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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 539-549
The Receptor Protein Tyrosine Phosphatase, PTP-RO, Is Upregulated
During Megakaryocyte Differentiation and Is Associated With the c-Kit
Receptor
By
Yoshitaka Taniguchi,
Roanna London,
Karin Schinkmann,
Shuxian Jiang, and
Hava Avraham
From the Division of Experimental Medicine, Beth Israel Deaconess
Medical Center, Harvard Institutes of Medicine, Boston, MA.
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ABSTRACT |
We have recently isolated a cDNA encoding a novel human
receptor-type tyrosine phosphatase, termed PTP-RO (for a protein
tyrosine phosphatase receptor omicron), from 5-fluorouracil-treated
murine bone marrow cells. PTP-RO is a human homologue of murine PTP and is related to the homotypically adhering  and µ receptor-type tyrosine phosphatases. PTP-RO is expressed in human megakaryocytic cell
lines, primary bone marrow megakaryocytes, and stem cells. PTP-RO mRNA
and protein expression are upregulated upon phorbol 12-myristate
13-acetate (PMA) treatment of the megakaryocytic cell lines CMS, CMK,
and Dami. To elucidate the function of PTP-RO in megakaryocytic cells
and its potential involvement in the stem cell factor (SCF)/c-Kit
receptor pathway, COS-7 and 293 cells were cotransfected with the cDNAs
of both the c-Kit tyrosine kinase receptor and PTP-RO. PTP-RO was found
to be associated with the c-Kit receptor in these transfected cells and
the SCF/Kit ligand induced a rapid tyrosine phosphorylation of PTP-RO.
Interestingly, these transfected cells demonstrated a decrease in their
proliferative response to the SCF/Kit ligand. In addition, we assessed
the association of PTP-RO with c-Kit in vivo. The results demonstrated
that PTP-RO associates with c-Kit but not with the tyrosine kinase
receptor FGF-R and that PTP-RO is tyrosine-phosphorylated after SCF
stimulation of Mo7e and CMK cells. Antisense oligonucleotides directed
against PTP-RO mRNA sequences significantly inhibited megakaryocyte
progenitor proliferation. Therefore, these data show that the novel
tyrosine kinase phosphatase PTP-RO is involved in megakaryocytopoiesis and that its function is mediated by the SCF/c-Kit pathway.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
TYROSINE PHOSPHORYLATION plays a crucial
role in the regulation of signal transduction during diverse cell
functions, including cell activation, proliferation, differentiation,
survival, and metabolic homeostasis.1,2 Tyrosine
phosphorylation is regulated by controlling the balanced and opposing
actions of protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases (PTPases). PTPases are classified into two groups,
membrane-spanning receptor-type (R-PTPases) and cytosolic-type.
Hematopoietic cells express a number of R-PTPases, such as CD45,
PTP , and PTP , which are highly expressed in hematopoietic
tissues.3-6 CD45 is required for T-cell development and
plays a positive role in both T- and B-cell receptor-mediated
signaling.3 PTP is expressed abundantly in immature
thymus and is suggested to control T-cell differentiation.5 PTP was shown to regulate hematopoietic differentiation using murine
embryonic stem cells.6 The cytosolic PTPase, SHP-1, expressed primarily in hematopoietic cells, negatively regulates signaling in T- and B-cell antigen receptors and c-Kit in hematopoietic progenitor cells.3,4,7 Another cytosolic PTPase, SHP-2, although expressed ubiquitously, regulates T-cell signaling negatively as well as hematopoietic cell development
positively.3,4,7,8 However, relatively little is known
about the role of PTPases in signaling in hematopoietic stem cells and megakaryocytes.
To examine the involvement of PTPases in hematopoietic stem cell and in
megakaryocytic cell growth and differentiation, we have characterized
the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone
marrow stem cells and have cloned the human cDNA of a novel R-PTPase
termed PTP-RO,9 which is similar to the recently discovered
hPTP-J.10 In addition, the mouse homologue of PTP-RO/PTP-J,
termed PTP/Ftp1, was also cloned recently.11,12 PTP-RO/PTP-J and PTP are related to the homotypically adhering and µ R-PTPases.13-19 The human PTP-RO cDNA clone encodes
a polypeptide of 1,439 amino acids and the predicted molecular mass of
the PTP-RO protein is 162 kD. PTP-RO/PTP-J and PTP proteins, as well
as PTP and PTPµ, consist of an extracellular segment containing an
MAM domain, an Ig domain, four fibronectin-type III (FN-III) repeats, a
transmembrane segment, and two tandem intracellular phosphatase domains
and are classified to be type IIB R-PTPases.20 Reverse
transcription-polymerase chain reaction (RT-PCR) and
Northern blot analyses showed that PTP-RO is expressed in human
CD34+ stem cells as well as in various human
tissues.9
In addition to homotypical binding, PTP, PTPµ, and PTP were
reported to interact with the cadherin/catenin
complex,11,21,22 which is essential for cadherin-mediated
cell-cell adhesion and association with the cytoskeleton. Furthermore,
 -catenin and E-cadherin were suggested to be substrates for PTP
and PTPµ, respectively.21,22 These data suggest that
PTP, PTPµ, and PTP are involved in the regulation of
cadherin/catenin-mediated cell-cell adhesion. However, the function of
type IIB R-PTPases in hematopoietic cells is largely unknown.
