Effects of Overexpression of PTP36, a Putative Protein Tyrosine
Phosphatase, on Cell Adhesion, Cell Growth, and Cytoskeletons in
HeLa Cells*
Masato
Ogata
,
Tsuyoshi
Takada,
Yoshiko
Mori,
Masatsugu
Oh-hora,
Yohzo
Uchida,
Atsushi
Kosugi§,
Kensuke
Miyake¶, and
Toshiyuki
Hamaoka
From the Department of Oncology, Biomedical Research Center, Osaka
University Medical School, Suita, Osaka 565-0871, the
§ School of Allied Health Science, Faculty of Medicine,
Osaka University, Suita, Osaka 565-0871, and the ¶ Department of
Immunology, Saga Medical School, Nabeshima, Saga 849, Japan
 |
ABSTRACT |
Non-receptor-type putative protein tyrosine
phosphatase-36 (PTP36), also known as PTPD2/Pez, possesses a domain
homologous to the N-terminal half of band 4.1 protein. To gain insight
into the biological function of PTP36, we established a HeLa cell line, HtTA/P36-9, in which the overexpression of PTP36 was inducible. PTP36
expressed in HeLa cells was enriched in the cytoskeleton near the
plasma membrane. There was little endogenous PTP36 detectable in
uninduced HtTA/P36-9 cells or in the parental HeLa cells. Upon induction of PTP36 overexpression, HtTA/P36-9 cells spread less well,
grew more slowly, and adhered to the extracellular matrix proteins less
well than uninduced cells. Moreover, decreases in the actin stress
fibers and the number of focal adhesions were observed. The tyrosine
phosphorylation of the focal adhesion kinase induced by
lysophosphatidic acid was suppressed in the HtTA/P36-9 cells
overexpressing PTP36. These results indicate that PTP36 affects
cytoskeletons, cell adhesion, and cell growth, thus suggesting that
PTP36 is involved in their regulatory processes.
 |
INTRODUCTION |
Among the diverse functions of protein tyrosine phosphatases
(PTPs),1 their roles in the
regulation of cell adhesion and motility are now emerging (1). It has
been reported that CD45, a transmembrane PTP (2), and YopH, a bacterial
PTP (3), negatively regulate the integrin-mediated cell adhesion or its
signal transduction. PTP1B, a cytoplasmic PTP, has been reported to
affect cadherin-actin linkage (4) and integrin-mediated signaling
pathways (5).
Recently, we have cloned a murine putative PTP, PTP36 (6). A human
homologue was also cloned and called PTPD2/Pez (7, 8). PTP36 is
expressed in various tissues and cell lines including 3T3 fibroblasts.
Besides a putative PTP domain, PTP36 has a band 4.1 homology domain
that has been found in various cytoskeletal proteins. The band 4.1 homology domains are responsible for the targeting of such cytoskeletal
proteins to the membrane-cytoskeleton interfaces. So far, five
mammalian PTPs with the band 4.1 homology domain have been reported.
These include PTPH1 (9), PTPMEG (10), PTPD1/PTP-RL10/rPTP2E (7, 11,
12), PTP36/PTPD2/Pez (6-8), and PTP-BAS/hPTP1E/PTPL1/FAP-1
(13-15).
The presence of the band 4.1 homology domain might imply the
involvement of these PTPs in the regulation of cytoskeletons and cell
adhesion. It was reported that, in COS-7 cells, overexpression of
PTPMEG inhibited cell proliferation, reduced the saturation density,
and blocked cell growth in soft agar (16). Therefore, it is possible
that PTPMEG is involved in the signal transduction of the cell adhesion
that regulates the cell growth. There is no evidence, however, that
PTPMEG has any effect on the cell adhesion signaling pathways.
