Characterization of a Myeloid Tyrosine Phosphatase, Lyp, and Its Role in the Bcr-Abl Signal Transduction Pathway*
Wenwen Chien
,
Nicola Tidow
,
Elizabeth A. Williamson,
Lee-Yung Shih ¶,
Utz Krug,
Arminja Kettenbach,
Anthony C. Fermin,
Chaim M. Roifman || ** and
H. Phillip Koeffler 
From the
Department of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA
School of Medicine, Los Angeles, California 90048,
¶Division of Hematology-Oncology, Chang Gung
Memorial Hospital and Chang Gung University, Taipei 105, Taiwan, and the
||Division of Immunology and Allergy, Department of
Pediatrics, University of Toronto and The Hospital for Sick Children, Toronto
M5G 1X8, Canada
Received for publication, May 1, 2003
 |
ABSTRACT
|
---|
The Bcr-Abl protein-tyrosine kinase is implicated in the development of
chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase
in the regulation of Bcr-Abl signaling was explored. First, expression
patterns of tyrosine phosphatases in leukemic cell lines were investigated
using degenerate primers for reverse transcription-PCR followed by cloning and
sequencing of the cDNA. Distinct patterns of distribution of phosphatase were
found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases
were ubiquitously expressed, others were limited to specific cell types.
Surprisingly, a previously cloned "lymphocyte-specific"
phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as
well as normal granulocytes and monocytes. Lyp was localized to the cytosol,
and overexpression of Lyp caused reduction in the phosphorylation levels of
multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl,
Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells
resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp
markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken
together, the data suggest that Lyp may play an antagonistic role in signaling
by the Bcr-Abl fusion protein.
 |
INTRODUCTION
|
---|
In Philadelphia chromosome-positive human chronic myeloid leukemia
(CML),1 malignant
transformation is mediated by a constitutively active tyrosine kinase Bcr-Abl
(1,
2). The abl gene
product is a 145-kDa protein encoding a non-receptor tyrosine kinase
(3). The protein contains three
src homology domains, SH1 with tyrosine kinase function, and SH2 and SH3
involved in protein-protein interaction. The enzymatic activity of Abl can be
regulated through protein-binding domains and stimulated by growth factors and
DNA damage (4,
5). The Philadelphia chromosome
results from a reciprocal translocation of the abl on chromosome 9
transposing to chromosome 22 in the break cluster region (bcr) gene
(6,
7). Interleukin 3-dependent
Ba/F3 cells infected with the fusion gene bcr-abl become growth
factor-independent and tumorigenic in nude mice
(8). The aberrant tyrosine
phosphorylation levels of Bcr-Abl activate a series of signaling pathways, and
a multitude of proteins exhibit a marked increase in their level of
phosphorylation including Bcr-Abl itself, CrkL (an adaptor protein), and
phosphatidylinositol 3-kinase
(9). Cellular Cbl is a 120-kDa
cytoplasmic protein that is ubiquitously expressed with high levels in
hematopoietic cells. Cbl can be phosphorylated in response to activation by a
variety of growth factors including epidermal growth factor, platelet-derived
growth factor, erythropoietin, as well as granulocyte-macrophage
colony-stimulating factor
(1012).
Tyrosine phosphorylation of Cbl is increased in Bcr-Abl transformed cells
(13). Interaction between Cbl
and CrkL is tyrosine phosphorylation-dependent, and the complex has been
implicated in Bcr-Abl mediated transformation
(14).
Protein-tyrosine phosphatases (PTPase) counter the activity of tyrosine
kinases by removing phosphate groups from proteins that have been
phosphorylated on tyrosyl residues. Whereas protein-tyrosine kinases have been
intensely studied over the past decade, the significance of PTPase has just
started to be recognized (15).
PTPases are involved in the regulation of cellular proliferation and
differentiation, as well as cell death
(16,
17). CD45 is one of the better
studied hematopietic PTPases and is a key regulator of lymphocyte functions
(18). Other hematopoietic
PTPases, such as SHP-1, are involved in cytokine receptor signaling
(19). PTPases display high
sequence homology in their catalytic domain
(20), which allows the
identification of PTPase family members by using degenerate primers in RT-PCR
amplification. We have employed this method to investigate the expression
pattern of PTPase in leukemic cells. Degenerate PTPase primers were designed
and used to amplify different PTPases from RNA isolated from leukemic cell
lines. PCR products were cloned, and individual clones were identified by DNA
sequencing. Specific PTPase expression patterns were obtained for each cell
line.