Stem cell factor (SCF) is a growth and differentiation factor for
hematopoietic progenitor cells.23,24 SCF is the ligand for
the transmembrane c-Kit receptor.25,26 Two forms of SCF, a
membrane-bound and a secreted form, have been
described.27-30 c-Kit subserves pivotal functions in
promoting the development, survival, and proliferation of hematopoietic
stem cells, neuronal crest-derived cells, and germ cells. Upon SCF
stimulation, the c-Kit receptor associates with and/or phosphorylates
other signal transduction molecules such as phosphatidylinositol
3'-kinase (PI-3 K), phospholipase C-1, Lyn, Csk homologous
kinase (CHK), Janus kinase 2 (JAK2), signal transducers and activators
of transcription 1 (STAT1), Tec kinase, and the tyrosine phosphatase
SHP-1.29-37 Recently, SHP-1 was reported to bind and
negatively modulate the c-Kit receptor by interaction with Tyrosine 569 in the juxtamembrane domain of the c-Kit receptor.38
In this report, we analyzed the expression of PTP-RO mRNA and protein
in a hematopoietic stem cell line and in various megakaryocytic cell
lines and elucidated the role of PTP-RO in SCF/c-Kit receptor signaling
pathways in megakaryocytes. Our results demonstrated that PTP-RO
expression is upregulated during differentiation of megakaryocytic
cells and that PTP-RO is constitutively associated with the c-Kit
receptor and is tyrosine-phosphorylated by the SCF/Kit ligand.
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MATERIALS AND METHODS |
Materials.
Phorbol 12-myristate 13-acetate (PMA) was purchased from Life
Technologies, Inc (Bethesda, MD) and stored at a concentration of 1 mg/mL in dimethyl sulfoxide (Sigma, St Louis, MO) at  80°C. SCF was kindly provided by Amgen (Thousand Oaks, CA). Calf intestine alkaline phosphatase (CIAP) was purchased from Boehringer Mannheim (Indianapolis, IN).
Antibodies.
Polyclonal antibodies against PTP-RO were raised in rabbits immunized
by injection of the N-terminus of the PTP-RO intracellular domain
(residues: 770-930) fused to glutathione S-transferase (GST; R5702) and
against the MAM domain of PTP-RO (residues: 21-189) fused to GST
(D2712). Both anti-PTP-RO antibodies were purified by protein
G-Sepharose (Pierce, Rockford, IL). Polyclonal anti-PTP antibodies
against the PTP extracellular domain ( ECD)11 were kindly provided by Dr Laurence A. Lasky (Genentech, Inc, South San
Francisco, CA). Monoclonal and polyclonal anti-Flag antibodies were
purchased from Kodak (Rochester, NY; M2) and Zymed (South San Francisco, CA), respectively. Antiphosphotyrosine antibody, 4G10,
was kindly provided by Dr Brian J. Druker (Oregon Health Sciences
University, Portland, OR). Another antiphosphotyrosine antibody, PY20, was purchased from Transduction Laboratories
(Lexington, KY). Anti--catenin and anticadherin antibodies were
purchased from Zymed and Transduction Laboratories, respectively.
Anti-c-Kit antiserum against the extracellular domain was kindly
provided by Amgen, and anti-c-Kit antibody against the C-terminus was
purchased from Transduction Laboratories.
Cell lines.
CTS, CMS, and CMK cells were kindly provided by Dr Takeyuki Sato (Chiba
University, Chiba, Japan). CTS, CMK, and Dami cells were
maintained in RPMI-1640 medium (Mediatech [Washington, DC] or Life
Technologies, Inc) containing 10% fetal calf serum (FCS; Life
Technologies, Inc). CMS, COS-7, and 293 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM; Mediatech or Life Technologies, Inc) containing 10% FCS. Mo7e cells were maintained in
RPMI-1640 medium containing 20% FCS, 10 ng/mL interleukin-3 (IL-3; R&D
Systems, Minneapolis, MN), and 10 ng/mL granulocyte-macrophage colony-stimulating factor (R&D Systems). Penicillin (100 IU/mL) and
streptomycin (10 µg/mL) were added to all media.
CTS cells are a recently established human leukemia cell line derived
from the peripheral blood of a 13-year-old girl suffering relapse of
acute myeloblastic leukemia (AML) and have the characteristics of
pluripotent stem cells.39 CMS cells have also been recently established as a megakaryoblastic cell line. CMK,40
Dami,41 and Mo7e42 cells have authentic
properties of megakaryocytic lineages.
Human bone marrow was obtained by aspiration from the iliac crests of
normal donors after informed consent was obtained, as described
previously.28,29 After two washes with sterile 1× phosphate-buffered saline (PBS), the cells were resuspended in RPMI-1640 medium with 7.5% platelet-poor plasma (PPP),
penicillin/streptomycin (P/S), and L-glutamine; seeded onto T-75 tissue
culture flasks (Corning, Corning, NY); and incubated at 37°C in 5%
CO2. CD34+ bearing marrow progenitor cells were
purified from heparinized bone marrow aspirates using immunomagnetic
beads coated with anti-CD34 monoclonal antibody as
described.28 The CD34+ cell population was 95%
to 98% pure as judged by labeling with fluorescein-conjugated CD34
antibodies after an overnight recovery in RPMI plus 7.5% PPP.
Antisense oligonucleotide synthesis and cell treatment.
Modified 18-mer oligonucleotides were synthesized by Genosys
Biotechnologies, Inc (The Woodlands, TX), precipitated, and resuspended in RPMI-1640. PTP-RO antisense AS1
(5'-CGTACTGGGCCTCCTTGAACA-3') corresponded to nucleotides
+4 to +24. All experiments were performed with the corresponding sense
(5'-AGTACAGCCAGGCCCAGTACG-3') and scrambled sequence
controls. CD34+ cells were incubated at a concentration of
1 × 106 cells/mL in serum-deprived medium. Medium
contained iron-saturated human transferrin (300 ng/mL), insulin (100 ng/mL), calcium chloride (28 µg/mL), deionized bovine serum albumin
(2%), 6.14 mg of oleic acid, and 7.4 mg of dipalmitoyl lecithin in 10 mL of RPMI. Incubation medium was supplemented with recombinant human
IL-3 (100 U/mL; R&D Systems). Oligonucleotides were used at a
concentration of 10 mmol/L (70 µg/mL). After 16 hours of incubation
at 37°C, 5 mmol/L of oligonucleotides was added. Cells were further
incubated for an additional 6 hours and then washed in RPMI-1640 before plating.
