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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 1922-1933
A Novel Spliced Form of SH2-Containing Inositol Phosphatase Is
Expressed During Myeloid Development
By
David M. Lucas and
Larry R. Rohrschneider
From the Fred Hutchinson Cancer Research Center, Seattle, WA.
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ABSTRACT |
SH2-containing Inositol Phosphatase (SHIP) is a 145 kD protein
expressed in hematopoietic cells. SHIP is phosphorylated on tyrosine
after receptor binding by several cytokines and has a negative role in
hematopoiesis. We cloned a murine complementary DNA (cDNA) sequence for
an isoform of SHIP with an internal 183 nucleotide deletion, encoding a
protein 61 amino acids shorter than 145 kD SHIP. This deletion
eliminates potential SH3-domain binding regions and a potential binding
site for the p85 subunit of Phosphatidylinositol 3-Kinase. Using
polyclonal anti-SHIP antibodies, we and others have previously observed
a 135 kD SHIP isoform that is coexpressed with 145 kD SHIP. Here, we
used monoclonal antibodies raised against the region deleted in the
spliced form to show that the product of the novel spliced SHIP cDNA is
antigenically identical to the 135 kD SHIP isoform. Like 145 kD SHIP,
135 kD SHIP expression was induced on differentiation of bone marrow cells. After macrophage colony-stimulating factor (M-CSF) stimulation of FDC-P1(Fms) myeloid cells, both 145 and 135 kD SHIP
forms were tyrosine phosphorylated and could be coimmunoprecipitated
with antibodies to Shc and Grb2. However, experiments showed only a weak association of 135 kD SHIP with p85. A potentially analogous 135 kD SHIP species also appears in human differentiated leukocytes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PRINCIPAL FACTORS THAT mediate the
survival and differentiation of hematopoietic progenitor cells include
colony stimulating factors (CSF), interleukins (IL), erythropoietin
(Epo), and stem cell factor (SCF).1 These receptor-ligand
systems have been used as models to investigate signal transduction
governing hematopoietic cell proliferation and differentiation, and
several molecules that become phosphorylated when these cytokines bind
their cognate receptors have been identified. One recent example is the
SH2-containing Inositol Phosphatase (SHIP).2,3 SHIP is a
signaling protein expressed in most, if not all, hematopoietic cells,
and is a negative regulator in the process of hematopoietic cell
development and function.2-7 SHIP contains an N-terminal
SH2 domain, a central inositol polyphosphate-5-phosphatase catalytic
region, two recognition sequences for phosphotyrosine binding (PTB)
domains, and several proline-rich potential SH3 domain-binding regions
near the C-terminus. We and others2-6,8-12 have shown
previously that SHIP is phosphorylated on tyrosine and associates with
the signaling molecule Shc after treatment of cells with a variety of
cytokines and growth factors including IL-3, macrophage CSF
(M-CSF), granulocyte macrophage CSF (GM-CSF), Epo, and SCF, or on
activation of several immunological receptors such as Fc receptors and
the B-cell-receptor Ig- and - chains. Recently, Helgason et
al7 produced mice with a targeted disruption in both
ship alleles. These mice, although viable, have a decreased
lifespan associated with multiple abnormalities in hematopoietic cell
development and cytokine responsiveness.7 Together, these
studies indicate a broad role for SHIP in hematopoiesis.
The protein interaction domains of SHIP potentially allow association
with a variety of different molecules. SHIP interaction with Shc occurs
via the PTB domain of Shc, which recognizes the tyrosine-phosphorylated
amino acid consensus sequence NPXY on SHIP.2,13,14 Two NPXY
motifs are present in the carboxy terminal region of SHIP. The tyrosine
in the first of these is followed by the amino acids IGM, which
constitutes a potential recognition site for SH2-mediated binding of
the p85 subunit of Phosphatidylinositol 3-Kinase (PI3K).15
Several studies have also shown a strong role for the SH2 domain
of SHIP in protein-protein interactions. Ono et al5 and
Osborne et al16 showed functional interactions of the SHIP
SH2 domain with immunoreceptor tyrosine-based activation or inhibitory
motifs (ITAMs or ITIMs), present in the cytoplasmic regions of many
receptors. Additionally, reports by Liu et al17 and Sattler
et al18 showed that SHIP interacts with the protein phosphatase SHP-2 through the SH2 domain of SHIP, and also that the SH2
domain is important for SHIP tyrosine phosphorylation and interaction
with Shc.19 Proline-rich stretches, present in the
C-terminal region of SHIP, may be recognized by proteins with SH3
domains.20 Earlier studies from several laboratories showed
that SHIP was found in a complex with Grb2,8,9 an interaction that may be governed by these polyproline motifs. Damen et
all3 and Osborne et al16 have shown specific
interactions of SHIP with the SH3 domains of PLC or Grb2 in vitro.
Evidence of an in vivo interaction was supplied by Odai et
al,12 who showed binding of SHIP with Grb2 in human
leukemia cells.
The existence of numerous interaction partners as well as
activating factors suggests multiple functions and regulatory
mechanisms for SHIP in vivo. Using immunoblot assays with anti-SHIP
antibodies, we and others2,5,7,21,26,30 have previously
detected isoforms of SHIP in addition to 145 kD SHIP that may be
important in the multiple activities and associations of this
molecule.* Kavanaugh et al21 reported that
three SHIP isoforms (SIP-110, -130, and -145) in human B cells are due
to alternative splicing of a single gene, and showed that the
differences between the forms are due to loss of complementary DNA
(cDNA) sequence encoding the N-terminal SH2 domain. In contrast, Damen
et al30 recently reported that isoforms of SHIP observed in
the murine hematopoietic cell line DA-ER are the result of C-terminal
proteolytic cleavages of 145 kD SHIP, possibly by calpain. In the
following report, we identify a cDNA sequence encoding an approximately
135 kD SHIP isoform that is coexpressed with 145 kD SHIP. However,
unlike the SIP proteins described by Kavanaugh or the SHIP cleavage
products described by Damen, this 135 kD version of SHIP results from a specific internal deletion that removes a polyproline-containing stretch between the two NPXY motifs. This 135 kD SHIP protein is
expressed under the same conditions as 145 kD SHIP, and appears to be
the result of alternative splicing of SHIP messenger RNA (mRNA). Our
studies also indicate that a 135 kD form of SHIP is produced in a human
myeloid leukemia cell line and human peripheral white blood cells, and
that a similar deletion in the SHIP message may be responsible for this
isoform as well. We further show that the murine 135 kD isoform is
phosphorylated in vivo and retains its ability to associate with the
signaling proteins Shc and Grb2 after cytokine stimulation. However,
our experiments showed only a weak association of 135 kD SHIP with the
C-terminal SH2 domain of p85 compared with the full-length 145 kD form.