Lyp, a PTPase that was previously shown to be expressed in lymphoid cells
(21), was identified in this
study as one of the major PTPases in myeloid leukemic cells. Lyp was localized
to the cytoplasm in the KCL22 CML cell line. Overexpression of Lyp in KCL22
CML cells caused reduction of total cellular phosphorylation levels of
proteins. Of particular interest, phosphorylation of Cbl and Bcr-Abl markedly
decreased in these cells, and this was associated with markedly decreased
levels of Bcr-Abl. Molecules that are substrates of Bcr-Abl, such as CrkL and
Erk1/2, also had a decrease in their phosphorylation levels, and amounts of
the Grb2 and Myc proteins decreased. Anchorage-independent clonal growth in
soft agar markedly decreased in KCL22 cells overexpressing Lyp. Our study
suggests a novel mechanism for Bcr-Abl regulation.
 |
EXPERIMENTAL PROCEDURES
|
---|
Cell Culture, Transfection, and CML Patient SamplesCell
lines were purchased from ATCC (Manassas, VA) except for the following: ML-1
cells were a gift from Dr. M. Kastan (The Johns Hopkins University, Baltimore,
MD). Kasumi-1 and Kasumi-3 cells were established by Dr. H. Asou (Hiroshima
University, Hiroshima, Japan). Adherent and suspension cells were grown in
Dulbecco's modified Eagle's medium and RPMI, respectively, supplemented with
10% fetal bovine serum. Transfection of KCL22 cells (5 x 107)
was performed by electroporation at 340 V with 20 µg of plasmid in RPMI
containing 50% fetal calf serum. Transfection of Cos-7 cells was carried out
using LipofectAMINE 2000 (Invitrogen) over 4 h according to the manufacturer's
protocol. Proteins and RNA were prepared from the bone marrow of CML patients
after their informed consent.
Reverse Transcription and Polymerase Chain ReactionTwo
µg of total RNA isolated with TRIzol reagent was reverse-transcribed with
Superscript II and random primers according to the manufacturer's protocol
(Invitrogen). PCR consisted of 2230 cycles of denaturation at 95 °C
for 1 min, annealing at 6264 °C for 1 min, and extension at 72
°C for 1 min. With degenerate primers, the annealing cycles were modified
by starting the annealing process at 37 °C and heating to 72 °C within
2 min. Primers used are:
-actin-specific,
5'-TACATGGCTGGGGTGTTGAA-3',
5'-AAGAGAGGCATCCTCACCCT-3'; Lyp-specific,
5'-TGGCCTCCAAGTGGTACCAG-3',
5'-CATCGGCAAGAAAGAAGGAC-3'. Degenerate primers for PTPase were
deduced from amino acid sequences: ACKCCNGCNSWRCARTG (upper strand) and
AGYGAYTAYATHAAYGC (lower strand). PCR products amplified from degenerate
primers were cloned into pBluescript for sequencing. DNA sequences were
compared with the NCBI data base using the BLAST program.
Separation of Peripheral Blood CellsPolymorphonuclear cells
(neutrophils) were isolated from anticoagulated blood using polymorphonuclear
neutrophil solution (Robbins Scientific, Sunnyville, CA) according to the
one-step density gradient centrifugation method. Briefly, whole blood was
layered over polymorphonuclear neutrophil solution and centrifuged for 25 min
at 500 x g. Mononuclear cells and neutrophils were separated
into two distinct bands, whereas erythrocytes pelleted to the bottom of the
tube. Neutrophils were obtained from the lower band and were washed twice with
serum-free medium. Monocytes were separated from the mononuclear cell fraction
by adherence to plastic cell culture dishes. CD3+ cells were
isolated from the mononuclear fraction by FACStar flow cytometer (BD
Biosciences) using monoclonal murine antibodies against CD3 conjugated to
fluorescein isothiocyanate and phosphatidylethanolamine, respectively (Dako,
Carpinteria, CA).
Hybridization with an Internal OligonucleotideGel-separated
PCR products were blotted onto nylon membrane (Amersham Biosciences) by
capillary transfer in 20x SSC. Prehybridization and hybridization were
performed at 42 °C in digoxigenin Easy-Hyb solution (Roche Applied
Science). The following internal oligonucleotides were used for hybridization
probes: Lyp-specific, 5'-AGTCAGCTGTACTAGCAACTGCTC-3';
-actin-specific: 5'-ATCGAGCACGGCATCGTCAC-3'. These were
end-labeled with digoxigenin using the digoxigenin 3' end labeling kit
(Roche Applied Science). Signal was detected by incubating with
chemiluminescent substrate CDP-Star (Tropix, Bedford, MA) followed by
autoradiography.
Fluorescent MicroscopyCells were transfected with Lyp-green
fluorescent protein (GFP), fixed at 24 h post-transfection with 2% neutral
buffered formaldehyde (2% formaldehyde, 20 mM NaPO4, pH
7.4) in Hanks' balanced salt solution for 15 min at 37 °C, washed with
phosphate-buffered saline three times, and examined under a fluorescent
microscope. The fluorescence data that are shown (see
Fig. 3) are representative of
multiple transfection experiments.

