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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2013-2024
Cloning and Characterization of a Lymphoid-Specific, Inducible
Human Protein Tyrosine Phosphatase, Lyp
By
Shai Cohen,
Harjit Dadi,
Ester Shaoul,
Nigel Sharfe, and
Chaim M. Roifman
From the Division of Immunology and Allergy, Department of
Paediatrics, Infection, Immunity, Injury and Repair Programme, Research
Institute, The Hospital for Sick Children and the University of
Toronto, Toronto, Ontario, Canada.
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ABSTRACT |
Protein tyrosine phosphatases act in conjunction with protein
kinases to regulate the tyrosine phosphorylation events that control
cell activation and differentiation. We have isolated a previously
undescribed human phosphatase, Lyp, that encodes an intracellular
105-kD protein containing a single tyrosine phosphatase catalytic
domain. The noncatalytic domain contains four proline-rich potential
SH3 domain binding sites and an NXXY motif that, if phosphorylated, may
be recognized by phosphotyrosine binding (PTB) domains. Comparison of
the Lyp amino acid sequence with other known proteins shows 70%
identity with the murine phosphatase PEP. The human Lyp gene was
localized to chromosome 1p13 by fluorescence in situ hybridization
analysis. We also identified an alternative spliced form of Lyp RNA,
Lyp2. This isoform encodes a smaller 85-kD protein with an alternative
C-terminus. The lyp phosphatases are predominantly expressed in
lymphoid tissues and cells, with Lyp1 being highly expressed in
thymocytes and both mature B and T cells. Increased Lyp1 expression can
be induced by activation of resting peripheral T lymphocytes with
phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively
associated with the proto-oncogene c-Cbl in thymocytes and T cells.
Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting
that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PROTEIN TYROSINE phosphorylation, a key
mechanism of cellular signal transduction, is regulated by the action
of both protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases (PTPases). Originally, PTKs were believed to control the
process of tyrosine phosphorylation, with a small number of PTPases
playing largely housekeeping roles. Unexpectedly, the structural
diversity of the growing number of PTPases has called this idea into
question, and it has become apparent that PTPases have important roles
in the regulation of growth and differentiation in both normal and neoplastic cells.1,2
All PTPases contain a catalytic domain of approximately 200 to 300 residues, including a subset of highly conserved amino acids that play
a role in substrate recognition and tyrosine
dephosphorylation.3 The PTPase family can be divided
broadly into two major classes: membrane-bound receptors and
intracellular phosphatases,4,5 both dividing into
subfamilies based on their sequence similarities and noncatalytic
domain structural motifs.6,7 The receptor PTPases may
contain one or two intracellular phosphatase domains and often Ig-like
and fibronectin-like extracellular regions6 that play a
role in cell-cell or cell-matrix interactions.8 Some
receptor PTPases appear to participate in homophilic and hetrophilic
binding interactions, which is suggestive of a role in cell guidance
and contact inhibition.7,8
The nonreceptor phosphatases display various intracellular
localizations determined by amino acid sequences outside the catalytic domain.9-11 Some also contain conserved noncatalytic
domains such as SH2 and SH3, allowing them to interact with tyrosine
phosphorylated proteins and proteins containing proline-rich sequences,
respectively.12,13 Cytoplasmic PTPases have been found
associated with a variety of PTKs, including Csk and the Jak kinases
and a number of cytokine and antigen receptors.7,13-15
Within the immune system several lines of evidence indicate that
PTPases are essential for lymphocyte development and activation. CD45,
a transmembrane phosphatase expressed exclusively in hematopoetic cells,16 is required for antigenic activation of B and T
lymphocytes.17,18 CD45-deficient mice show that CD45 also
plays a pivotal role in thymic development and T-cell
apoptosis.19,20 Recent studies have shown that the
hematopoetic-specific intracellular phosphatase SHP-1 negatively
regulates signaling through the B-cell receptor, Fc RIIB1,21 and the inteleukin-3 (IL-3) receptor  chain.22 SHP1 also participates in T-cell signaling events
through dephosphorylation of the T-cell receptor, p56lck, and
Zap-70.23 Mutations in the murine motheaten locus,
encoding the SHP1 protein, result in severe combined immunodeficiency
and systemic autoimmunity, as well as many other hematopoietic
abnormalities.24 Furthermore, expression of HePTP, a
hematopoetic-specific cytoplasmic PTPase, is induced in lymphocytes
stimulated by phytohemagglutinin (PHA), concavalin A,
lipopolysaccharide, and anti-CD3,25 which is suggestive of a role in mature lymphocyte signaling pathways. All of these studies suggest a critical role for phosphatases not only in regulating signaling, but also in the development of the immune system.
Our search for PTPases involved in lymphocyte growth and development
led to the isolation of a novel human cytoplasmatic phosphatase that is
predominantly expressed in lymphoid cells, designated lymphoid
phosphatase (Lyp). The Lyp gene was localized to human chromosome 1p13.
The Lyp protein is closely related (70% homology) to the murine
phosphatase PEP.12 Lyp is expressed in both immature and
mature B and T cells. The level of Lyp protein expression was found to
increase significantly upon stimulation of resting peripheral T
lymphocytes with PHA or anti-CD3. We also describe an isoform of Lyp
(Lyp2) that is the product of C-terminal alternative RNA splicing and
that demonstrates a different pattern of RNA expression to Lyp1. In
contrast to Lyp1, Lyp2 protein could only be detected in resting T cells.
Upon anti-CD3 stimulation of human thymocytes, a 116-kD phosphorylated
protein was found to become associated with Lyp1. This protein was
subsequently identified as the molecular adapter protein c-Cbl.26 Furthermore, expression of Lyp1 in COS cells
results in a reduction in endogenous cbl phosphorylation, suggesting a role for Lyp in regulating the function of cbl in the TCR signaling pathway.