This 135 kD SHIP isoform may therefore be involved in alternative
protein associations or functions of SHIP.
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MATERIALS AND METHODS |
Cell culture.
The WEHI-3 monocyte cell line was obtained from the American Type
Culture Collection (ATCC, Rockville, MD) and was maintained in RPMI
1640 medium supplemented with penicillin and streptomycin, plus 10%
fetal bovine serum (FBS) (Summit Labs, Fort Collins, CO). FDC-P1
myeloid cells expressing the murine M-CSF receptor Fms
(Genbank accession number FMSCR) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, plus 10% WEHI-3
conditioned media as a source of IL-3, as described.2 For
M-CSF stimulation, cells were first incubated for 5 hours in 1% FBS
without IL-3. Cells were pelleted by centrifugation and resuspended in
one half the pellet volume of PBS (control) or of M-CSF (70,000 U/mL
final concentration). Cells were incubated for 2 minutes at room
temperature, then lysed in cold NP-40 lysis buffer [50 mmol/L NaCl,
100 mmol/L HEPES, 30 mmol/L
Na4P2O7, 50 mmol/L NaF, 5 µmol/L
ZnCl2, 0.5% NP-40, 1 mmol/L Na3VO4
(all from Sigma, St Louis, MO), pH 7.0]. 293T and Rat2 fibroblast cell
lines were also maintained in DMEM, plus 5% FBS and 5% calf serum
(Hyclone, Logan, UT). Bone marrow cells (BMCs) were obtained from the
femurs of mature C57-BL6 mice and plated in DMEM plus 10% FBS for 6 hours. At this point, nonadherent cells were recovered and either lysed
in NP-40 lysis buffer or were replated and incubated for 7 days in DMEM
with 10% FBS, IL-3, and M-CSF (2000 U/mL). After 4 days, nonadherent
cells were removed by washing with PBS and the adherent BM-derived
macrophages (BMM) were lysed in NP-40 lysis buffer. The ML-1 cell line
was cultured in RPMI 1640 plus 10% FBS as described.26
Normal human peripheral white blood cells were purified from whole
blood using Lymphoprep (Nycomed, Oslo, Norway) according to the
manufacturer's instructions.
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was isolated using TRIzol reagent (GIBCO-BRL, Grand Island,
NY) according to the manufacturer's instructions. To ensure that there
was no genomic DNA contamination, the RNA preparation was treated with
10 U RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN) at room
temperature for 20 minutes in the presence of 3.75 mmol/L Tris-HCl (pH
7.5), 10 mmol/L MgCl2, 1 mmol/L dithiothreitol (DTT). This
reaction was terminated with 5 µL of 0.5 mol/L EDTA and the RNA was
extracted with chloroform and reprecipitated. RT-PCR assays were
performed as previously described.22 Briefly, single-stranded cDNA was synthesized from 10 µg purified total RNA
after priming with 10 pmol oligo dT15, using 200 U
Superscript (GIBCO BRL) according to the manufacturer's instructions.
The final reaction volume was diluted to 100 µL in water, and 2 µL of the cDNA sample was used as a template in each 40 µL PCR. The amplification conditions were: 1 minute at 94°C, 1 minute at
60°C, and 1 minute at 72°C, for varying cycles. These
conditions were also used to perform competitive PCR experiments with
the 2666 to 3247 primer set. Primer sequences used were as follows:
numbers refer to the murine SHIP cDNA sequence, Genbank accession
number U51742: 73 (sense): 5'-GACCCAGTCCAGGAGACCC-3'; 800 (antisense): 5'-GCAGAGACTCCAGGGATG-3'; 2042 (sense):
5'-CTGACCCGGGACAAGTATGC-3'; 2703 (antisense):
5'-GGATTCATCCCGCTCTGTCT-3'; 2666 (sense):
5'-CAGGGCAAGATGAGGGAGAA-3'; 3247 (antisense):
5'-CGATGCTGGGTGATGAGATT-3'. Primer sequences for human SHIP
were as follows; numbers refer to SHIP cDNA sequence, Genbank accession
number U84400: 2615 (sense): 5'-GCCACTTCCAGGGGGAGATCA-3'; 3212 (antisense): 5'-GCAGGGCGGGGGTTCCTTCC-3'.
Genomic DNA sequencing.
A murine genomic DNA library (using Lambda DASH II; Stratagene, La
Jolla, CA) was constructed by Dr Zhi Chen and was the kind gift of Dr
Phil Soriano, Fred Hutchinson Cancer Research Center. Approximately 1.5 × 106 phage plaques were screened using full-length
SHIP cDNA labeled with [-32P] dCTP (RTS RadPrime DNA
Labeling System, GIBCO BRL). Thirty positive clones were isolated and
rescreened with the 581 bp product of the 2666-3247 PCR primer set,
using a slot-blot apparatus (Schleicher & Schuell, Keene, NH). Positive
clones were digested with EcoRI and/or BamHI
and subjected to southern analysis with the same probe. Bands that
hybridized were purified, subcloned into pBluescript (Stratagene), and
sequenced using an automated sequencer (Perkin-Elmer, Norwalk, CT).
SHIP cDNA cloning.
A cDNA library was constructed from FDC-P1 cells,2 using
the Lambda-ZAP kit (Stratagene). This library was probed using full-length SHIP cDNA labeled with [-32P] dCTP.
Thirty-four positive clones were obtained, and these were reprobed
using [-32P] dCTP-labeled cDNA representing
nucleotides 38-502 of the SHIP cDNA sequence. Clones that were positive
for this probe (5' region of SHIP) were identified, and the
membrane was stripped and rehybridized using a probe representing
nucleotides 2842-3019 (183 nucleotide deletion) of SHIP cDNA. Clones
that hybridized with both probes, as well as clones that hybridized to
the first probe and not the second probe, were treated as described in
the manufacturer's instructions to excise the plasmid pBK-CMV
containing the insert.