View larger version (54K):
[in this window]
[in a new window]
|
FIG. 3. Subcellular localization of Lyp in KCL22 and Cos-7 cells. KCL22 CML
and Cos-7 cells were transiently transfected with Lyp-GFP; 48 h later, cells
were fixed, and Lyp-GFP protein was detected by green fluorescence
microscopy.
|
|
Real-time Quantitative RT-PCRIsolated RNA was
reverse-transcribed as described above. Real-time PCR was performed on iCycler
thermal cycler (Bio-Rad). PCR reaction contained a total volume of 25 µl
and consisted of 5 µl of cDNA, 500 nM of either Lyp or
-actin-specific primer pairs, HotStart TaqDNA polymerase
(Qiagen, Valencia, CA), 0.25 mM dNTPs, and 1 µl of 1:60,000
SYBRgreen I ® (Molecular Probes, Eugene, OR). Triplicate samples were
analyzed for each cDNA. Expression of Lyp was normalized to
-actin
expression.
Western Blot AnalysisCells were lysed with
immunoprecipitation buffer (50 mM Tris, pH 7.6, 5 mM
EDTA, 300 mM NaCl, 1 mM dithiothreitol, and 0.1% Nonidet
P-40) containing protease inhibitors (0.2 mM phenylmethylsulfonyl
fluoride and 1 µg/ml each of leupeptin, pepstatin, and aprotinin). For
immunoprecipitation, 1 mg of lysate was incubated with anti-abl
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 2 h and
binding with protein A/G-Sepharose (Calbiochem) at 4 °C for 1 h. The beads
were washed with lysis buffer and resuspended in the sample buffer before
loading onto a 10% SDS-polyacrylamide gel. For blotting, various antibodies
were used followed by ECL detection (Amersham Biosciences).
Soft Agar Colony AssaysEqual numbers of cells (2,000 cells)
were plated in 0.3% agar containing 20% fetal bovine serum and RPMI on top of
0.5% agar in 24-well plates. Colony formation was counted and photographed
after 2 weeks of culture. Data represent results of triplicate dishes.
 |
RESULTS
|
---|
Distribution of Protein-tyrosine Phosphatase Expression in Myeloid
Leukemic Cell LinesThe distribution of PTPase in myeloid cells was
assessed by RT-PCR using degenerate PTPase primers and cloning of individual
protein-tyrosine phosphatases. Over 371 individual clones were sequenced from
three myeloid cell lines, K562, HL-60, and ML-1. The analysis of a large
number of PTPase-containing clones should provide a good reflection of the
expression pattern of PTPase in human myeloid leukemic cells and may provide
the opportunity to identify a unique PTPase. The K562 cells are
erythroleukemic cells, the ML-1 cells are early myeloblasts, and the HL-60
cells are late myeloblasts. CD45 was the most prominently expressed PTPase,
representing 29, 46, and 57% of the clones isolated from K562, HL-60, and
ML-1, respectively (Table I).
In K562 cells, both CD45 and the PTPase PEST were equally abundant with both
being expressed in the same number of clones (41 of a total of 140). PEST was
the second most prominent PTPase in ML-1 cells (22%). In total, between 8 and
12 different PTPases were identified per cell line with HL-60 myeloblasts
showing the greatest diversity in expression (12 different PTPases). Each cell
line displayed a distinct set of PTPases. PTP
, PTPN9, LCPTP, and PTP-1B
could only be identified in K562 cells. PTYPH, PTPRF, and PTPRO were only
found in HL-60 cells. PTPN7 and PTPD1 were only identified in ML-1 cells,
whereas CD45, PEST, and Lyp were present in all three cell lines.
Expression Pattern of LypLyp has recently been described as
a lymphoid-specific PTPase
(21); thus, we were surprised
to find it to be one of the more frequently expressed PTPases in the three
myeloid cell lines used in this study
(Table I, Fig. 1). Lyp represented 30% of
the PTPase-containing clones in HL-60 cells and 1012% of clones in K562
and ML-1 cells. Therefore, we studied Lyp expression in a panel of human
leukemic cell lines as well as in normal peripheral blood lymphocytes using
semiquantitative RT-PCR with Lyp-specific primers. In the myeloid cell lines,
we observed high levels of Lyp mRNA in KG-1 (very early myeloblasts), HL-60
and ML-1 (myeloblasts), U937 (myelomonoblasts), as well as SK-NO1 and KCL22
(CML blasts) (Fig. 1). The
expression was comparable with that seen in the Jurkat T-cell line
(Fig. 1). In contrast, Lyp was
less robustly expressed in THP-1 (monoblasts), K562 (erythroblasts), Kasumi-1
(mature myeloblasts expressing the AML1-ETO fusion protein), and Kasumi-3
(very early myeloblasts) cells (Fig.
1). We also analyzed a series of colon cancer cell lines and found
very low or undetectectable expression of Lyp (data not shown). Next, we
isolated different types of normal human peripheral white blood cells to
analyze Lyp expression. We found that Lyp was expressed at a higher level in
neutrophils and monocytes as compared with CD3+ T lymphocytes
(Fig. 2).

View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2. Expression of Lyp in peripheral blood white cells. Subpopulations of
human peripheral white blood cells were separated and analyzed by
semiquantitative RT-PCR using either -actin- or Lyp-specific primers.
