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J Biol Chem, Vol. 275, Issue 11, 7887-7893, March 17, 2000
From the Erythroid cells terminally differentiate in
response to erythropoietin binding its cognate receptor. Previously we
have shown that the tyrosine kinase Lyn associates with the
erythropoietin receptor and is essential for hemoglobin synthesis in
three erythroleukemic cell lines. To understand Lyn signaling events in
erythroid cells, the yeast two-hybrid system was used to analyze
interactions with other proteins. Here we show that the
hemopoietic-specific protein HS1 interacted directly with the SH3
domain of Lyn, via its proline-rich region. A truncated HS1, bearing
the Lyn-binding domain, was introduced into J2E erythroleukemic cells
to determine the impact upon responsiveness to erythropoietin.
Truncated HS1 had a striking effect on the phenotype of the J2E
line Erythropoiesis, the process of red blood cell development, is
primarily controlled by erythropoietin
(epo).1 Several model systems
have been used to study erythropoiesis in vitro. Although
primary erythroid cells provide the ideal cell type for analysis,
heterogeneity in preparations and insufficient numbers can preclude
biochemical analysis of epo signaling. A number of erythroid cell lines
have been derived which provide useful models for analyzing epo-induced
signaling cascades, including the SKT6 and J2E lines (1, 2). The J2E
cell line was used in this study because it proliferates, remains
viable, produces hemoglobin and undergoes morphological maturation in
response to epo (2, 3). Following epo stimulation of J2E cells,
phosphorylation changes to the epo receptor, janus kinase-2 (JAK2),
signal transducer and activator of transcription-5 (STAT5),
ras-GTPase activating protein, phosphatidylinositol
3-kinase, phospholipase C Previously, we reported that epo-initiated signaling was disrupted in a
J2E subclone (J2E-NR), which remained viable in the presence of epo but
did not differentiate or undergo enhanced proliferation following
hormonal stimulation (4). The tyrosine kinase Lyn was shown to be
severely reduced in the J2E-NR cells, and reintroduction of Lyn
restored the ability of the cells to terminally differentiate (5). Lyn
pre-associated with the epo receptor in parental J2E cells, and
inhibition of its activity suppressed differentiation (5). Chin
et al. (6) confirmed the binding of Lyn to the epo receptor
and demonstrated that it may play a role in regulating the JAK/STAT
pathway. Lyn is a member of the Src family of membrane-associated
tyrosine kinases, which is present mainly in lympho/hemopoietic cells
and is involved in signal transduction from numerous receptors
(7-15).
Lyn is most closely related to the tyrosine kinase Lck which plays an
essential role in T cell activation and development (16). The SH2
domain of Lck binds to tyrosine-phosphorylated CD45 and ZAP-70, whereas
its SH3 domain associates with phosphatidylinositol 3-kinase, p120, and
HS1 (17-21). HS1, or LckBP1, is a 75-kDa intracellular protein
expressed mainly in hemopoietic and lymphoid cells (22) and is a major
substrate for several Src family kinases (21, 23, 34). It contains a
proline-rich region, an SH3 domain, an acidic In this study we attempted to identify downstream effectors of Lyn in
erythroid cells using a yeast two-hybrid screen of wild type and a
kinase inactive mutant (Y397F) of Lyn. Of the seven Lyn-interacting
proteins identified, we report here on the interaction between Lyn and
HS1, and the crucial role of HS1 for epo-induced differentiation of
erythroid cells.
Cell Culture--
Cells were grown in Dulbecco's modified
Eagle's medium, 5% fetal calf serum (FCS). Differentiation of J2E
(2), clone 11, and clone 24 (30) as well as ME17 (31) cell lines was
initiated with epo (5 units/ml) or sodium butyrate (0.5 mM), whereas murine erythroleukemia (MEL) cells were
stimulated with Me2SO (1.5%). Viability was determined by
eosin dye exclusion (4) and hemoglobin synthesis by benzidine staining
(32). Note that benzidine staining levels reported here were higher
than in previous reports (3) because of batch variations in FCS. Cell
morphology was examined by cytocentrifugation onto glass slides and
Wright-Geimsa staining (3). Proliferation was assayed by
[3H]thymidine incorporation as described previously
(3).
Molecular Biology Techniques--
Total RNA was extracted by the
method of Chomczynski and Sacchi (33), from which poly(A)+
RNA was isolated using the Poly(A)-Tract mRNA isolation system (Promega). Northern blots were performed essentially as described by
Sambrook et al. (34). Restriction enzyme digestions
(Promega) and ligation reactions (Life Technologies, Inc.,
Gaithersburg, MD) were performed as recommended by the manufacturers.
