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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3734-3745
Lyn Physically Associates With the Erythropoietin Receptor and May
Play a Role in Activation of the Stat5 Pathway
By
Hiroshi Chin,
Ayako Arai,
Hiroshi Wakao,
Ryuichi Kamiyama,
Nobuyuki Miyasaka, and
Osamu Miura
From the First Department of Internal Medicine and School of Allied
Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan; and
the Helix Research Institute, Chiba, Japan.
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ABSTRACT |
Protein tyrosine phosphorylation plays a crucial role in signaling
from the receptor for erythropoietin (Epo), although the Epo receptor
(EpoR) lacks the tyrosine kinase domain. We have previously shown that
the Jak2 tyrosine kinase couples with the EpoR to transduce a growth
signal. In the present study, we demonstrate that Lyn, a Src family
tyrosine kinase, physically associates with the EpoR in Epo-dependent
hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression
experiments in COS7 cells further showed that Lyn induces tyrosine
phosphorylation of the EpoR and that both LynA and LynB, alternatively
spliced forms of Lyn, bind with the membrane-proximal 91-amino acid
region of the EpoR cytoplasmic domain. In vitro binding studies
using GST-Lyn fusion proteins further showed that the Src homology
(SH)-2 domain of Lyn specifically binds with the
tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells,
whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic
phosphopeptide competition assays further indicated that the Lyn SH2
domain directly binds to the tyrosine-phosphorylated EpoR, most likely
through its interaction with phosphorylated Y-464 or Y-479 in the
carboxy-terminal region of the EpoR. In vitro binding studies also
demonstrated that the Lyn SH2 domain directly binds to
tyrosine-phosphorylated Jak2. In vitro reconstitution
experiments in COS7 cells further showed that Lyn induces tyrosine
phosphorylation of Stat5, mainly on Y-694, and activates the
DNA-binding and transcription-activating abilities of Stat5. In
agreement with this, Lyn enhanced the Stat5-dependent transcriptional
activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn
was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in
vitro. These results suggest that Lyn may play a role in activation of
the Jak2/Stat5 and other signaling pathways by the EpoR.
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INTRODUCTION |
ERYTHROPOIETIN (Epo) is a hematopoietic
growth factor that regulates the growth and differentiation of
erythroid progenitor cells through activation of its specific receptor
expressed on the cell surface.1-3 The receptor for Epo
(EpoR) belongs to the cytokine receptor family and lacks a tyrosine
kinase domain. However, we previously showed that protein tyrosine
phosphorylation plays a pivotal role in the EpoR-mediated growth
signaling4,5 and further demonstrated that Jak2, a member
of the JAK family of tyrosine kinases, physically associates with the
EpoR membrane-proximal domain, which contains the Box1 and Box2 motifs
conserved in the cytokine receptor family, and becomes activated upon
Epo stimulation.6,7 Epo stimulation also induces tyrosine
phosphorylation of the EpoR itself4 and various signaling
molecules, such as Stat5, Shc, SHP-2, Vav, Cbl, CrkL, and SHIP, which
are directly or indirectly recruited to the tyrosine-phosphorylated
EpoR through the interaction between phosphotyrosines in the EpoR
cytoplasmic domain and the Src homology (SH)-2 domain, a conserved
modular domain that binds to phosphotyrosine-containing sequences, of
these signaling molecules.8 Upon tyrosine phosphorylation,
Stat5 dissociates rapidly from the EpoR to form a homodimer through the
reciprocal SH2 domain-phosphotyrosine interaction and is then
translocated to the nucleus, where it activates target genes by binding
to specific regulatory sequences.9 Epo also induces the
recruitment of other SH2-containing signaling molecules, including the
p85 regulatory subunit of phosphatidyl inositol 3 -kinase (PI3K)
and SHP-1, although their tyrosine phosphorylation state does not show
any significant change after Epo stimulation.8 Previously,
we showed that Jak2 plays a critical role in inducing tyrosine
phosphorylation of the EpoR as well as the various cellular substrates,
because, in cells expressing mutant EpoRs that failed to associate with
and thus activate Jak2, Epo failed to induce tyrosine phosphorylation
of the mutant receptors or any of these cellular
substrates.5-7,10,11 However, it has remained to be known
whether Jak2 is the only tyrosine kinase that is directly involved in
Epo-induced tyrosine phosphorylation of the EpoR as well as the variety
of cellular substrates.