PMN, polymorphonuclear neutrophils; MNC, peripheral blood
mononuclear cells; Adh. cells, adherent cells (monocytes);
CD3+, peripheral blood T-lymphocytes (which express CD3
antigen).
|
|
The subcellular localization of the Lyp phosphatase was determined. The
Cos-7 and KCL22 CML myeloid blast cells were transiently transfected with an
expression vector of Lyp fused to GFP and examined by fluorescence microscopy
48 h after transfection. We found that Lyp was predominantly cytoplasmic
(Fig. 3).
Role of Lyp in Myeloid CellsThe Lyp phosphatase has
previously been shown to interact with and regulate the tyrosine
phosphorylation of Cbl in T lymphocytes
(21). Cbl itself has been
demonstrated to be a target for tyrosine phosphorylation by the Bcr-Abl
tyrosine kinase (22). Thus, we
were interested to determine whether Lyp might have a role in
Bcr-Abl-expressing CML cells, such as KCL22. Human Lyp cDNA was inserted into
a Zn2+-inducible vector, and the resulting Lyp
expression vector (pMT-Lyp) and the empty vector (pMT-neo) were transfected
separately into KCL22 cells. Stable cell lines were isolated by G418
selection. The induction of Lyp was measured by quantitative real-time PCR for
up to 4 days of exposure to ZnSO4. We found a 3-fold induction of
Lyp mRNA when comparing KCL22 stably transfected with pMT-Lyp to the empty pMT
vector alone after 2 and 3 days of zinc induction
(Fig. 4A). Only after
4 days in the presence of zinc did we observe more than 810-fold of Lyp
mRNA induction. Total cellular proteins were isolated from 2 and 4 days of
zinc induction, and the total cell lysates were analyzed for Lyp protein
expression by Western immunoblotting (Fig.
4B). Because Bcr-Abl is a major protein involved in the
transforming phenotype of KCL22, we looked at the Bcr-Abl protein levels in
these Lyp-overexpressing cells. We found that after 4 days of zinc exposure,
Bcr-Abl protein level is decreased by more than 10-fold
(Fig. 4B).

View larger version (26K):
[in this window]
[in a new window]
|
FIG. 4. Inducible overexpression of Lyp in KCL22 cells. KCL22 cells were
transfected with either pMT-neo or pMT-Lyp expression vector. Single cell
clones were selected with G418. As shown in A, clones were cultured
in the presence of 100 µM ZnSO4 for up to 4 days.
Total cellular RNA was prepared after cells were harvested at day 1, 2, 3, and
4 of zinc induction, and Lyp expression was detected by quantitative real-time
RT-PCR. The endogenous Lyp expression level in KCL22 is designated as
1. Other clones were also studied with results similar to those shown
on Figs. 6,
7,
8. As shown in B,
protein expression of Lyp and Bcr-Abl was detected by sequential Western
blotting with anti-Lyp and anti-Abl antibodies. The anti-Abl antibody
recognized the p210 protein of Bcr-Abl. GAPDH antibody was used as loading
control. Neo, neomycin.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 6. Expression of Lyp in CML patient samples. A, Western blot
analysis of Lyp in CML samples. Lysates were prepared from bone marrow samples
of CML patients, and Western analyses were performed to detect Lyp expression.
CP, chronic phase; A, accelerated phase; BC, blast
crisis phase of CML. Expression of Lyp protein levels on Western blots were
quantitated by densitometry and normalized to GAPDH expression from the same
blots. Samples number 1, 2, and 3 designate paired specimens
from the same patient at different stages of CML. B, real-time PCR
analysis on RNA prepared from CML samples. Lyp expression was normalized to
levels of -actin.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
FIG. 7. Dephosphorylation and degradation of Bcr-Abl caused by expression of
Lyp, associated with decreased levels of Grb2 and Myc. Cos-7 cells were
co-transfected with Bcr-Abl (p185) cDNA and either the GFP or Lyp-GFP
expression vector. Total lysates were prepared 48 h after transfection. In
A, antibodies against GFP were used to demonstrate expression of
either GFP or Lyp-GFP. In B, anti-Lyp antiserum was used to detect
Lyp expression. In D, total lysates were quantitated with anti-GAPDH
to ensure equal amounts of protein in each lane. In C, E, and
F, antibodies against Abl (for Bcr-Abl detection), Grb2, and Myc,
respectively, were used separately to detect the expression of the
protein.
|
|

View larger version (41K):
[in this window]
[in a new window]
|
FIG. 8. Decrease in the phosphorylation levels of CrkL and Erk1/2 after
overexpression of Lyp. Phosphorylation levels of the CrkL and Erk1/2
proteins were examined sequentially using anti-CrkL and anti-phosphorylated
Erk1/2 antibodies. The anti-CrkL antibody recognizes both phosphorylated and
non-phosphorylated CrkL. The results as shown used Cos7 cells; similar results
were obtained using KCL22 cells (data not shown).
|
|
Since Lyp dephosphorylates tyrosine residues, we first probed a Western
blot from the protein lysates with an anti-phosphotyrosine antibody to
determine whether overexpression of Lyp affected the overall level of tyrosine
phosphorylation as compared with the control cells. We observed a decrease in
the level of protein tyrosine phosphorylation upon Lyp overexpression
(Fig. 5A). Of
particular interest was the marked reduction of tyrosine phosphorylation for
proteins of 120 and 210 kDa (Fig.