Colony assays.
Cells were placed in the fibrin clot culture system as
described.28,29 Cells were seeded at a concentration of 500 cells/0.5 mL in culture containing 10% PPP and IL-3 (100 U/mL).
Cultures were incubated for 12 days. Fibrin clots were fixed for 5 minutes with 10% neutral formalin and reacted with platelet
glycoprotein IIIa (GpIIIa) fluorescein-conjugated monoclonal mouse
antibodies to human GpIIIa (1:1000 dilution; Dako, Carpinteria, CA) for
30 minutes. The numbers of positive megakaryocyte colony-forming units
(CFU-MK) were counted.
Flow cytometric analysis of surface protein expression.
To detect the potential surface binding proteins that bind PTP-RO, we
used flow cytometric analysis (FACS staining). Cells were washed with
sterile PBS, and 1 × 106 cells, untreated or treated
with PMA for 24 hours, were resuspended in 0.1 mL of PBS. Cells were
incubated with 10 µL of the PTP-RO antibodies or with GpIIIa
antibodies as a positive control, mouse IgG as a negative control
(Immunotech Inc, Westbrook, ME), or PBS at 4°C for 20 minutes.
Fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG or goat
antirabbit IgG was added at a final dilution of 1:500 and followed by
incubation for 20 minutes at 4°C. Cells were washed twice and
resuspended in 0.5 mL of 1% (vol/vol) paraformaldehyde in PBS. Cells
were then analyzed by flow cytometry.
Generation of a PTP-RO expression construct.
A pcDNA3/Flag expression vector was constructed by inserting a short
DNA fragment encoding an 8-amino acid Flag peptide into a pcDNA3
expression vector (Invitrogen, San Diego, CA) at the EcoRI
site.43 A PTP-RO cDNA was then subcloned into the
pcDNA3/Flag vector to generate a PTP-RO expression vector
(pcDNA3/PTP-RO-Flag), where the Flag sequence was added downstream of
the PTP-RO cDNA.
Transfection analysis.
COS-7 and 293 cells were transfected using SuperFect (Qiagen, Valencia,
CA) according to the manufacturer's protocol. In the cotransfection
system, pcDNA3/c-Kit44 and pcDNA3/PTP-RO-Flag were
transfected simultaneously. In some experiments,
pcDNA3.1()/Myc-His/lacZ (Invitrogen) was also
transfected simultaneously to determine transfection efficiency. After
48 to 72 hours of transfection, cells were either lysed or
serum-starved. The amounts of extract used for immunoprecipitation were
normalized by measurement of -galactosidase activity by an
O-nitrophenyl -D-galactopyranoside reporter assay (Promega,
Madison, WI).
Immunoprecipitation and Western blotting.
Cells were washed with PBS and were lysed in modified RIPA buffer (50 mmol/L Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL
aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mmol/L NaF, and
1 mmol/L Na3VO4) or in Triton X-100 lysis
buffer (10 mmol/L Tris-HCl, pH 7.4, 1% Triton X-100, 50 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL
aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mmol/L NaF, and
1 mmol/L Na3VO4). In some experiments, as
indicated, cells were lysed in modified RIPA buffer without NaF and
Na3VO4. After centrifugation at 14,000 rpm for
15 minutes, supernatant was used as total cell lysate. Protein
concentrations were determined using a colorimetric protein assay kit
(Bio-Rad, Hercules, CA).
Total cell lysates were incubated with different antibodies for 4 hours
or overnight at 4°C. Protein G-Sepharose (Pierce) was added for 1 hour at 4°C. Precipitates were collected by centrifugation and
washed four times with the modified RIPA buffer. Proteins were
separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Micron Separations, Inc, Westboro, MA). After blocking with
nonfat dry milk (Carnation) or bovine serum albumin
(Boehringer Mannheim), the membranes were probed with primary
antibodies. Immunoreactive bands were visualized using horseradish
peroxidase (HRP)-conjugated secondary antibodies and the enhanced
chemiluminescent (ECL) system (Amersham Pharmacia,
Piscataway, NJ) or Renaissance Western blot chemiluminescent
reagent (NEN, Boston, MA). In some experiments, as indicated, total
cell lysates were applied directly on SDS-PAGE gels.
Proliferation assay.
For proliferation assays, 104 cells were plated in 96-well
microliter plates and cultured in RPMI plus 0.5% FCS for 4 hours. SCF/Kit ligand was added at a final concentration of 10 ng/100 µL,
and cell proliferation was evaluated every 24 hours by a Cell Titer 96 nonradioactive assay (Promega) based on the conversion of a tetrazolium
salt to formazan. Proliferation was quantitated by measuring the amount
of formazan at 570 nm with a microtiter cell reader.
Northern blot analysis.
Poly(A+) RNA was isolated using an Oligo(dT)-cellulose
column (Amersham Pharmacia or Life Technologies, Inc) or a Fast Track 2.0 Kit (Invitrogen). Six micrograms of poly(A+) RNA of
each sample were run on a 1% agarose gel containing formamide and then
transferred to a nylon membrane (Hybond-N; Amersham Pharmacia). Hybridization and washing were performed using standard procedures. Radiolabeled DNA probes of the PTP-RO 3' untranslated region and 5' region of the cDNA were generated by PCR. Blots were assessed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or an actin-specific probe as a control.