To study the biological functions of PTP36, we established a HeLa cell
line in which the overexpression of PTP36 was inducible. PTP36
expressed in HeLa cells was enriched in the cytoskeleton near the
plasma membrane. When the overexpression of PTP36 was induced, we
observed morphological change (17), reduced cell growth and cell
adhesion, decreases in actin stress fibers and focal adhesions, and
reduced phosphorylation of focal adhesion kinase (FAK) induced by
lysophosphatidic acid (LPA). Our results suggest that PTP36 is involved
in the regulation of cytoskeletons, cell adhesion, and cell growth.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Enzymes were purchased from Life Technologies,
Inc., New England Biolabs (Beverly, MA), Promega (Madison, WI), and
Stratagene (La Jolla, CA). Anti-actin (AC-40, mouse IgG2a),
anti-vimentin (V9, mouse IgG1), anti-vinculin (hVIN-1, mouse IgG1),
phalloidin-tetramethylrhodamine isothiocyanate (P1951),
doxycycline (D-9891), puromycin (P-7255), and lysophosphatidic acid
(L-7260) were purchased from Sigma. Anti-FAK (06-367, rabbit polyclonal
IgG), anti-
-tubulin (DM1A, mouse IgG1), and PY20 (mouse IgG2b) were
from Upstate Biotechnology Inc. (Lake Placid, NY), Cedarlane
Laboratories (Westbury, NY), and Transduction Laboratories (Lexington,
KY), respectively. The rat anti-PTP36 monoclonal antibody, 21-4, was
established in our laboratory using a glutathione
S-transferase fusion protein containing amino acids 399-738
of the PTP36 polypeptide (GST-P36/399-738) as an antigen.
Construction of Expression Plasmids and Transfection--
The
expression construct pEFP36HA, which encoded influenza virus
hemagglutinin (HA)-tagged PTP36 (PTP36HA) was created as follows. The
stop codon of the PTP36 cDNA was replaced by a sequence encoding a
tandem repeat of the HA epitope. The cDNA encoding HA-tagged PTP36
was inserted into the ClaI/XbaI site of the
mFas-Fc plasmid (18). Alternatively, the cDNA was subcloned
into pUHD10-3, which contained a tet operator
sequence (19). The resulting plasmid, pTREP36HA, and pNYpgk-pc, a
plasmid possessing a puromycin resistance gene, were introduced into
HeLa cells expressing a tetracycline-controlled transactivator (HtTA)
using Tfx-20TM reagent (Promega, Madison, WI). Positive
clones were selected in DMEM containing 10% fetal calf serum, 0.4 µg/ml puromycin, 0.1 mg/ml geneticin, and 5 ng/ml doxycycline.
Immunoblotting Analysis and Immunoprecipitation--
After
SDS-polyacrylamide gel electrophoresis, proteins were electroblotted
onto polyvinylidene fluoride membranes. The membranes were blocked in
T-TBS (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.1% Tween 20) containing 2% casein for 1 h
at room temperature or overnight at 4 °C and incubated with the
primary antibodies. To detect the primary antibodies, horseradish
peroxidase-conjugated sheep anti-mouse IgG, or protein A, and the ECL
system (Amersham Pharmacia Biotech) were used. To detect
tyrosine-phosphorylated proteins using PY20, the low salt buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween
20) was used for incubation and washing, and 5% bovine serum albumin
(BSA) was used for the blocking.
For immunoprecipitation, cells were lysed in 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF,
and 5 mM sodium pyrophosphate. The lysate was centrifuged
at 15,000 × g for 10 min at 4 °C, and the
supernatant was precleared by the addition of 10 µl (pellet volume)
of protein A-Sepharose (Amersham Pharmacia Biotech) followed by
incubation at 4 °C for 30 min. Then, the lysate was incubated with
10 µl (pellet volume) of protein A-Sepharose preloaded with 2 µg of
the rabbit anti-FAK antibody for 1 h.