Transfections.
pBK-CMV containing SHIP cDNA or  183 SHIP cDNA was transfected into
293T fibroblast cells using a calcium phosphate precipitation protocol.23 Briefly, 10 µg of DNA was diluted in 500 µL
of 250 mmol/L CaCl2 before being mixed with an equal volume
of a 2× HeBS phosphate buffer (280 mmol/L NaCl, 10 mmol/L KCl,
1.5 mmol/L Na2 HPO4, 12 mmol/L glucose, 50 mmol/L HEPES, pH 7.06). This solution was incubated at room temperature
for 5 minutes, then added to a 150 mm plate of 293T fibroblast cells at
approximately 75% confluence. The cells were incubated for 48 hours
before being washed and processed for immunoblotting.
Antibodies.
The anti-SHIP polyclonal antibody #5340 was made using SHIP amino acids
670-868, and the #5369 antibody was made using amino acids
889-1046.2 The anti-Shc polyclonal antibody was produced using a GST fusion protein with the SH2 domain of Shc,
amino acids 374-469 (Genbank accession number U15784). The anti-p85
polyclonal antibody #06-195 was purchased from Upstate Biotechnology
(Lake Placid, NY). Polyclonal antibodies were used at a dilution of 1:5000. Monoclonal antibodies (MoAbs) to SHIP were produced using BALB/C mice immunized with histidine-tagged proteins representing either amino acids 4-126 of SHIP (P6H8), or amino acids 866-1020 of
SHIP (P2C6, P1D7, and P1C1). The 866-1020 peptide includes both NPXY
motifs and the intervening polyproline stretch, as well as the
region deleted in the 183 SHIP cDNA sequence (amino acids 920-980).
Immunizations and fusions were performed by Dr Elizabeth Wayner, Fred
Hutchinson Hybridoma Shared Resource. These clonal hybridoma
supernatants were used at a dilution of 1:100. The antiphosphotyrosine MoAb 4G10 (the kind gift of Dr Brian Druker, Dana Farber Cancer Institute, Boston, MA) was used at a dilution of
1:10,000.
SDS-PAGE, immunoprecipitations, and immunoblots.
These assays were performed as described by Lioubin et al9
and Carlberg et al.24 All cells were lysed in NP-40 lysis buffer containing 1 mmol/L Na3VO4, and cell
debris was removed by centrifugation. Total protein amount was measured
by spectrophotometry using Protein Assay Reagent (BioRad, Hercules,
CA), and equal amounts of protein were loaded into each lane. SDS-PAGE
was run using a 6% acrylamide gel, and protein molecular weights were compared using unstained Perfect Protein Markers (Novagen, Madison, WI). For immunoprecipitations, 400 µg of total protein was used in
each reaction along with 40 µL of Protein A (or Protein G for the
4G10 MoAb) conjugated to sepharose beads (Pharmacia, Piscataway, NJ).
Proteins were precipitated with 2 µL of anti-Shc, 1 µL of antiphosphotyrosine, or 2 µL of #5340 polyclonal anti-SHIP antibody. The lysate was mixed at 4°C for 6 hours, and beads were washed five
times in cold NP-40 buffer. 2× sample loading buffer (100 mmol/L
Tris pH 6.8, 200 mmol/L DTT, 4% SDS, 20% glycerol, 0.1% bromophenol
blue) was added, and the sample was heated for 2 minutes at 100°C.
SDS-PAGE, transfer, and detection were performed as described by
Carlberg.24 For GST pull-down experiments, cDNA fragments
encoding either the N-terminal or C-terminal SH2 domains from p85 were
cloned into the pGEX-3X vector (Pharmacia), and protein was expressed
and purified as described by Carlberg.24 Fifty µg of
purified GST-SH2 protein was used in each reaction, plus 40 µL of a
50% slurry of GST-agarose beads (Sigma). The experiments were
performed identically to the immunoprecipitations, except that the
binding and washing buffer composition was as described in Joos
et al.28
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RESULTS |
Identification of 183 SHIP.
Previous immunoblot data from our laboratory and
others2,5,7,21,26 showed the expression of several proteins
that react with anti-SHIP polyclonal antibodies. The polyclonal
anti-SHIP antibodies #5340 and #5369 detect the 145 kD SHIP protein in
the myeloid cell line FDC-P1. These antibodies also react with a larger SHIP-related form, approximately 160 kD, two smaller proteins of
approximately 135 and 130 kD, and under certain conditions a 110 kD
protein.26 We performed RT-PCR analysis to detect
alternative SHIP transcripts that might produce these additional
protein forms. We generated oligonucleotide primers that annealed to
several regions of SHIP sequence (Fig 1A)
and performed RT-PCR using RNA from either WEHI-3 or FDC-P1 myeloid
cell lines, which produce identical expression patterns of SHIP
isoforms or SHIP-like proteins in immunoblot assays. A primer set
flanking the region of SHIP cDNA encoding the SH2 domain (nucleotides
73-800, amino acids 1-237), as well as a primer set flanking the region
encoding the epitope for antibody #5340 (nucleotides 2042-2703, amino
acids 651-871) resulted in amplification of the predicted product sizes only. However, primers flanking nucleotides 2666-3247 (amino acids 866-1059) amplified a product at the predicted size of approximately 600 nucleotides, plus a smaller product of approximately 400 nucleotides (Fig 1B). A larger product of approximately 700 nucleotides
was also visible in some experiments.
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| Fig 1.
SHIP RT-PCR. (A) The 3,570 nucleotide ORF of murine SHIP
cDNA is shown with structural and potential functional domains labeled.
The NPXY and YIGM motifs are depicted ( ), as well as the
proline-rich potential SH3 domain binding regions ( ). Also shown are
the binding sites for the PCR primer sets used. Nucleotide numbering
refers to the cDNA sequence of murine SHIP, Genbank accession number
U51742. (B) RT-PCR was conducted using RNA from the WEHI-3 and FDC-P1
myeloid cell lines and the primer sets shown in Fig 1A. The products
were separated by electrophoresis in a 1% agarose gel and stained with
ethidium bromide. Amplification of cDNA with primers to GAPDH was
included to verify cDNA quality.