5A). We proposed that these proteins were most likely Cbl
and Bcr-Abl, respectively, based on their molecular sizes. Therefore, the
Western immunoblot was reprobed with antibodies against these two proteins,
which confirmed that the proteins at 120 and 210 kDa were indeed Cbl and
Bcr-Abl (Fig. 5, B and
C). A comparison of the Lyp-overexpressing KCL22
versus control KCL22 cells showed that the overall amount of Cbl
protein was unaffected by the induction of Lyp
(Fig. 5B). However,
when the immunoblot was reprobed with an antibody against Abl, we observed
that overexpression of Lyp resulted in a decrease in Bcr-Abl protein
expression (Fig. 5C),
similar to what was observed above (Fig.
4B).

View larger version (57K):
[in this window]
[in a new window]
|
FIG. 5. Reduction of tyrosine phosphorylation of proteins in KCL22 cells which
overexpressed Lyp. Total proteins were prepared from KCL22 CML cells after
4 days of exposure to 100 µM ZnSO4. Lysates were
analyzed by SDS-PAGE followed by sequential immunoblotting with antibody
against phosphotyrosine (pTyr), Cbl, and Abl and then visualized by ECL.
Neo, neomycin.
|
|
We also examined the expression of Lyp protein in the leukemic cells from
patients with CML. CML has a chronic phase, a short accelerated phase,
evolving into the short-lived blast crisis. Lyp was expressed in these samples
at various levels, and no correlation was noted between the amount of
expression of Lyp protein and stage of the CML
(Fig. 6A). Similar
observations were made when looking at Lyp RNA levels in CML patients by
real-time PCR, although the number of cases was small
(Fig. 6B).
To investigate further the role of Lyp in the regulation of Bcr-Abl, we
transiently co-transfected Cos-7 cells with an expression vector for Bcr-Abl
and either Lyp-GFP or GFP alone. Total cellular proteins were isolated and
analyzed by Western immunoblots. The use of an anti-GFP antibody and Lyp
antiserum demonstrated the expression of the Lyp phosphatase in these cells
co-transfected with Lyp-GFP and Bcr-Abl
(Fig. 7, A and
B). Negligible Bcr-Abl could be detected from the Cos-7
cells co-transfected with Bcr-Abl and the Lyp-GFP
(Fig. 7C). GAPDH
immunoblotting demonstrated that equal amounts of protein lysates were used
(Fig. 7D). These
results suggest that the expression of Lyp causes a decrease in Bcr-Abl
protein expression, presumably via regulating its degradation.
Activation of the Ras signaling pathway by Bcr-Abl involves a series of
molecular events including direct interaction between the adaptor protein Grb2
and the phosphorylated Bcr-Abl
(23), induction of Myc protein
(24), and phosphorylation of
CrkL and Erk1/2. We found that when Lyp was expressed, levels of Bcr-Abl and
Grb2 markedly decreased, and a slight reduction of Myc protein levels was
observed (Fig. 7E).
Also, a prominent decrease of CrkL and Erk1/2 phosphorylation occurred in
these Lyp-expressing cells (Fig.
8).
The effect of Lyp overexpression on anchorage-independent clonal growth in
soft agar of KCL22 CML cells was investigated. KCL22 pMT-neo formed robust
colonies in soft agar (Fig. 9).
In comparison, KCL22 pMT-Lyp cells had markedly decreased clonal growth, both
in number and size of the colonies, suggesting that overexpression of Lyp
inhibited the transforming potential of KCL22 CML cells
(Fig. 9).

View larger version (17K):
[in this window]
[in a new window]
|
FIG. 9. Overexpression of Lyp inhibits transforming potential of KCL22 CML cells
in soft agar. An equal number of either KCL22-pMT-Lyp or KCL22-pMT-neo
were seeded in 1% agar containing 10% fetal bovine serum, RPMI culture media,
and 100 µM ZnSO4 on top of 1.5% agar. Colony
formation was counted, and dishes were photographed 2 weeks after plating the
cells. Colonies containing 100 cells were enumerated. Data represent the
mean ± S.D. of triplicate plates.
|
|
 |
DISCUSSION
|
---|
In this study, we identified a number of PTPases in three human myeloid
leukemic cell lines. Of these, CD45, PEST, and Lyp were the most prevalent.