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MATERIALS AND METHODS |
Polymerase chain reaction (PCR) and subcloning of PTPases clones.
Total RNA was prepared from thymocytes using Trizol reagent (GIBCO-BRL,
Gaithersburg, MA). First-strand cDNA synthesis was performed with oligo-dT primer and Superscript II RT (GIBCO-BRL). This
was used as a template for PCR amplification with Taq DNA polymerase
(Perkin Elmer Cetus, Norwalk, CT) and the following degenerate primers: PTP1, GCGGATCCTCIGA(C/T)TA(C/T)AT(A/C/T)AA(T/C)GC (sense); and PTP2, GCGAATTCCCIACICCIGC(A/G)CT(G/A)CA(G/A)TG
(antisense). These degenerate primers are designed to match two highly
conserved sequences within PTPase catalytic domains, XDYINA and
HCSAGI/VG, respectively. PCR was performed as follows: five cycles of
60 seconds at 94°C, 30 seconds at 37°C, and 60 seconds at
72°C and a further 25 cycles with an annealing temperature of
45°C. The PCR products (~400 bp) were isolated, cloned, and sequenced.
Isolation and sequencing of Lyp1 and Lyp2 cDNA clones.
An oligo-dT-derived  gt10 DNA library from human thymocytes was
screened with a 32P-labeled 430-bp Lyp1 fragment obtained
by PCR. Plaques were transferred to ICN Biotrans nylon filters
(ICN, Costa Mesa, CA) and screened by hybridization at
65°C in 5× SSC, 5× Denhart's solution, 0.1% sodium
dodecyl sulfate (SDS).27 Phage DNA was prepared from
positive plaques, and cDNA inserts were excised, subcloned into pUC19,
and sequenced. To obtain the complete Lyp1 cDNA, secondary and tertiary
library screenings were performed with 1.3-kb and 0.6-kb partial Lyp
cDNA clones isolated in the first screening
(Fig 1). One clone (P5) from the second
screening was found to contain the carboxy-terminal sequence of the
alternatively spliced form of Lyp1 (Lyp2).
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| Fig 1.
Schematic diagram of Lyp1 and Lyp2 deduced from the cDNA
clones. Boxes indicate the open reading frame, with thin lines
representing the 5' and 3' untranslated regions. The PTPase
catalytic domain is colored in gray. The six overlapping cDNA clones
obtained from a human thymus cDNA library (bold black lines) are shown
under the schematic structures of the cDNAs.
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Fluorescence in situ hybridization (FISH) detection and image
analysis.
A 1.8-kb Lyp cDNA fragment was used as a probe to examine the
chromosomal location of the human Lyp gene. Biotinylated Lyp probe was
prepared by nick translation for FISH to normal human lymphocyte
chromosomes (counterstained with propidium iodide and 4',6-diamidin-3-phenylindol-dihydrochloride [DAPI] according to published methods28,29). The probe was detected with
avidin-fluorescein isothiocyanate (FITC), followed by biotinylated
antiavidin antibody and avidin-FITC. Images of metaphase preparations
were captured by a thermoelectrically cooled charge coupled camera
(Photometrics, Tucson, AZ). Separate images of DAPI-banded
chromosomes30 and FITC-targeted chromosomes were obtained
and merged electronically using image analysis software (courtesy of
Tim Rand and David Ward, Yale University, New Haven, CT) and
pseudo-colored blue (DAPI) and yellow (FITC) as described by Boyle et
al.29 The band assignment was determined by measuring
the fractional chromosome length and by analyzing the banding pattern
generated by the DAPI counterstained image.31
Northern blot analysis.
Total RNA was extracted from thymocytes using Trizol reagent
(GIBCO-BRL). Poly A+ RNA was isolated by two passages
through an oligo(dT) column. Two micrograms of Poly A+ RNA
per lane was electrophoresed in a 1% agarose formaldehyde gel and
capillary blotted onto nitrocellulose filters. Filters and human
multiple tissue poly A+ RNA membrane (Clontech, La Jolla,
CA) were hybridized overnight at 42°C with
32P-labeled Lyp cDNA probes in 50% formamide, 5×
SSC, 5× Denhart's solution, 0.1% SDS, 50 mmol/L
Na2HPO4, pH 6.5, and denatured Salmon sperm DNA
(100 µg/mL). After hybridization, the final wash was performed in
0.2% SSC, 0.1% SDS at 55°C.27
Lymphocyte isolation.
Thymuses were obtained from children undergoing open heart surgery.
Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation. Adherent cells were removed by incubation to
plastic dishes for 60 minutes at 37°C. The resulting thymocytes are
typically greater than 95% CD3+. Lymphocytes were
isolated from tonsil tissue or from peripheral blood of healthy
volunteers by Ficoll-Hypaque gradient centrifugation, followed by rosetting with neuraminidase-treated sheep red blood cells
(RBCs) to isolate T lymphocytes. After isolating rosettes on
Ficoll-Hypaque, T cells were released by ACT treatment (0.75% NH4Cl in 20 mmol/L Tris, pH 7.2) of the rosettes to lyse
the RBCs. The buffy layer, containing the B cells, was washed three
times with phosphate-buffered saline (PBS). The resultant T lymphocytes are typically 98% to 99% CD3+ and the B
lymphocytes from tonsils are typically 85% to 90% CD19+.
To induce activation and maturation of peripheral T lymphocytes, 25 × 106 T cells were stimulated with 2.5 µg/mL of
anti-CD3 (Calbiochem) or 10 µg/mL of PHA (GIBCO-BRL) for 24 to 48 hours at 37°C in RPMI (10% fetal calf serum [FCS]).
Cell lines.