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Subcloning and sequencing of the two major bands produced by the
2666-3247 primers showed the known full-length SHIP cDNA and SHIP
cDNA with a deletion of 183 nucleotides
(Fig 2A). This deletion occurs within the
region amplified by the 2666-3247 primers; the primer binding sites are
unaffected, and thus the amplification of this smaller band was not due
to improper annealing of the primers. At the protein level, this
"183" sequence predicts an in-frame deletion of 61 amino acids
in the proline-rich C-terminal end of SHIP producing an isoform with a
calculated size of 127 kD.
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| Fig 2.
SHIP deletion. (A) The two bands produced using the
2666-3247 primer set, shown in Fig 1B, were subcloned and
sequenced. The larger (581 bp) band
corresponds to the published SHIP sequence. The smaller (398 bp) band
has a deletion of 183 nucleotides at the site shown, but is otherwise
identical. (B) SHIP genomic DNA was isolated from a lambda genomic DNA
library. Phage clones which hybridized with the 581 bp product of the
2666-3247 PCR primers were sequenced to show the NPXY-containing exon.
Exon sequence is in upper case letters, and the sequence of the  183
deletion is in bold type. The verified splice acceptor and donor are
shown in shaded boxes, and the potential splice sites that would
produce the 183 deletion as an intron are shown in open boxes.
Numbering refers to the SHIP cDNA sequence as in Fig 1.
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This deleted form of SHIP cDNA could be the result of alternative RNA
splicing. We therefore sequenced murine genomic DNA to map the
intron/exon boundaries and investigate this possibility. Our genomic
sequencing of ship showed that the deleted 183 nucleotide section occurs entirely within a single 276 nucleotide exon. However, the 183 nucleotide segment contains a consensus splice donor and acceptor site at either end, and thus could act as an intron (Fig 2B).
183 SHIP mRNA expression.
It has previously been shown that SHIP message is present in a wide
variety of hematopoietic cells.2,10 Using RT-PCR with the
2666-3247 primer set, we have detected 183 SHIP message along with
full-length SHIP message in the macrophage cell lines WEHI-3 and RAW
264.7, the B-cell progenitor line BaF3, and the T-cell line CTLL-2 (not
shown), in addition to the myeloid progenitor cell line FDC-P1 from
which 183 SHIP was cloned. No SHIP mRNA was detected in the
fibroblast cell lines NIH 3T3 (mouse), Rat2 (rat), or 293T (human) (not
shown). We were unable to detect either full-length or 183 SHIP
message in untreated murine BMC RNA by RT-PCR using the primers to
nucleotides 2666-3247. However, using primers to nucleotides 73-800, we
detected a band of the predicted size for SHIP cDNA
(Fig 3A). When BMCs were differentiated to macrophages using M-CSF, we readily detected both full-length and
183 SHIP mRNA using the 2666-3247 primer set, and full-length SHIP
using the 73-800 primer set. This finding correlates with our
immunoblot analysis using untreated BMC lysates or IL-3 plus M-CSF-differentiated BMM shown in Fig 3B. In untreated BMC
lysates, a SHIP-like protein of approximately 110 kD was expressed as
reported by us previously,26 but full-length SHIP was not
detected (Fig 3B). Within 4 days of incubation in media containing IL-3
and M-CSF, adherent BMM cells expressed the 145 kD and 135 kD SHIP proteins. The band produced using the 73-800 primer set in RT-PCR analysis of untreated BM may represent this approximately 110 kD SHIP
protein form, which may lack part of the region encoded by the
nucleotides 2666-3247 and thus is not recognized by our MoAbs.
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| Fig 3.
SHIP expression in BM and BMM. (A) Murine BM was isolated
and treated as described in Materials and Methods. Total RNA was
purified from undifferentiated BM or from BMM. RT-PCR was performed
using the primer sets shown in Fig 1A. (B) Untreated murine BMCs or BMM
were lysed in NP-40 lysis buffer. Equal amounts (40 µg) of total
protein were loaded into each lane of a 6% polyacrylamide gel. The
gels were processed as described and protein was detected with the
anti-SHIP polyclonal antibody #5340.
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We hypothesized that the smaller PCR product of the primer set
2666-3247, observed in Fig 1B and 3A, may in fact be a minor component
of SHIP message detected only because of the sensitivity of RT-PCR. To
address this, we performed competitive PCR analysis to compare the rate
of amplification of the two products, as well as the level of 183
SHIP cDNA relative to the full-length product. Using the 2666-3247 primer set, cDNA from WEHI-3 cells, FDC-P1 cells, and BMM was amplified
for varying cycles under the conditions described in Materials and
Methods. The resulting PCR products were transferred to a membrane and
hybridized to the radiolabeled probe made from SHIP cDNA (nucleotides
2666-2795) that anneals to a region common to both products. The signal
of each band was measured using a Phosphorimager and plotted against
cycle number (Fig 4A).
As can be observed, the rate of amplification of the two products is
similar; no preferential amplification of either product is detected.
We then compared the lower (183 SHIP) band with the upper
(full-length SHIP) band in the same lane in each cell type. Within the
linear range of amplification (cycles 26 through 32), 183 SHIP cDNA
was present in amounts 2 to 10 times greater than full-length SHIP cDNA
(Fig 4B).
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| Fig 4.
SHIP competitive PCR. (A) cDNA from WEHI-3 cells, FDC-P1
cells, and BMM was amplified for the specified number of cycles using
the 2666-3247 primer set. Products were transferred to a membrane and
hybridized using a 32P-labeled SHIP cDNA sequence
common to both. (B) The signal of each band was measured using a
Phosphorimager and plotted against cycle number. Within the linear
range of amplification, cycles 26 through 32, the 398 bp 183 SHIP
band ( ) from each cDNA type was compared with the 581 bp
full-length SHIP band ( ) in the same lane.
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SHIP cDNA cloning.
We used full-length SHIP cDNA to probe a library constructed from
FDC-P1 cell mRNA. Positive clones were analyzed on a slot blot, using a
probe to the 5' untranslated region of SHIP cDNA (nucleotides
38-502), to determine whether they contained the beginning of the open
reading frame (ORF) of 145 kD SHIP protein. This slot blot was also
probed with SHIP cDNA representing the 183 nucleotides deleted in the
PCR products (nucleotides 2842-3019). Clones that hybridized to both
probes, as well as clones that hybridized to the 5' probe but not
to the 183 probe were subjected to sequence analysis. In this
screen, we obtained two complete full-length SHIP cDNA clones and three
complete 183 SHIP cDNA clones. These 183 SHIP clones were
identical to full-length SHIP cDNA except for the 183 nucleotide
deletion described.