Other than the JAK family of tyrosine kinases, Lyn, a member of the Src
family tyrosine kinases, is abundantly expressed in the hematopoietic
cells,12,13 including erythrocytes.14 The Src
family kinases have a common domain organization. The N-terminal segment contains a myristylation site, which is required for membrane localization, and a unique domain of 5 to 70 residues that does not
show any significant similarity among the family members. The SH3, SH2,
and catalytic (SH1) domains follow in order. The 56-kD and 53-kD forms
of Lyn, designated as LynA and LynB, respectively, are translated from
alternatively spliced transcripts and expressed simultaneously in
hematopoietic cells. LynB lacks the amino acid residues 24 through 44 in the unique domain of LynA.13 Recent studies on
lyn / mice15,16 have demonstrated that
Lyn plays crucial roles in signaling mediated through the B-cell
antigen receptor and the high affinity receptor for Fc . Lyn has also been implicated in signaling from the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IL-5,17-22 which belong to the cytokine receptor
superfamily and share a common signal-transducing subunit,
 c,23 as well as from the granulocyte colony-stimulating
factor (G-CSF) receptor.24 Although abnormalities of
hematopoiesis have not been reported in lyn-deficient mice, the
role of Lyn might have been complemented by the redundantly expressed
other Src family members. Consistent with this idea, single knock-out
experiments of any Src kinases have not been reported to result in
abnormalities of murine hematopoiesis.25 Interestingly, the
receptors sharing c also activate Jak2 and induce tyrosine
phosphorylation of a similar set of cellular proteins with the
EpoR.8 In addition, a very recent study has suggested that
Lyn may play an important role in Epo-induced
differentiation.26 In the present study, we thus examined
the physical interaction of Lyn with the EpoR and its involvement in
the EpoR-mediated signaling.
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MATERIALS AND METHODS |
Cells and reagents.
IL-3-dependent 32D cells and a clone of 32D cells expressing the
wild-type murine EpoR (32D/EpoR-Wt)5 were previously
described and maintained in RPMI 1640 medium supplemented with 10%
fetal calf serum (FCS) and 10% WEHI-3 conditioned medium as a source of IL-3. An Epo-dependent human erythroleukemia cell line,
F36E,27 was obtained through the Riken Gene Bank (Ibaraki,
Japan) and cultured in RPMI 1640 medium supplemented with 10% FCS and
4 U/mL of Epo. COS7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS. Recombinant human Epo was kindly provided by Chugai Pharmaceutical Co Ltd (Tokyo, Japan). Recombinant murine IL-3 was purchased from PeproTech Inc (Rocky Hill, NJ).
Expression plasmids for the murine wild-type and mutant EpoRs were
described previously.4,28 A -casein promoter luciferase construct, pZZ1,29 and cDNA clones coding for murine
Jak2,30 murine Stat5A,31 murine
LynA,13 murine LynB,13 wild-type ovine
Stat5,32 and mutant ovine Stat5 with a substitution of Tyr694 with Phe29,33 have also been described previously.
Control Renilla luciferase plasmids pRL-TK and pRL-SV40 were purchased from Promega (Madison, WI).
A rabbit antiserum raised against a glutathione S-transferase (GST)
fusion protein containing amino acids 257 to 441 of the EpoR
cytoplasmic domain was previously described.34 Rabbit
antibodies against Lyn, Stat5A, SHP-2, and the carboxy-terminal region
of EpoR (M-20) and a monoclonal antibody against GST were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against phosphotyrosine (4G10) and the influenza virus hemagglutinin (HA) epitope were from Upstate Biotechnology, Inc (Lake Placid, NY) and
from Boehringer Mannheim (Indianapolis, IN), respectively.
Immunoprecipitation and immunoblotting.
Cells were washed free of IL-3, cultured overnight, and left
unstimulated as a negative control or stimulated with Epo or IL-3 at a
saturating concentration, unless otherwise described. Cells were
solubilized with a lysis buffer composed of 1% Triton X-100, 20 mmol/L
Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L sodium
orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL aprotinin, and 10 µg/mL leupeptin. Cell lysates were
subjected to immunoprecipitation as described previously.10 For double immunoprecipitation, immunoprecipitates were eluted with
1× Laemmli's sodium dodecyl sulfate (SDS) sample
buffer, diluted 100-fold with lysis buffer supplemented with 0.1%
bovine serum albumin, and subjected to reimmunoprecipitation with a
second antibody or normal rabbit serum, as indicated. For immunoblot analysis of total cell lysates, an aliquot of the clarified supernatant was directly mixed with equal volumes of 2× Laemmli's sample
buffer and heated at 100°C for 5 minutes. Samples were separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrotransferred to Immobilon P membranes (Millipore, Bedford, MA).
The membranes were probed with a relevant antibody followed by
detection using enhanced chemiluminescence Western blotting detection
system (Amersham, Buckinghamshire, UK). For reprobing of the membranes,
they were treated with stripping buffer composed of 100 mmol/L
2-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris-HCl (pH 6.7) at
50°C for 30 minutes and subsequently probed with a different
antibody.
In vitro binding studies using GST-Lyn fusion proteins.