CD45, also known as the leukocyte common antigen, is an integral membrane
protein expressed on all nucleated hematopoietic cells
(25,
26). It regulates the activity
of the Src family of kinases
(27). Since CD45 has been
shown to be expressed on all hematopoietic lineages and stages of development,
we were not surprised that it was the predominant PTPase detected in our
study. PEST, a major PTPase identified in K562 cells, has been described as an
important regulator of cell migration
(28) and has been suggested to
have a role in leukemia through an interaction with paxillin
(29). The third major PTPase
detected in the myeloid cells was Lyp. Previously, Lyp was identified as a
lymphocyte-specific PTPase
(21). We found that Lyp
represented 31% of the 155 clones obtained from the late myeloblast HL-60
cells, and a substantial amount of Lyp expression was found in several myeloid
cell lines including myelomonoblastic U937 cells and the very early
myeloblastic KG-1 cells. Expression of Lyp can also be found in peripheral
blood granulocytes and monocytes, suggesting a potential function of Lyp in
myeloid cell development.
Lyp has cytoplasmic expression and some perinuclear staining in Jurkat
cells (21). We showed in KCL22
CML cells that Lyp is localized to the cytoplasm, where it may interact with
Bcr-Abl and play a role in regulating the signaling of this transforming
fusion protein. We found that overexpression of Lyp resulted in a decrease in
the expression of Bcr-Abl as well as a reduction in the phosphorylation levels
of Cbl. The decrease in Cbl tyrosine phosphorylation is probably the result of
Lyp phosphatase activity independent of Bcr-Abl function since Cbl has been
shown to be a target of Lyp activity in T cells not containing Bcr-Abl
(21). In T cells, Lyp
regulates T cell receptor signaling by associating with and dephosphorylating
Cbl. Alternatively, Cbl is a substrate of Bcr-Abl tyrosine kinase, and
down-regulation of Bcr-Abl would cause a reduction in the level of Cbl
phosphorylation.
CML is caused in part by activation of various signaling pathways by the
aberrant tyrosine kinase activity of the Bcr-Abl fusion protein. This active
tyrosine kinase phosphorylates a number of signaling proteins, including
STAT5, SHC, Cbl, Grb2, paxillin, and CrkL. The interaction of Bcr-Abl with Cbl
probably causes activation of the phosphatidylinositol 3-kinase pathway
(14), and interaction between
Bcr-Abl with Grb2 can lead to the activation of Ras signaling. Stimulation of
both of these pathways is important for Bcr-Abl-induced transformation. Either
a dominant-negative form of Grb2
(30) or Bcr-Abl with mutation
in the Grb2-binding SH2 domain can suppress Bcr-Abl transformation
(31) and leukemogenesis
(32). More recently, Lyp has
also been shown to interact with the adaptor molecule Grb2
(33). In this instance,
wild-type Lyp, but not a catalytically inactive Lyp, had a negative regulatory
role in T cell signaling via regulation of interactions of Grb2 with the T
cell receptor.
In KCL22 CML cells, lower Grb2 expression levels were found when Lyp was
overexpressed. The SH3 domain of Grb2 interacts with Sos (the guanine
nucleotide releasing factor son of sevenless) and stimulates Ras and the MAPK
kinase pathways. Inhibition of CML blast cell proliferation can be induced by
disruption of Grb2-Sos complexes
(34). Potentially, the
degradation of Grb2 can be a direct effect of overexpression of Lyp, and
disruption of the Grb2-Sos complex leads to inactivation of Erk1/2 and
consequently contributes to the loss of the transforming potential of KCL22
cells in the soft agar assay. Decreased phosphorylation of CrkL may partially
contribute to the loss of transforming potential of Lyp-overexpressing cells.
Multiple tyrosine residues are phosphorylated in CrkL when activated by
Bcr-Abl, and mutations or deletions in CrkL diminish cell transformation and
adhesion in both fibroblasts and hematopoietic cells
(35).
To date, a major focus of basic CML research has focused on the aberrant
tyrosine kinase activity of Bcr-Abl and the consequences of tyrosine
phosphorylation. However, intracellular levels of tyrosine phosphorylation are
controlled by the opposing actions of kinases and phosphatases. Bcr-Abl can
interact with and regulate phosphatases. In some instances, Bcr-Abl
phosphorylates these phosphatases, and subsequently, these phosphatases can
dephosphorylate Bcr-Abl, resulting in a decrease in Bcr-Abl kinase activity
(36). For example, Bcr-Abl
up-regulates the expression of PTP-1B PTPase, which in turn dephosphorylates
Bcr-Abl, resulting in the inhibition of Grb2 binding and suppression of Ras
activity (37). Bcr-Abl has
been shown to interact with and to regulate the expression of SHIP, an
SH2-containing inositol phosphatase that regulates the phosphatidylinositol
3-kinase signaling pathway
(38). Bcr-Abl can directly
inhibit the expression of SHIP phosphatase by down-regulating the amount of
mRNA as well as decreasing the half-life of the protein
(39). Since SHIP
down-regulates the activity of the phosphatidylinositol 3-kinase signaling
pathway, direct control of this negative regulator by Bcr-Abl can result in
increased myeloid proliferation, as was observed in SHIP/ mice
(40). In previous studies that
showed an interaction of a phosphatase such as PTP1B with Bcr-Abl, the Bcr-Abl
kinase activity decreased, but no change occurred in the overall expression of
the fusion protein (36,
37). Our study is, to our
knowledge, the first description of a phosphatase regulating Bcr-Abl signaling
via alteration of the levels of Bcr-Abl protein. The mechanism by which this
occurs has yet to be determined. We attempted experiments to determine whether
Bcr-Abl was ubiquitinated by Lyp. The results of these experiments showed that
Bcr-Abl was not ubiquitinated by co-expression of Lyp (data not shown). In
K562 cells, the molecular chaperone Hsp90 forms a complex with Bcr-Abl,
extending the half-life of Bcr-Abl
(41). By interfering with the
function of Hsp90, geldanamycin or its analog 17-allyamnogeldanamycin can
induce Bcr-Abl protein degradation
(41). Perhaps overexpression
of Lyp disrupts the association between the Hsp90 and Bcr-Abl complex and
promotes Bcr-Abl degradation.
