEN2A cells (data
not shown). Conversely, a small number of colonies of
RET-MEN2A/LAR cells were observed under the same conditions,
demonstrating that cells expressing LAR, but not LAR-
TP, became more
anchorage-dependent. Thus, the effect of LAR expression on
RET-MEN2A receptor phosphorylation seems to become translated into the
effect on cell growth in soft agar (Table
I). The LAR expression did not
significantly affect the colony formation of RET-MEN2B cells, although
their colony size was smaller than that of the RET-MEN2A cells (Table
I, Fig. 8).
Finally, to examine the ability of the transfectants to form tumors, we
inoculated NIH 3T3, RET-MEN2A/LAR, and RET-MEN2A/LAR-
TP cells
subcutaneously into scid mice. At 7 days after injection, small tumors
were formed in the mice injected with RET-MEN2A/LAR-
TP cells.
Sixteen days later, both RET-MEN2A/LAR and RET-MEN2A/LAR-
TP cells
formed palpable tumors, although RET-MEN2A/LAR-
TP cells yielded
rapidly growing tumors than RET-MEN2A/LAR cells (Table I). These
results provide evidence that RET-MEN2A/LAR cells are less oncogenic
than RET-MEN2A/LAR-
TP cells.
 |
DISCUSSION |
In vitro, it has been proved that intracellular PTPs
such as PTP1B and low molecular weight PTP, and transmembrane PTPs such as LAR, RPTP
, and PTPT
, have substantial activity toward
receptor protein-tyrosine kinases. LAR, RPTP
, and PTPT
have
been shown to regulate insulin signaling via effects on the activated
receptor (40-42). It has been postulated that LAR functions as an
inhibitor of receptor protein-tyrosine kinase signaling in
vivo by direct dephosphorylation of certain growth factor
receptors (1), and that LAR could be a tumor suppressor because of its
PTP activity, tissue distribution, and chromosome location (43, 44).
Because the LAR gene is localized to human chromosome
1p32, which is frequently deleted in tumors developed in MEN2 (26, 27),
it was interesting to investigate whether LAR modulates the functions
of RET-MEN2 proteins. Thus, we cotransfected LAR or
phosphatase domain deletion mutant LAR
(LAR-
TP) with RET-MEN2A (Cys-634
Tyr) or RET-MEN2B (Met-918
Thr) into NIH 3T3 cells,
and demonstrated that the constitutive tyrosine autophosphorylation of
RET-MEN2A but not RET-MEN2B became much reduced by LAR expression. The
in vitro RET-MEN2A tyrosine kinase activity was also
markedly decreased (~70-80%). The observations that high levels of
LAR protein expression significantly reduced proliferation of
RET-MEN2A/LAR cells in vitro as well as tumor growth
in vivo clearly emphasize potentially important roles for
LAR in the regulation of neoplastic cell proliferation and
tumorigenicity. This is the first report, to our knowledge, documenting
suppression of biological activities of the RET-MEN2A protein by
LAR.
To elucidate the mechanism by which LAR suppresses tumor growth
of cells expressing RET-MEN2A, it was essential to investigate the
association of LAR with RET-MEN2A. The significant reduction in the
proliferation rates of RET-MEN2A/LAR cells probably depends on the LAR
function, because cell proliferation is regulated by the
phosphotyrosine state in these cells. One possibility is that RET-MEN2A
itself is a target substrate for LAR. It has been reported that LAR was
able to dephosphorylate autophosphorylated epidermal growth factor
receptor in vitro (45) and that LAR and the insulin receptor
was able to be coimmunoprecipitated after chemical cross-linking on the
surface of rat hematoma cells (46). In this report, we have shown that
the level of RET-MEN2A tyrosine phosphorylation was significantly
reduced by overexpression of LAR although the level of RET expression
was unchanged. Like other PTPs, LAR contains a conserved 11-amino acid
motif ((I/V)HCXAGXXR(S/T/G)) within the
catalytic domain, which constitutes the active site of the phosphatase
(43). The invariant cysteine within this motif is absolutely required
for catalysis. N-terminal to this region is a conserved aspartic acid
residue (47). A mutation of the cysteine or aspartic acid residue to
serine or alanine, respectively, resulted in inactivation of PTPs but
the mutant PTPs retained their ability to bind
tyrosine-phosphorylated substrates and have successfully been used for
the identification of candidate PTP substrates (48). Our results
demonstrated that the LAR-CS mutant protein interacts directly with
RET-MEN2A as well as RET-MEN2B using the glutathione S-transferase LAR-CS fusion proteins, suggesting that the
normal interaction between LAR and RET is likely transient and that RET may be quickly dissociated from LAR after dephosphorylation. Thus, this
transient interaction may not have been detected by
coimmunoprecipitation even in the vanadate-treated cells (data not shown).