Immunofluorescence Study--
Immunofluorescence microscopy was
performed as described (20). Briefly, cells on a 34-mm tissue culture
dish were washed with the cytoskeleton buffer (100 mM MES,
pH 6.5, 1 mM MgCl2, 1 mM EGTA, and
4% polyethylene glycol 8000) and fixed with 3.7% formaldehyde in the
cytoskeleton buffer for 10 min at room temperature. After
permeabilization by 0.1% Triton X-100, cells were blocked by 10 mg/ml
goat globulin in phosphate-buffered saline (PBS) and incubated with the
primary antibodies in PBS containing goat globulin. For the detection
of the primary antibodies, 25 µg/ml fluorescein isothiocyanate-conjugated goat anti-mouse IgG (catalog no. 115-095-068, Jackson ImmunoResearch) was used. The polymerized actin was visualized using 0.2 µg/ml phalloidin-tetramethylrhodamine isothiocyanate in
PBS.
Cell Culture and Cell Adhesion Analysis--
HtTA/P36-9 and
HtTA/P36-18 cells were maintained in DMEM containing 10% fetal calf
serum, 0.3 µg/ml puromycin, 0.1 mg/ml geneticin, and 5 ng/ml
doxycycline. Overexpression of PTP36 was induced by incubating the
cells in the culture medium without doxycycline. After 5 days, cells
were collected by trypsinization and incubated in fresh culture medium
overnight. Then cells were detached by incubating them in PBS
containing 5 mM EDTA and 0.1% BSA. After washing, cells
were used for the adhesion assay as described (21). Briefly, cells were
labeled by incubation in serum-free DMEM containing 5 µM
5-carboxylfluorescein diacethoxymethyl ester (BCECF)-AM (catalog no. FM-0011-50, Dojin) at 37 °C for 1 h, followed by washing. The labeled cells (1.5 × 104) in 100 µl of DMEM
containing 0.1% BSA were incubated in 96-well plates precoated with
various extracellular matrix proteins. After a 20-min incubation at
37 °C, each well was filled with DMEM, and the plate was centrifuged
upside down at 100 × g for 10 s. Cells attached
to the plate were lysed in PBS containing 1% Nonidet P-40, and the
fluorescence intensity (530 nm) was measured (excitation wavelength at
490 nm). The wells of 96-well microtiter plates (catalog no. 25860, Corning Glass) were coated with various concentrations of extracellular
matrix proteins in 50 µl of PBS at 37 °C for 1 h. The total
protein concentrations were adjusted to 10 µg/ml by the addition of
BSA. The plates were blocked with PBS containing 0.5% heat-treated
(80 °C for 15 min) BSA at 37 °C for 1 h.
Cytoskeletal Fractionation--
The cytoskeletal fractions were
prepared as described (22). Cells were lysed in the cytoskeleton
stabilizing buffer (10 mM PIPES, pH 6.8, 250 mM
sucrose, 3 mM MgCl2, 120 mM KCl, 1 mM EGTA) containing 0.15% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, 25 mM NaF, and 5 mM sodium pyrophosphate for 5 min at 4 °C (total
lysate). The total lysate was centrifuged at 14,000 × g for 10 min at 4 °C yielding the pellet, which contained the polymerized actin and the intermediate filaments, and the supernatant, which contained the depolymerized actin and tubulin.
 |
RESULTS |
Conditional Overexpression of PTP36 in HeLa Cells--
To
investigate the biological functions of PTP36, we established a cell
line in which the expression level of PTP36 was regulatable. A HeLa
cell line, HtTA, that expressed the tetracycline-controlled transactivator was transfected with an inducible construct containing the cDNA of PTP36 tagged with the HA epitope. Two cell lines, HtTA/P36-9 and HtTA/P36-18, were established, and similar results were
obtained with them. HtTA/P36-9 cells were maintained in medium containing 5 ng/ml doxycycline and expressed little PTP36 protein (Fig.
1A,
lane 1). Overexpression was induced by removing doxycycline from the culture. PTP36 was detectable after overnight culture and
reached its plateau after 3 days (Fig. 1A, lane
2). In the parental HtTA cells, PTP36 was undetectable in the
presence or absence of doxycycline (data not shown).