In conclusion, we showed that Lyp is expressed in the myeloid cell lineage,
and Lyp overexpression in KCL22 cells induces Bcr-Abl and Grb2 degradation, as
well as dephosphorylation of Cbl, CrkL, and Erk1/2. Overexpression of Lyp
inhibits transforming potential of KCL22 cells. Our data suggest that Lyp may
behave as a tumor suppressor.
 |
FOOTNOTES
|
---|
* This work was supported in part by grants from the National Cancer
Institute of Canada and the NCI, National Institutes of Health, as well as the
C. and H. Koeffler Fund and the Parker Hughes Fund. The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
A recipient of a fellowship of the Deutsche Forschungsgemein-schaft. 
** Holds the Donald and Audrey Campbell Chair of Immunology in the Hospital
for Sick Children in Toronto, Canada. 

Holds the Mark Goodson Chair in Oncology Research and is a member of the
Molecular Biology Institute and the Jonsson Cancer Center of UCLA. 
To whom correspondence should be addressed: Dept. of Hematology/Oncology,
Cedars-Sinai Medical Center, 110 George Burns Rd., D5065, Los Angeles, CA
90048. Tel.: 310-423-7759; Fax: 310-423-0225; E-mail:
chienw{at}cshs.org.
1 The abbreviations used are: CML, chronic myeloid leukemia; PTPase,
protein-tyrosine phosphatase; Erk, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein kinase; SHIP, Src homology 2 domain containing
inositol phosphatase; GFP, green fluorescent protein; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription. 
 |
REFERENCES
|
---|
- Lugo, T. G., Pendergast, A. M., Muller, A. J., and Witte, O. N.
(1990) Science
247,
10791082[Medline]
[Order article via Infotrieve]
- Daley, G. Q., Van Etten, R. A., and Baltimore, D.
(1990) Science
247,
824830[Medline]
[Order article via Infotrieve]
- Laneuville, P. (1995) Semin.
Immunol. 7,
255266[CrossRef][Medline]
[Order article via Infotrieve]
- Plattner, R., Kadlec, L., DeMali, K. A., Kazlauskas, A., and
Pendergast, A. M. (1999) Genes Dev.
13,
24002411[Abstract/Free Full Text]
- Ito, Y., Pandey, P., Mishra, N., Kumar, S., Narula, N., Kharbanda,
S., Saxena, S., and Kufe, D. (2001) Mol. Cell.
Biol. 21,
62336242[Abstract/Free Full Text]
- Nowell, P. C., and Hungerford, D. A. (1960)
Science 132,
1497
- Rowley, J. D. (1973) Nature
243,
290293[Medline]
[Order article via Infotrieve]
- Daley, G. Q., and Baltimore, D. (1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
93129316[Abstract]
- Deininger, M. W., Goldman, J. M., and Melo, J. V.
(2000) Blood
96,
33433356[Free Full Text]
- Odai, H., Sasaki, K., Iwamatsu, A., Hanazono, Y., Tanaka, T.,
Mitani, K., Yazaki, Y., and Hirai, H. (1995) J. Biol.
Chem. 270,
1080010805[Abstract/Free Full Text]
- Barber, D. L., Mason, J. M., Fukazawa, T., Reedquist, K. A.,
Druker, B. J., Band, H., and D'Andrea, A. D. (1997)
Blood 89,
31663174[Abstract/Free Full Text]
- Levkowitz, G., Klapper, L. N., Tzahar, E., Freywald, A., Sela, M.,
and Yarden, Y. (1996) Oncogene
12,
11171125[Medline]
[Order article via Infotrieve]
- Salgia, R., Sattler, M., Pisick, E., Li, J. L., and Griffin, J. D.
(1996) Exp. Hematol.
24,
310313[Medline]
[Order article via Infotrieve]
- Sattler, M., Salgia, R., Okuda, K., Uemura, N., Durstin, M. A.,
Pisick, E., Xu, G., Li. J. L., Prasad, K. V., and Griffin, J. D.
(1996) Oncogene
12,
839846[Medline]
[Order article via Infotrieve]
- Tonks, N. K., and Neel, B. G. (1996)
Cell 87,
365368[Medline]
[Order article via Infotrieve]
- Fischer, E. H. (1999) Adv. Enzyme
Regul. 39,
359369[CrossRef][Medline]
[Order article via Infotrieve]
- Li, L., and Dixon, J. E. (2000) Semin.