The OCI/AML3 cell line was kindly provided by Dr Mark D. Minden
(Ontario Cancer Institute, Toronto, Ontario, Canada). The origin and properties of this myeloid cell line have been previously described.32 The G2 pre-pre-B-cell line was derived from a
patient with acute lymphocytic leukemia.33 All of the other
cell lines used for this study were obtained from the American Type
Culture Collection (Rockville, MD). All cells were
maintained in RPMI 1640 containing 10% FCS.
Antibodies.
Rabbit polyclonal antibodies were raised to a mixture of two peptides
of LyP with the amino acid sequences RTKSTPFELIQQR and SKMSLDLPEKQDG.
These peptides were chosen from a potentially exposed area, as
predicted by Hopp and Woods,33a in the
noncatalytic domain. A second polyclonal antibody was raised to a
bacterial fusion protein of the catalytic domain of LyP (Pet vector;
Novagen, Madison, WI). After careful testing, these
antibodies were used for immunoprecipitation and Western blotting. T7
antibody was purchased from Novagen (WI); anti-cbl, anti-Jak3, and
anti-p110 were purchased from Santa Cruz Biotech (Santa Cruz, CA); and
antiphosphotyrosine was purchased from from UBI (Lake Placid, NY).
Transfection of COS-7 cells.
COS-7 cells (0.5 × 106) were transfected with 5 µg
plasmid DNA in 50 µL of Lipofectamine (GIBCO-BRL) for 5 hours
according to the manufacturer's instructions. Forty-eight hours after
transfection, the COS-7 cells were harvested and solubilized in cold
lysis buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA,
1% NP 40, and 1 mmol/L phenylmethyl sulfonyl fluoride
[PMSF]).
Immunoprecipitation and Western blotting.
One percent NP-40 cell lysates were precleared by centrifugation.
Immunoprecipitation of T7-tagged Lyp was performed by the addition of 1 µg of T7 antibody or by the addition of 5 µL of the lyp antiserum
followed by the addition of 20 µL of a 50:50 suspension of protein G
sepharose (Pharmacia, Uppsala, Sweden) and incubation
overnight at 4°C. Immunoprecipitates were washed three times with
lysis buffer and separated by 6% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). The separated proteins were
electrophortically transferred to Hybond C Super nitro-cellulose
membrane (Amersham Life Science, Arlington Heights, IL).
Membranes were blocked with 5% nonfat milk and blotted with anti-T7
(1:10,000) or with anti Lyp (1:800). Detection was performed with
horseradish peroxidase-conjugated second antibodies from Amersham Life
Science and chemiluminescence reagent from Kirkeggard & Perry
Laboratories (Gaithersburg, MD).
Intracellular localization by indirect immunofluorescence.
Lyp1 and Lyp2 were inserted into the pCDNA3 eucaryotic expression
vector (Invitrogen, San Diego, CA) and a T7 tag or HA
epitope (YPYDVPDYA), as a three-tandem repeat, was inserted at the
5' end of the coding sequences. Constructs were verified by
sequencing. COS-7 cells were transfected with 2 µg DNA and 17 µL of
lipofectamine for 5 hours, incubated on sterile cover slips in 6-well
plates (0.3 × 106/plate) in Dulbecco's modified
Eagle's medium (DMEM) containing 10% FCS, and stained 48 hours
posttransfection. The COS-7 cells were then washed in PBS and fixed for
30 minutes at room temperature in 2% paraformaldehyde. Cell
permeabilization was performed with 0.1% Triton X100 and, after
blocking nonspecific sites with 5% donkey serum, the cells were
incubated with monoclonal anti-HA (1:1,000) from Baco-Berkely
(Richmond, CA) for 60 minutes at room temperature.
The cells were washed and exposed for 45 minutes to
cy3-conjugated affinipure Donkey antimouse IgG (1:1000 in PBS) from Jackson Immunoresearch Laboratories, Inc. (West Grove,
PA). After 3 to 4 washes, immunoreactivity was detected
by fluorescence microscopy.
Phosphatase assay.
The synthetic peptide Raytide was phosphorylated according to the
method described by Guan et al34 on tyrosine by p60src (Oncogene Research Products, Cambridge, MA) as follows:
10 µg Raytide in 50 mmol/L HEPES, pH 7.5, 10 mmol/L
MgCl2, 0.067% -mercapto-ethanol, 0.05 mmol/L ATP was
incubated with 300 µCi 33P ATP/mL and 2 µg p60src in
a final volume of 30 µL. The reaction was allowed to proceed for 30 minutes at 30°C and was stopped by the addition of 120 µL 10%
phosphoric acid.
The sample was spotted onto two 1 × 1 cm sheets of P81
phosphocellulose paper and extensively washed with 0.5% phosphoric acid. Phosphorylated peptide was eluted twice with 1 mL 500 mmol/L (NH4)2CO3, lyophilized, and resuspended in 100 µL H2O.
The phosphorylated substrate was used in the phosphatase assay as
described by Stueli et al.35 The phosphatase assay mixture (50 µL) contains 5 µL of ×10 phosphatase buffer (250 mmol/L
HEPES, pH 7.3, 50 mmol/L EDTA, 100 mmol/L dithiothreitol), 5 µL of
radioactive substrate (Raytide), and 5 µL of sample (Lyp
immunoprecipitate) and H2O to final volume.
The assay was allowed to proceed at 30°C for the indicated time and
the reaction was terminated by the addition of 750 µL of a charcoal
mixture (0.9 mol/L HCl, 90 mmol/L sodium pyrophosphate, 2 mmol/L
NaH2PO4, 4% vol/vol Norit A). After
centrifugation, the free 33P in the supernatant was measured.
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RESULTS |
Isolation of novel human PTPases.