PCR reactions were performed using either Pfu (Stratagene)
or Taq (Promega) polymerases on a PTC-200 Peltier Thermal
Cycler (MJ Research, Watertown, MA). DNA was sequenced using the
ABI-Prism method (PE Applied Biosystems, Branchburg, NJ).
Yeast Two-hybrid Analysis--
The yeast two-hybrid procedures
used were essentially as described by Vojtek et al. (35),
using the Saccharomyces cerevisiae L40 strain
(MATa, his3
Plasmids expressing LexA fusions of the unique (pBTM116-Un), SH2
(pBTM116-SH2), and SH3 (pBTM116-SH3) domains of Lyn for the yeast
two-hybrid system were generated by ligating PCR fragments into
pBTM116. The generation of plasmid pBTM116- In Vitro Binding Assay--
Plasmids expressing glutathione
S-transferase (GST) fusion proteins of Lyn (pGEX-Lyn), Lip-1
(pGEX-Lip-1), and Lip-2 (pGEX-Lip-2) were generated. GST fusion
proteins were expressed and purified, with minor modifications of the
method described by Smith and Johnson (37). Cells were lysed in PBS
containing 1% Triton X-100, 10 mg/ml lysozyme, 10 units/ml DNase I, 1 mM benzamidine for 2 h at 37 °C, centrifuged, and
processed as described by Smith and Johnston (37). Soluble Lyn was
produced by thrombin (Sigma) cleavage of the GST fusion. Binding
experiments were performed by the addition of purified soluble Lyn
(~100 ng) to GST, GST-Lip-1, or GST-Lip-2 (~500 ng) attached to
glutathione-agarose beads in PBS containing 1% Triton X-100, 5 mM dithiothreitol, 5 mM EDTA and incubated at
4 °C for 2 h. Bound Lyn was detected by SDS-polyacrylamide gel
electrophoresis and Western blotting.
Immunoprecipitation and Western Blot Analysis--
Cells were
lysed in 20 mM Tris-HCl, pH 8.0, 120 mM NaCl,
1.0% Nonidet P-40, 10 mM Indirect Immunofluorescence Microscopy--
Cells were
cytocentrifuged onto slides, fixed in 50% methanol, 50% acetone, and
then processed essentially as described by Harlow et al.
(41) for indirect immunofluorescence using anti-Lyn (SC-15, Santa Cruz
Biotechnology Inc.) or anti-HS1 (21) antibodies and a fluorescein
isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody
(Silenus, Melbourne, Australia). DNA was counterstained with Hoechst
33258. Slides were mounted in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 2.5%
1,4-diazabicyclo-{2.2.2} octane and visualized using a Bio-Rad
MRC-1000/1024 UV laser scanning confocal microscope (Bio-Rad, Hercules, CA).
Retroviral Infection of Cells--
Sense and antisense truncated
HS1 cDNAs, encoding amino acids 271-409, were generated by PCR.
The fragments were subcloned into the pMSCV2.2 neo vector (42) creating
pMSCV-Ds and pMSCV-Da for the sense and antisense orientation,
respectively. The packaging cell lines PA317 and Flow Cytometry--
Cells (106) were incubated with
antibodies to the epo-R (189; Ref. 38), transferrin receptor (R17 208;
Ref. 46), c-kit (47), or Ter119 (48) for 30 min on ice, washed three
times in PBS, 2% FCS followed by incubation with a secondary antibody conjugated to FITC (Silenus) for 30 min on ice, then washed three times
in PBS-2% FCS before analysis on an Epics XL/MCL flow cytometer (Beckman-Coulter, Palo Alto, CA). Cells incubated in the absence of
primary antibody were analyzed as controls.
Lyn Interacts with HS1--
The yeast two-hybrid system was used
to identify molecules that could associate with wild type or a dominant
negative Lyn (Y397F). Two screens, using Lyn and LynY397F, of
107 clones each yielded 67 and 82 HIS3 positive
clones, respectively, of which 34 and 68 were also The SH3 Domain of Lyn Binds the Proline-rich Region of
HS1--
The ability of HS1 to interact with the various domains of
Lyn was assessed. Deletion of the kinase domain of Lyn (Lyn
The sections of HS1 encoded by Lip-1 and Lip-2 encompass the SH3 domain
and part, or all, of the proline-rich region. Fig. 1E shows
that a section of the proline-rich region containing two consensus
SH3-binding sites, P1 (amino acids 324-329) and P2 (amino acids
345-350), was able to interact with Lyn. Taken together, these data
suggest that the SH3 domain of Lyn associates with the consensus
SH3-binding sites (P1 and P2) of HS1.
