From the Departments of Internal Medicine and
§ Biological Chemistry, University of Michigan Medical
School, Ann Arbor, Michigan 48109
Received for publication, September 12, 2000, and in revised form, April 2, 2001
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ABSTRACT |
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It has been shown previously that the
Huntingtin interacting protein 1 gene (HIP1) was
fused to the platelet-derived growth factor Chronic myelomonocytic leukemia is a type of myelodysplastic
syndrome characterized by dysplastic monocytosis, variable bone marrow
fibrosis, and progression to acute leukemia. Chromosomal translocations
involving the platelet-derived growth factor The TEL/PDGF We have recently examined in more detail via mutational analysis the
roles of both HIP1 and the PDGF During this mutational analysis, we found that regardless of the
transforming HIP1/PDGF During purification of p130 from
transformed cells, p130 was found to comigrate on SDS-PAGE gels with
one of the in vivo processed forms of the SH2-containing
inositol 5-phosphatase SHIP1. SHIP1 is an enzyme that catalyzes the
hydrolysis of both PtdIns(3,4,5)P3 and
Ins(1,3,4,5)P4, resulting in the formation of
PtdIns(3,4)P2 and Ins(1,3,4)P3 respectively. It
is phosphorylated on tyrosyl residues in response to growth factor
stimulation and activation of immune receptors (12-14) and is
expressed solely in hematopoietic and developing spermatogonial tissues
(15, 16). During murine development, SHIP1 is first expressed at
embryonic day 7.5, coincident with the onset of hematopoiesis. This is
consistent with the phenotype of mice homozygous for a SHIP1
deletion, which, although viable and fertile, overproduce granulocytes
and macrophages and suffer from progressive splenomegaly, massive
myeloid infiltration of the lungs, and a shortened lifespan (17). The
granulocyte/macrophage progenitors from these mice are more sensitive
to many cytokines compared with similar cells from their wild type littermates.
Based on the phenotype of SHIP1 gene disruption and
the hydrolysis of PtdIns(3,4,5)P3 and
Ins(1,3,4,5)P4, SHIP1 is likely to be a negative regulator
of cellular signaling involving PtdIns 3-kinase. Consistent with this
hypothesis, it has recently been shown that SHIP1 protein levels were
reduced in primary neoplastic cells from patients with chronic
myelogenous leukemia, as might be expected with the myeloproliferative
phenotype observed in SHIP We demonstrate here that the p130 phosphorylated protein observed in
HIP1/PDGF Cell Lines and Culture--
Ba/F3, 32D, and FL5.12 cells were
grown in RPMI 1640 and 10% fetal calf serum with or without IL-3 (1 ng/ml). 293T cells were grown in Dulbecco's modified Eagle's medium
and 10% fetal calf serum.
Immunoprecipitations--
Protein extracts (500 µg of total
protein) were incubated at 4 °C for 1 h with 4 µl of 4G10
anti-Tyr(P) antibody (Upstate Biotechnology, Inc.), 16 µl of
anti-SHIP antibody (Pharmingen), 5 µl of anti-tail PDGF Western Blotting--
Extracts (100 µg of total protein) or
immunoprecipitates were separated on 7% SDS-PAGE, transferred to
nitrocellulose (APB; Hybond-ECL), and blocked with TBST, 5% bovine
serum albumin. Primary antibodies (1:1000-1:5000 dilutions depending
on antibody) were incubated with the blocked membrane in 5% bovine
serum albumin/TBST. The membranes were washed with TBST, and then
anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary
antibodies (1:5000 in TBST) were used to develop the blots with ECL
(Amersham Pharmacia Biotech).
DNA Constructs--
A Myc-tagged SHIP1a DNA insert in
pVL1393 (kindly provided by Philip Majerus, Washington University, St.
Louis) was digested with EcoRI. The released Myc-SHIP
fragment was ligated into the EcoRI site of pcDNA3.
HIP1/PDGF Stable Expression of Fusion Proteins in Ba/F3 Cells--
All
PDGF Transient Expression in 293T Cells--
Murine 293T cells were
passaged 1:2 onto 15-cm plates in Dulbecco's modified Eagle's medium,
10% fetal calf serum, 10 units/ml penicillin/streptomycin. When
confluent, cells were passaged at a dilution of 1:4 onto 10-cm plates.
Approximately 48 h after plating, cells were transfected with 15 µg of DNA in 450 µl of Dulbecco's modified Eagle's medium and 90 µl of Superfect transfection reagent as described by the manufacturer
(Qiagen). When two plasmids were co-transfected, 7.5 µg of each
plasmid were used. Protein extracts were prepared from cells harvested
48 h post-transfection.
Antibody Production--
The pGEX-3'HIP1 construct was used to
express and purify recombinant protein as described by the supplier of
the pGEX vector (Amersham Pharmacia Biotech). The purified protein was
dialyzed to remove glutathione and treated with thrombin to release
glutathione S-transferase, and the free glutathione
S-transferase was removed by adding a second aliquot of
glutathione-Sepharose and collecting the unbound fraction as antigen.
For the initial immunization, 100 µg of purified antigen were
dissolved in complete Freund's adjuvant and injected subcutaneously
into a rabbit at multiple sites. Purified antigen (50 µg) was mixed
with incomplete Freund's adjuvant and used for the secondary immunizations.
Enzyme Assays--
The phosphatase activity of
immunoprecipitates derived from the Ba/F3 cell line and 293T cells was
assessed using Ins(1,3,4,5)P4 as a substrate. Protein
extract (1 mg of total protein) used for the immunoprecipitations was
prepared as described above. Prior to use in phosphatase assays,
immunoprecipitates were washed three times with 1 ml of phosphatase
assay buffer (50 mM Tris, pH 7.5, 10 mM
MgCl2) to remove phosphatase inhibitors. Phosphatase assays were carried out at 37 °C as described for SHIP2 (20) using a
malachite green-based assay system (21).
HPLC--
For analysis of products formed after incubation of
Ins(1,3,4,5)P4 with immunoprecipitates, the reactions were
carried out using 3H-labeled substrate and separated by
anion HPLC using a Partisphere 5-SAX column. Separations were achieved
using an ammonium phosphate gradient consisting of buffer A (10 mM ammonium phosphate, pH 3.8) and buffer B (1.0 M ammonium phosphate, pH 3.8) as follows: 0-10 min, 0% B;
10-40 min, 0% B to 12.5% B; 40-80 min, 12.5% B to 100% B; 80-89
min, 100% B; 89-90 min, 100% B to 0% B; 90-115 min, 0% B at a
flow rate of 1 ml/min. An online radioisotope detector was used with
Beckman Ready Flow III scintillant.