To identify novel members of the PTPase gene family that are expressed
in thymocytes, we used a PCR-based approach with degenerate oligonucleotides directed at conserved regions of the PTPase catalytic domain. We amplified a fragment of approximately 400 bp from thymocyte cDNA and identified clones corresponding to seven different
phosphatases. Six clones were identical to previously isolated human
phosphatases: PTP-PEST,36 PTP1B,37
TCPTP,38 HPTP ,6 CD45, and
PTPMEG2.39 A seventh clone had no known human homologue but
was 90% homologous to a portion of the catalytic domain of the murine
phosphatase PEP.12 This clone was used to screen a human
thymocyte cDNA library. The first screening isolated two overlapping
clones: P1 and P2 (Fig 1). Clone P2 was used to isolate a further three overlapping clones, P3, P4, and P5, from the cDNA library. Assembly of
the five overlapping clones showed a single cDNA of 2,300 bp containing
an open reading frame (ORF) of 2,076 bp, predicting a protein of 692 amino acids. The sequence surrounding the putative ATG/methionine start
codon contained a purine (A) at position  3 and G at +4, both
regarded as important criteria for an eucaryotic initiation
site.40 The N-terminal region of the amino acid sequence (Fig 2) contained a single PTPase catalytic
domain characterized by the conserved sequence motif (I/V)HCXXGXXRS/T.
This sequence is thought to form the phosphate binding pocket for
substrate, is found in all PTPases, and is essential for their
enzymatic activity.3 In addition to the five overlapping
clones, a single 1-kb clone was isolated (P6; Fig 1) with the 200 bp of
its 5'-end overlapping nucleotides 1950 through 2055 of the
complete cDNA previously isolated. However, this was followed by an
alternative 700 bp, encoding an ORF totalling 2,424 bp (808 amino
acids). The long and short forms share nucleotides 1 through 2097 but contain alternative C-terminal sequences. We have designated these forms Lyp1 (long) and Lyp2 (short). We suggest that LyP2 is an alternative spliced isoform of Lyp1.
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| Fig 2.
Alignment of Lyp1 and PEP amino acid sequences. The
PTPase domain is indicated by brackets. An arrow indicates the end of
the amino acid sequence shared by Lyp1 and Lyp2 and the beginning of
the unique C-terminal sequence of Lyp1. The NXXY motif is indicated by
a line above the sequence. The four potential SH3 domain binding sites
are also indicated (asterisks). A consensus sequence is shown below the
alignment. The unique seven amino acids of Lyp2 are shown in the box
below the alignment.
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Hydropathy analysis indicates that Lyp contains no obvious signal
sequence or hydrophobic segments and is therefore likely to be an
intracellular protein. The N-terminal regions of Lyp1 and Lyp2 contain
a single phosphatase catalytic domain. Amino acid sequence analysis
showed an overall identity of 70% between Lyp1 and the murine
phosphatase PEP (89% within the catalytic domain and 61% within the
noncatalytic portion), whereas the homology between the phosphatase
domain of Lyp1 and other PTPases varies between 30% and 60%. On the
basis of this analysis, Lyp1 and PEP certainly belong to the same
phosphatase family and may possibly be homologues.
Chromosomal mapping of the Lyp gene.
FISH hybridization was performed using a Lyp 1.8-kb cDNA probe to
determine the chromosomal localization of the Lyp gene. The regional
assignment of this cDNA probe was determined by the analysis of 40 well-spread metaphases. Positive hybridization signals at the short arm
of human chromosome 1 in region p13 (shown schematically in
Fig 3) were noted in approximately 10% of
the cells. The band assignment was determined by measuring the
fractional chromosome length and by analyzing the banding pattern
generated by DAPI counterstained image. The low frequency of
hybridization obtained with this probe is commonly seen with small cDNA
probes of this size. Signals were visualized on both homologues in 90% of the positive spreads (Fig 3). No fluorescence signal was seen on any
other chromosome, implying that the human LyP gene is located on
chromosome 1 in the p13 region.
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| Fig 3.
Regional mapping of the Lyp gene by FISH to normal human
lymphocyte chromosomes counterstained with DAPI. Biotinylated cDNA
probe was detected with avidin-FITC. Separate images of DAPI
counterstained metaphase chromosomes and of LyP cDNA probe
hybridization signals were captured and overlaid electronically as
described in Materials and Methods. Part of a representative metaphase
preparation is shown to indicate the position of the Lyp probe FISH
signals that are visible as two yellow fluorescent spots on the p arm
of chromosome 1. A DAPI banded chromosome 1 together with schematic
ideogram is shown to indicate that the Lyp1 probe hybridizes to band
1p13.
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Lyp2 is produced by alternative RNA splicing of the Lyp1 message.
To confirm the hypothesis that LyP2 was produced by alternative
splicing of Lyp1 RNA, three oligonucleotide-matching sequences around
the putative splicing site were used in PCR amplifications on a genomic
DNA template (Fig 4A). Oligonucleotide 1 corresponded to
the common nucleotides 2076-2097 of Lyp1 and Lyp2 (Fig 4), olignucleotide 2 to Lyp2 untranslated area adjacent to the stop codon
(nucleotides 2150-2168), and olignucleotide 3 to sequence immediately
downstream of primer 1 in lyp1 (nucleotides 2098-2120). The resultant
PCR products are shown in Fig 4B. PCR with primers 1 and 3 created an
approximately 3.5-kb fragment, suggesting the presence of an intron
between the primers. However, PCR with primers 1 and 2 resulted in a
much smaller fragment of 100 bp, the size expected from Lyp2 cDNA
sequence. Upon sequencing, the 5' end of the 3.5-kb fragment was
found to contain the alternative C-terminus, stop codon, and
untranslated nucleotide sequence of Lyp2 (Fig 4C). This clearly
demonstrated that LyP1 and LyP2 are the alternatively spliced
transcripts of a single gene. Whereas the 3.5-kb intron is spliced out
of the Lyp1 form, this does not occur in the Lyp2 isoform, and as a
result only 7 amino acids are added and an alternative stop codon is
used.