Lyn and HS1 Interact in Erythroid Cells--
To demonstrate an
in vivo association between Lyn and HS1, Lyn was
immunoprecipitated from several erythroid cell lines. Fig. 2A shows that HS1
co-immunoprecipitated with Lyn in each of these lines. Although HS1
associated with Lyn in erythroid cells, it also interacted with Lck and
Fyn, but not with Hck or Src (Fig. 2B). To determine whether
the association between Lyn and HS1 was altered after epo stimulation,
J2E cells were exposed to the hormone and HS1 was immunoprecipitated.
Interestingly, maximal association with Lyn occurred between 15 and 30 min of epo stimulation (Fig. 2C). However, no
phosphorylation of HS1 was detected after epo stimulation.
Indirect immunofluorescence microscopy showed that Lyn and HS1 have
distinct, but overlapping, subcellular localizations in J2E cells (Fig.
2D). Lyn had fairly uniform cytoplasmic staining, with some
concentration around the cell membrane and a small amount in the
nucleus. While HS1 also localized primarily in the cytoplasm, the
staining was more punctate than Lyn; some HS1 staining was present in
the nucleus. Thus, the intracellular co-localization of Lyn and HS1
support the in vitro binding data (Fig. 1) and the
co-immunoprecipitation results (Fig. 2, A-C).
Truncated HS1 Alters the Phenotype of Erythroid Cells--
In an
attempt to determine the biological role of HS1 in erythroid cells, a
truncated form of HS1 (tHS1) encompassing the proline-rich Lyn-binding
domain (amino acids 271-409) was introduced into J2E cells, which were
then called JDs cells. It was anticipated that tHS1 would bind Lyn and
disrupt signaling via endogenous HS1. The corresponding reverse
orientation construct was also expressed in these cells (termed JDa)
and used as a control. Numerous clones were isolated, expressing
comparable amounts of sense and antisense tHS1 RNA. Each of the clones
had the same phenotype, and a representative example is shown in Fig.
3. J2E cells and vector-alone control
(JM5) cells grew in large clusters in culture and had a distinctive
proerythroblast morphology (Fig. 3, A and B).
However, when cells expressed tHS1 a significant change in morphology
was observed. Not only did the cells grow in isolation, but they also
developed the appearance of basophilic erythroblasts (Fig. 3,
A and B). The JDs cells were noticeably smaller,
with a more condensed cytoplasm than the proerythroblastoid JM5 and JDa
lines (Fig. 3, B and C). Unlike the JM5 and JDa
lines, cells expressing tHS1 displayed no signs of morphological
maturation when epo was added to the cultures (Fig. 3B).
Moreover, the JDs cells grew much slower than the JM5 or JDa lines,
with almost twice the doubling time (data not shown). While the cell
surface expression of Ter119, c-kit, and TfR were unaltered (data not shown), the level of surface epo receptors was approximately half in
the JDs cells (Fig. 3D). These alterations were observed in each of the JDs clones analyzed, indicating that the tHS1 construct had
a significant impact upon the cells.
Truncated HS1 Inhibits Erythroid Differentiation and
Proliferation--
Because tHS1 had a major effect on the phenotype of
J2E cells, the effect of this mutant on erythroid differentiation was examined. Strikingly, hemoglobin synthesis was severely repressed in
each of the JDs clones studied (Fig.
4A). Production of the oxygen
carrier was inhibited in both uninduced and epo-treated JDs cells.
Similarly, the capacity to manufacture hemoglobin in response to sodium
butyrate was almost extinguished (Fig. 4A). Thus,
introduction of tHS1 had a significant inhibitory effect on the
biochemical differentiation of J2E cells. These observations are
commensurate with the inability of JDs to undergo morphological maturation (Fig. 3B). In contrast, tHS1 had no effect on the
Me2SO-induced differentiation of MEL cells (data not
shown).
The impact of tHS1 on proliferation and viability of J2E cells was
investigated next. As JDs cells grew much slower than JM5 and JDa
controls, it was not surprising to observe reduced thymidine incorporation by these cells (Fig. 4B). While the JM5 and
the JDa cells displayed the characteristic modest increase in DNA synthesis after epo stimulation (3), the increased proliferation rate
of JDs cells remained well below that of the controls. However, tHS1
had no effect on the viability of J2E cells (data not shown). Together
these data demonstrate that tHS1 had a dramatic impact on the
proliferation and differentiation of J2E cells.