To prepare GST-Lyn fusion proteins, the fragments of LynA cDNA coding
for amino acid residues 1 through 230 (Lyn1-230) and 231 through 512 (Lyn231-512) were amplified by the polymerase chain reaction (PCR)
method using Pwo DNA polymerase (Boehringer Mannheim, Mannheim,
Germany). Using restriction enzyme recognition sites artificially added
in the 5- and 3-primers, the PCR-generated fragments were
subcloned into pGEX-4T-3 (Pharmacia, Uppsala, Sweden). In addition, the
fragment coding for Lyn1-230 was digested with Bal I, and the
two fragments coding for amino acid residues 1 through 118 (Lyn1-118)
and 119 through 230 (Lyn119-230) were separately subcloned into
pGEX-4T-3. The expression plasmids were transformed into
Escherichia coli DH5 , and the recombinant fusion proteins were purified by affinity chromatography on glutathione-Sepharose beads
(Pharmacia).
In vitro binding of cellular proteins to GST-Lyn fusion proteins was
examined essentially as described previously.10 In brief,
parental 32D or 32D/EpoR-Wt cells were lysed in the lysis buffer
described above, mixed with GST fusion proteins on
glutathione-Sepharose beads, and incubated at 4°C for 2 hours. The
beads were washed twice with the lysis buffer, and proteins bound to
the beads were eluted by boiling in 1× SDS sample buffer and
examined by immunoblotting with indicated antibodies. For competition
assays with previously described EpoR-derived phosphotyrosine
peptides,35,36 200 µmol/L or indicated concentrations of
a synthetic peptide was added to cell lysate from Epo-stimulated cells
before being subjected to binding analysis using GST-Lyn119-230. For
Far-Western blotting, GST-Lyn fusion proteins were eluted from
glutathione-Sepharose beads with a buffer composed of 50 mmol/L
Tris-HCl (pH 8.0) and 5 mmol/L reduced glutathione. After
immunoprecipitates were separated by SDS-PAGE and electrotransferred to
Immobilon P membranes, the membrane was first incubated overnight at
4°C with an indicated GST-Lyn fusion protein followed by detection
with anti-GST immunoblotting.
Affinity purification of DNA-binding proteins.
Cells were solubilized with a lysis buffer composed of 0.5% NP-40, 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1 mmol/L EDTA, 10 mmol/L
NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L dithiothreitol, 1 mmol/L
PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin. The oligonucleotide sequence used was derived from the prolactin-inducible element (PIE) of the ovine -casein gene
(5-AGATTTCTAGGAATTCAAT CC-3).29,32 After
preclarification, 500 µL of cell lysates was incubated at 4°C for
2 hours with 1 µg of double-stranded, 5-biotinylated
PIE-oligonucleotide and 10 µL of streptavidin-agarose (Pierce,
Rockford, IL). After washing twice with the lysis buffer, bound
proteins were eluted by heating at 100°C for 5 minutes in Laemmli's sample buffer and analyzed by immunoblotting with
antiphosphotyrosine or anti-Stat5.
Transient expression in COS7 cells.
Transfection of expression plasmids into COS7 cells was performed using
the Lipofectamin reagent (GIBCO-BRL, Grand Island, NY) according to the
manufacturer's instructions. Two days after transfection, cells were
stimulated with Epo (or left unstimulated), solubilized, and subjected
to immunoprecipitation or affinity purification with the
PIE-oligonucleotide, followed by immunoblotting with indicated
antibodies as described above.
Luciferase reporter assays.
COS7 cells were cotransfected with the expression vector for Jak2
(pcDNA-Jak2) or Lyn (pXM-LynA and pXM-LynB) or with pcDNA3 as a control
along with the Stat5-responsive luciferase reporter construct (pZZ1), a
control Renilla luciferase plasmid (pRL-TK), and the expression
plasmids for murine Stat5 (pRK-Stat5) and the EpoR (pXM-EpoR-Wt). Two
days after transfection, transfected cells were incubated with or
without Epo (5 U/mL) for 5 hours and harvested for the luciferase assay
using Dual-Luciferase Reporter Assay System (Promega) according to the
manufacturer's instructions. For transient overexpression experiments
in 32D/EpoR-Wt, cells were electroporated at 960 µF and 300 V with
the expression vector for Jak2 (pcDNA-Jak2) or Lyn (pXM-LynA and B) or
pcDNA3 along with pZZ1 and a control Renilla luciferase plasmid,
pRL-SV40. After a recovery period of 1 day, cells were starved
overnight, incubated for 5 hours in medium with or without 4 U/mL of
Epo, and harvested for the luciferase assay.
In vitro kinase assays.
The in vitro kinase assay of anti-Lyn immunoprecipitates was performed
as described previously.7,13 In brief, anti-Lyn immunoprecipitates were subjected to the in vitro kinase
reaction in kinase buffer (10 mmol/L HEPES, pH7.5, 50 mmol/L NaCl, 5 mmol/L MgCl2, 5 mmol/L MnCl2, 100 µmol/L
sodium orthovanadate) containing [ -32P]-ATP, with or
without rabbit enolase added as an exogenous substrate, were resolved
by SDS-PAGE, and were analyzed by autoradiography.