183 SHIP protein expression.
To more precisely identify SHIP and its potential isoforms, we produced
MoAbs to SHIP. The first immunogen used included SHIP amino acids
866-1020, encompassing the region eliminated in the 183 SHIP cDNA
(Fig 1) and resulted in the MoAbs P2C6, P1D7, and P1C1. The second
immunogen included amino acids 4-126, encompassing the N-terminal SH2
domain, and resulted in the MoAb P6H8. Immunoprecipitation and
immunoblot analyses showed that the 145 kD band recognized by each of
these anti-SHIP MoAbs was also recognized by the anti-SHIP polyclonal
antibodies #5340 and #5369 previously produced, and thus is a closely
related if not identical protein (Fig 5).
To examine the products of the SHIP cDNA clones that were isolated, 293T fibroblast cells were transfected with an expression vector containing either full-length or 183 SHIP cDNA. Lysates from these
cells, along with lysates from WEHI-3 and FDC-P1 myeloid cells, were
then analyzed by immunoblot assay (Fig 5). Although untransfected 293T
fibroblast cells showed no bands using any of the anti-SHIP antibodies
(not shown), 293T cells transfected with either full-length or 183
SHIP cDNA produced bands of the predicted size that were recognized by
the anti-SHIP polyclonal antibody #5340 and the anti-SHIP MoAbs P2C6
and P6H8 (Fig 5, lanes 2 and 3). The MoAb P1D7 did not recognize the
product of transfected 183 SHIP cDNA, nor did it recognize the 135 kD protein in either WEHI-3 or FDC-P1 myeloid cells. However, MoAb P1D7
recognized the 145 kD SHIP protein in the hematopoietic cells and in
293T cells transfected with full-length SHIP cDNA. This result shows that the epitope recognized by the P1D7 antibody exists within the 61 amino acids eliminated in 183 SHIP. Furthermore, the migration characteristics of 135 kD SHIP and the translated product of 183 SHIP cDNA were indistinguishable in higher resolution SDS-PAGE experiments. The P1C1 antibody recognition pattern was the same as that
of the P2C6 and P6H8 antibodies (not shown). Taken together, these data
provide evidence that 183 SHIP and the 135 kD protein observed in
hematopoietic cells are identical, and that this protein differs from
full-length SHIP in the region encoded by the deleted 183 nucleotides.
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| Fig 5.
SHIP immunoblots. Equal amounts (40 µg) of total
protein from the cell type indicated was loaded into each lane of
four identical 6% polyacrylamide gels. The blots were
processed as described and incubated with the anti-SHIP
polyclonal antibody #5340 or the anti-SHIP MoAbs as indicated.
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In both 293T fibroblasts (Fig 5) and Rat2 fibroblasts2
transfected with full-length SHIP cDNA, we observed a SHIP
antibody-reactive protein of approximately 130 kD (Fig 5, lane 2). This
band was present in fibroblasts that were transfected with only
full-length SHIP cDNA. The 183 SHIP sequence could not be detected
in these cells by RT-PCR analysis, although full-length SHIP cDNA is
readily amplified (not shown). Unlike the translated product of 183
SHIP cDNA, shown in lane 3 of Fig 5, this approximately 130 kD band in
full-length SHIP-transfected fibroblasts is recognized by all our
anti-SHIP antibodies and also migrates slightly faster than the
hematopoietic 135 kD SHIP protein on SDS-PAGE. In the WEHI-3 and FDC-P1
cells we also detected additional smaller bands including the 130 kD
band observed in SHIP-transfected fibroblasts (Fig 5, #5340 panel), but
these bands were inconsistently detected and none shared the size or
antibody recognition pattern of 183 SHIP.
Human hematopoietic cells produce a 135 kD isoform of SHIP.
Western blotting experiments of tetradecanoyl phorbol acetate
(TPA)-treated ML-1 cells using polyclonal SHIP antibodies
#5340 and #5369 show that a protein of 135 kD is produced in addition to 145 kD SHIP (Fig 6A). This 135 kD
protein is also detectable in human peripheral white blood cells, but
at a very low level. Human SHIP is recognized poorly by P2C6, P1D7, and
P6H8. The P1C1 MoAb, produced using the same immunogen as for P1D7 and
P2C6, recognizes human 145 kD SHIP well, but does not recognize human 135 kD SHIP (not shown).
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| Fig 6.
Human SHIP 135 kD protein. (A) ML-1 human monocytic
leukemia cells were treated for 2 days with TPA, and adherent cells
were lysed and subjected to SDS-PAGE. Blots were incubated with SHIP
polyclonal antibodies #5340 or #5369. (B) RNA from ML-1 cells with the
same TPA treatment was analyzed by RT-PCR using primers to human SHIP
2615-3231 (Genbank accession number U84400). Two bands were detected at
approximately 600 and 350 bp. These were purified and subjected to DNA
sequencing, and the results are depicted as in Fig 2A.
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RT-PCR experiments using human hematopoietic cells detected a smaller
product in addition to the full-length band, but the level was
significantly lower than 183 SHIP cDNA expression in murine cells
and was not easily detected with ethidium bromide staining. This 334 bp
product was amplified from both TPA-treated ML-1 cells and normal human
peripheral white blood cells, using primers to the human SHIP sequence
analogous to the murine SHIP primers 2666-3247 (Fig 6B). Sequence
analysis showed the same in-frame 282 bp deletion in samples from both
ML-1 cells and peripheral white blood cells, encoding 94 amino acids
with a calculated molecular weight of 10 kD. The human 282 deletion
is in a region of SHIP overlapping the murine 183 deletion region,
but is not identical. However, both ends of the deleted segment in
human SHIP correspond perfectly to intron-exon boundaries we detected
in murine genomic DNA, and are preceded by the required consensus
splice acceptor nucleotides AG.
183 SHIP is tyrosine phosphorylated and interacts
with Shc and Grb2 in response to M-CSF.