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RESULTS |
Expression of PTP-RO mRNA and protein in stem cell and megakaryocytic
cell lines.
We examined PTP-RO mRNA and protein expression in a hematopoietic stem
cell line and various megakaryocytic cell lines by Northern analysis
using purified Poly(A+) RNA and Western blot analysis using
total cell lysates. Northern analysis showed the existence of one (4.4 kb) or two mRNAs (5.5 and 4.4 kb) in the CTS, CMK, CMS, and Dami cell
lines (Fig 1). Western blot analysis of
CMS, CMK, Dami, and Mo7e cells using two anti-PTP-RO antibodies
against different regions of PTP-RO (the MAM domain and the
intracellular domain) showed several PTP-RO proteins ranging from 190 to 250 kD (Fig 2). These results suggest that the megakaryocytic cell lines express several isoforms of PTP-RO,
including the full-length PTP-RO. Additionally, Western blot analysis
showed the presence of a possible proteolytic product of PTP-RO (~100
kD).
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| Fig 1.
Expression of PTP-RO mRNA in megakaryocytic cell lines. 6 µg of Poly(A+) mRNA extracted from the indicated human
megakaryocytic cell lines (A) and 100 nmol/L PMA-treated (6 and 18 hours) CMK cells (B) were electrophoresed in a denatured 1%
agarose-formaldehyde gel and transferred to nylon membranes.
Hybridization was performed with a 32P-labeled PTP-RO probe
(upper panel), followed by hybridization with a 32P-labeled
GAPDH probe or actin probe (lower panel). The positions of PTP-RO,
GAPDH, and actin mRNAs are indicated.
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| Fig 2.
Upregulation of PTP-RO protein expression in
megakaryocytic cell lines. (A) Total cell lysates containing
equal amounts of protein (100 µg) from CTS, CMK, Dami, and Mo7e cells
were separated on 7.5% SDS-PAGE. Immunoblotting was performed using
anti-PTP-RO antibodies (R5702; 7 µg/mL) or control preimmune
antibody (7 µg/mL). (B and C) CMS, CMK, and Dami cells were cultured
in RPMI-1640 or DMEM medium (referred to in Materials and Methods)
containing 10% FCS in the presence of 100 nmol/L PMA at a
concentration of 106 cells/mL. After culture for 0, 24, and
48 hours, cells were collected and lysed with the RIPA buffer. One
hundred micrograms of protein from clarified cell lysates were analyzed
by 4% to 12% SDS-PAGE followed by Western blotting using two
anti-PTP-RO antibodies R5702 (7 µg/mL; B) and D2712 (8 µg/mL; C)
as well as control preimmune antibodies (8 µg/mL). The positions of
migration of full-length PTP-RO and putative proteolytic PTP-RO are
indicated.
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The approximately 100-kD polypeptide was recognized by the R5702
antibodies (against the N-terminus of PTP-RO fused to GST), but not by
the D2712 antibodies (against the MAM domain of PTP-RO fused to GST;
see Materials and Methods for more details). This protein was also
recognized by polyclonal anti-PTP antibodies (the mouse homologue of
PTP-RO; ECD) against its extracellular domain (data not shown),
suggesting that the approximately 100-kD protein is a proteolytic
product containing the intracellular domain and the extracellular
domain of PTP-RO without the MAM domain.
Upregulation of PTP-RO mRNA and protein expression in megakaryocytic
cell lines upon PMA stimulation.
Our analyses confirmed that, in CMS cells, the primitive hematopoietic
marker CD34 was downregulated, whereas the megakaryocyte-specific markers, CD42b (GPIb) and CD61 (GPIIIa), were upregulated by PMA treatment, as analyzed by flow cytometric analysis (data not shown). CTS, CMK, and Dami cells differentiate into mature megakaryocytes upon
stimulation with PMA.39-41 Northern blot analysis
demonstrated a time-dependent upregulation of PTP-RO mRNA expression
upon PMA stimulation (Fig 1B). We have examined whether PTP-RO protein expression levels are also regulated by PMA treatment in these megakaryocytic cell lines. The full-length PTP-RO was significantly upregulated by PMA treatment (Fig 2B and C). Upregulation was observed
after 24 hours of PMA treatment.
Immunofluorescence staining showed that more than 58% of the CMK cells
stained positive with PTP-RO antibodies. Upon PMA treatment, more than
89% of the CMK cells were stained positive with PTP-RO antibodies. No
staining was observed with control antibody. Taken together, these data
along with the Western blot analysis indicate positive expression of
PTP-RO on megakaryocytic cells, an expression that is upregulated upon
PMA treatment.
Cell surface expression of PTP and PTPµ has been reported to be
upregulated by cell-cell contact due to high cell
density.22,45 To examine whether upregulation of PTP-RO is
due to cell-cell contact, we seeded CMK cells at three independent
concentrations (1 × 106, 3.3 × 105,
and 1.1 × 105 cells/mL). After 48 hours, cells became
approximately 100%, 90%, and 60% confluent, respectively. We
analyzed PTP-RO protein expression by Western blotting. No significant
difference in PTP-RO expression was observed among the three cell
densities (data not shown), indicating that upregulation of PTP-RO was
not due to cell-cell contact.
CMK cells became more adherent to culture flasks upon stimulation with
PMA. Under normal conditions, without any treatments, CMK cells grow in
in vitro culture in which approximately 70% of the cells are grown in
suspension and approximately 30% are adherent. To examine whether cell
adhesion is related to upregulation of PTP-RO, we compared PTP-RO
protein expression of the nontreated CMK cells growing in suspension
with that of the adherent CMK cells. Expression of PTP-RO was similar
in both populations of CMK cells, indicating that PTP-RO upregulation
was not due to cell adhesion (data not shown).