Subcellular Fractionation--
Crude homogenates of 293T cells
were made by sonication at 4 °C in lysis buffer without the Triton
detergent. The first centrifugation step precipitated cellular debris
and nuclei for 5 min at 1,500 × g. The 1,500 × g supernatant was subsequently centrifuged for 30 min at
142,000 × g. This was designated the total membrane fraction. The remaining supernatant was defined as the cytosolic fraction.
Aliquots of the total membrane fraction were suspended twice at 2 mg/ml
in 0.5 M NaCl, 10 mM Tris (pH 7.4), and
protease inhibitors. The membrane suspensions were incubated on ice for
30 min and then centrifuged at 142,000 × g for 30 min.
The supernatant from this was designated the peripheral membrane
fraction. The pellet was designated the integral membrane fraction.
Peripheral and cytosolic fraction protein concentration was determined
by Bradford assay. The integral membrane fraction was solubilized in a
minimal volume of 1% SDS, 3 M urea, and 0.1 mM
dithiothreitol and Tris-buffered saline and sonicated. Samples were
diluted at least 1× with 5× sample buffer and loaded onto SDS-PAGE
gels after boiling. Western blotting was performed as described above.
p130 Is Immunologically Related to the 125-kDa Form of
SHIP1--
Recently, Sattler et al. (18) demonstrated that
SHIP1 RNA and protein levels were down-regulated in BCR/ABL-transformed Ba/F3 cells. Based on these results, we wished to determine whether SHIP1 protein levels were similarly altered in
HIP1/PDGF
Following this observation, p130 immunoprecipitation by anti-SHIP1
antibodies was tested. As shown in Fig.
2, anti-SHIP1 antibodies specifically
immunoprecipitated p130 from Ba/F3 cells transformed with the
HIP1/PDGF
Fig. 2 also demonstrates that anti-SHIP1 antibodies were able to
immunoprecipitate p130 in the H/P(F8)-transformed cells but not in the
H/P(KI)-expressing nontransformed cells grown in IL-3. H/P(F8) is a
mutant of the HIP1/PDGF SHIP1 Is Tyrosine-phosphorylated and Associates with HIP1/PDGF
We next tested whether SHIP1 could physically associate with the
HIP1/PDGF
To address the specificity of the interaction between SHIP1 and the
PDGF SHIP1 Inositol 5-Phosphatase Activity Is Not Altered by Tyrosine
Phosphorylation in HIP1/PDGF
The data from these activity studies further support the
identification of p130 as SHIP1, since the anti-phosphotyrosine
antibody immunoprecipitates from HIP1/PDGF SHIP1, HIP1/PDGF
In addition to the data presented above, which suggests that
phosphorylated SHIP1 binds directly to the transforming fusion proteins, we determined the cellular localization of SHIP1 and the
PDGF In this paper, we provide evidence that a previously unidentified
130-kDa phosphorylated protein, from Ba/F3 cells transformed by
PDGF We also find that the enzymatic activity of SHIP1 is not altered by
tyrosine phosphorylation in transformed cells, leading us to propose
that SHIP1 may be physically sequestered away from activated receptor
tyrosine kinases such as the IL-3 receptor and therefore unable to
negatively regulate signaling via PtdIns(3,4,5)P3 or
Ins(1,3,4,5)P4. This would lead to constitutive activation of cell growth signaling pathways. In support of this hypothesis, we
find that the PDGF It is also noteworthy that cells transformed with the H/P(F8) mutant,
in which eight recognized SH2-binding phosphotyrosines are mutated to
phenylalanine, still exhibit tyrosine phosphorylation of SHIP1. Since
the H/P(F8) mutant is recognized by the anti-Tyr(P) antibody, it is
likely that the association of SHIP1 with the fusion protein is
mediated through an as yet unidentified autophosphorylation site in the
PDGF We did not find substantial changes in SHIP1 protein levels, Akt
phosphorylation, or association of either Grb2 or Shc with SHIP1c
(p130) in the HIP1/PDGF However, the lack of constitutive or enhanced Akt activation in
HIP1/PDGF It is relevant that Phee et al. (22) have found that the
SHIP1-mediated decrease in endogenous PtdIns(3,4,5)P3 and
resultant inhibition of
PtdIns(3,4,5)P3-dependent kinases depends on
recruitment of the constitutively active SHIP1 enzyme to a
tyrosine-phosphorylated plasma membrane growth factor receptor. Our
results suggest that the PDGF In addition to the absence of Akt activation, we did not detect an
increase in PtdIns(3,4,5)P3 in resting or IL-3-stimulated PDGF It has been proposed by several groups that SHIP1 5-phosphatase
activity is stimulated when SHIP1 is tyrosine-phosphorylated by
activated receptor tyrosine kinases. However, tyrosine phosphorylation of SHIP1 has in fact not been consistently shown to increase or decrease enzymatic activity (24). For example, one study in yeast has
shown that co-expression of Lck with SHIP1 results in phosphorylation
of SHIP1 and a 3-fold decrease in 5-phosphatase activity (14).
Additionally, activity measurements of SHIP1 immunoprecipitates from
cytokine-activated B6YTA1 cells (24) and FDC-P1 cells (27) show no
difference in hydrolysis of PtdIns(3,4,5)P3 or
Ins(1,3,4,5)P3. This is consistent with our finding that
tyrosine-phosphorylation does not alter enzymatic activity. It remains
possible that in each of these studies in vitro assays may
not be sensitive enough to detect differences in enzymatic activity
when only a very small amount of tyrosine-phosphorylated SHIP1 is
present in the samples.
Finally, the molecular basis and significance of the differential
tyrosine phosphorylation of the 125-kDa SHIP1 isoform, but not other
SHIP1 isoforms, in our transformed cells remains unclear. It is
possible that the 125-kDa form has a binding site for the fusion
proteins that is masked on the 145- or 135-kDa forms or that may be
truncated in the 110-kDa form. It has been previously suggested that
the truncated forms may have different signaling properties. For
example, the 110-kDa form was found to be exclusively localized to the
cytoskeleton (24).