To determine whether the effects of tHS1 could be extended to
non-transformed cells, fetal liver cells were exposed to the retrovirus
containing this construct, and CFU-E development was enumerated. Fig.
4C shows that colony numbers for these erythroid progenitors
were also reduced by tHS1. It was concluded that HS1 plays an important
role in erythroid differentiation, and the truncated molecule had a
dominant negative effect on maturation.
Intracellular Alterations in JDs Cells--
Having observed
phenotypic differences in JDs cells, and an inhibition of proliferation
and differentiation, biochemical alterations were then investigated.
Initially, the expression of several Src family kinases was examined in
these cells. Fig. 5A shows the presence of tHS1 in the JDs cells but not the control JM5 or JDa lines.
Interestingly, the level of endogenous HS1 was unaltered in cells
bearing either the truncated or antisense constructs. In contrast, the
amount of Lyn was reduced by approximately 80% in JDs cells, although
other Src kinases were not appreciably affected by the presence of the
truncated HS1 (Fig. 5A). The reduction in Lyn levels may
account in part for the overall decrease in tyrosine phosphorylation
within JDs cells (Fig. 5B).
As erythroid transcription factors are crucial for the development of
red blood cells, several DNA-binding proteins known to be involved in
erythroid maturation were studied. Data presented in Fig. 5C
show that transcription factors EKLF and NF-E2 were not markedly
affected by the introduction of tHS1. In contrast, GATA-1, a key
regulator of erythropoiesis, was barely detectable in the JDs cells.
Globin protein content was not significantly effected by the tHS1
construct. Therefore, tHS1 affected the amount of Lyn and GATA-1 in J2E
cells, as well as the phosphorylation status of the cells. In addition,
tHS1 inhibited endogenous HS1 association with Lyn (Fig.
5D), which supports the proposition that tHS1 plays a
dominant negative role.
To investigate the effect of tHS1 on epo receptor and STAT5 activation,
cells were stimulated with epo and phosphorylation of the receptor, and
STAT5 was assessed. Fig. 6 demonstrates
that, despite reduced surface receptor numbers (Fig. 3D),
the epo receptor was phosphorylated with typical kinetics. However, the
activation of STAT5 was delayed in JDs cells. These results suggest
that tHS1 did not affect receptor activation but did alter the kinetics of STAT5 phosphorylation.
Epo-mediated Proteolysis of Endogenous HS1 and Lyn in JDs
Cells--
It has been suggested that HS1, with its helix-turn-helix
features, may translocate to the nucleus following ligand activation of
receptors (24). To determine whether this occurred in erythroid cells,
the JM5, JDa, and JDs lines were exposed to epo and subjected to
indirect immunofluorescence and confocal microscopy (Fig.
7A). However, no translocation
from the cell membrane to the nucleus was observed. Significantly, HS1
protein virtually disappeared from JDs cells 60 min after epo
stimulation (Fig. 7A). Immuno-blot analysis was then
conducted to measure the diminution of HS1 in these cells. Fig.
7B confirmed that the protein content of endogenous HS1 in
JDs cells decreased markedly following ligand binding, but this was not
evident in the other cell lines. Furthermore, the levels of Lyn were
also diminished 120 min after epo activation.
The fine punctate appearance of HS1 in epo-treated JDs cells (Fig.
7A) indicated that the protein might be degraded in
endosomes/lysosomes. To examine this possibility, JDs cells were
treated with NH4Cl/chloroquine to inhibit
endosomal/lysosomal degradation. Fig. 7C shows that, in the
presence of the inhibitors, HS1 degradation was impeded dramatically.
These data demonstrate that tHS1 facilitates endosomal/lysosomal degradation of its endogenous counterpart following exposure to epo.
In this manuscript we have shown that the known Src kinase
substrate HS1 associates with Lyn in erythroid cells. We have also demonstrated that a truncated HS1 (tHS1) markedly interferes with the
phenotype of erythroid cells and impairs their ability to proliferate
and differentiate. By disrupting the Lyn/HS1 interaction, we have
generated a cascade of events which had a profound effect on erythroid
maturation. These data indicate that HS1 plays a pivotal role in
regulating intracellular signaling within erythroid cells and support
the recent prediction by Nagata et al. (29) that
" ... HS1 is likely to be involved in erythroid proliferation and differentiation. . . . . "
Introduction of a truncated form of HS1 into erythroid cells produced
significant morphological, biochemical, and functional perturbations.