As we reported previously, specific phosphotyrosine residues in RET
were required for the mitogenic and transforming activity (29, 49, 50).
Upon RET activation, the autophosphorylated tyrosine residues, Tyr-905,
Tyr-1015, Tyr-1062, and Tyr-1096, act as docking sites for the signal
transduction effectors, Grb7/Grb10, PLC
, SHC/Enigma, and Grb2,
respectively (29, 51-56). This is consistent with the data indicating
that several signaling pathways, including phosphatidylinositol
3-kinase (PI3K), RAS/ERK, PLC
, and c-Jun N-terminal kinase
(JNK) pathways are activated by RET. Among these, activation of
the RAS/ERK, PI3K/AKT, and JNK signaling pathways depended on
phosphorylation of tyrosine 1062 (39, 57). In addition, we demonstrated
that a mutation of tyrosine 1062 markedly decreased the transforming
activity of both RET-MEN2A and RET-MEN2B (29, 49, 50). The recruitment
of the GRB2·SOS complex to SHC bound to tyrosine 1062 was
involved in the activation of RAS/ERK signaling pathway, whereas
association of SHC with GRB2-associated binder 1 appeared to be
essential for the activation of PI3K pathway (39, 58). If RET is the
primary target of LAR action, it is expected that the
LAR-dependent reduction in its tyrosine kinase activity
would be paralleled by reduction in substrate phosphorylation.
Consistent with this view, we showed the decreased levels of
phosphorylation of AKT, ERK1/2, and PLC
in the RET-MEN2A/LAR cells.
Furthermore, immunoblotting with anti-phospho-RET (tyrosine
1062)-specific antibody revealed the decreased tyrosine-phosphorylation of RET-MEN2A, suggesting that tyrosine 1062, a binding site for SHC
adaptor protein, is one of the sites affected by LAR action. Similarly,
decreased phosphorylation of PLC
in the RET-MEN2A/LAR cells
suggested that tyrosine 1015 is another target site for LAR.
In this study, another interesting finding is that LAR neither
reduces the phosphorylation and the kinase activity of RET-MEN2B nor
decreases the tumor growth of RET-MEN2B cells. As we and others reported previously, RET-MEN2A is dimerized through the formation of
disulfide bonds between unpaired cysteine residues in the extracellular domains of two molecules, and the levels of its autophosphorylation and
tyrosine kinase activity are elevated in parallel (59, 60). On the
other hand, the RET-MEN2B mutation does not provoke
constitutive dimerization of the RET protein, and activation of
RET-MEN2B results from an altered conformation of the kinase domain
that appears to lead to altered substrate specificity (30, 49, 58, 60, 61). We therefore hypothesize that the dimerization state of RET-MEN2A
may be preferable for LAR action. In addition, we showed that RET
dimerization and phosphorylation were significantly reduced in
RET-MEN2A/LAR cells as compared with those in RET-MEN2A/LAR-
TP cells. This result suggests that LAR expression on the cell surface may interfere with the RET dimerization, resulting in the decrease in
its oncogenic activity. In this respect, it is interesting to note that
we recently succeeded in generating transgenic mice expressing the
RET-MEN2A proteins that showed tissue-specific tumor development. In
transgenic mice, no tumor development was observed in several tissues
despite the high levels of expression of the RET-MEN2A proteins. We
found that RET-MEN2A dimerization was undetectable in these tissues,
suggesting that certain cell surface proteins could interfere with the
RET dimerization (62). Thus, it is possible that LAR expression plays
an inhibitory role in the tumor development in vivo,
affecting the RET-MEN2A dimerization as well as its kinase activity,
although further analyses are necessary to elucidate the exact
mechanism of RET-MEN2A inactivation by LAR.
In summary, this study indicated that LAR specifically suppresses the
biological activities of RET-MEN2A by reducing its kinase activity and
down-regulating activation of its downstream signaling pathways. Thus,
the deletion of the LAR gene that is localized to
human chromosome 1p32 may affect biological behaviors of medullary thyroid carcinoma and pheochromocytoma developed in MEN2A patients.