Phosphorylation of SHIP1 in cells transformed with the PDGF receptor gene
(PDGF
R) in leukemic cells of a patient with
chronic myelomonocytic leukemia. This resulted in the expression of the
chimeric HIP1/PDGF
R protein, which oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the Ba/F3 murine hematopoietic cell line to interleukin-3-independent growth. Tyrosine phosphorylation of a 130-kDa protein (p130) correlates with transformation by HIP1/PDGF
R and related transforming mutants. We report here that the
p130 band is immunologically related to the 125-kDa isoform of the Src
homology 2-containing inositol 5-phosphatase, SHIP1. We have
found that SHIP1 associates and colocalizes with the HIP1/PDGF
R fusion protein and related transforming mutants. These mutants include
a mutant that has eight Src homology 2-binding phosphotyrosines mutated
to phenylalanine. In contrast, SHIP1 does not associate with H/P(KI),
the kinase-dead form of HIP1/PDGF
R. We also report that
phosphorylation of SHIP1 by HIP1/PDGF
R does not change its 5-phosphatase-specific activity. This suggests that phosphorylation and
possible PDGF
R-mediated sequestration of SHIP1 from its substrates (PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4) might
alter the levels of these inositol-containing signal transduction
molecules, resulting in activation of downstream effectors of cellular
proliferation and/or survival.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor gene
(PDGF
R) are recurring cytogenetic
abnormalities associated with chronic myelomonocytic leukemia. These
translocations result in cellular expression of fusion proteins such as
TEL/PDGF
R (1), CEV1/PDGF
R (2), and HIP1/PDGF
R (1, 3) that contain dimerizing amino-terminal motifs fused to the transmembrane and
tyrosine kinase domains of PDGF
R.
R and HIP1/PDGF
R fusions both exhibit constitutive
kinase activity due to dimerization mediated through the TEL and HIP1
domains. Expression of these fusion proteins transforms the
IL-31 dependent murine
hematopoietic cell line, Ba/F3, to IL-3-independent growth and induces
hematopoietic malignancies in murine models of leukemia (3-5). Both
the kinase activity of the PDGF
R portion and the oligomerization
motifs of the TEL or HIP1 portions of the fusions are necessary for transformation.
R in the fusion protein-mediated transformation (6). For the HIP1 portion, stepwise deletions were made
to find the minimal oligomerization domain. For the PDGF
R portion,
we tested the effect of several tyrosine to phenylalanine substitutions
that abrogate binding of signal transduction molecules, including
PtdIns 3-kinase and PLC
. These studies demonstrated that
dimerization and autophosphorylation alone were insufficient for
transformation. Furthermore, key mitogenic pathways mediated by PLC
and PtdIns 3-kinase, that are normally activated by native PDGF
R,
were not necessary for transformation by HIP1/PDGF
R. This raised the
question of which signaling pathways, activated by HIP1/PDGF
R, are
necessary for transformation.
R mutant used, cells contained a
hyperphosphorylated 130-kDa protein (p130) and constitutively active
STAT5. Since STAT5 is known to promote DNA synthesis and cell division
(7-9), its constitutive activation might result in a transformed
phenotype. In addition, there is strong evidence that constitutive
STAT5 activation is not only correlated with transformation but is also necessary for leukemogenesis (10, 11). To further clarify our
understanding of the molecular events leading to cellular transformation by HIP1/PDGF
R, we have focused in this study on the
identification and characterization of p130.
/
mice (18).
Furthermore, ectopic expression of BCR/ABL in Ba/F3 cells led to
a rapid reduction in the level of SHIP1 protein. The involvement of
SHIP1 in the regulation of hematopoietic proliferation, along with the
electrophoretic comigration of p130 with SHIP1 in our transforming
HIP1/PDGF
R extracts led us to pursue the idea that p130 might be
identical or related to SHIP1.
R-transformed Ba/F3 cells is indeed related to the 125-kDa
isoform of SHIP1. Although SHIP1 protein levels were not consistently
altered in these cells, tyrosine phosphorylation of SHIP1 and its
physical association with the HIP1/PDGF
R fusion proteins might
contribute to transformation, sequestering SHIP1 from its normal
inositol-containing substrates and altering their cellular levels.
Since SHIP1 is only expressed in hematopoietic tissue and developing
spermatogonia, we predict it would be an excellent therapeutic target
for hematologic malignancies such as chronic myelomonocytic leukemia.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R antibody
(Pharmingen), or 5 µl of anti-HIP1 immune serum (see below). Protein
G-Sepharose (Amersham Pharmacia Biotech) was used to precipitate the
immune complexes. The protein G beads were washed three times with 500 µl of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, protease inhibitors, 30 mM sodium pyrophosphate, 50 mM NaF, and 100 µM sodium orthovanadate).
R, TEL/PDGF
R, and the various mutants have been
previously described (6). The glutathione S-transferase-HIP1
fusion construct that was used to generate anti-HIP1 rabbit
serum contained glutathione S-transferase fused in
frame to HIP1 amino acid sequences starting at the internal EcoRI site (nucleotide 1250) and ending at the native stop
codon (nucleotide 3010). pcDNA3(HIP1) was digested with
EcoRI, and the 3-kilobase pair HIP1 fragment was inserted
into the EcoRI site of pGEX 4T-1 (Amersham Pharmacia
Biotech) to obtain a construct designated pGEX-3'HIP1.
R fusion constructs were cloned into MSCVneo as described (6).
293T cells (the kind gift of G. Gilliland, Harvard Medical School,
Boston) were transfected with Superfect transfection reagent as
instructed by the supplier (Qiagen). A 48-h post-transfection supernatant (1 ml) was then added to 106 Ba/F3, 32D, or
FL5.12 cells (1 ml) (all kindly provided by W. Pear, University of
Pennsylvania, Philadelphia) in the presence of polybrene as described
previously (19). Cells with stable expression were selected in the
presence of G418 and IL-3 as described (4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R-transformed Ba/F3, 32D, and FL5.12 cell lines. Levels of
SHIP1 protein were measured in these cells by immunoblotting with
various anti-SHIP1 antibodies. In contrast to the situation with
BCR/ABL, we found that all four SHIP1 isoforms (designated as 110-, 125-, 135-, and 145-kDa isoforms) showed similar levels in control and
HIP1/PDGF
R-transformed cells (Fig.
1A). However, one of the four
SHIP1 protein bands migrated in a similar position to our previously
unidentified p130 (6), as shown in three independent pairs of
HIP1/PDGF
R and vector-expressing Ba/F3 cell lines in Fig. 1. The
same phosphorylation pattern was seen in HIP1/PDGF
R-expressing 32D
and FL5.12 hematopoietic cell lines (data not shown).