PMA suppresses CMK cell growth and induces cell
differentiation.40 To analyze whether PTP-RO upregulation
is due to cell growth suppression, we maintained CMK cells in
serum-free medium for 48 hours and then harvested the cells. PTP-RO
expression was not upregulated by serum starvation (data not shown).
This suggests that PTP-RO upregulation by PMA treatment was not due to
cell growth suppression, but to megakaryocyte differentiation.
Taken together, these results strongly suggest that PTP-RO upregulation
is observed during megakaryocyte differentiation and is not mediated by
cell-cell contact, cell adhesion, and/or cell growth suppression.
PTP-RO function in megakaryocytic cells is not related to its
interaction with the cadherin/catenin complex.
To examine whether PTP-RO is involved in the cadherin/catenin
complex-like PTPs , µ, and ,11,21,22 we analyzed
the expression of cadherin and -catenin proteins in CTS, CMS, CMK,
and Dami cells using Western blot analysis. Neither the cadherin nor
-catenin proteins were detected in CTS, CMK, and Dami cells
(Fig 3). Interestingly, untreated CMS cells
did not express cadherin and -catenin, whereas PMA treatment did
induce expression of cadherin as shown by Western blot analysis (Fig
3B). -Catenin was not observed in PMA-treated CMS cells. Taken
together, the cadherin/catenin complex was not found in the CTS cells
and the megakaryocytic Dami and CMK cell lines examined, suggesting
that PTP-RO function is not related to its interaction with this
well-known complex in megakaryocytes, contrary to observations in other
cells such as epithelial cells.
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| Fig 3.
A hematopoietic stem cell line and megakaryocytic cell
lines do not express -catenin and cadherin. Western blot analysis of
total cell lysates (100 µg protein per lane) from CTS, CMS, CMK, and
Dami cells untreated or PMA (100 nmol/L)-treated for 48 hours was
performed using anti--catenin antibody (1 µg/mL; A) and
anticadherin (1 µg/mL) antibody (B). COS-7 cells were used as a
positive control. The positions of migration of -catenin and
cadherin are shown.
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Association of the c-Kit receptor with PTP-RO.
To address the role of PTP-RO in signal transduction and cell growth,
we characterized the association of PTP-RO and the c-Kit tyrosine
kinase receptor in two cell lines (Mo7e and CMK) known to express high
levels of c-Kit receptor and to proliferate in response to SCF
stimulation. CMK and Mo7e cells were starved and stimulated with 500 ng/mL SCF. After stimulation, cells were lysed and immunoprecipitated
with anti-c-Kit antibodies against the extracellular domain of c-Kit.
The precipitates were immunoblotted with antiphosphotyrosine
antibodies. Tyrosine phosphorylation of the c-Kit receptor was observed
upon SCF stimulation of CMK and Mo7e cells
(Fig 4A and C). Next, the blots were
reprobed with anti-PTP-RO antibodies (R5702). Interestingly, the
approximately 190-kD protein, corresponding to the full-length PTP-RO,
was observed specifically in the immunoprecipitates using anti-c-Kit
receptor antibodies from both unstimulated and SCF-stimulated CMK and
Mo7e cells (Fig 4A and C). These results suggest a constitutive
association of PTP-RO and the c-Kit receptor in CMK and Mo7e cells.
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| Fig 4.
In vivo association of c-Kit and PTP-RO in megakaryocytic
cells. (A and B) CMK cells were starved in serum-free RPMI-1640 medium
for 6 hours and incubated with or without 500 ng/mL SCF or FGF for 5 minutes at 37°C. Reaction was stopped by adding one-third volume of
ice-cold PBS, followed by rapid centrifugation. After an additional
wash with PBS, cells were lysed with the Triton X-100 lysis buffer. The
clarified cell lysates were immunoprecipitated with either anti-c-Kit
antiserum (Amgen; 10 µL), normal rabbit serum (NRS), or anti-FGF-R
antiserum. The precipitates were immunoblotted with the 4G10 antibody
(1:3,000; upper panel), anti-PTP-RO antibodies (R5702; 7 µg/mL;
[A], middle panel; [B], lower panel), anti-c-Kit antibody (Santa
Cruz, Santa Cruz, CA; 1 µg/mL; [A], lower panel) or
anti-FGF-R antibody (Santa Cruz; [B], middle panel). (C and D) Mo7e
cells were starved in RPMI-1640 medium containing 0.5% bovine serum
albumin for 6 hours and stimulated with 500 ng/mL SCF for 10 minutes.
Additional procedures were the same as in the case of CMK cells, except
that cells were lysed with the modified RIPA buffer, and PY20 antibody
(1:800) was used in (C) instead of the 4G10 antibody. Lysates were
immunoprecipitated with anti-c-Kit or anti-PTP-RO antibodies (10 µL) or normal rabbit serum (NRS). The immunoprecipitates were
immunoblotted with the 4G10 antibody (1:800), anti-PTP-RO antibodies
(R5702), or anti-c-Kit antibodies, as indicated.
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The potential association of PTP-RO with another tyrosine kinase
receptor was examined in CMK cells. Stimulation of CMK cells with FGF
induced a rapid, but transient, tyrosine phosphorylation of a number of
cellular proteins, including FGF-R. In contrast to the results with
c-Kit, PTP-RO was not detected in FGF-R immunoprecipitates at any of
the times examined after FGF stimulation (Fig 4B). These results
indicate that PTP-RO selectively associates with c-Kit in
megakaryocytic cells.