The truncated mutant spanned the carboxyl-terminal, Lyn-binding region
of HS1 that is sequentially phosphorylated by kinases (49), but it did
not include the amino-terminal Hax1-binding region (50). Strikingly,
cells expressing tHS1 were smaller, more basophilic, and replicated
much more slowly. In addition, epo-induced differentiation was almost
totally blocked as the cells failed to mature morphologically and did
not synthesize hemoglobin. These data demonstrate that tHS1 acted in a
dominant negative fashion and emphasize the importance of a fully
functional HS1 to erythroid maturation.
The tHS1 mutant had several biochemical effects on the erythroid cells,
which may account for its dominant negative activity. First, it greatly
reduced the level of Lyn protein within the cells, and as a consequence
a marked decrease in tyrosine phosphorylation of intracellular proteins
was detected. This result is compatible with our previous observation
that the J2E-NR subclone expressed low levels of Lyn, and tyrosine
phosphorylation of proteins was substantially reduced (5). As Lyn
associates with the epo receptor (5, 6), transmission of signals from
the receptor via this kinase would be significantly diminished in the
transfected cells. Second, the GATA-1 content fell dramatically in
these cells. GATA-1 is a key transcription factor involved with
erythroid development (51, 52), and in its absence erythroid precursors
arrest at the proerythroblast stage (53). Thus, the loss of GATA-1
protein may play a significant role in the inability of cells bearing tHS1 to mature morphologically or to produce hemoglobin.
In addition to reducing the levels of Lyn and GATA-1, tHS1 had a
significant effect on endogenous HS1. While the epo receptor was
phosphorylated normally in cells containing the mutant HS1 (Fig. 6),
endogenous HS1 was degraded rapidly in endosomes/lysosomes after
exposure to epo (Fig. 7), indicating that the truncated mutant promoted
this proteolysis. It is likely that degradation of endogenous HS1
prevented transmission of signals by this molecule. Thus, tHS1
uncovered an unexpected mechanism for dominant negative action. This
observation warrants further investigation with the recent association
of signaling molecules with degradation viz. the SOCS family
of negative regulators of cytokine action target Janus kinases for
degradation by associating with elongins (54, 55), and c-cbl
regulates receptor ubiquitination and endocytosis (56, 57).
In this study HS1 was identified as a Lyn-binding protein through a
yeast two-hybrid screen, which was confirmed by direct association
in vitro, together with co-immunoprecipitation in vivo. In addition to intracellular co-localization within
erythroid cells, the proline-rich region of HS1 was shown to bind the
SH3 domain of Lyn, similar to the Lck/HS1 interaction reported
previously (21). Lyn has been shown to associate with HS1 in B and T
cells (45), but this is the first report that we are aware of where Lyn
interacts with HS1 in erythroid cells. Although HS1 and Lyn were
constitutively associated in erythroid cells, the interaction increased
appreciably after epo activation (Fig. 2C), which is consistent with previous observations in T cells where the SH3 domain
of Lck, or Lyn, binds to HS1 in the absence of stimulation, then the
SH2 domain of these kinases associates with HS1 upon receptor
activation, increasing the interaction (25).
HS1 is linked with several kinases. Here we demonstrated HS1
co-immunoprecipitated with Lyn, and two other Src kinases (Lck and Fyn)
in J2E cells (Fig. 2B); tHS1 could potentially interfere with signaling from these molecules. HS1 also associates with the novel
hemopoietic kinase HPK1 in the erythroid SKT6 cell line (29). In
addition, Takemoto et al. (45) suggested that the HS1
association with the SH3 domain of Grb2 may regulate the Grb2 and Src
signaling pathways. Together these results suggest that HS1 may mediate
signals emanating from several kinases and play a crucial role in
transmitting intracellular signals within erythroid cells.
Assistance with confocal microscopy was
kindly provided by P. Rigby and S. Codey, University of Western
Australia. Recombinant human epo (Eprex) was a generous gift from Dr.
J. Adams (Jansen-Cilag). We also thank Drs. S. Cory, J. Adams, and T. Metz for kindly providing the cell lines ME17, clone 11, and clone 24.
*
This work was supported by grants from the National Health
Medical Research Council (99-0596 and 98-0610), and the Cancer Foundation of Western Australia.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.