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Fig. 1.
p130 co-migrates with a band recognized by
SHIP1 antibodies. Lysates from the transformed HIP1/PDGF R (H/P)
and nontransformed (vector) Ba/F3 cell lines were separated on 7%
SDS-PAGE and blotted onto nitrocellulose. Proteins were detected using
horseradish peroxidase-conjugated secondary antibodies and ECL. The
primary antibodies used were anti-SHIP N-1 from Santa Cruz
Biotechnology (A) and anti-phosphotyrosine (4G10) from
Upstate Biotechnology (B). H/P, HIP1/PDGF
R
stably transfected IL-3-independently growing Ba/F3 extracts.
Vector, MSCVneo vector-transfected
IL-3-dependently grown Ba/F3 cell extracts.
R fusion protein. When the anti-phosphotyrosine blot of the
anti-SHIP1 immunoprecipitates was stripped and reprobed with anti-SHIP1
antibodies, p130 co-migrated with the 125-kDa SHIP1 isoform. Fig. 2
also shows that p130 was cleared from the supernatant with anti-SHIP1
antibodies after three serial immunoprecipitations of the supernatant.
On average, ~50% of p130 was cleared from the supernatant when most
of the SHIP1 125-kDa isoform was cleared, suggesting that this band may
contain another phosphoprotein. It also remains a possibility that
SHIP1 associates with a currently unidentified co-migrating
phosphoprotein. We have previously eliminated CBL, RAS-GAP, CAS,
JAK1-3, TYK2, interleukin-3
receptor, PLC
, PtdIns 3-kinase,
and focal adhesion kinase as candidates for p130 (6) and more recently
tested to see if the SIRP
/
p130 protein was part of the
phospho-p130 in our transformed extracts. A monoclonal antibody
specific to SIRP
/
was used to immunoprecipitate p130, and while
all of SIRP
was precipitated from the supernatant, all of p130
remained unprecipitated (data not shown).
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Fig. 2.
p130 is immunoprecipitated by SHIP1-specific
antibodies. Lysates from transformed (H/P and H/P(F8)) and
nontransformed (expressing kinase-dead H/P(KI)) Ba/F3 cell lines were
immunoprecipitated (IP) with 16 µl of anti-SHIP1 antibody
(Pharmingen). The supernatants (S1, S2, and
S3) were subjected to further immunoprecipitation until they
were cleared of SHIP1 reactivity. Samples were separated on 7%
SDS-PAGE, and transferred onto nitrocellulose. The proteins were first
blotted with anti-phosphotyrosine antibody and then stripped (15 min at
50 °C in 1% SDS, 10 mM Tris, pH 8.5, and 2 mM -mercaptoethanol) and reblotted with the anti-SHIP1
antibodies. In this experiment, the antibody recognized the 145-, 125-, and 110-kDa isoforms much better than the 135-kDa isoform of SHIP1.
P1, first pellet from serial immunoprecipitations with
anti-SHIP1 antibodies. S3, half of the supernatant from the
third serial immunoprecipitation. H/P,
pcDNA3-HIP1/PDGF
R; H/P(F8), pcDNA3-H/P(F8);
H/P(KI), pcDNA3-H/P(KI) kinase-dead mutant (6)
R fusion protein that has eight of the
SH2-binding site tyrosines mutated to phenylalanines. H/P(KI) is a
construct that has a point mutation at arginine 634 of the PDGF
R,
where the substituted lysine leads to a kinase-dead mutant (6). As
shown previously (6) as well as in Fig. 1, p130 was not detectable in
cells transfected with vector alone, suggesting that the tyrosine
phosphorylation of SHIP1 can be attributed directly to expression of
the fusion protein. Additionally, p130 co-migrated with SHIP1 and was
recognized by SHIP1 antibodies in all of the previously described
HIP1/PDGF
R- and TEL/PDGF
R-transformed lines (6) (data not shown).
This included the H/P(F8) mutant (Fig. 2).
R
and TEL/PDGF
R Fusion Proteins When Co-expressed in 293T
Cells--
To determine whether the tyrosine phosphorylation of SHIP1
is mediated by the HIP1/PDGF
R and TEL/PDGF
R fusion
proteins, we initially analyzed protein extracts derived from cells
heterologously co-expressing Myc-tagged SHIP1 with either HIP1/PDGF
R
or TEL/PDGF
R. As shown in Fig.
3A, Myc-SHIP1 was
tyrosine-phosphorylated when co-expressed with HIP1/PDGF
R or
TEL/PDGF
R (lanes 5 and 6). SHIP1 phosphorylation was not detected when it was expressed alone
(lane 2) or in cells transfected with empty
vector (lane 1). It should be noted that
endogenous SHIP1 phosphorylation was not detected in 293T cells
transfected with fusion constructs alone (lanes 3 and 4) because SHIP1 was expressed in these cells.
Expression of the recombinant SHIP1 was confirmed by immunoblotting
with anti-Myc antibody as shown in Fig. 3B (lanes
2, 5, and 6). These results strongly
suggest that tyrosine phosphorylation of SHIP1 was catalyzed directly
by the PDGF
R kinase domain of the fusion proteins or a kinase
activated by the fusion proteins. As illustrated in Fig. 3A
(lanes 3 and 4), both fusion proteins
undergo autophosphorylation on tyrosine residues as expected.
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Fig. 3.
SHIP1 is tyrosine-phosphorylated
in cells heterologously expressing HIP1/PDGF R
or TEL/PDGF
R. A, cell extracts
from transiently transfected 293T cells were immunoblotted with the
anti-phosphotyrosine antibody 4G10. B, cell extracts from
the same cells were immunoblotted with anti-Myc antibodies.
Lane 1, protein extracts of cells transfected
with vector (pcDNA3); lane 2,
pcDNA3-MycSHIP1; lane 3, pcDNA3-H/P;
lane 4, pcDNA3-T/P; lane
5, pcDNA3-H/P plus pcDNA3-MycSHIP1; lane
6, pcDNA3-T/P plus pcDNA3-MycSHIP1. H/P,
HIP1/PDGF
R; T/P, TEL/PDGF
R; *, background band.