To determine whether c-Kit engagement in these cells is associated with
the tyrosine phosphorylation of c-Kit and PTP-RO, the proteins were
individually immunoprecipitated from resting and SCF-stimulated Mo7e
and CMK cells, and their phosphorylation status was assessed by
antiphosphotyrosine immunoblotting analysis. As shown in Fig 4C and D,
PTP-RO and c-Kit are both tyrosine-phosphorylated after c-Kit
engagement by SCF. These findings suggest that PTP-RO associates with
the c-Kit receptor and is tyrosine-phosphorylated upon SCF/Kit ligand stimulation.
To further confirm the in vivo association of the c-Kit receptor with
PTP-RO, we performed a transient transfection of both the c-Kit
receptor and PTP-RO in COS-7 and 293 cells. An expression vector for
the c-Kit receptor was transfected together with the construct for
PTP-RO-Flag or a control vector, pcDNA3/Flag.
pcDNA3.1()/Myc-His/LacZ, the expression vector for
-galactosidase, was also cotransfected to determine the transfection
efficiency in these sets of experiments. After starvation, cells were
stimulated with SCF and then lysed. The expression amount of the c-Kit
receptor was normalized by measuring -galactosidase activity. Cell
lysates showing approximately the same -galactosidase activity were
immunoprecipitated with anti-c-Kit antibodies. SCF-induced tyrosine
phosphorylation of c-Kit was observed in both c-Kit-transfected and
PTP-RO/c-Kit-cotransfected COS-7 cells
(Fig 5) and in 293 cells (data not shown),
indicating that c-Kit was expressed as a functional receptor. Kinetic
studies indicated that c-Kit phosphorylation levels were similar
between PTP-RO-transfected and untransfected cells. The 190-kD protein corresponding to PTP-RO, because of its reactivity to anti-Flag antibody, was observed clearly only in the immunoprecipitates from the
293 cells (data not shown) and COS-7 cells expressing PTP-RO (Fig 5).
These results indicate that PTP-RO was coimmunoprecipitated by
anti-c-Kit antibodies, suggesting constitutive association of c-Kit
and PTP-RO.
View larger version (60K):
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| Fig 5.
In vivo association of c-Kit and PTP-RO in transfected
COS-7 cells. COS-7 cells were cotransfected with c-Kit and PTP-RO cDNAs
or c-Kit cDNA and control vector together with
pcDNA3.1()/Myc-His/LacZ. Cells were starved with DMEM
containing 0.5% FCS for 15 hours, followed by serum-free DMEM for 4 hours. SCF stimulation (500 ng/mL) was performed for the indicated time
points and then cells were lysed with the modified RIPA buffer. After
measuring -galactosidase activity, clarified total cell lysates
containing equal amounts of c-Kit were immunoprecipitated with
anti-c-Kit antiserum (10 µL; Amgen). The precipitates were
immunoblotted with the 4G10 antibody (1:3,000; A), anti-Flag antibody
(2 µg/mL; Zymed; B), anti-c-Kit antibody (1 µg/mL; Santa Cruz; C),
or anti-PTP-RO antibodies (D).
|
|
SCF induces tyrosine phosphorylation of PTP-RO.
To examine whether PTP-RO phosphorylation is regulated by the SCF/c-Kit
pathway, we used 293 cells and COS-7 cells cotransfected with c-Kit and
PTP-RO-Flag cDNAs. After serum starvation, COS-7 transfectants were
stimulated with SCF and then harvested at the indicated times
(Fig 6). Cells were lysed and
immunoprecipitated with anti-Flag antibody. The precipitates were then
immunoblotted with the PY20 antibody. SCF stimulation induced the
appearance of four tyrosine-phosphorylated bands with molecular masses
of approximately 190, 145, 100, and 80 kD (Fig 6A). The 145-kD
band corresponded to c-Kit. The phosphorylation level of
all proteins increased at 5 minutes, peaked at 15 minutes, and then
decreased at 20 minutes. To confirm the identity of these proteins, the membrane was stripped and reprobed with anti-Flag antibody (Fig 6B),
anti-PTP-RO antibodies (R5702) against the N-terminus of the
intracellular domain (Fig 6C), and anti-PTP antibodies (ECD) reactive to the extracellular domain of PTP-RO (Fig 6D). All antibodies recognized the 190-kD band as the full-length PTP-RO
protein as well as the proteolytic products of PTP-RO. Similar results
were obtained with transfected 293 cells (data not shown). These
results indicate that SCF induced tyrosine phosphorylation of the
full-length PTP-RO (190 kD) and the proteolytic forms of PTP-RO (100 and 80 kD). Interestingly, using the anti-Flag antibody, a
non-tyrosine-phosphorylated 73-kD band was detected in addition to
those of 190, 90, and 80 kD. Because the 73-kD polypeptide did not
react with the R5702 antibodies, it must be another proteolytic product
of PTP-RO containing only the intracellular domain possessing a
deficiency in its N-terminus (Fig 7).
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| Fig 6.
SCF-induced tyrosine phosphorylation of PTP-RO in COS-7
cells transfected with c-Kit and PTP-RO cDNAs. Serum-starved COS-7
cells expressing exogenous c-Kit and PTP-RO-cFlag were incubated with
500 ng/mL SCF for the indicated times. Cells were lysed with the
modified RIPA buffer and immunoprecipitated with 5 µg of anti-Flag
antibody (Zymed). The immunoprecipitates were used for Western blotting
using the antiphosphotyrosine antibody, PY20 (1:800; A). The immunoblot
was stripped and reprobed with anti-Flag antibody (M2; 1:2,000; B),
anti-PTP-RO antibodies (R5702; 7 µg/mL; C), and anti-PTP
antibodies (D), sequentially.
|
|
View larger version (19K):
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| Fig 7.