In vitro tyrosine phosphorylation of the EpoR and Stat5 by Lyn was
examined by using GST-EpoR and GST-Stat5 fusion proteins. A GST-EpoR
fusion protein containing the cytoplasmic domain of the EpoR (amino
acid residues 257 through 483) has been described previously.36 To prepare a GST-Stat5 fusion protein, the
portion of the murine Stat5A cDNA coding for amino acid residues 515 through 793 was amplified by the PCR and subcloned into the pGEX-4T-3 plasmid, as described above. The GST-EpoR and GST-Stat5 fusion proteins, eluted from glutathione-Sepharose beads, were incubated at
room temperature for 30 minutes with anti-Lyn immunoprecipitates from
unstimulated or Epo-stimulated 32D/EpoR-Wt cells in the kinase buffer supplemented with or without 1 mmol/L cold ATP. After the kinase
reaction, the reaction products were mixed with equal volumes of
2× Laemmli's sample buffer, heated at 100°C for 5 minutes, and subjected to antiphosphotyrosine immunoblotting followed by reprobing with anti-EpoR or anti-Stat5.
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RESULTS |
Lyn physically associates with the EpoR in hematopoietic cells.
To investigate whether Lyn is involved in signaling through the EpoR,
we stimulated 32D/EpoR-Wt cells, a clone of IL-3-dependent 32D cell
line that expresses the EpoR, with Epo or IL-3 and examined anti-Lyn
immunoprecipitates with antiphosphotyrosine blotting. As shown in
Fig 1A, neither Epo nor IL-3 induced any
significant effect on the tyrosine phosphorylation status of two
alternatively spliced forms of Lyn, 56-kD LynA and 53-kD LynB. The
kinase activity of Lyn, as determined by the immune complex in
vitro kinase assays, was also not significantly changed after
Epo or IL-3 stimulation in repeated experiments (negative data not
shown). However, as indicated with an asterisk in Fig 1A, a
tyrosine-phosphorylated 72-kD protein was found to be
coimmunoprecipitated with Lyn only after Epo stimulation. The
association of the 72-kD phosphotyrosyl protein with Lyn was also
observed in an Epo-dependent human erythroleukemia cell line, F36E,
after stimulation with Epo for 1 or 5 minutes (Fig 1B). Because both
the EpoR and SHP-2 are inducibly tyrosine phosphorylated after Epo
stimulation and comigrate on SDS-PAGE as 72-kD proteins, the anti-Lyn
immunoprecipitates from 32D/EpoR-Wt cell lysates were subjected to a
second immunoprecipitation with anti-EpoR, anti-SHP-2, or preimmune
rabbit serum and then examined by antiphosphotyrosine blotting. As
shown in Fig 1C, the 72-kD phosphotyrosyl protein was
reimmunoprecipitated with anti-EpoR but not with anti-SHP-2. These
results indicate that Lyn is associated directly or indirectly with the
tyrosine-phosphorylated EpoR in Epo-stimulated hematopoietic cells.
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| Fig 1.
Lyn associates with the tyrosine-phosphorylated EpoR in
hematopoietic cell lines. (A) 32D/EpoR-Wt cells, a clone of
IL-3-dependent 32D cell line expressing the transfected wild-type
murine EpoR cDNA, were starved overnight and left unstimulated
() or stimulated with 100 U/mL of Epo (Ep) or 25 ng/mL of
IL-3 (IL3) for 5 minutes at 37°C before solubilization. Cell
lysates were immunoprecipitated with anti-Lyn. Immunoprecipitates were
resolved by SDS-PAGE and subjected to immunoblotting with an
antiphosphotyrosine monoclonal antibody, 4G10 (PY). (B) A human
erythroleukemic cell line, F36E, was starved overnight and stimulated
with 100 U/mL Epo for the indicated times. Cells were then lysed and
analyzed as described above. (C) 32D/EpoR-Wt cells were left
unstimulated or stimulated with Epo, as indicated. Cells were then
lysed and immunoprecipitated with anti-Lyn. Anti-Lyn immunoprecipitates
were then subjected to a second immunoprecipitation with anti-EpoR,
anti-SHP-2, or preimmune serum (PI), as indicated, and analyzed by
antiphosphotyrosine (PY) immunoblotting. A 72-kD phosphotyrosyl
protein that coimmunoprecipitates with Lyn from Epo-stimulated cell
lysates is indicated with an asterisk. The positions of EpoR, Lyn, and
the Ig heavy chain (IgH) are indicated. The molecular weight markers
are indicated and given in kilodaltons.
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Lyn induces tyrosine phosphorylation of the EpoR and binds to the
membrane-proximal cytoplasmic region of the EpoR in COS7 cells.