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Fig. 1.
Conditional overexpression and subcellular
localization of PTP36 in HeLa cells. A, induction of
PTP36 overexpression in HtTA/P36-9. HtTA/P36-9 cells were cultured in
the presence (lane 1) or absence (lane 2) of 5 ng/ml doxycycline (Dox) for 3 days, lysed, and analyzed by
immunoblotting using an anti-PTP36 monoclonal antibody (Ab),
21-4. B, cytoskeletal fractionation of HeLa cells. HeLa
cells transiently transfected with a plasmid encoding PTP36 were lysed
in the cytoskeleton stabilizing buffer at 4 °C and centrifuged at
14,000 × g yielding the pellet (P) and the
supernatant (S). The equal cell equivalent of each fraction
was analyzed. Actin-based microfilaments and intermediate filaments
containing vimentin were recovered in the cytoskeletal P fraction,
whereas actin monomers were recovered in the cytosolic S fractions.
PTP36, actin, and vimentin in each fraction were detected by
immunoblotting analysis.
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|
Then the subcellular distribution of PTP36 in HeLa cells was analyzed.
PTP36 was found in the cytoskeletal fraction (P) containing actin and
vimentin (Fig. 1B, lane 1). The
immunofluorescence study revealed that PTP36 and PTP36 fused to green
fluorescent protein (PTP36-GFP) were enriched in the region near the
cytoplasmic membrane (Fig. 2,
a-c and e-g). Thus, we
concluded that PTP36 in HeLa cells was enriched in the cytoskeleton
near the plasma membrane.

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Fig. 2.
Confocal microscopy analysis of HeLa cells
expressing PTP36 and PTP36-GFP. HeLa cells transiently transfected
with the plasmid encoding PTP36HA (a-c) or PTP36-GFP
(e-g) were analyzed 2 days after the transfection. PTP36HA
was detected by the anti-HA monoclonal antibody (12CA5),
followed by fluorescein isothiocyanate-conjugated goat anti-mouse
IgG. Mock transfection (d) or cells transfected with the GFP
plasmid (h) are shown as controls. Cells were imaged by
confocal microscopy. Images of the bottom (a and
e), middle (b and f), and
top planes (c and g) are shown.
|
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Morphology and Growth of HtTA/P36-9 Cells Induced to Overexpress
PTP36--
HtTA/P36-9 cells and the parental HeLa cells (HtTA) were
cultured in the presence (off, the noninducing condition) or
absence (on, the inducing condition) of doxycycline. The
morphology of the uninduced HtTA/P36-9 cells did not differ
significantly from that of the parental HtTA cells (Fig.
3, upper panels). After 3 days
of induction, however, HtTA/P36-9 cells became more rounded and spread
less well than the uninduced cells (Fig. 3, lower panels). Similar results were obtained with HtTA/P36-18 cells (data not shown).
No change in the morphology was detectable when the parental HtTA cells
were cultured with or without doxycycline. In addition, HtTA/P36-9
cells (Fig. 4, A and
B) and HtTA/P36-18 cells (data not shown) induced to express
PTP36 grew slower than the uninduced cells. The growth inhibition was
mild but very reproducible. In six independent experiments, the cell
number was reduced to 66.3 ± 7.3% (50.5-72.9%) of that of
control (uninduced) cells. No difference in the viability was observed
using the dye exclusion method. Doxycycline revealed no effect on the
growth of the parental HtTA cells (Fig. 4A).

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Fig. 3.
Morphology of HtTA/P36-9 cells induced to
overexpress PTP36. HtTA/P36-9 cells or parental HtTA cells were
cultured in the presence (off) or absence (on) of
doxycycline for 2-4 days as indicated.
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Fig. 4.