Schematic presentation of the full-length and proteolytic
forms of PTP-RO in COS-7 cells. MAM, MAM domain; Ig, Ig domain; PTP 1 and 2, protein tyrosine phosphatase domains 1 and 2, respectively; N,
N-terminus; C, C-terminus.
|
|
Effect of PTP-RO antisense oligonucleotides on in vitro
megakaryocytopoiesis.
To address the role of PTP-RO in the regulation of
megakaryocytopoiesis, we exposed purified bone marrow CD34+
cells to PTP-RO antisense and sense oligonucleotides. This approach has
been successfully used to address the function of C-myb in megakaryocytopoiesis. C-myb antisense oligonucleotide
(5'-GTGCCGGGGTCTTCGGCC-3') served as a positive control due
to its known inhibitory effects on the generation of megakaryocyte
colonies (CFU-MK).46 The CD34+ cells were
isolated using immunomagnetic beads. CD34+ cells were
incubated at a concentration of 1 × 106 cells/mL in
serum-deprived medium containing growth factors and synthetic sense or
antisense oligonucleotides. The generation of megakaryocyte colonies
(CFU-MK) from CD34+ progenitor cells treated with PTP-RO
antisense was reduced significantly (~50%) compared with the
sense-treated CD34+ and control untreated cells
(Table 1). These results indicate that
PTP-RO antisense oligonucleotides specifically inhibited in vitro
megakaryocytopoiesis using primary bone marrow progenitor cells.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of Oligonucleotide Treatment of Bone
Marrow-Derived CD34+ Cells On In Vitro CFU-MK Colony
Formation
|
|
| |
DISCUSSION |
In this report, we have shown that a type IIB R-PTPase, PTP-RO, was
expressed in megakaryocytic cell lines and that its expression was
upregulated during differentiation by PMA treatment. In addition, PTP-RO was constitutively associated with the c-Kit receptor and tyrosine-phosphorylated upon SCF stimulation of megakaryocytic cells.
Full-length PTP-RO was expressed as multiple isoforms (Fig 2). Two main
categories of PTP-RO isoforms were found: the high (~220 to ~250
kD) and the low (~190 to ~ 200 kD) molecular mass proteins.
Isoforms of PTP-RO might be formed by alternative processing. Alternatively, these various PTP-RO isoforms might result from differences in their glycosylation. In addition to the full-length PTP-RO, we observed a putative proteolytic product of PTP-RO (~100 kD), consistent with previous data about type IIB R-PTPases and other
R-PTPases. It is a common phenomenon that R-PTPases are digested within
the fourth FN-III repeat at a sequence, RLRR,11 that exists
within PTP-RO sequences. Determining whether alternative processing is
taking place and identifying whether different glycosylation forms of
PTP-RO exist remain to be addressed in future studies.
In addition, we have observed that PMA treatment upregulates PTP-RO
mRNA and protein expression in megakaryocytic cells (Figs 1B and 2B and
C). This upregulation was related to megakaryocyte differentiation but
not to cell-cell contact, cell adhesion, and cell growth suppression.
Interestingly, our data are in contrast with hPTP-J expression in
Jurkat cells, in which hPTP-J was downregulated by PMA
stimulation.10 hPTP-J was also downregulated by calcium ionophore, A23187. It is likely that signal transduction mechanisms by
PMA stimulation are different between T cells, such as Jurkat cells,
and megakaryocytic cells. Transforming growth factor (TGF), an
inhibitor of proliferation of human keratinocyte cells, upregulated
PTP expression.46 Cell-cell contact upregulated PTP and µ expression in mink lung epithelial cells and mammary carcinoma
cells, respectively.22,45 Thus, the type IIB R-PTPases can
be regulated by different mechanisms in various cell types.
Because type IIB R-PTPases such as PTPs , µ, and play a role
in the regulation of cell-cell contact and adhesion mediated by the
cadherin/catenin complex,11,21,22,47 we analyzed whether PTP-RO has similar functions. In the megakaryocytic cell lines, CMS,
CMK, and Dami, expression of PTP-RO was upregulated by treatment with
PMA. In parallel, PMA treatment induced these cells to aggregate (CMS)
or to be adherent (CMK and Dami). However, neither -catenin nor
cadherin was expressed in these cell lines, except for in CMS cells
treated with PMA (Fig 3). There are no reports showing that cadherin
controls cell-cell adhesion without catenin. Thus, if PTP-RO regulates
cell contact and adhesion in megakaryocytes, it may be due to the
direct interaction of PTP-RO via its extracellular domain and not
through the -catenin/cadherin complex.
Interestingly, we have observed that PTP-RO is tyrosine-phosphorylated
upon SCF stimulation and is associated with c-Kit independently of SCF
stimulation (Figs 4, 5, and 6). Various proteins, such as CHK, the p85
subunit of PI-3 K, phospholipase C-1, Fyn, Lyn, Shc, Grap, SHP-1,
and STAT1,31,32,34,36,37,48 have been reported to be
associated with c-Kit upon SCF stimulation via their SH2 domains.
Tec-kinase was constitutively associated with c-Kit via its SH3
domain.33 However, PTP-RO contains neither an SH2 nor an
SH3 domain, suggesting that, unlike other signal transduction proteins,
the binding mechanism of PTP-RO to c-Kit is not via SH2 and SH3 domains.