To explore the possibility that Lyn may induce the tyrosine
phosphorylation of the EpoR, the EpoR was coexpressed with Lyn or Jak2
in COS7 cells. When the EpoR was expressed alone, Epo did not induce
any detectable tyrosine phosphorylation of the EpoR
(Fig 2). On the other hand, the
coexpression of Lyn or Jak2 induced a prominent tyrosine
phosphorylation of the EpoR even without Epo stimulation (Fig 2). In
accordance with this, Jak2 was tyrosine phosphorylated and thus
activated without Epo stimulation when overexpressed with the EpoR in
COS cells (data not shown). In repeated experiments, Lyn and Jak2
induced comparable levels of tyrosine phosphorylation of the EpoR,
which were only marginally enhanced by Epo stimulation. In addition, a
phosphotyrosyl protein corresponding in size to Lyn was
coimmunoprecipitated with the EpoR from cells coexpressing the EpoR and
Lyn, as indicated by an asterisk in Fig 2.
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| Fig 2.
Lyn induces tyrosine phosphorylation of the EpoR in COS7
cells. In COS7 cells, the EpoR was transiently coexpressed with Lyn or
Jak2, as indicated. Transfected cells were either stimulated with 100 U/mL Epo for 5 minutes or left unstimulated, as indicated (Epo st. + or , respectively). Cells were lysed and immunoprecipitated with
anti-EpoR. Immunoprecipitates were analyzed by antiphosphotyrosine (PY) immunoblotting followed by reprobing with anti-EpoR, as indicated. A coimmunoprecipitated phosphotyrosyl protein that corresponds in size to Lyn is indicated with an asterisk.
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We next examined the binding of Lyn with the previously characterized
EpoR mutants,4 as shown in Fig
3A, in COS7 cells. The H- and S-mutant EpoRs lack 72- and 142-amino
acid residues, respectively, by carboxy-terminal truncation, whereas
the PB-mutant EpoR lacks 20-amino acid residues by an internal deletion
within the membrane proximal cytoplasmic domain required for the
activation of Jak2.6,7 As demonstrated in Fig 3B, anti-EpoR
blotting of anti-Lyn immunoprecipitates from transfected COS7 cells
indicated that all the three mutant EpoRs as well as the wild-type EpoR physically associated with Lyn when coexpressed. Anti-Lyn
immunoblotting of anti-EpoR immunoprecipitates, shown in Fig 3C,
confirmed the Lyn association with the wild-type and mutant EpoRs and
further indicated that both LynA and LynB associated with the EpoR. To confirm this, LynA and LynB were individually coexpressed with the
wild-type EpoR in COS7 cells. Anti-Lyn blotting of anti-Lyn immunoprecipitates demonstrated that LynA and LynB were expressed as
56- and 53-kD proteins, respectively, although slower-migrating minor
species were observed for both LynA and LynB (Fig 3D, lower panel).
Anti-EpoR blotting of anti-Lyn immunoprecipitates (Fig 3D, upper panel)
as well as anti-Lyn blotting of anti-EpoR immunoprecipitates (Fig 3E,
upper panel) confirmed the physical association of both forms of Lyn
with the EpoR in transfected COS7 cells. In repeated experiments, the
binding of Lyn with the EpoR in COS7 cells was observed independently
of Epo stimulation (data not shown). These data demonstrate that both
LynA and LynB bind with the membrane-proximal 91-amino acid region of
the EpoR cytoplasmic domain. However, amino acids 281 to 300 within
this region were dispensable for the association of Lyn, which is in
contrast to the binding of Jak2.6,7 In addition, Lyn was
found to induce tyrosine phosphorylation of the EpoR in COS7 cells.
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| Fig 3.
Binding of LynA and LynB to various mutant EpoRs in COS7
cells. (A) Schematic representation of EpoR mutants used in binding studies in COS7 cells. The transmembrane domain (TM) is represented with a hatched box, whereas the conserved sequence motifs, Box1 and
Box2, are represented with solid boxes. The positions of eight tyrosine
residues in the cytoplasmic region are indicated with vertical lines.
Numbers in parentheses indicate the amino acid number at the carboxy
terminus of the EpoR or at the sites of truncation. EC, extracellular
region; IC, intracellular region. (B and C) The wild-type (W)
or mutant EpoRs (H, S, or PB) were coexpressed with both LynA and LynB
in COS7 cells, as indicated. Cells were lysed and immunoprecipitated
with anti-Lyn or anti-EpoR, as indicated. Immunoprecipitates were
subjected to immunoblotting with indicated antibodies. (D and E) LynA
(A) or LynB (B) was coexpressed with the wild-type EpoR in COS7 cells,
as indicated, and examined as described above.
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In vitro binding of GST-Lyn fusion proteins to the EpoR.