Growth of HtTA/P36-9 cells induced to
overexpress PTP36. A, 5 × 104
HtTA/P36-9 cells or parental HtTA cells were grown in a 6-cm tissue
culture dish for 6 days in the presence (off) or absence
(on) of doxycycline. B, HtTA/P36-9 cells were
cultured in 96-well microtiter plates in the presence (off, open
circles) or absence (on, closed circles) of doxycycline
for 6 days. Then the proliferation was measured by
[3H]thymidine incorporation. Each well of triplicate
wells was pulsed with 20 kBq of [3H]thymidine for 4 h. The data shown are the mean values ± S.E.
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Effects of PTP36 Overexpression on the Adhesion of HtTA/P36-9 Cells
to Extracellular Matrix Proteins--
The HtTA/P36-9 cells induced to
overexpress PTP36 seemed to attach loosely to the culture dish.
Therefore, we investigated the adhesiveness of HtTA/P36-9 cells to
various extracellular matrix proteins. A representative result from
four independent experiments is shown in Fig.
5. The HtTA/P36-9 cells induced to overexpress PTP36 adhered to type IV collagen (Fig. 5A) and
laminin (Fig. 5B) less well than uninduced cells. No
difference was observed when the parental HtTA cells were cultured in
the presence or absence of doxycycline. As shown in Fig. 5C,
the adhesion of HtTA/P36-9 cells to these matrix proteins was blocked
almost completely by an anti-
1 integrin antibody, SG/19
(23). The expression levels of
1 integrin subunits on
the surface of the induced and uninduced HtTA/P36-9 cells were almost
identical (Fig. 5D).

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Fig. 5.
Adhesion of HtTA/P36-9 cells induced to
overexpress PTP36 to extracellular matrix proteins. A,
adhesion to type IV collagen. HtTA/P36-9 cells cultured in the presence
(off) or absence (on) of doxycycline were
incubated in a 96-well plate precoated with various concentrations of
type IV collagen. After a 20-min incubation at 37 °C, cells attached
to the plate were measured as described under "Experimental
Procedures." The data shown are the mean values ± S.E.
B, adhesion to laminin was measured as described in
A. C, effects of anti- 1 integrin
antibody (Ab) on the adhesion. During the assay of adhesion
to type IV collagen (0.8 µg/ml)-coated wells (left panel)
or laminin (0.63 µg/ml)-coated wells (right panel), cells
were incubated with no antibody, 4 µg/ml mouse IgG, or an
anti- 1 integrin antibody (SG/19). D,
immunofluorescence staining of 1 integrin subunits. The
HtTA/P36-9 cells cultured in the presence (off) or absence
(on) of doxycycline were stained with SG/19 followed by
fluorescein isothiocyanate-labeled goat anti-mouse IgG and analyzed by
fluorescence-activated cell sorter. The sample stained without the
primary antibody was shown as a negative control (c).
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Cytoskeletons and Focal Adhesions in HtTA/P36-9 Cells Induced to
Overexpress PTP36--
The cytoskeletons could have profound effects
on the cell shape and the cell adhesion. Thus, we examined the
cytoskeletons in the HtTA/P36-9 cells induced (on) or not
induced (off) to overexpress PTP36. There was no difference
found in the microtubules and the intermediate filaments containing
vimentin (Fig. 6, i-l). In
contrast, the actin stress fibers were greatly decreased in the induced HtTA/P36-9 cells (Fig. 6b). Furthermore, the number of focal
adhesions was reduced (Fig. 6f). In the parental HtTA cells,
doxycycline revealed little effect on the actin stress fibers and focal
adhesions (Fig. 6, c, d, g, and h).

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Fig. 6.
The cytoskeletons and focal adhesions in
HtTA/P36-9 cells induced to overexpress PTP36. HtTA/P36-9 cells or
parental HtTA cells were cultured in the presence (off) or
absence (on) of doxycycline for 6 days, fed with fresh
culture medium, and fixed after 20 min. Cells were permeabilized, and
polymerized actin (a-d), focal adhesions
(e-h), microtubules (i and
j), and vimentin (k and l) were
visualized using phalloidin-tetramethylrhodamine isothiocyanate, hVIN-1
(anti-vinculin), DM1A, and V9, respectively, in combination with the
appropriate second reagents.