The abbreviations used are:
epo, erythropoietin;
epo-R, epo-receptor;
HS1, hematopoietic lineage cell-specific protein;
tHS1, truncated HS1;
SH, Src homology;
GST, glutathione
S-transferase;
PBS, phosphate-buffered saline;
HS1 Interacts with Lyn and Is Critical for Erythropoietin-induced
Differentiation of Erythroid Cells*
,,,,, and
Laboratory for Cancer Medicine, Department
of Biochemistry, the University of Western Australia and Royal Perth
Hospital, WA 6001, Western Australia, Australia, the
§ Institute for Gene Therapy and Molecular Medicine,
Mount Sinai School of Medicine, New York, New York 10030, and the
¶ Molecular Biology Department, Tsukuba Research Laboratories,
Nippon Glaxo Ltd., 43, Wadai, Tsukuba-shi, Ibaraki 300-42, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
the cells were smaller, more basophilic than the parental
proerythoblastoid cells and had fewer surface erythropoietin receptors.
Moreover, basal and erythropoietin-induced proliferation and
differentiation were markedly suppressed. The inability of cells
containing the truncated HS1 to differentiate may be a consequence of
markedly reduced levels of Lyn and GATA-1. In addition, erythropoietin
stimulation of these cells resulted in rapid, endosome-mediated
degradation of endogenous HS1. The truncated HS1 also suppressed the
development of erythroid colonies from fetal liver cells. These data
show that disrupting HS1 has profoundly influenced the ability of
erythroid cells to terminally differentiate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and MAP-kinase are identical to the
kinetics reported in other cell systems (4, 5).
-helix, and a basic
segment resembling the DNA-binding motif of the helix-turn-helix
family, suggesting it could play a role in both signal transduction and
transcriptional regulation (21). HS1 is phosphorylated following
activation of B cell and T cell receptors (21, 23-25) but not after
stimulation of IL-3, GM-CSF, or SCF receptors (26). Significantly, Lyn
has been shown to associate with HS1 in B and T cells (23, 24). Like
Lyn knockout mice, studies on HS1-deficient mice have revealed a
central role for HS1 in B cell responsiveness (27, 28). While this manuscript was in preparation, HS1 was shown to bind the novel hematopoietic progenitor kinase (HPK1) in erythroid cells (29).
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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200,
trp1-901, leu2-3,
112, ade2,
LYS2::(lexAop)4-HIS3, URA3::(lexAop)8-lacZ,
GAL4). The 1.5-kilobase wild-type Lyn and a dominant
negative Lyn (Y397F) were subcloned into pBTM116 (35) to generate LexA
DNA binding domain Lyn and LynY397F fusions. The L40 strain was
transformed separately with pBTM116-Lyn and pBTM116-LynY397F. Both
yeast strains were then used as the "bait" in two yeast two-hybrid
screens of a cDNA library in the VP16 transcriptional activation
domain fusion plasmid (pVP16) made from mRNA derived from the
lymphohemopoietic progenitor cell line EML C.1 (36). The pVP16 plasmids
from the Lyn and LynY397F-specific His+/Lac+
colonies were then rescued into Escherichia coli and
sequenced. These plasmids were subsequently co-transformed with
pBTM116-Lyn, pBTM116-Lyn Y397F, or pBTM116-HLS7 into the yeast L40
strain before performing HIS3 and 
-gal assays.
243Lyn expressing a LexA
fusion of the first 243 amino acids of Lyn has been described previously (5). Plasmids were also generated for pGAD-HS1 and pGAD-P1-P2 expressing transcription activation domain fusions of
full-length HS1 and the P1-P2 region of HS1 (amino acids 324-350), respectively.
-glycerophosphate, 10 mM NaF, 1 mM Na3VO4, 2 mM EDTA, 10 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin. For
co-immunoprecipitations, clarified cell lysates were incubated with
antibodies to Lyn, Lck, Src, Hck, Fyn (SC-15G, SC-15, SC-13, SC-19,
SC-72, SC-16, Santa Cruz Biotechnology Inc., Santa Cruz, CA), HS1 (21),
epo-R (#187; Ref. 38) or STAT5a and STAT5b (Santa Cruz Biotechnology
Inc., SC-1081, SC-835) for 2 h at 4 °C, then collected with
protein A-Sepharose beads for 16 h before washing and analyzing by
Western blotting. Additional antibodies used in Western blotting
directed against MAP-kinase, v-Raf, phosphotyrosine, and GATA-1 were
from Santa Cruz Biotechnology Inc. (SC-154, SC-133, SC-7020, SC-265).