A 200-kD transmembrane glycoprotein was reported to be associated with
c-Kit by SCF stimulation of Mo7e cells.49 Although 200 kD
is very close to the molecular mass of PTP-RO, the 200-kD protein
failed to associate with c-Kit without SCF stimulation, which is in
contrast with our data concerning PTP-RO. Thus, our analysis suggested
that the 200-kD protein is not PTP-RO. Importantly, in CMK and Mo7e
cells, only the low molecular mass (~200 kD) isoforms of PTP-RO were
immunoprecipitated by anti-c-Kit antibody. This suggests that
different full-length PTP-RO isoforms may play various roles such as
some other R-PTPases, such as CD45 isoforms, which differently affect
T-cell development.50
PTP-RO was tyrosine-phosphorylated by SCF stimulation, suggesting that
PTP-RO is a signaling molecule involved in the SCF/c-Kit-mediated signal transduction pathway. Some R-PTPases, LAR, PTP , and DEP-1, were tyrosine-phosphorylated in TrkA-dependent and EGF-R-dependent manners, respectively.51,52 TrkA, the nerve growth factor
(NGF) receptor, and EGF-R are receptor-type tyrosine kinases that have similar characteristics in signal transduction as c-Kit, such as
dimerization, tyrosine phosphorylation, and binding to several SH2-containing proteins (ie, Shc, the p85 subunit of PI-3 K, and phospholipase C-1) upon ligand stimulation.53,54
However, the mechanisms of tyrosine phosphorylation of DEP-1 by EGF and PTP-RO by SCF are likely to be different. Whereas relatively long-time stimulation (30 minutes) by EGF was necessary to tyrosine phosphorylate DEP-1,52 PTP-RO phosphorylation was observed after only 5 minutes of SCF stimulation (Fig 6). These results suggest that, whereas DEP-1 is not a direct target of EGF-R, PTP-RO might be a direct target
of c-Kit.
At present, the biological importance of the tyrosine phosphorylation
of PTP-RO is not known. One possibility is that the tyrosine
phosphorylation of PTP-RO regulates its catalytic activity. There are
some reports suggesting tyrosine phosphorylation-induced phosphatase
activity. For instance, tyrosine phosphorylation of SHP-2, PTP1B, and
CD45 correlated with an enhancement in their catalytic
activity.55-57 Although we detected possible in vivo phosphatase activity of PTP-RO in COS-7 cells overexpressing PTP-RO (data not shown), we could not detect in vitro catalytic activity. Catalytic activity of PTP-RO may require modification of PTP-RO such as
its tyrosine phosphorylation. The other possibility is that its
tyrosine phosphorylation site(s) is a target(s) for other proteins. Our
next interest is the identification of molecules that bind to the
phosphorylated tyrosine residues of PTP-RO. CD45, upon phosphorylation
on tyrosine by treatment with a phosphatase inhibitor, was reported to
bind to Lck kinase.56 PTP is tyrosine-phosphorylated constitutively in vivo and is associated with the adaptor protein, Grb2, via its SH2 domain.58,59 Although there are 30 tyrosine residues in the cytoplasmic region of PTP-RO, not all of them are known as potential SH2 binding sites.
A schematic presentation of the full-length and proteolytic forms of
PTP-RO in COS-7 transfectants are shown in Fig 7. The immunoreactivity
of these PTP-RO isoforms to three different antibodies and their
tyrosine phosphorylation are also shown. All constructs contained the
C-terminus of PTP-RO, because they were recognized by anti-Flag
antibody. Because the 100- and 80-kD proteins bound to WGA-Sepharose,
they should have been glycosylated, implying that they have the
C-terminus of the extracellular domain of PTP-RO. The failure of PTP-RO
to react with anti-PTP antibodies (ECD) of the 80-kD protein
indicates that it contains only a very small part of the extracellular
domain close to the membrane. The 73-kD protein has only an
intracellular domain deficient in its N-terminus. Interestingly, only
the 73-kD protein was not tyrosine-phosphorylated. This suggests that
phosphorylated tyrosine residues exist within the epitope of the R5702
antibodies. Thus, within the amino acid residues 770 to 930, there is a
possible epitope in which there are six tyrosine residues responsible
for PTP-RO phosphorylation.
Our results demonstrating that the function of PTP-RO is mediated by
the SCF/c-Kit pathway and that antisense oligonucleotides directed
against PTP-RO mRNA sequences significantly inhibit megakaryocyte progenitor proliferation indicate that PTP-RO is involved in
megakaryocytopoiesis. We propose a novel biological function of PTP-RO,
a type IIB R-PTPase, in the regulation of SCF/c-Kit signaling in
megakaryocytes. We suggest that PTP-RO negatively regulates c-Kit
signaling and thereby mitigates the signaling events linking c-Kit
engagement to hemopoietic cell proliferation and differentiation.
Future studies will determine the binding site of PTP-RO to c-Kit and
the molecular mechanisms by which c-Kit signaling is regulated by
PTP-RO.
| |
ACKNOWLEDGMENT |
The authors thank Dr Brian J. Druker (Oregon Health Sciences
University) for providing the antiphosphotyrosine antibody, 4G10; Dr
Brian Bennett (Amgen Inc) for providing anti-c-Kit antiserum and SCF;
Dr Laurence A. Lasky (Genentech, Inc) for providing anti-PTP antibodies; and Dr Takeyuki Sato (Chiba University) for providing CTS,
CMS, and CMK cell lines. We also thank Yigong Fu for DNA sequencing. We
thank Drs Shalom Avraham and Daniel Price for their much appreciated
advice and reading of this manuscript. We are grateful to Tee Trac and
Peter Park for typing this manuscript, Janet Delahanty for editing, and
Nancy DesRosiers for preparation of the figures.
| |
FOOTNOTES |
Submitted December 29, 1998; accepted March 22, 1999.
Supported in part by National Institutes of Health Grant No. HL 51456 and the Toray Research Center Inc.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hava Avraham, PhD, Division of Experimental
Medicine, Harvard Institutes of Medicine-BIDMC, 4 Blackfan Circle,
Boston, MA 02115; e-mail: havraham{at}caregroup.harvard.edu.
| |
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INTRODUCTION