R or TEL/PDGF
R fusion proteins when co-expressed in 293T
cells. We surmised that association of SHIP1 with autophosphorylated HIP1/PDGF
R or TEL/PDGF
R, via its N-terminal SH2 domain, might play a role in regulating its phosphorylation by these proteins. To
address this question, immunoprecipitations were performed from lysates
of 293T cells expressing the various combinations of SHIP1 and
the fusion proteins, as described above, using an antibody directed
toward the C terminus of the PDGF
R. As seen in Fig.
4A, tyrosine-phosphorylated
SHIP1 co-immunoprecipitates with either HIP1/PDGF
R or TEL/PDGF
R
(lanes 5 and 6), and
immunoprecipitation with anti-Myc antibodies showed that Myc-SHIP1 was
only phosphorylated in HIP1/PDGF
R and TEL/PDGF
R co-expressing
extracts (Fig. 4B, lanes 5 and
6). SHIP1 expression in extracts of all cells transfected with pcDNA3-MycSHIP1 was confirmed by immunoblotting using anti-Myc antibody as shown in Fig. 3B (lanes 2,
5, and 6).
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Fig. 4.
Association of
HIP1/PDGF R and
TEL/PDGF
R with SHIP1.
Immunoprecipitations (IP) were performed from 293T
cell extracts using anti-PDGF
R antibodies (A) or anti-Myc
antibody (B). Samples were washed with lysis buffer,
separated on 7% SDS-PAGE, and blotted onto nitrocellulose. The blots
were probed with anti-phosphotyrosine 4G10 monoclonal antibody.
Lane 1, protein extracts of cells transfected
with pcDNA3; lane 2, pcDNA3-MycSHIP1;
lane 3, pcDNA3-H/P; lane
4, pcDNA3-T/P; lane 5,
pcDNA3-H/P plus pcDNA3-MycSHIP1; lane 6,
pcDNA3-T/P plus pcDNA3-MycSHIP1. H/P, HIP1/PDGF
R;
T/P, TEL/PDGF
R.
R fusion proteins, we next tested the transforming H/P(F8) and
nontransforming kinase-inactive R634K also known as H/P(KI) mutants of
HIP1/PDGF
R for their ability to associate with and promote
phosphorylation of SHIP1. The H/P(F8) mutant, which has eight known SH2
phosphotyrosine binding sites mutated to phenylalanine, still
associated with and promoted tyrosine phosphorylation of SHIP1 (Fig. 2, right panel,
lane 2; Fig. 5A, lane
4; and Fig. 6A,
lane 4), consistent with its previously reported transforming properties (6). The binding site for SHIP1 on the fusion
protein may be an autophosphorylation site, since the H/P(KI) mutant of
the HIP1/PDGF
R fusion neither promoted phosphorylation (Fig. 2,
lanes 3, and Fig. 5A, lane
6) nor associated with SHIP1 (Fig. 6A,
lane 6). Expression of SHIP1 and H/P(KI) was
confirmed (Fig. 5B, lane 6 for SHIP1;
Fig. 5C, lane 6 for H/P(KI)), and all
of the HIP1/PDGF
R mutants were immunoprecipitated equally well by
the anti-HIP1 antibody (Fig. 6C, lanes
3-6). The data also indicate that the anti-HIP1 antibody is
specific for the fusion protein or fusion protein complexes, since no
SHIP1 was immunoprecipitated by the HIP1 antibody (Fig. 6B,
lanes 2 and 6). Although the levels of
expression of the fusion protein and SHIP1 in 293T cells were
nonphysiologic, the lack of association between the kinase-dead fusion
protein, H/P(KI), and SHIP1 provides evidence that the interaction of
SHIP1 with HIP1/PDGF
R was phosphorylation-dependent and
specific and suggests that there was direct phosphorylation of SHIP1 by
the transforming fusion protein. The exact phosphorylation sites of
SHIP1 and interaction site(s) of both proteins remain to be determined,
since the known SH2-binding domains of the PDGF
R do not appear to be
involved.
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Fig. 5.
SHIP1 is tyrosine-phosphorylated in cells
heterologously expressing the HIP1/PDGF R(F8)
mutant but not the kinase-dead HIP1/PDGF
R(KI)
mutant. A, cell extracts from 293T cells were separated
by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with 4G10
anti-phosphotyrosine antibody. Lanes 3 and
4, phosphorylation of H/P(F8); lane 4,
phosphorylation of SHIP1. B, extracts from 293T cells were
separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted
with anti-Myc antibodies to show expression of SHIP1 in all of the
pcDNA3-MycSHIP1-transfected cells. C, 293T extracts were
separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted
with anti-PDGF
R tail antibody to show expression of the fusion
proteins in pcDNA3-H/P(F8)- as well as
pcDNA3-H/P(KI)-transfected cells. Lane 1,
protein extracts from cells transfected with pcDNA3;
lane 2, pcDNA3-MycSHIP1; lane
3, pcDNA3-H/P(F8); lane 4,
pcDNA3-H/P(F8) plus pcDNA3-MycSHIP1; lane
5, pcDNA3-H/P(KI); lane 6,
pcDNA3-H/P(KI) plus pcDNA3-MycSHIP1. H/P,
HIP1/PDGF
R; T/P, TEL/PDGF
R.
View larger version (22K):
[in a new window]
Fig. 6.
Association of H/P(F8) but not H/P(KI) with
SHIP1. Immunoprecipitations were performed on 293T cell extracts
with anti-HIP1 serum. Samples were washed with lysis buffer, separated
on 7% SDS-PAGE, and blotted onto nitrocellulose. A,
immunoprecipitates were blotted with anti-phosphotyrosine 4G10
monoclonal antibody. B, immunoprecipitates were blotted with
anti-Myc monoclonal antibody to confirm that SHIP1 is in the H/P(F8)
coexpressed precipitate. C, immunoprecipitates were blotted
with anti-PDGF R tail antibody to confirm that the anti-HIP1 antibody
immunoprecipitated H/P(F8) as well as H/P(KI). Lane
1, immunoprecipitates from cells transfected with
pcDNA3; lane 2, pcDNA3-MycSHIP1;
lane 3, pcDNA3-H/P(F8); lane
4, pcDNA3-H/P(F8) plus pcDNA3-MycSHIP1;
lane 5, pcDNA3-H/P(KI); lane
6, pcDNA3-H/P(KI) plus pcDNA3-MycSHIP1.
H/P, HIP1/PDGF
R; T/P, TEL/PDGF
R.
