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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2013-2024
By
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.
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.
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 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.
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
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% 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
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.
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.
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.
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).
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.
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).
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.
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 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.
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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CD45-null transgenic mice reve |
TOP
ABSTRACT
INTRODUCTION
RIIB1,
chain.
gt10 DNA library from human thymocytes was
screened with a 32P-labeled 430-bp Lyp1 fragment obtained
by PCR. 
,
3 and G at +4, both
regarded as important criteria for an eucaryotic initiation
site.