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| Fig 4.
Lyp2 is a result of alternative splicing of the Lyp1
gene. (A) A schematic map of the PCR strategy used. Primer 1 corresponds to the last 20 nucleotides shared by both the Lyp1 and Lyp2
sequences, primer 2 to Lyp2 untranslated area, and primer 3 to the
beginning of the unique Lyp1 sequence, immediately downstream of primer
1 (see also [C]). (B) The results of the PCR amplification on genomic
DNA. Lane 1, DNA ladder; lane 2, a product of 3.5 kb was amplified with
primers 1 and 3; lane 3, a product of 100 bp was amplified with primers
1 and 2. (C) Schematic map of Lyp1 splicing. The sequences before the
vertical line represent the splice donor site, whereas the nucleotide
sequences after it are the Lyp1 intronic sequence that code for the
unique C-terminal seven amino acids, stop codon (asterisk), and
untranslated sequence (lower case letters) of Lyp2. An open box
represents the common cDNA sequence shared by Lyp1 and Lyp2; the solid
and the light gray boxes represent the unique sequences of Lyp1 and
Lyp2, respectively.
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Predominantly lymphoid expression of Lyp RNA and differences between
Lyp1 and Lyp2.
Northern blot analysis of mRNA from various human tissues using a Lyp
cDNA probe common to both Lyp isoforms showed a major transcript of
approximately 4.4 kb in all of the lymphoid tissues examined
(Fig 5A). Substantial levels of Lyp mRNA
were detected in spleen, thymus, tonsil, and B and T lymphocytes. In
contrast, Lyp transcripts were not detected in prostate, ovary, testis, or colon tissues (or other human tissues, including heart, lung, brain,
placenta, or liver; data not shown). However, a low level of Lyp
expression could be detected in small intestine and appendix mucosa,
probably due to the presence of contaminating lymphocytes. From its
expression pattern in normal human tissues and cells, Lyp appears to be
a predominantly lymphoid phosphatase, although a low level of
expression could also be detected in the monocyte cell line, U937.
Myeloid (OCI/AML3) and erythrolukemia (K562) cell lines displayed
little to no expression.
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| Fig 5.
Expression profile of Lyp1 and Lyp2 transcripts. (A) Two
micrograms of poly A+ RNA from various human tissues and
cell lines (OCI/AML3, acute myeloblastic leukemia cell line; K562,
erythroleukemia cell line; and U937, monocytes cell line) were
hybridized with a 1.3-kb cDNA probe common to both Lyp1 and Lyp2
(exposure time, 7 days) and with actin (exposure time, 24 hours). (B)
RNA from immune relevant human tissues (Clontech) was blotted first
with a cDNA probe from the unique 280-bp 3' nucleotides sequence
of Lyp2 (including the untranslated sequence) and then, after
stripping, with a 600-bp cDNA probe from the unique 3' nucleotide
sequence of Lyp1 (exposure time, 7 days) and with actin (exposure time,
24 hours). The sizes of the RNA markers are indicated in kilobases.
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To further characterize the expression of the Lyp isoforms, Northern
blots were performed with a Lyp2-specific cDNA probe on human mRNA from
lymphoid and hematopoietic tissues. This showed a single 5.2-kb
transcript in all of the tissues examined, with the highest level of
expression found in fetal liver (Fig 5B). Subsequent blotting of the
same membrane with a Lyp1-specific probe showed the dominant 4.4-kb
transcript previously observed. Lyp1 demonstrated a high level of
expression not only in the mature lymphoid tissues, but also in the
thymus. In contrast to Lyp2, Lyp1 mRNA could not be detected in fetal
liver and only a low level of expression could be seen in bone marrow.
Characterization and intracellular localization of the Lyp1 and Lyp2
proteins.
To determine the actual size of the Lyp1 and Lyp2 proteins, the
full-length cDNAs were cloned by PCR from oligo-dT selected mRNA,
tagged with a T7 epitope, and transfected into COS-7 cells. The deduced
amino acid sequences of Lyp1 and Lyp2 predict molecular weights of 92 and 78 kD, respectively. Immunoprecipitation of the transfected
proteins with anti-T7 or anti-LyP antibodies and blotting with the T7
antibody showed the protein Lyp2 to have an apparent molecular weight
of 85 kD, which is slightly higher than the predicted molecular weight.
Two proteins with apparent molecular weights of 96 and 105 kD were
observed in COS-7 cells transfected with the Lyp1 cDNA
(Fig 6). Both of these proteins were
recognized by the T7 and Lyp antibodies. The lower molecular weight
product probably represents the result of proteolytic degradation, whereas the 105-kD protein is intact Lyp1. When immunoprecipitated from
lymphoid cell lines, the native Lyp1 protein has an apparent molecular
weight of 105 kD, in agreement with the size observed in transfected
COS-7 cells (see Fig 8).
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| Fig 6.
Transfection of Lyp1 and Lyp2 cDNA. T7-tagged Lyp1 (A)
and Lyp2 (B) were transfected into COS-7 cells and immunoprecipitated
with anti-LyP or anti-T7 antibody and blotted with anti-T7. (A) Lyp1
transfection results in a transfected protein of 105 kD and a probable
degradation product of 96 kD, whereas (B) shows LyP2 as a protein of 85 kD.