R-expressing Cells--
Since SHIP1 is
known to function as a negative regulator of cell growth and
antiapoptotic signal transduction pathways, we tested the possibility
that its phosphatase activity might be inhibited by tyrosine
phosphorylation in HIP1/PDGF
R-transformed Ba/F3 cells. To address
this issue, in vitro phosphatase assays were performed with
SHIP1 immunoprecipitated from Ba/F3 transformed cells, using
Ins(1,3,4,5)P4 as a substrate. Unexpectedly, we found that
SHIP1 activity was not significantly affected by tyrosine phosphorylation. The in vitro phosphatase activity of SHIP1
immunoprecipitated with anti-SHIP1 antibodies exhibited no significant
changes in transformed cells when compared with cells transfected with
vector alone, suggesting that tyrosine phosphorylation was not a
principal mechanism of SHIP1 regulation (data not shown). Even when
SHIP1 along with HIP1/PDGF
R or TEL/PDGF
R was overexpressed in
293T cells, tyrosine phosphorylation did not result in a dramatic
change in SHIP1 phosphatase activity (Fig.
7A). In Fig. 7A,
~20% of the Ins(1,3,4,5)P4 phosphatase activity was
eliminated with the anti-SHIP1 antibody in all extracts (HIP1/PDGF
R,
TELl/PDGF
R, or control extracts) and ~5% with the
anti-phosphotyrosine antibody in the HIP1/PDGF
R- and
TEL/PDGF
R-expressing extracts as compared with 0.5% in the control
extracts. These results suggest that the increase in 5-phosphatase
activity found in the anti-phosphotyrosine immunoprecipitates of the
HIP1/PDGF
R- or TEL/PDGF
R-expressing cells was a result of
increased immunoprecipitation of SHIP1 mass due to increased tyrosine
phosphorylation rather than stimulation of enzyme-specific activity.
View larger version (20K):
[in a new window]
Fig. 7.
SHIP1 5-phosphatase activity is not altered
by tyrosine phosphorylation. A, an in vitro
phosphatase assay using Ins(1,3,4,5)P4 as a substrate was
performed using 293T cell extracts, immunoprecipitated with
anti-phosphotyrosine 4G10 (anti-pTyr), anti-SHIP1, or
anti-Myc antibodies, from cells transfected with MycSHIP1 alone or
MycSHIP1 with H/P or T/P. B, HPLC elution profile of
reaction products generated by the anti-SHIP1 or anti-phosphotyrosine
4G10 immunoprecipitates using 3H-labeled
Ins(1,3,4,5)P4 as a substrate. The extracts used for the
immunoprecipitations were those from vector-transfected Ba/F3 cells
(anti-SHIP1 antibody, middle panel) or
H/P-transfected IL-3-independently growing Ba/F3 cells (4G10 antibody,
lower panel). The upper
panel is a standard run for reference purposes.
R-expressing cells
contained more intrinsic inositol 5-phosphatase activity than extracts
from control cells. Consistent with the substrate specificity of SHIP1, the HPLC elution positions of the reaction products generated by these
anti-phosphotyrosine immunoprecipitates using radiolabeled Ins(1,3,4,5)P4 as a substrate demonstrate that the
phosphatase activity is specific for the 5' position in both
transformed and nontransformed cells (Fig. 7B). The product
was exclusively Ins(1,3,4)P3 with no additional or
alternative hydrolysis of other positions on the inositol ring.
R, and TEL/PDGF
R Are Colocalized in
Vivo--
Recently, Phee et al. suggested that the
enzymatic activity of SHIP1 is regulated by its localization to the
plasma membrane (22). These authors also reported that neither
phosphorylation of SHIP1 by Lyn (a Src-related protein tyrosine kinase)
and Syk, nor receptor binding of SHIP1 changed its intrinsic enzymatic activity. Rather, it was SHIP1 membrane localization that significantly reduced PtdIns(3,4,5)P3 levels. Phee et al. (22)
concluded that membrane localization of SHIP1 is required for its
negative regulation of cellular signaling pathways. Our work extends
these findings and explains how phosphorylation of SHIP1 might
contribute to transformation. We suggest that in the
HIP1/PDGF
R-transformed cells, physical association of the fusion
protein with SHIP1 sequesters SHIP1 from binding to tyrosine kinase
receptors that are known to provide docking sites for the SH2 domain of
SHIP1. This could deny SHIP1 access to its substrates,
PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, thus
prolonging their steady-state levels.
R fusions. Consistent with this hypothesis, we found that the
majority of SHIP1 in 293T cells was localized to the cytoplasm rather
than the membrane (Fig. 8). Although
HIP1/PDGF
R was co-localized with SHIP1 to the cytoplasm,
HIP1/PDGF
R was distributed equally in the peripheral membrane and
cytosol, whereas SHIP1 was mostly cytosolic. Fig. 8 also shows
that co-expression of SHIP1 and the fusion proteins did not
dramatically alter either of their subcellular locales. Similar
co-localization results were obtained when TEL/PDGF
R and SHIP1 were
co-expressed in 293T cells (data not shown), suggesting that
localization of SHIP1 to a cellular environment deficient in
PtdIns(3,4,5)P3 (i.e. the cytosol) may
contribute to cellular transformation by both of these fusion
proteins.
View larger version (66K):
[in a new window]
Fig. 8.
Biochemical fractionation of SHIP1,
HIP1/PDGF R, and various mutants expressed in
293T cells. 293T cells were transfected with 1 µg each of the
various constructs designated at the top of the
lanes. Each protein fraction (50 µg) was analyzed on 7.5%
SDS-PAGE. Fractions were obtained by differential centrifugation of
HIP1 (upper blots) and Myc-SHIP1
(lower blots) into cytosolic and peripheral
membrane fractions. Neither protein was detected in the integral
membrane or nuclear fractions (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R fusion proteins, appears highly related, if not identical, to
the 125-kDa isoform of SHIP1. We show that HIP1/PDGF
R and TEL/PDGF
R physically associate with SHIP1, suggesting that SHIP1 phosphorylation may be catalyzed by the PDGF
R tyrosine kinase moiety
of these fusion proteins. This conclusion is further supported by the
observation that the kinase-dead mutant form of the fusion, H/P(KI),
which is not phosphorylated in the H/P(KI)-expressing cells, does not
associate with or promote phosphorylation of SHIP1. This is consistent
with our previous experiments showing that the H/P(KI) mutant is
nontransforming and that H/P(KI)-expressing Ba/F3 cells do not contain
phospho-p130 (6).
R fusion proteins coimmunoprecipitate with SHIP1
and that SHIP1 and the fusion proteins are co-localized in the cell.