To explore the mechanisms of Lyn binding with the EpoR, in vitro
binding of GST-Lyn fusion proteins, shown in
Fig 4A, with the EpoR was examined. First,
cell lysates from parental 32D cells or 32D/EpoR-Wt cells were
incubated with the GST-Lyn fusion proteins, and cellular proteins bound
to these fusion proteins were examined by anti-EpoR blotting. As shown
in Fig 4B, GST-Lyn231-512, which encompasses the Lyn tyrosine kinase
domain, bound with the 62- to 66-kD forms of the EpoR in lysates from
32D/EpoR-Wt cells. In contrast, GST-Lyn119-230, which contains the Lyn
SH2 domain, bound specifically with the 72-kD form of the EpoR from
Epo-stimulated cells, which represents a small fraction of the receptor
that was transported to the cell surface and tyrosine phosphorylated after Epo stimulation.4,37 In accordance with this,
antiphosphotyrosine blotting of the proteins bound to GST-Lyn119-230
confirmed that the 72-kD EpoR is the predominant phosphotyrosyl protein
that binds the Lyn SH2 domain (Fig 4C, upper and middle panels). On the
other hand, GST-Lyn1-119, which comprises the unique and SH3 domains of
Lyn, did not show any binding with the EpoR (Fig 4B). Antiphosphotyrosine blotting of proteins bound to the GST-Lyn fusion
proteins also showed that a phosphotyrosyl 130-kD protein in
Epo-stimulated cell lysate specifically bound with GST-Lyn119-230 (Fig
4C, upper panel). This phosphotyrosyl protein was identified as Jak2 by
reprobing with anti-Jak2 (Fig 4C, lower panel).
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| Fig 4.
In vitro binding studies with GST-Lyn fusion proteins.
(A) Schematic representation of LynA and recombinant Lyn
proteins used in in vitro binding studies. The SH3 and SH2
domains are represented by hatched and solid boxes, respectively. Amino
acid numbers are shown under Lyn. (B) Cell lysate from parental 32D
(EpoR ) or 32D/EpoR-Wt (EpoR +) cells, which were unstimulated or
stimulated with Epo as indicated (Epo st. or +, respectively),
was incubated with GST-Lyn fusion proteins indicated below the panel.
Affinity-purified proteins were resolved by SDS-PAGE and subjected to
anti-EpoR immunoblotting. The tyrosine-phosphorylated 72-kD EpoR as
well as the 62- to 66-kD forms of the EpoR is indicated. (C)
Cell lysate from Epo-stimulated or unstimulated 32D/EpoR-Wt was
incubated with GST (C) or GST-Lyn fusion proteins indicated below the
panel. Affinity-purified proteins were subjected to antiphosphotyrosine immunoblotting followed by reprobing with anti-EpoR and anti-Jak2, as
indicated. (D) 32D/EpoR-Wt cells were starved overnight and stimulated
with 100 U/mL of Epo for 5 minutes or left unstimulated, as indicated,
before solubilization. Cell lysates were immunoprecipitated with
anti-EpoR or anti-Jak2 (as indicated), resolved by SDS-PAGE, and
electrotransferred onto a PVDF membrane. The membrane was probed with
GST-Lyn119-230 followed by detection with anti-GST immunoblotting. The
membrane was then reprobed with GST-Lyn1-118, GST-Lyn231-512, GST (C),
antiphosphotyrosine (PY), and the antibody used for
immunoprecipitation, as indicated. (E) Cell lysate from Epo-stimulated
32D/EpoR-Wt was mixed with synthetic phosphopeptides (200 µmol/L)
corresponding to potential tyrosine phosphorylation sites, as
indicated, and incubated with GST-Lyn119-230. Proteins bound to
GST-Lyn119-230 were then subjected to immunoblotting with anti-EpoR.
(F) Cell lysate from Epo-stimulated 32D/EpoR-Wt was mixed with
indicated concentrations of the Y-464 and Y479 phosphopeptides, as
indicated, or the unphosphorylated equivalent of Y-479 polypeptide
(479DP) and analyzed as described above.
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To examine whether the Lyn SH2 domain binds directly to the EpoR and
Jak2, the binding was next examined by Far-Western blotting. As shown
in Fig 4D, GST-Lyn119-230 specifically bound to the
tyrosine-phosphorylated 72-kD form of EpoR from Epo-stimulated
32D/EpoR-Wt cells, whereas GST-Lyn1-118 and GST-Lyn231-512 failed to
show any binding to the EpoR. Similarly, tyrosine-phosphorylated Jak2
bound specifically with GST-Lyn119-230 in Far-Western blotting (Fig 4D,
lower panels). These data indicate that the Lyn SH2 domain directly
binds to the tyrosine-phosphorylated forms of EpoR and Jak2.