After 5 hours of incubation in media containing 1% serum without IL-3,
FDC-P1 cells were centrifuged and either left untreated or were treated
with M-CSF for 2 minutes. Lysates from these cells were
immunoprecipitated with #5340 anti-SHIP antibody, and one third of the
resulting supernatant was loaded onto each of four identical
polyacrylamide gels for SDS-PAGE as described. The blots were incubated
with the phosphotyrosine-specific antibody 4G10 or the anti-SHIP MoAbs
P2C6 or P1D7 as indicated (Fig 7). As can be observed in the SHIP IP lanes 3 and 4, comparable amounts of all
SHIP proteins were immunoprecipitated from untreated or M-CSF-treated lysates using the polyclonal anti-SHIP antibody #5340. The 135 kD band
was readily observed with MoAb P2C6 as expected, and was not visible
using MoAb P1D7 (SHIP IP lanes 5 and 6). In this experiment we also
detected a band at 130 kD (SHIP IP lane 5), but its recognition by the
P1D7 antibody shows that it is distinct from the 183 SHIP isoform
and likely represents proteolytic degradation of 145 kD SHIP. Both the
145 kD and the 135 kD bands are detected by the 4G10 antibody in the
SHIP immunoprecipitations using lysates from M-CSF-treated cells,
showing they are both tyrosine phosphorylated in response to M-CSF
treatment (SHIP IP lane 2).
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| Fig 7.
Immunoprecipitations. Lysates from FDC-P1 cells, either
untreated ( ) or treated with M-CSF (+), were mixed with protein A
sepharose beads and each of the antibodies indicated on the left (IP).
Immunoprecipitation and immunoblotting analyses were performed as
described, and three identical gels were prepared. These gels were
incubated with the antiphosphotyrosine antibody 4G10, or the SHIP MoAbs
P2C6 or P1D7 (IB). The positions of 145 kD and 135 kD SHIP are indicted
with arrows.
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|
As can be observed in the Shc IP lane 2, immunoprecipitations using the
anti-Shc antibody contain tyrosine-phosphorylated proteins of 145 and
135 kD in lysates from M-CSF-treated FDC-P1 cells. These proteins
comigrate exactly with the SHIP isoforms and are recognized by the MoAb
P2C6. The 145 kD, but not the 135 kD, isoform is also detected with
P1D7 antibody, as was shown in Fig 5. As we and others have previously
described,8-10 145 kD SHIP is found in a complex with both
Shc and Grb2. To address whether 135 kD SHIP is also involved in this
interaction, we performed immunoprecipitations using a polyclonal
anti-Grb2 antibody and lysates from the untreated or M-CSF-treated
FDC-P1 cells. As can be observed in the Grb2 IP lane 2, tyrosine-phosphorylated proteins are present in Grb2 immunoprecipitates
that migrate identically to 145 kD and 135 kD SHIP. Additionally, both
bands are recognized by the MoAb P2C6, but the 135 kD band is not
recognized by the antibody P1D7 (Grb2 IP lanes 3-6). The Grb2
immunoprecipitation experiments required longer exposure times than the
Shc or SHIP experiments, suggesting a weaker interaction of Grb2 with SHIP.
The presence of the YIXM motif in SHIP suggests that it may bind the
SH2 domain of p85, the regulatory subunit of PI3K.27 Previous experiments in our laboratory showed that immunoprecipitates using anti-p85 polyclonal antibody contained SHIP, but it was not clear
whether this was a direct interaction or the result of a larger complex
formation.24 To more specifically detect p85-SHIP
interactions, we performed pull-down experiments using either the
N-terminal or the C-terminal SH2 domains of p85 fused to GST
(Fig 8). An immunoblot of the GST-SH2
domain pull-down eluates, incubated with the anti-SHIP MoAb P1D7,
showed a clear phosphorylation-dependent interaction of the p85
C-terminal SH2 domain with 145 kD SHIP (Fig 8, lanes 5 and 6).
Identical immunoblots incubated with the monoclonal anti-SHIP antibody
P2C6 also indicated an interaction of the p85 C-terminal SH2 domain
with 145 kD SHIP, and a much weaker interaction of the SH2 domain with
183 SHIP (lanes 11 and 12). Comparisons of 145 kD SHIP and 183 kD
SHIP bands in GST pull-downs (lanes 11 and 12) and SHIP
immunoprecipitations (lanes 7 and 8) showed that the ratio of 145 kD
SHIP to 183 SHIP was approximately 3:1 in the SHIP
immunoprecipitates, but was at least 10:1 in the SH2 domain GST
pull-down experiments. In each case, the p85 N-terminal SH2 domain
showed only a faint SHIP antibody-reactive band at 145 kD, which was
similar in the unstimulated and the M-CSF-stimulated lysates (lanes 3, 4, 9, and 10). This may indicate a weak phosphorylation-independent
interaction between SHIP and the N-terminal SH2 domain.
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| Fig 8.
GST pull-downs. Lysates from FDC-P1 cells were either
untreated () or treated with M-CSF (+) as for Fig 7. Lysates were
immunoprecipitated with anti-p85 or anti-SHIP #5340 polyclonal
antibodies (IP), or were mixed with GST-agarose beads and purified
peptides representing either the N-terminal or C-terminal SH2 domains
of p85 fused to GST (GST). Beads were incubated and washed and the
eluted proteins subjected to immunoblotting with MoAbs P1D7 or P2C6.
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| |
DISCUSSION |
In this report, we describe a novel spliced form of the 145 kD inositol
phosphatase SHIP that is expressed in hematopoietic cells. Our
experiments show that this 135 kD form, 183 SHIP, is produced along
with 145 kD SHIP at both the mRNA and protein levels in
M-CSF-differentiated BMCs and in various hematopoietic cell lines. In
the report by Helgason et al,7 a targeted disruption of
ship in mice eliminated both the 135 kD and the 145 kD forms of
SHIP protein, showing that these two isoforms are the product of a
single gene. Therefore, 183 SHIP expression is not likely to be the
product of a separate allele or transcriptional regulatory mechanism.
Also, the 183 nucleotide region deleted in 183 SHIP cDNA is flanked
by consensus intron splice donor and acceptor sequences, and the
deletion results in the elimination of 61 amino acids from within the
protein sequence. This finding suggests that the appearance of this 135 kD form is the result of alternative mRNA splicing and is not the
result of proteolytic cleavage or other post-translational modification
of SHIP protein.