Antibodies to EKLF (39), NF-E2 (40), and globin (#55447, Cappel
Research, Organon Technika, Belguim) were also used for Western
blotting. Secondary antibodies were coupled to horseradish peroxidase
(Amersham Pharmacia Biotech, Uppsala, Sweden) and detected by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
2 were transfected,
and supernatants containing amphotropic and ecotropic retroviruses,
respectively, were collected. Amphotropic retroviruses were used to
infect J2E and MEL cells, clones were isolated, and viral integration
was confirmed by Southern analysis while expression levels were
determined by Northern analysis (5). Ecotropic viruses infect at least
50% of fetal liver erythroid progenitors (43) that emerge in colony
assays (44).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-gal positive.
After curing and rescuing, several clones emerged, which were termed
Lyn interacting proteins (Lip).
Here we report on Lip-1 and Lip-2 (Fig.
1A), which were identical to
HS1 between amino acids 268-486 (Lip-1) and 346-486 (Lip-2) (28). To
determine whether the association between Lyn and Lip-1/Lip-2 was via a
direct interaction, in vitro binding was performed with
purified immobilized GST, GST-Lip-1, or GST-Lip-2 and with purified,
soluble Lyn. A clear association was seen between Lip-1 or Lip-2 and
Lyn, with no appreciable binding of Lyn to GST alone (Fig. 1,
B and C). These data show that Lyn and HS1 can
interact directly in vitro.
View larger version (33K):
[in a new window]
Fig. 1.
Lyn interacts with HS1 in yeast and in
vitro. A, yeast strain L40 was
co-transfected with the plasmids pBTM116 (Vector),
pBTM1116Lyn (Lyn-wt), or pBTM116-LynY397F
(LynY397F) and pVP16-Lip1 (Lip-1) or pVP16-Lip2
(Lip-2). The resultant colonies were replated onto
Leu 
/Trp/His plates and
assayed for HIS3 activity. B, in vitro
binding assay of wild-type Lyn and GST-Lip-1. C, in
vitro binding assay of wild-type Lyn and GST-Lip-2.
D, yeast strain L40 was co-transfected with the plasmids
pBTM1116Lyn (Lyn), pBTM1116LynY397F (LynY397F),
pBTM1116Lyn243 (Lyn243), pBTM1116LynUn
(LynUn), pBTM1116LynSH3 (LynSH3), or
pBTM1116LynSH2 (LynSH2), and pGAD-HS1 (HS1).
LexA represents the vector, whereas Un represents
the unique region of Lyn. The domain boundaries are indicated
graphically and by residue number. The resultant colonies were replated
onto Leu/Trp plates, and -gal activity
was determined by liquid assay. E, yeast strain L40 was
co-transfected with the plasmids pGAD-HS1 (HS1), pVP16-Lip-1
(Lip-1), pVP16-Lip2 (Lip-2), or pGAD-P1-P2
(P1-P2), and pBTM116-Lyn (Lyn). VP16 is the
vector, while HtH represents the helix-turn-helix motif of
HS1, and PP represents the proline-rich domain. The domain
boundaries are indicated graphically and by residue number. The
resultant colonies were replated onto
Leu/Trp plates, and -gal activity was
determined by liquid assay.
243) did
not prevent the interaction (Fig. 1D). This observation is compatible with the yeast two-hybrid data, where Lip-1 and Lip-2 bound
both Lyn and LynY397F (Fig. 1A), indicating a functional kinase domain was not required for the association. However, the interaction was eliminated when only the unique domain of Lyn was
retained. This result suggested that the SH2 and/or SH3 domains of Lyn
were required for the association. However, Fig. 1D shows that the SH3, but not the SH2, domain was responsible for the interaction between Lyn and HS1.
View larger version (26K):
[in a new window]
Fig. 2.
In vivo association of Lyn and
HS1. A, co-immunoprecipitation of Lyn and HS1 from
erythroid cell lines J2E, C11, C24, MEL, and ME17 (in logarithmic
growth phase) following immunoprecipitation of Lyn and immunoblotting
with antibodies to HS1. B, co-immunoprecipitation of HS1
with Lyn, Lck, and Fyn but not with Hck or Src, in J2E cells. Src
family members were immunoprecipitated with the antibodies shown and
then immunoblotted with antibodies to HS1. C,
co-immunoprecipitation of Lyn and HS1 is maximal 15-30 min after epo
stimulation of J2E cells. Lysates from epo-treated J2E cells were
immunoprecipitated with anti-HS1 antibodies and probed with antibodies
to Lyn. D, overlapping subcellular localization of Lyn and
HS1 in uninduced J2E cells. The cell nuclei were stained with Hoechst
33258 (red), while Lyn and HS1 were identified with
FITC-conjugated antibodies (green).