Next, to determine which tyrosine residues in the EpoR are involved in
binding with the Lyn SH2 domain, in vitro binding competition assays
were performed using synthetic phosphopeptides corresponding to the
potential tyrosine phosphorylation sites in the EpoR cytoplasmic domain. When added at 200 µmol/L to cell lysate from Epo-stimulated 32D/EpoR-Wt, the phosphopeptide containing Y-464 almost completely inhibited the binding of GST-Lyn119-230 with the EpoR, as shown in Fig
4E. The Y-479 containing peptide also showed a prominent inhibitory
effect on the Lyn SH2 binding to the EpoR, whereas the other
phosphopeptides had less significant or no effects. Various
concentrations of the Y-464 and Y-479 peptides were then added to cell
lysate to determine the inhibitory efficiency, as shown in Fig 4F.
Densitometric scanning of the immunoblot showed that less than 50 µmol/L of the Y-464 peptide was required to inhibit half of the Lyn
SH2 binding to the EpoR. On the other hand, the half-inhibitory
concentration of the Y-479 peptide was between 50 and 100 µmol/L and
thus was higher than that of the Y-464. Nevertheless, the inhibition
was shown to be dependent on phosphorylation of Y-479, because the
unphosphorylated peptide with the identical sequence did not show any
inhibition at 200 µmol/L. These results thus indicate that Y-464 and
Y-479 in the carboxy-terminal region of the EpoR may be involved in
binding with the Lyn SH2 domain.
Lyn induces phosphorylation of Stat5 on Y-694 and activates its
DNA-binding activity in COS7 cells.
To explore the possibility that Lyn may be involved in activation of
the down-stream signaling pathways from the EpoR, we next examined the
possible effect of Lyn on the Stat5 activation. The effect of Lyn on
the tyrosine phosphorylation of Stat5 was first examined by
reconstituting the EpoR signaling pathways in COS7 cells. As shown in
Fig 5A, when ovine Stat5, tagged with the
HA epitope alone was coexpressed with the EpoR, a moderate tyrosine
phosphorylation of Stat5 was observed only after Epo stimulation, as
demonstrated by antiphosphotyrosine blotting of anti-Stat5A
immunoprecipitates. When Jak2 was additionally coexpressed, the
tyrosine phosphorylation of Stat5 was observed constitutively but was
significantly augmented by Epo stimulation. On the other hand, the
coexpression of Lyn induced a strong constitutive tyrosine phosphorylation of Stat5, which was comparable with that attained in
the Epo-stimulated Jak2-coexpressing cells and was not increased by Epo
stimulation (Fig 5A, upper panel). Anti-HA blotting of the anti-Stat5A
immunoprecipitates confirmed that ovine Stat5 was comparably expressed
under each condition (Fig 5A, lower panel).
View larger version (32K):
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| Fig 5.
Lyn induces tyrosine phosphorylation of Stat5 and
activates its DNA-binding ability in COS7 cells. (A) The EpoR and ovine Stat5 tagged with an epitope recognized with anti-HA were transiently coexpressed with Jak2 or with both LynA and LynB, as indicated, in COS7
cells. Cells were left unstimulated () or stimulated (+) with Epo
for 5 minutes before solubilization, as indicated. Cell lysates were
subjected to immunoprecipitation with anti-Stat5A followed by
immunoblotting with the indicated antibodies. (B) The EpoR and
wild-type ovine Stat5 (W) or its mutant with a substitution of Tyr694
with Phe (M) were coexpressed in COS7 cells with Jak2 or Lyn, as
indicated. Cell lysates were immunoprecipitated with anti-Stat5A and
subjected to immunoblotting with indicated antibodies. (C) The EpoR and
ovine Stat5 were coexpressed with Jak2 or Lyn in COS7 cells, as
indicated. Cells were left unstimulated () or stimulated (+) with
Epo for 15 minutes before solubilization, as indicated. Cell lysates
were subjected to affinity purification with a PIE oligonucleotide.
Total cell lysates (TCL) or affinity-purified proteins (PIE) were
analyzed by immunoblotting with indicated antibodies.
|
|
To determine whether Lyn induces the phosphorylation of Stat5 on Y-694,
which is required for homodimerization and activation of Stat5, we next
examined the effect of Lyn on tyrosine phosphorylation of an ovine
Stat5 mutant in which Y-694 is replaced with a Phe residue.29,33 As shown in Fig 5B, the tyrosine
phosphorylation of mutant Stat5 was induced by the coexpression of Lyn
but not by that of Jak2 in COS7 cells coexpressing the EpoR.
Densitometric analysis showed that the expression and tyrosine
phosphorylation levels of wild-type Stat5 coexpressed with Lyn were 1.8 and 3.7 times higher, respectively, than those of mutant Stat5.
Accordingly, the intensity of tyrosine phosphorylation of wild-type
Stat5 induced by Lyn was about two times higher than that of mutant
Stat5, thus suggesting that Lyn phosphorylates Y-694 as well as other
tyrosine residues.