R kinase domain. Tyrosine phosphorylation of SHIP1 in the
absence of PtdIns 3-kinase and PLC
activation offers a possible
explanation for the ability of the H/P(F8) mutant to transform Ba/F3
cells (6, 9). For example, although we have previously found that
PtdIns 3-kinase is not constitutively activated in the H/P(F8)
transformed cells, SHIP1 could be inhibited from negatively regulating
PtdIns(3,4,5)P3-dependent signals. Although normally generated by activated PtdIns 3-kinase phosphorylation of
PtdIns (4,5)P2, sequestration of SHIP1 away from its
PtdIns(3,4,5)P3 substrate provides an alternative mechanism
by which to increase the PtdIns(3,4,5)P3 growth signaling
in the absence of elevated PtdIns 3-kinase activity. A similar
explanation applies to PLC
activation, which ultimately generates
the Ins(1,3,4,5)P4 signal by generating
Ins(1,4,5)P3 from PtdIns(4,5)P2.
Ins(1,4,5)P3 is then converted to
Ins(1,3,4,5)P4 by an Ins (1,4,5)P3-specific 3-kinase. Inhibition of Ins(1,3,4,5)P4 degradation by SHIP1
phosphorylation/sequestration away from its Ins(1,3,4,5)P4
substrate provides an alternative pathway to increase levels of
Ins(1,3,4,5)P4, thus making constitutive activation of
PLC
unnecessary for transformation.
R- or TEL/PDGF
R-transformed Ba/F3 cells
(data not shown). The SHIP1 polypeptide has been shown to exist in at
least four different molecular weight forms. The largest are the 145- and 135-kDa forms, which have C-terminal tyrosine phosphorylation sites
that bind the SH2-domain of the adaptor protein Shc (23). In addition,
proline-rich sequences found within the carboxyl-terminal of the larger
SHIP1 isoforms bind the SH3 domain of Grb2 (24). Shc and Grb2 are
adaptor proteins that were first identified as important in growth
factor receptor signaling through the RAS/mitogen-activated protein
kinase pathway. The mitogen-activated protein kinase pathway was not
constitutively activated in our HIP1/PDGF
R-transformed cells,
despite multiple attempts to show this (6). The inability of Grb2 and
Shc to bind p130 (possibly 125-kDa SHIP1) is not unexpected, since the C-terminal region of the 145- and 135-kDa SHIP1, to which these proteins bind, is not present in the 125-kDa isoform of SHIP1. Hence,
the lack of a high affinity association of p130 with Grb2 and Shc is
consistent with the possible identification of p130 as the 125-kDa form
of SHIP1.
R transformed Ba/F3 cells observed in our experiments was
unexpected. Enhanced Akt phosphorylation was expected, since Akt
activation was observed in response to IL-3 in SHIP1 null mice. In that
study, it was predicted that SHIP1 would function as a negative
regulator of Akt kinase activity by lowering cellular levels of
PtdIns(3,4,5)P3, the primary activator of Akt, and that SHIP1 deletion would lead to Akt activation (25). However, in HIP1/PDGF
R-transformed Ba/F3 cells, we have not detected excess Akt
activation either constitutively or as a hyperresponsive activation in
response to IL-3. We see similar Akt activation in response to IL-3
stimulation in all cell lines whether transformed with HIP1/PDGF
R or
not. Hence, the mechanism by which SHIP1 phosphorylation in
HIP1/PDGF
R-transformed cells confers IL-3-independent growth remains
unclear. It is also possible that SHIP1 phosphorylation in response to
IL-3 may not be sufficient to alter Akt activation but may be
sufficient to mediate positive effects on proliferation by, for
example, altering levels of its water-soluble substrate (Ins(1,3,4,5)P4) or reaction product
(Ins(1,3,4)P3).
R fusion proteins may sequester SHIP1
from binding to activated receptor tyrosine kinases, as suggested by
Phee et al. (22). This provides a mechanism whereby the
fusion proteins could abrogate negative signaling via SHIP1.
R fusion-transformed cells compared with control cells (data not
shown). However, this observation could be due to low sensitivity of
our assay, since we find very low and variable levels of
PtdIns(3,4,5)P3 in IL-3-stimulated Ba/F3 cells. Therefore,
detecting altered PtdIns(3,4,5)P3 levels in these cells
after IL-3 stimulation or constitutively may require more refined and
as yet undeveloped techniques. Alternatively, IL-3-mediated changes in
cellular levels of other phosphoinositides may play a role in Akt
activation. This is supported by data from a previous study that
identified Akt activation as a downstream signaling event in
IL-3-stimulated Ba/F3 cells. This activation required PtdIns 3-kinase
activity (26). However, this effect was not demonstrated to be mediated
directly through PtdIns(3,4,5)P3, suggesting the
possibility that SHIP1 may function as a regulator of distinct
phosphoinositide effectors other than PtdIns(3,4,5)P3.
R fusions
is a novel finding. We have reported previously that PtdIns 3-kinase
and PLC
were phosphorylated in HIP1/PDGF
R-transformed cells.
However, Tyr to Phe point mutants that prevent PtdIns 3-kinase and
PLC
phosphorylation by the PDGF
R fusions did not affect cell
transformation (6). The difference described herein is that SHIP1
phosphorylation is seen in all transformed cells examined to date.
Additional studies using SHIP1 dominant negative constructs and SHIP1 null mice will be necessary to clarify whether
SHIP1 phosphorylation is necessary for transformation. Future work in our laboratory will focus on identifying the exact mechanism and role(s) of both SHIP1 phosphorylation and its physical interaction with
the fusion tyrosine kinase oncoproteins in cellular transformation.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Sean Morrison, Gabriel Nunez, Mark Benson, Eric Fearon, Dinesh Rao, Jenny Chang, Ikuko Mizukami, and Glennda Smithson for critical review of this work. We especially thank Jack E. Dixon for reagent contributions as well as for critical review of this work and Denise Poirier for outstanding secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a National Institutes of Health (NIH) Endocrinology and Metabolism Training Grant (to G. S. T.), Grant KO8 CA76025-01 (to T. S. R.), and Grant RO1 CA82363-01A1 (to T. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ A postdoctoral research fellow supported by an Endocrinology and Metabolism Training Grant from the Michigan Diabetes Research and Training Center.
Supported by the Cancer Research Fund of the Damon
Runyon-Walter Winchell Foundation Award DRS-22. To whom correspondence may be addressed: University of Michigan Cancer Center, 4217 CCGC Box
0936, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-615-5509; Fax: 734-647-9654; E-mail: tsross@umich.edu.