To further study the SHIP isoforms, we developed several MoAbs. In the
hybridoma screening process, MoAbs were selected that recognized only
145 kD SHIP (P1D7), but we also obtained antibodies that recognized
both 145 kD SHIP and a 135 kD form (P2C6, P1C1, and P6H8), and these
specificities were not separable by repeated cloning of the hybridomas.
The recognition of both 145 and 135 kD SHIP bands by the MoAbs P2C6,
P1C1, and P6H8, as well as both the anti-SHIP polyclonal antibodies,
shows that the two species are closely related. Furthermore, the 135 kD
form of SHIP lacks the epitope recognized by the MoAb P1D7. Our
comparison of SHIP isoforms using these antibodies, together with our
RT-PCR analyses, provides a powerful means of detecting specific
differences in the various SHIP proteins.
Initially, we considered that the 135 kD band recognized by our
anti-SHIP antibodies may be a proteolytic cleavage product of 145 kD
SHIP, as was recently described by Damen et al.30 However,
treatment of cell lysates with protease inhibitors or lysing cells
directly into SDS-PAGE sample loading buffer did not affect the
appearance of the 135 kD band in immunoblotting experiments.
Furthermore, our data showed that the epitope recognized by the MoAb
P1D7 is absent in the 135 kD isoform. To remove this epitope by normal
proteolysis, more than 160 amino acids (about 19 kD) must be deleted
from the C terminus. Our SDS-PAGE size estimates show that full-length
and 135 kD SHIP differ by less than 10 kD, making it unlikely that the
epitope recognized by P1D7 is eliminated by proteolytic degradation.
We obtained complete cDNA sequences for both the 145 kD and the 135 kD
forms of SHIP using an FDC-P1 murine cDNA library. 293T fibroblasts
transfected with this full-length SHIP cDNA in an expression vector
produce SHIP protein of the expected size and also a protein
approximately 15 kD smaller (Fig 5, lane 2). However, this smaller (130 kD) band in the full-length SHIP transfectants is recognized using the
MoAb P1D7 that does not recognize either the translated product of
183 SHIP in the same cell lines, or the 135 kD SHIP in hematopoietic
cells. RT-PCR analysis of full-length SHIP-transfected 293T cells
using the 2666-3247 primer set readily detects full-length SHIP cDNA as
expected, but is unable to detect 183 SHIP cDNA. These results
suggest that the smaller bands observed in fibroblasts transfected with
SHIP cDNA are the result of proteolysis or other modification of SHIP
perhaps as described by Damen et al,30 but that these
modifications are not responsible for the appearance of 183 SHIP/135
kD protein. We also observe the 130 kD protein in some, but not all,
preparations of hematopoietic cells (Fig 7, SHIP IP, lane 5).
Considering the specificity of the MoAbs, this 130 kD SHIP protein must
differ from full-length SHIP in a region other than that deleted in
183 SHIP/135 kD protein.
Our competitive PCR experiments have shown that in the cell types we
have tested, 183 SHIP cDNA is present in higher abundance than
full-length SHIP cDNA. This is not the result of preferential amplification of the shorter product, because as can be observed in Fig
4, the rate of amplification for both products is nearly identical. In
contrast, our immunoblot assays show that 145 kD SHIP protein appears
to be present in at least three-fold greater abundance than 183 SHIP
protein. Reasons for this difference may include lesser affinities of
the anti-SHIP antibodies for 183 SHIP, an increased rate of protein
degradation, or a decreased efficiency of translation for the 183
SHIP isoform. We are conducting experiments to distinguish between
these or other possibilities. As we have observed in murine BMCs, the
expression pattern of SHIP or SHIP isoforms appears to be based on the
maturational stage of the cell, and further experimentation is
necessary to determine the details of this change in regulation. In
future studies, the competitive PCR strategy we describe will allow us to compare ratios of 183 SHIP to full-length SHIP message in the
course of hematopoietic cell development to determine if there is a
difference in abundance related to cell differentiation.
The human myeloid leukemia cell line ML-1 produces a SHIP isoform of
approximately 110 kD. When these cells are treated with TPA to induce
differentiation toward a macrophage phenotype, SHIP forms of
approximately 135 and 145 kD are expressed and production of the 110 kD
form is diminished26 (Fig 6A). This pattern parallels what
we have observed in this study using murine BMCs treated with IL-3 plus
M-CSF, and suggests that human cells may produce SHIP splice forms as
well. Our experiments also showed that normal human peripheral white
blood cells express a 135 kD SHIP protein in addition to 145 kD SHIP,
but the expression of this form is low relative to the 145 kD form.
Using RT-PCR, we detected a shorter species of SHIP cDNA, as well as
full-length SHIP cDNA, in both TPA-treated ML-1 cells and in normal
human peripheral white blood cells. The smaller cDNA species was
amplified in addition to the expected product using primers to the
human SHIP sequence analogous to the murine SHIP 2666-3247 primers, and
may represent a human SHIP splice form similar to 183 SHIP. Our
genomic and cDNA sequencing results show that the 282 bp deletion in
this potential splice form overlaps the site of the 183 SHIP
deletion. Also, the segment deleted is preceded on each end by a
consensus splice acceptor, and corresponds perfectly to intron-exon
boundaries we determined for murine genomic DNA. Thus, 282 SHIP is
likely to arise from mRNA splicing. The deleted segment encodes 10 kD,
and may be responsible for the size difference between the 145 and 135 kD forms observed in our immunoblotting experiments. Interestingly, the
deletion in the human SHIP cDNA has the same result as the 183 SHIP
deletion, which is to either eliminate or alter the specificity of the
proximal NPXY motif and to remove potential SH3-binding motifs from the SHIP protein.
Kavanaugh et al21 have identified SHIP isoforms of
predicted molecular weight 110, 130, and 145 kD (SIP-110, SIP-130, and SIP-145) in human B cells. According to this study, these SIP/SHIP species vary from each other in the N-terminal region and do not contain a deletion in the C-terminal region. Also, our P6H8 MoAb, made
to the SH2 domain of SHIP, recognizes both the 145 and 135 kD forms of
SHIP (Fig 5). Thus, 183 SHIP and the murine homologs of the human
SIP isoforms, if present in these myeloid cells, are distinct.