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Effects of PTP36 Overexpression on the Phosphorylation of
FAK--
Several sources of stimulation are known to induce the
formation of focal adhesions or actin stress fibers. The
integrin-mediated adhesion of cells to extracellular matrix proteins
induces the formation of focal adhesions and the phosphorylation of
various proteins including FAK. It is also known that LPA activates the small GTPase, Rho, to trigger the formation of focal adhesions and
actin stress fibers as well as the phosphorylation of FAK. Defects in
these signal transduction cascades might result in the reduction of
actin stress fibers and focal adhesions. Thus, we investigated the
phosphorylation of FAK induced by the integrin engagement or LPA.
Induction of PTP36 overexpression had little effect on the tyrosine
phosphorylation of FAK induced by cell adhesion to fibronectin (Fig.
7A, lanes 2 and
4). On the contrary, the LPA-induced phosphorylation of FAK
was suppressed in the cells induced to overexpress PTP36 (Fig.
7B, lanes 2 and 4).

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Fig. 7.
Phosphorylation of FAK in HtTA/P36-9 cells
induced to overexpress PTP36. A, phosphorylation of FAK
induced by integrin engagement. After a 6-day culture in the presence
(off, lanes 1 and 2) or absence
(on, lanes 3 and 4) of doxycycline,
HtTA/P36-9 cells were harvested (lanes 1 and 3)
and replated on a fibronectin-coated dish (10 µg/ml) for 60 min
(lanes 2 and 4). Cell lysates were prepared, and
FAK was immunoprecipitated (IP) by a rabbit anti-FAK
polyclonal antibody. The phosphorylation level of FAK was detected by
the immunoblotting analysis using the anti-phosphotyrosine antibody
PY20 (p-Y). B, phosphorylation of FAK induced by
LPA. HtTA/P36-9 cells cultured in the presence (off,
lanes 1 and 2) or absence (on,
lanes 3 and 4) of doxycycline were serum-starved
for 2 h and then stimulated with (lanes 2 and
4) or without (lanes 1 and 3) 10 µg/ml LPA for 20 min (left panel). The means ± S.E. of
the relative phosphorylation level of FAK from two separate experiments
are shown (right panel).
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 |
DISCUSSION |
The presence of a band 4.1 domain in the band 4.1 superfamily PTPs
may suggest their involvement in the regulation of cytoskeletons and
cell adhesion. However, little was known about their roles. In this
study, we demonstrate that the overexpression of PTP36 has effects on
cell growth, cell adhesion, cytoskeletons, and LPA-induced
phosphorylation of FAK in the HeLa cells. The expression level of PTP36
in the induced HtTA/P36-9 cells was only 2-5-fold higher than the
level of endogenous PTP36 in the 3T3 cells. In addition, no increase in
the total phosphatase activity was detectable (data not shown). These
results might support the possibility that the effects of PTP36
observed are not artifacts of overexpression.
In HeLa cells, PTP36 is enriched in the membrane-associated
cytoskeletal fraction. This distribution pattern is similar to that of
PTPMEG, which localizes primarily to the membrane and cytoskeletal
fractions (24).
Although we have found various effects of overexpression, this does not
necessarily mean that multiple target molecules of PTP36 are present.
Focal adhesions are the sites at which cells adhere to the
extracellular matrix via integrin family receptors. At their
cytoplasmic face, focal adhesions are linked to actin stress fibers. In
addition, various signal transducing molecules have been identified in
the focal adhesions (25, 26). Because focal adhesions are involved in
both cell adhesion and signal transductions, their changes might
affect cell morphology, adhesiveness to extracellular matrix
proteins, and cell growth. The target molecule of PTP36 remains to
be determined.