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To determine the intracellular localization of the lyp phosphatases,
the distribution of Lyp1 and Lyp2 was determined by indirect immunofluorescence in transiently transfected COS-7 cells. An HA
epitope was attached as a three tandem repeat to the 5' end of
both the Lyp1 and Lyp2 cDNAs in the pCDNA3 eucaryotic expression vector. Forty-eight hours after transfection, cells were fixed and
incubated with antibodies to the HA epitope. COS-7 cells transfected with either Lyp1 or Lyp2 displayed prominent perinuclear and
cytoplasmatic staining, but no staining of the nucleus
(Fig 7B). No fluorescence was observed in
COS-7 cells transfected with vector alone (Fig 7A). The pattern of
staining suggests that both of the phosphatases are predominantly
cytoplasmatic.
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| Fig 7.
Localization of Lyp1 and Lyp2 in transiently transfected
COS-7 cells by immunofluorescence. COS-7 cells were transiently
transfected with HA-tagged LyP1 and LyP2 cDNAs in pcDNA3 and
immunofluorescent detection was performed with a monoclonal antibody
against the HA tag (original magnification × 1,000). (A) Cells
transfected with HA-Lyp2 cDNA. (B) Cells transfected with HA-LyP1
cDNA.
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Characterization of Lyp protein expression.
Two rabbit polyclonal antibodies were raised against the Lyp protein.
After careful testing, an antibody raised against two peptides from a
common C-terminal area of the Lyp isoforms was used for
immunoprecipitation, because this antibody appeared to precipitate more
efficiently than an antibody raised against a catalytic domain fusion
protein. Both antibodies appeared to Western blot equally well. These
polyclonal antibodies were first used to characterize the expression of
Lyp proteins in human hematopoietic cell lines
(Fig 8). A single band of 105 kD could be
seen in both T-cell (Jurkat) and B-cell lines (Daudi and Ramos), the
same size as observed upon transfection of Lyp1 cDNA into COS-7 cells
(Fig 6). Lyp1 expression could not be detected in either the monocytic (U937) or myeloid (K562) cell lines, whereas low levels of expression could be seen in pre-B cells (G2 and A1). This pattern of protein expression correlates well with that of Lyp1 mRNA observed by Northern
blotting. We could not detect a protein of the predicted size of Lyp2
(85kD) in any of the cell lines examined.
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| Fig 8.
Lyp protein expression in lymphoid and myeloid cell
lines. Lyp was immunoprecipitated from cell lines (107
cells) and blotted with Lyp antibodies. A protein band of 105 kD
corresponding to LyP1 could be detected in Jurkat, Daudi, Ramos, A1,
and G2 cells, whereas U937 and K562 do not appear to have detectable
amounts of Lyp. (PB), preimmune serum control.
|
|
Having observed the predominantly lymphoid pattern of Lyp mRNA
expression, we also examined expression of the lyp protein in primary
lymphoid cells (Fig 9A). Both thymocytes
and tonsil T lymphocytes expressed Lyp1, whereas resting T cells from
peripheral blood, in addition to expressing low levels of Lyp1, also
expressed an 85-kD protein recognized by both polyclonal Lyp
antibodies. This is the predicted molecular weight of Lyp2, the shorter
alternatively spliced form of Lyp1.
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| Fig 9.
Expression of Lyp proteins in resting and activated T
cells. (A) Lyp was precipitated from thymocytes (80 × 106
cells), peripheral blood T cells (25 × 106 cells), and
tonsil T cells (10 × 106 cells) and immunoblotted with
anti-Lyp. Preimmune serum controls (PB) are presented in each case. A
band of 105 kD is present in each sample and a band of 85 kD can be
seen only in resting peripheral T cells. (B) Lyp was immunoprecipitated
from peripheral blood T cells (25 × 106 cells) before and
after stimulation with anti-CD3 (2.5 µg/mL) or PHA over a period of
48 hours. There is increased 105-kD Lyp1 expresssion, whereas the 85-kD
protein appears to be downregulated.
|
|
To determine whether expression of the lyp proteins may be regulated by
activation in T cells, normal peripheral blood T lymphocytes were
incubated with either PHA or anti-CD3 and harvested after 24 or 48 hours (Fig 9B). An increase in the level of Lyp1 protein expression was
observed after 24 hours of either stimulus, with a further increase
seen after 48 hours with anti-CD3. Interestingly, the 85-kD protein
could no longer be detected after 24 hours of incubation with either
PHA or anti-CD3.
Lyp phosphatase activity.
To determine whether Lyp1 possessed a catalytically active tyrosine
phosphatase domain, COS cells were transfected with T7-Lyp cDNA, the
protein immunoprecipitated with anti-T7 and used to dephosphorylate a
labeled synthetic peptide, Raytide, in an in vitro phosphatase assay.
Raytide peptide was 33P labeled on tyrosine residues in
vitro using the tyrosine kinase p60src and purified on phosphocellulose
paper. Release of 33P over time was measured in the
phosphatase assay and compared with controls from untransfected cells.
The results showed a sevenfold increase in 33P release from
the substrate incubated with Lyp immunoprecipitates compared with
control immunoprecipitates (Fig 10),
demonstrating that Lyp does indeed possess tyrosine phosphatase
activity. This activity could be completely inhibited by pervanadate
(not shown).
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| Fig 10.
Measurement of lyp1 phosphatase activity. Anti-lyp
immunoprecipitates from untransfected and pcDNA3-lyp1-transfected
cells were prepared in pervanadate-free lysis buffer and incubated with
33P-labeled substrate Raytide. At the indicated time
points, reactions were stopped by the addition of charcoal and the free
33P released from the peptide and now present in the
supernatant was measured by liquid scintillation counting.
|
|
Involvement of Lyp1 in TCR signaling.