Similarly, a recent report from Pesesse et al25 describes
an additional member of the SHIP family in human cells, SHIP2. We do
not believe that the 135 kD SHIP protein we have examined is the murine
version of SHIP2. Our results show that the 135 kD band observed in
SHIP immunoblots of myeloid cells is identical to the predicted
character of translated 183 SHIP cDNA, which in turn is identical in
sequence to full-length SHIP except for the 183 nucleotide deletion.
SHIP2, in contrast, is less than 50% identical to human 145 kD SHIP by
amino acid comparison, and is larger than SHIP by approximately 10 kD.
In some instances, our results contradict the recent findings of Damen
et al,30 who showed that a 135 kD protein, as well as
smaller forms, are produced by C-terminal cleavage of full-length SHIP.
For example, they show that 145 kD, but not 135 kD, SHIP associates
with Shc after IL-3 treatment of DA-ER cells. It is possible that the
phosphorylation pattern of SHIP isoforms differs between FDC-P1 and
DA-ER cells, or that IL-3 treatment of DA-ER cells causes the
phosphorylation of 145 kD SHIP and not of 135 kD SHIP. Another
explanation is that our antibodies detect a different SHIP form than
the 135 kD cleavage product observed by Damen et al. As discussed
above, we also observe a separate SHIP form at approximately 130 kD in
some experiments that is distinct from 135 kD SHIP; this may be the
same form described by Damen et al. Additionally, the 135 kD protein
observed by Damen et al could be the product of mRNA splicing as for
183 SHIP cDNA. Their results show that 135 kD SHIP is recognized by
their C-terminal polyclonal antibody. The immunogen used to produce
this antibody includes the amino acids 1157-1178. Therefore, this
antibody could not recognize a proteolytic cleavage product of 145 kD
SHIP lacking more than 4 kD from the C-terminal end. Their results,
however, are consistent with our data showing that a deletion occurs
internally and not at the C-terminal end of SHIP. This explanation
would require splicing of message from the transfected SHIP cDNA, which we did not find in our experiments using Rat2 fibroblasts, but that may
occur in murine hematopoietic cells. It should be determined whether
DA-ER cells actually produce the message for 183 SHIP, and whether
135 kD SHIP in these cells has the same migration and antibody
recognition pattern as the 183/135 kD SHIP we have described.
An important question raised by this work is the biological reason for
expression and function of two similar forms of SHIP in the same cell
types under the same conditions. It is likely that the 61 amino acid
region deleted in 183 SHIP is critical in certain protein-protein
interactions, and loss of this region alters SHIP association with
other signaling intermediates while allowing normal expression of the
SHIP SH2 and catalytic domains. We have shown in this report that like
145 kD SHIP, 183 SHIP is phosphorylated on tyrosine after M-CSF
stimulation in the myeloid cell line FDC-P1. Furthermore, both 135 kD
and 145 kD SHIP isoforms interact with Shc and Grb2 after M-CSF
treatment in FDC-P1 cells; binding of these molecules to SHIP is not
affected by the 183 deletion.
GST pull-down experiments using the two SH2 domains of the p85
regulatory subunit of PI3K showed a potentially important difference in
the association of the two isoforms with PI3K. The deletion in 183
SHIP occurs within the amino acid sequence YIGM, which when
phosphorylated has been shown to be the preferred binding target of
p85.15 The 183 deletion eliminates the methionine at the
+3 position, crucial for recognition by the p85 SH2
domain,27 and changes the sequence to YIAN. Although the
145 kD SHIP form bound well to the C-terminal SH2 domain of p85 in our
experiments, 183 SHIP bound poorly. Thus, the 183 deletion
appears to eliminate interaction of 183 SHIP with PI3K, possibly
through this YIGM motif. PI3K is vital in phosphatidylinositol
signaling, and is activated by many extracellular factors that also
promote its recruitment to the cell membrane.15 PI3K has
been shown to activate other protein tyrosine kinases, as well as to be
involved in vesicular trafficking and actin cytoskeletal
rearrangements. The enzymatic activities of SHIP and PI3K can affect
the same phosphatidylinositol intermediates. Therefore, SHIP and PI3K
might associate in a pathway and together regulate levels of specific
phosphatidylinositols. 183 SHIP may escape this interaction with
PI3K to alternately affect inositol levels. A likely target of this
regulatory mechanism is the serine-threonine kinase Akt/protein kinase
B, active in growth-factor mediated signal transduction.29
Further experiments, including overexpression of 183 SHIP in
hematopoietic cells, are under way to provide evidence for this hypothesis.
| |
ACKNOWLEDGMENT |
The authors thank all the members of the Rohrschneider laboratory for
many helpful discussions and comments throughout this work. We would
especially like to thank Dr Paul Algate who constructed the FDC-P1 cDNA
library, Susan Geier and Carol Romero for expert technical assistance,
and Dr Elizabeth Wayner in the FHCRC Hybridoma Shared Resource.
| |
FOOTNOTES |
Submitted July 14, 1998; accepted November 2, 1998.
Supported by Public Health Service Grant No. CA20551 and Fred
Hutchinson Cancer Research Center funding to L.R.R., and a National Research Service Award (F32 DK09774-01) to D.M.L.
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.
*
Full-length SHIP, with a calculated molecular weight of 133 kD,
has been described previously as a 145 kD protein due to its slower
migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) analyses. To maintain consistency, we will refer to the
apparent molecular weights of SHIP throughout this report.
Address correspondence to David M. Lucas, Division of Basic Sciences,
Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, B2-152, PO
Box 19024, Seattle, WA 98109-1024.
| |
REFERENCES |
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Dorshkind K:
Regulation of hemopoiesis by bone marrow stromal cells and their products.
Annu Rev Immunol
8:111, 1990[Medline]
[Order article via Infotrieve]
2.
Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold R, Rohrschneider LR:
p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity.
Genes Dev
10:1084, 1996[Abstract/Free Full Text]
3.
Damen JE, Liu L, Rosten P, Humphries RK, Jefferson AB, Majerus PW, Krystal G:
The 145-kD protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase.
Proc Natl Acad Sci USA
93:1689, 1996 |
TOP
ABSTRACT
INTRODUCTION