Published, JBC Papers in Press, April 3, 2001
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ABBREVIATIONS |
---|
The abbreviations used are:
IL-3, interleukin-3;
PAGE, polyacrylamide gel electrophoresis;
PtdIns, phosphatidylinositol;
PtdIns(3, 4,5)P3, phosphatidylinositol
3,4,5-trisphosphate;
PtdIns(3, 4)P2, phosphatidylinositol
3,4-bisphosphate;
PtdIns(4, 5)P2, phosphatidylinositol
4,5-bisphosphate;
Ins(1, 3,4,5)P4, inositol
1,3,4,5-tetraphosphate;
Ins(1, 3,4)P3, inositol
1,3,4-trisphosphate;
PLC, phospholipase C
;
SH2, Src homology 2;
HPLC, high pressure liquid chromatography.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Golub, T. R., Barker, G. F., Lovett, M., and Gilliland, D. G. (1994) Cell 77, 307-316[Medline] [Order article via Infotrieve] |
2. |
Abe, A.,
Emi, N.,
Tanimoto, H.,
Terasaki, H.,
Marunouchi, T.,
and Saito, H.
(1997)
Blood
90,
4271-4277 |
3. |
Ross, T. S.,
Bernard, O. A.,
Berger, R.,
and Gilliland, D. G.
(1998)
Blood
91,
4419-4426 |
4. |
Carroll, M.,
Tomasson, M. H.,
Barker, G. F.,
Golub, T. R.,
and Gilliland, D. G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14845-14850 |
5. |
Tomasson, M. H.,
Williams, I. R.,
Hasserjian, R.,
Udomsakdi, C.,
McGrath, S. M.,
Schwaller, J.,
Druker, B.,
and Gilliland, D. G.
(1999)
Blood
93,
1707-1714 |
6. |
Ross, T. S.,
and Gilliland, D. G.
(1999)
J. Biol. Chem.
274,
22328-22336 |
7. | D'Ambrosio, D., Hippen, K. L., Minskoff, S. A., Mellman, I., Pani, G., Siminovitch, K. A., and Cambier, J. C. (1995) Science 268, 293-297[Medline] [Order article via Infotrieve] |
8. | de Groot, R. P., Coffer, P. J., and Koenderman, L. (1998) Cell Signal. 10, 619-628[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Tomasson, M. H.,
Sternberg, D. W.,
Williams, I. R.,
Carroll, M.,
Cain, D.,
Aster, J. C.,
Ilaria, R. L., Jr.,
Etten, R. A.,
and Gilliland, D. G.
(2000)
J. Clin. Invest.
105,
423-432 |
10. | Wilbanks, A. M., Mahajan, S., Frank, D. A., Druker, B. J., Gilliland, D. G., and Carroll, M. (2000) Exp. Hematol. 28, 584-593[CrossRef][Medline] [Order article via Infotrieve] |
11. | Schwaller, J., Parganas, E., Wang, D., Cain, D., Aster, J. C., Williams, I. R., Lee, C. K., Gerthner, R., Kitamura, T., Frantsve, J., Anastasiadou, E., Loh, M. L., Levy, D. E., Ihle, J. N., and Gilliland, D. G. (2000) Mol. Cell 6, 693-704[Medline] [Order article via Infotrieve] |
12. | Chacko, G. W., Tridandapani, S., Damen, J. E., Liu, L., Krystal, G., and Coggeshall, K. M. (1996) J. Immunol. 157, 2234-2238[Abstract] |
13. |
Damen, J. E.,
Liu, L.,
Rosten, P.,
Humphries, R. K.,
Jefferson, A. B.,
Majerus, P. W.,
and Krystal, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1689-1693 |
14. |
Osborne, M. A.,
Zenner, G.,
Lubinus, M.,
Zhang, X.,
Songyang, Z.,
Cantley, L. C.,
Majerus, P.,
Burn, P.,
and Kochan, J. P.
(1996)
J. Biol. Chem.
271,
29271-29278 |
15. |
Geier, S. J.,
Algate, P. A.,
Carlberg, K.,
Flowers, D.,
Friedman, C.,
Trask, B.,
and Rohrschneider, L. R.
(1997)
Blood
89,
1876-1885 |
16. |
Liu, Q.,
Shalaby, F.,
Jones, J.,
Bouchard, D.,
and Dumont, D. J.
(1998)
Blood
91,
2753-2759 |
17. |
Helgason, C. D.,
Damen, J. E.,
Rosten, P.,
Grewal, R.,
Sorensen, P.,
Chappel, S. M.,
Borowski, A.,
Jirik, F.,
Krystal, G.,
and Humphries, R. K.
(1998)
Genes Dev.
12,
1610-1620 |
18. |
Sattler, M.,
Verma, S.,
Byrne, C. H.,
Shrikhande, G.,
Winkler, T.,
Algate, P. A.,
Rohrschneider, L. R.,
and Griffin, J. D.
(1999)
Mol. Cell. Biol.
19,
7473-7480 |
19. |
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396 |
20. |
Wisniewski, D.,
Strife, A.,
Swendeman, S.,
Erdjument-Bromage, H.,
Geromanos, S.,
Kavanaugh, W. M.,
Tempst, P.,
and Clarkson, B.
(1999)
Blood
93,
2707-2720 |
21. | Maehama, T., Taylor, G. S., Slama, J. T., and Dixon, J. E. (2000) Anal. Biochem. 279, 248-250[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Phee, H.,
Jacob, A.,
and Coggeshall, K. M.
(2000)
J. Biol. Chem.
275,
19090-19097 |
23. |
Lamkin, T. D.,
Walk, S. F.,
Liu, L.,
Damen, J. E.,
Krystal, G.,
and Ravichandran, K. S.
(1997)
J. Biol. Chem.
272,
10396-10401 |
24. |
Damen, J. E.,
Liu, L.,
Ware, M. D.,
Ermolaeva, M.,
Majerus, P. W.,
and Krystal, G.
(1998)
Blood
92,
1199-1205 |
25. |
Liu, Q.,
Sasaki, T.,
Kozieradzki, I.,
Wakeham, A.,
Itie, A.,
Dumont, D. J.,
and Penninger, J. M.
(1999)
Genes Dev.
13,
786-791 |
26. | del Peso, L., Hernandez, R., Esteve, P., and Lacal, J. C. (1996) J. Cell. Biochem. 61, 599-608[CrossRef][Medline] [Order article via Infotrieve] |
27. | Lioubin, M. N., Algate, P. A., Tsai, S., Carlberg, K., Aebersold, A., and Rohrschneider, L. R. (1996) Genes Dev. 10, 1084-1095[Abstract] |