One of the earliest events after TCR stimulation of T cells is the
induction of tyrosine phosphorylation. To determine whether Lyp played
a role in TCR signaling, human thymocytes were stimulated with anti-CD3
for various periods of time, Lyp immunoprecipitated, and blotted with
antiphosphotyrosine. This showed that, whereas Lyp itself is not
detectably tyrosine phosphorylated, a heavily phosphorylated protein of
116 to 120 kD coprecipitates with lyp, appearing within 1 minute of
stimulation (Fig 11A). Once activated, the phosphorylation level of this protein remained constant over a
period of 20 minutes. We attempted to identify the 116-kD
phosphorylated protein by Western blotting of Lyp immunoprecipitates
from CD3-stimulated thymocytes with antibodies to various candidate
proteins. The 116-kD protein associated with LyP1 was found to be c-Cbl
(Fig 11B), but not p125Fak, p116 Jak3, or p110 PI3-kinase. No
alteration in the amount of Cbl coimmunoprecipitating with lyp could be
detected upon anti-CD3 stimulation, suggesting that Lyp1 and Cbl are
constitutively associated, although Cbl can be inducibly
phosphorylated. This interaction was also observed in the mature T-cell
line Jurkat (not shown) and further confirmed by transfection of Lyp1
into COS-7 cells and examining its association with the endogenous Cbl
protein (Fig 11C). Lyp1 was found not only to coprecipitate with cbl in
COS cells, but also to significantly reduce the basal level of cbl
tyrosine phosphorylation (Fig 11D). This suggests that lyp1 may serve
to regulate cbl function in lymphoid cells and possibly that of
Cbl-associated proteins.
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| Fig 11.
Involvement of Lyp1 in TCR signaling. (A) Lyp
immunoprecipitates from thymocytes (80 × 106
cells) stimulated with anti-CD3 were blotted with antiphophotyrosine. A
single phosphorylated band of 116 kD was detected coimmunoprecipitating
with Lyp. Lyp protein loading was quantitated by anti-Lyp Western blot
after stripping. (B) Immunoblotting with anti-Cbl identified the 116-kD
phosphorylated protein as Cbl, whereas immunoblotting with anti-FAK or
anti-p110 (subunit of PI-3 kinase) showed them not to be associated
with Lyp. (C) Lyp1 was transfected into COS-7 cells and Cbl
immunoprecipitates prepared from these and untransfected cells. Western
blotting was performed with Lyp antibodies. The position of lyp is
indicated by an arrow. Cbl immunoprecipitates were also prepared and
blotted with antiphosphotyrosine (D) and then anti-cbl after
stripping.
|
|
| |
DISCUSSION |
We have isolated two novel intracellular phosphatase cDNA sequences
from a human thymus cDNA library: Lyp1 and its splice variant, Lyp2.
Sequence analysis of Lyp1 showed significant homology with the murine
phosphatase PEP, an intracellular PTPase widely expressed in
hematopoietic tissues.12 Lyp1 shares an overall 70% amino
acid identity with PEP (Fig 2). Although there is 89% identity between
the catalytic domain of Lyp1 and PEP, significantly less homology is
observed within the noncatalytic portions (61%). This degree of
homology is lower than any other currently described pair of
murine-human phosphatase homologues. Within this low homology area Lyp1
contains four proline-rich sequences, which are also present in PEP
(see Fig 2), forming putative PXXP and class II (XPPLPXR) SH3 domain
binding motifs.12,41 A recent study has demonstrated
association between one of the proline-rich motifs of PEP (PPPLPERTP,
also present in Lyp) and the SH3 domain of the protein tyrosine kinase
p50csk.15 The same motif occurs in the phosphatase
PTP-PEST, where it is also responsible for binding to
csk.16 Preliminary experiments also show Lyp1 to associate
with csk in T cells (not shown). The Lyp1 noncatalytic domain also
contains a large area of unique sequence, including an NXXY motif (see
Fig 2). When tyrosine phosphorylated, this motif may be recognized by a
phosphotyrosine binding (PTB) domain42 found in adaptor
proteins such as IRS, Shc, and cbl.
The lyp-related murine phosphatase PEP also has several PEST consensus
sequences.12 These contain an unusually high percentage of
proline (P), glutamic/aspartic acid (E/D), serine (S), and threonine
(T) residues; have been identified in many proteins; and were proposed
to confer a high susceptibility to rapid degradation.43 Early studies on the rapidly degraded enzyme ornithine decarboxylase provided support for this hypothesis,44 but such a claim
could not be substantiated for PEP11 or for other
PEST-containing proteins.45,46 Analysis of the Lyp1 protein
sequence using the program PEST-FIND (PC analysis software; Oxford
Molecular Group, Oxford, UK) indicated the presence of
only a single PEST motif (amino acids 702-736), whereas five were
confirmed in PEP.
The relatively high degree of homology between the catalytic domains of
Lyp1 and PEP suggests that Lyp1 may be the human homologue of PEP.
However, there are significant differences between the other domains
and, although the function of both phosphatases remains unclear, it is
difficult to determine whether they are in fact homologues.
Using FISH analysis, the Lyp gene was localized to chromosome 1p13 (Fig
3). This region is of particular interest, because it is a common site
of chromosomal rearrangement in both solid and hematopoietic
cancers.47-52 Several lines of evidence already suggest
that PTPases may act as tumor suppression genes.2,53 Knowledge of the precise chromosomal locus, along with comparative studies in normal and malignant cells, will help to establish whether
Lyp may play a role in tumorigenesis.
The shorter form of Lyp mRNA isolated by library screening, Lyp2, was
found to be derived from alternative splicing of a Lyp gene intron. A
3.5-kb intronic sequence of Lyp1 contains an alternative exon coding
for the C-terminal 7 amino acids of Lyp2 and at least part of its
3' untranslated sequence (Fig 4). Consequently, the 116 amino
acid C-terminal of Lyp1 is replaced by 7 alternative amino acids in
Lyp2. Precisely the same mechanism of generating an alternative
C-terminal has been observed in the receptor phosphatase PTP-P1.53 An intronic sequence of PTP-P1 was found to
contain an alternative exon coding for the C-terminal 26 amino acid of PTP-PS and its 3'-untranslated region. Several other studies have also shown that diversity can be generated by alternative splicing in
cytoplasmic PTPases.54-57 Thus, the creation of C-terminal
diversity by alternative RNA splicing appears to be a general mechanism of generating functional diversity in phosphatases.