Stimulation through integrins, growth factor receptors, and G
protein-coupled receptors leads to the formation of focal adhesions and
stress fibers (25-27). The tyrosine phosphorylation of proteins including FAK seems to be the essential step for these responses (25,
26, 28-31). The overexpression of PTP36 inhibits the phosphorylation of FAK induced by LPA. LPA stimulates a G protein-coupled receptor and
induces the formation of focal adhesions and stress fibers through the
activation of Rho, a small GTPase (32). Thus, it is possible that the
target molecule of PTP36 might be in the signaling cascade involving
Rho. On the contrary, the overexpression of PTP36 reveals no inhibitory
effect on the phosphorylation of FAK induced by the cell adhesion to
fibronectin. It was reported that the early, but not late,
phosphorylation of FAK induced by integrin engagement occurred
independently of Rho family GTPases (31). This result suggests that
PTP36 might not inhibit the early integrin signals leading to the FAK
phosphorylation. Clearly, further study is necessary to elucidate the
mechanism of the inhibition.
So far, there is no evidence that PTP36 is playing the role of a PTP in
HeLa cells. Among the band 4.1 superfamily PTPs, PTP36/PTPD2/Pez (6-8)
and PTPD1/PTP-RL10/rPTP2E (7, 11, 12) share a high sequence similarity.
The amino acid sequence in the PTP domain of PTPD1/PTP-RL10/rPTP2E has
a substitution of glutamic acid for the conserved aspartic acid. This
aspartic acid acts as a general acid in the catalytic reaction (33).
This substitution was reported to reduce the
kcat of a PTP about 600-fold (34). There is a substitution of histidine for the aspartic acid in murine PTP36 but not
in human PTPD2/Pez. To test the enzymatic activity, PTP domains of
PTP36 and PTPD2/Pez were expressed as glutathione
S-transferase fusion proteins, and the catalytic activities
were measured using p-nitrophenyl phosphate as a substrate.
No significant, or very marginal, activity was detected for PTP36 and
PTPD2/Pez, respectively.2
These observations raise the possibility that some members of band 4.1 superfamily PTP, including PTP36, might have noncatalytic functions. In
line with this notion, it was reported that the catalytic activity of
PTPMEG, a member of band 4.1 superfamily PTP, was not essential for the
functions of PTPMEG (10, 16, 24).
Nonetheless, the PTP domain might be important for the function of
PTP36. A deletion mutant of PTP36 (lacking amino acids 803-1184) was
expressed in HeLa cells. This mutant possessed the band 4.1 domain but
not the phosphatase domain and revealed little inhibitory effect on the
adhesion of the cells to
laminin.2 It was reported
that the mutation of the invariant aspartic acid converted PTP into a
catalytically inactive form, which could make a stable complex with
tyrosine-phosphorylated substrates (34). It is possible that PTP36
might interact with signaling molecules through its multiple domains,
including PTP and the band 4.1 domains, and function as an adaptor molecule.
 |
ACKNOWLEDGEMENT |
We thank Drs. H. Bujard and S. Takeda for
kindly providing pUHD10-3 plasmid and for helpful information.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science, and
Culture, Japan and by the Japan Research Foundation for Clinical
Pharmacology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Biomedical Research
Center, Osaka University Medical School C6, 2-2 Yamadaoka, Suita, Osaka
565-0871, Japan. Tel.: 81-6-6879-3982; Fax: 81-6-6879-3989; E-mail:
mogata{at}ongene.med.osaka-u.ac.jp.
2
M. Ogata and T. Hamaoka, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
PTP, protein
tyrosine phosphatase;
FAK, focal adhesion kinase;
LPA, lysophosphatidic
acid;
HA, hemagglutinin;
HtTA, HeLa cells expressing a
tetracycline-controlled transactivator;
DMEM, Dulbecco's modified
Eagle's medium;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
GFP, green fluorescent protein;
MES, 4-morpholineethanesulfonic acid;
PIPES, 1,4-piperazinediethanesulfonic
acid;
PTPMEG, cytosolic megakaryocyte protein tyrosine
phosphatase.
 |
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