View larger version (38K):
[in a new window]
Fig. 3.
Truncated HS1 alters the phenotype of J2E
cells. A, suspension cultures showing that JM5 and JDa1
cells formed large clumps, while JDs12 cells grew in isolation.
B, Wright-Geimsa-stained cells showing the
proerythroblastoid morphology of JM5 and JDa1 cells, while the JDs12
cells were more basophilic with darker condensed cytoplasms. After
48 h in the presence of epo, JM5 and JDa1 cells underwent
morphological maturation (arrows) which included the
appearance of reticulocytes (asterisk), while JDs12 cells
showed no noticeable changes. C, flow cytometric analysis of
forward scatter (FS) and side scatter (SS) shows
that JDs12 cells were smaller and less heterogeneous than JM5 and JDa1
cells. D, saturation epo-receptor binding assay of JM5,
JDa1, and JDs12 cells using 125I-epo. Each bar
represents the mean ± S.D. (n = 3).
View larger version (25K):
[in a new window]
Fig. 4.
Truncated HS1 inhibits differentiation and
alters proliferation of erythroid cells. A, epo and
sodium-butyrate-induced hemoglobin production by JM5, and four
independent JDa and JDs clones measured by benzidine staining after
48 h. B, proliferation rate with, and without, epo of
JM5, JDa, and JDs clones as measured by [3H]thymidine
incorporation. C, epo-induced erythroid colony formation
(CFU-E) in methylcellulose after infection of fetal liver
cells with MSCV-Da or MSCV-Ds. Each point is the mean ± S.D.
(n = 3).
View larger version (76K):
[in a new window]
Fig. 5.
Intracellular alterations in JDs cells.
A, immunoblot analysis of tHS1, HS1, Lyn, Lck, Fyn, Hck, and
Src expression in JM5, JDa1, and JDs12 cells. v-raf was used as a
loading control. B, anti-phosphotyrosine Western blot of
total cell extracts from JM5, JDa1, and JDs12 cells. C,
immunoblot analysis of EKLF, GATA-1, NF-E2, and globin expression in
JM5, JDa1, and JDs12 cells. MAP-kinase (MAPK) was used as a
loading control because of the separation required to detect globin
proteins. D, immunoblot analysis of HS1 and Lyn from Lyn
immunoprecipitates of JM5, JDa1, and JDs12 cells. All protein lysates
were from unstimulated cells. Note that 10-fold more protein was used
in the immunoprecipitates from JDs12 cells to compensate for the lower
Lyn expression.
View larger version (41K):
[in a new window]
Fig. 6.
Truncated HS1 retards epo-induced STAT5
phosphorylation. Anti-phosphotyrosine immunoblot of epo-R and
STAT5 immunoprecipitates from JM5, JDa1, and JDs12 cells stimulated
with epo for the indicated times.
View larger version (28K):
[in a new window]
Fig. 7.
Epo-mediated proteolysis of endogenous HS1
and Lyn in JDs cells. A, indirect immunofluorescence
analysis of HS1 in JM5, JDa1, and JDs12 cells stimulated with epo over
60 min. Cell nuclei were stained with Hoechst 33258 (red),
and HS1 was identified with FITC-conjugated antibodies
(green). B, immunoblot analysis of HS1, Lyn, and
MAP-kinase (MAPK) protein levels in JM5, JDa1, and JDs12
cells stimulated with epo for 120 min. Protein loading of JDs12 lysates
was adjusted 10-fold to compensate for the lower level of Lyn.
C, immunoblot analysis of HS1 levels in JDs12 cells
stimulated with epo for 120 min with, or without, the endosome/lysosome
inhibitors NH4Cl (5 mM) and chloroquine (5 mM).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Laboratory for
Cancer Medicine, Level 6, MRF Bldg., Rear 50 Murray St., Perth, WA
6001, Western Australia. Tel.: (61-8)-9224-0333; Fax: (61-8)-9224-0322; E-mail: pklinken@cyllene.uwa.edu.au.
ABBREVIATIONS
-gal, -galactosidase;
FCS, fetal calf serum;
PCR, polymerase chain
reaction;
FITC, fluorescein isothiocyanate;
MEL, murine
erythroleukemia;
MAP-kinase, mitogen-activated protein kinase.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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