Immunol. 12,
7584[CrossRef][Medline]
[Order article via Infotrieve]
- Alexander, D. R. (2000) Semin.
Immunol. 12,
349359[CrossRef][Medline]
[Order article via Infotrieve]
- Kim, H., and Baumann, H. (1999) Mol. Cell.
Biol. 18,
53265338
- Andersen, J. N., Mortensen, O. H., Peters, G. H., Drake, P. G.,
Iversen, L. F., Olsen, O. H., Jansen, P. G., Andersen, H. S., Tonks, N. K.,
and Moller, N. P. (2001) Mol. Cell. Biol.
21,
71177136[Free Full Text]
- Cohen, S., Dadi, H., Shaoul, E., Sharfe, N., and Roifman, C. M.
(1999) Blood
93,
20132024[Abstract/Free Full Text]
- de Jong, R., ten Hoeve, J., Heisterkamp, N., and Groffen, J.
(1995) J. Biol. Chem.
270,
2146821471[Abstract/Free Full Text]
- Pendergast, A. M., Quilliam, L. A., Cripe, L. D., Bassin, C. H.,
Dai, Z., Li, N., Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and
Gishizky, M. L. (1993) Cell
75,
175185[Medline]
[Order article via Infotrieve]
- Cortez, D., Kadlec, L., and Pendergast, A. M. (1995)
Mol. Cell Biol. 15,
55315541[Abstract]
- Streuli, M., Hall, L. R., Saga, Y., Schlossman, S. F., and Saito,
H. (1987) J. Exp. Med.
166,
15481566[Abstract]
- Sasaki, T., Sasaki-Irie, J., and Penninger, J. M.
(2001) Int. J. Biochem. Cell Biol.
33,
10411046[CrossRef][Medline]
[Order article via Infotrieve]
- Ishikawa, H., Tsuyama, N., Abroun, S., Liu, S., Li, F. J.,
Taniguchi, O., and Kawano, M. M. (2002)
Blood 99,
21722178[Abstract/Free Full Text]
- Garton, A. J., and Tonks, N. K. (1999) J.
Biol. Chem. 274,
38113818[Abstract/Free Full Text]
- Shen, Y., Lyons, P., Cooley, M., Davidson, D., Veillette, A.,
Salgia, R., Griffin, J. D., and Schaller, M. D. (2000)
J. Biol. Chem. 275,
14051413[Abstract/Free Full Text]
- Gishizky, M. L., Cortez, D., and Pendergast, A. M.
(1995) Proc. Natl. Acad. Sci. U. S. A.
92,
1088910893[Abstract]
- Goga, A., McLaughlin, J., Afar, D. E., Saffran, D. C., and Witte,
O. N. (1995) Cell
82,
981988[Medline]
[Order article via Infotrieve]
- Million, R. P., and Etten, R. A. (2000)
Blood 96,
664670[Abstract/Free Full Text]
- Hill, R. J., Zozulya, S., Lu, Y. L., Ward, K., Gishizky, M., and
Jallal, B. (2002) Exp. Hematol.
30,
2372344[CrossRef][Medline]
[Order article via Infotrieve]
- Kardinal, C., Konkol, B., Lin, H., Eulitz, M., Schmidt, E. K.,
Estrov, Z., Talpaz, M., Arlinghaus, R. B., and Feller, S. M.
(2001) Blood
98,
17731781[Abstract/Free Full Text]
- Senechal, K., Heaney, C., Druker, B., and Sawyers, C. L.
(1998) Mol. Cell. Biol.
18,
50825090[Abstract/Free Full Text]
- LaMontagne, K. R., Jr., Hannon, G., and Tonks, N. K.
(1998) Proc. Natl. Acad. Sci. U. S. A.
95,
1409414099[Abstract/Free Full Text]
- LaMontagne, K. R., Jr., Flint, A. J., Franza, B. R., Jr.,
Pandergast, A. M., and Tonks, N. K. (1998) Mol. Cell.
Biol. 18,
29652975[Abstract/Free Full Text]
- Tauchi, T., Feng, G. S., Shen, R., Song, H. Y., Donner, D., Pawson,
T., and Broxmeyer, H. E. (1994) J. Biol.
Chem. 269,
1538115387[Abstract/Free Full Text]
- Sattler, M., Salgia, R., Shrikhande, G., Verma, S., Choi, J. L.,
Rohrschneider, L. R., and Griffin, J. D. (1997)
Oncogene 15,
23792384[CrossRef][Medline]
[Order article via Infotrieve]
- Liu, Q., Sasaki, T., Kozieradzki, I., Wakeham, A., Itie, A.,
Dumont, D. J., and Penninger. J. M. (1999) Genes
Dev. 13,
786791[Abstract/Free Full Text]
- An, W. G., Schulte, T. W., and Neckers, L. M. Cell
Growth & Differ. (2000)
11,
355360[Abstract/Free Full Text]
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.