The significance of the alternative C-terminal sequences of Lyp1 and
Lyp2 remains unclear, but there are several differences that may be key
to showing functional divergence. The C-terminus of Lyp1, but not Lyp2,
contains a consensus sequence XS/TPXK/R (741KTPGK
745) recognized by the p34cdc2 kinase,58
a cell cycle regulatory kinase,59 suggesting that Lyp1 may
be phosphorylated in a cell cycle-dependent manner. Lyp1 also contains
four potential SH3 domain binding sites, compared with a single motif
in Lyp2, suggesting that the isoforms may interact with different sets
of SH3 domains.
The pattern of Lyp1 expression observed by Northern blot showed it to
be preferentially expressed in lymphoid cells (Fig 5), particularly in
thymocytes and mature B and T cells. A low level of Lyp1 expression was
also seen in tissues rich in lymphoid infiltrates, such as the small
intestine and appendix. The pattern of Lyp1 protein expression detected
by antibodies in human hematopoietic cell lines correlated well with
Lyp1 mRNA expression (Fig 8). This pattern suggests that Lyp1 may play
a role in aspects of both the early and late stages of T-cell
differentiation. The lack of expression in fetal liver tissue, which
contains a large population of pre-B cells, suggests a different role
in the biology of B-cell development. However, it should be noted that
Northern blot analysis indicated significant expression of Lyp2 in
fetal liver tissue.
We have differentiated between the mRNA expression of Lyp1 and its
isoform, Lyp2, by the use of specific cDNA probes (Fig 5B). The two
isoforms demonstrated different patterns of RNA expression. Although
Lyp2 was present at lower levels then LyP1 in all the lymphoid tissue
examined, its expression was higher in fetal liver tissue. This clearly
indicates that there are differences in the regulation of expression
between the two isoforms. None of the cell lines examined (Fig 8)
expressed a protein of the expected size of Lyp2 (85 kD), as observed
in lysate from Lyp2 COS-7 cell transfectants (Fig 7). Only resting
peripheral T lymphocytes demonstrated expression of an 85-kD protein
recognized by the LyP-specific antibodies. Interestingly, stimulation
of T lymphocytes with PHA or anti-CD3 resulted in the induction of the
Lyp1 protein, with a simultaneous downregulation of the 85-kD protein
(Fig 9B). The 85-kD protein is believed to be Lyp2 on the basis of its
apparent molecular weight and the fact both Lyp antibodies can
recognize it. This finding suggests that Lyp2 may play an important
role in resting cells, because thymocytes, tonsil T cells, and lymphoid cell lines, which are activated cells, do not express the protein. Further investigation of its role will require the generation of
Lyp2-specific antisera directed against its unique C terminal sequence.
However, these observations also suggest that the two isoforms may play
significantly different roles in T-cell development.
While examining antigen receptor- and cytokine-mediated activation of T
cells, anti-CD3 stimulation of thymocytes was found to induce the
association of a 116-kD phosphorylated protein with Lyp1. Western
blotting of Lyp immunoprecipitates identified the phosphorylated band
as the proto-oncogene c-Cbl. Although inducibly phosphorylated, cbl was
found to be constitutively associated with Lyp1. From previous studies
it is known that Cbl is heavily tyrosine phosphorylated after TCR
stimulation60 and can associate with the Syk and ZAP
tyrosine kinases, negatively regulating their activities.61-65 Treatment of Jurkat cells with the
phosphatase inhibitor pervanadate leads to a marked increase in the
phosphorylation of Cbl,63 suggesting that tyrosine
phosphatases keep Cbl in a basally dephosphorylated state. We have
shown that Lyp1 is basally associated with Cbl in thymocytes and
confirmed this interaction in Jurkat (not shown) and in COS cells by
transfection, where Cbl phosphorylation is also reduced by Lyp1
overexpression (Fig 11). This strongly suggests that Lyp may play a
role in regulating Cbl activity through modulation of its tyrosine
phosphorylation status. Because Cbl is an adaptor protein that
associates with numerous protein tyrosine kinases, it is also
conceivable that Lyp may play a role in the regulation of these
proteins.64 Although we could not detect tyrosine
phosphorylation of Lyp1, a minor variant (EPNY) of the Cbl PTB domain
consensus binding motif (D(N/D)XpY) is present in the noncatalytic
domain that could form the basis for interaction. Alternatively, in the
absence of other identifiable interactive domains in either protein, a
multiple SH3 domain adaptor protein such as Grb2 may serve to link Lyp
and Cbl.
| |
ACKNOWLEDGMENT |
The authors are indebted to Sergio Grinsten for helping with the
cellular localization of Lyp, to Barbara Beatty for assisting with
chromosomal localization of Lyp, and to Michal Shahar for excellent
technical assistance.
| |
FOOTNOTES |
Submitted June 24, 1998; accepted November 4, 1998.
Supported by the Medical Research Council of Canada, the National
Cancer Institute, and The Lotte and John Hecht Memorial Foundation.
The GenBank accession number for Lyp1 cDNA is AF 001846 and for Lyp2 is
AF001847.
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 Chaim M. Roifman, MD, Division of
Immunology/Allergy, Infection, Immunity, Injury and Repair Programme,
The Hospital for Sick Children, 555 University Ave, Toronto, Ontario,
Canada M5G 1X8.
| |
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