The ability of Lyn to activate the DNA-binding activity of Stat5 was
then examined. For this purpose, proteins that bound to the PIE
oligonucleotide, which contains the Stat5-binding sequence, were
affinity purified from COS7 cells in which the EpoR signaling pathway
was reconstituted by coexpressing the EpoR, HA-tagged ovine Stat5, and
Jak2 or Lyn. In accordance with the previous results shown in Fig 5A,
antiphosphotyrosine blotting of total cell lysates showed that Stat5
was constitutively and prominently tyrosine phosphorylated in COS7
cells coexpressing Jak2 or Lyn, whereas Epo stimulation was required to
induce any detectable tyrosine phosphorylation of Stat5 when these
kinases were not coexpressed (Fig 5C, left upper panel).
Antiphosphotyrosine and anti-HA blotting of proteins bound to the PIE
oligonucleotide showed that the DNA-binding activity of Stat5 closely
correlated with its tyrosine phosphorylation state in these transfected
cells (Fig 5C). It was thus demonstrated that Lyn and Jak2 comparably activated the DNA-binding activity of Stat5, which agrees with the fact
that Lyn induces phosphorylation of Stat5 mainly on Y-694.
Lyn enhances Stat5-mediated transcriptional activation of the
-casein promoter in COS7 and 32D/EpoR-Wt cells.
To investigate whether Lyn activates Stat5 to induce transcription from
responsive genes, we coexpressed Lyn or Jak2 in COS7 cells along with
the EpoR, Stat5, and a Stat5-responsive luciferase reporter plasmid,
pZZ1, in which the expression of the luciferase gene is controlled by
the -casein promoter containing two Stat5-binding sites.
Figure 6A shows that the coexpression of
Lyn significantly increased the transcriptional activation of the
-casein promoter with or without Epo stimulation. To further confirm
that Lyn activates the transactivation potential of Stat5, Lyn was next
transiently overexpressed in 32D/EpoR-Wt cells along with the
Stat5-responsive reporter plasmid. As shown in Fig 6B, the
overexpression of Lyn or Jak2 increased the basal transcription level
of the -casein promoter as well as the level obtained after Epo
stimulation. These results are in agreement with the idea that Lyn
enhances Epo-induced gene transcription through the action of Stat5.
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| Fig 6.
Lyn enhances expression of a Stat5-responsive reporter
plasmid. (A) COS7 cells were cotransfected with either pcDNA3
(Cont.), pcDNA-Jak2 (Jak2), or pXM-LynA and pXM-LynB (Lyn), as
indicated, along with the Stat5-responsive luciferase reporter pZZ1
plasmid, the control Renilla luciferase pRL-TK plasmid, pRK5-Stat5, and pXM-EpoR-Wt. Two days after transfection, cells were either left unstimulated or stimulated with 5 U/mL for 5 hours, as indicated, and
harvested for the dual-luciferase assay. The luciferase activity was
normalized by the Renilla luciferase activity and expressed in
arbitrary units. The data represent averages ± standard deviations of
three independent experiments. (B) 32D/EpoR-Wt cells were transiently transfected with either pcDNA3 (Cont.), pcDNA-Jak2 (Jak2), or pXM-LynA
and pXM-LynB (Lyn), as indicated, along with pZZ1 and pRL-SV40. One day
after transfection, cells were starved overnight, incubated for 5 hours
in medium with or without 4 U/mL of Epo (as indicated), and harvested
for the dual-luciferase assay.
|
|
Lyn phosphorylates the EpoR and Stat5 on tyrosines in vitro.
To confirm that the EpoR and Stat5 are substrates for Lyn, we next
examined whether Lyn phosphorylates the EpoR and Stat5 in vitro. For
this purpose, a GST-EpoR fusion protein containing the EpoR
intracellular domain or a GST-Stat5 protein containing a
carboxy-terminal portion of Stat5 including Y-694 was incubated, in the
presence or absence of ATP, with Lyn immunoprecipitated from
32D/EpoR-Wt cells. As shown in Fig 7,
antiphosphotyrosine blotting showed that the tyrosine phosphorylation
of these fusion proteins was induced only in the presence of both Lyn
and ATP. On the other hand, Lyn did not induce any detectable tyrosine phosphorylation of the GST protein in vitro (negative data not shown). These data indicate that the EpoR and Stat5 are substrates for
tyrosine phosphorylation by Lyn in vitro. In the experiment shown in Fig 7, the phosphorylation of the EpoR and Stat5 induced by
Lyn immunoprecipitated from Epo-stimulated cells was less prominent than that induced by Lyn from nonstimulated cells. However, in a
repeated experiment, Epo stimulation of 32D/EpoR-Wt cells before solubilization did not have any significant effect on the ability of
Lyn to phosphorylate these fusion proteins.
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| Fig 7.
Lyn phosphorylates the EpoR and Stat5 on tyrosine in
vitro. 32D/EpoR-Wt cells were left unstimulated or stimulated with Epo, as indicated. Cells were then lysed and immunoprecipitated with anti-Lyn (Lyn +) or with normal rabbit serum (Lyn ). GST-EpoR (upper panels) and GST-Stat5 (lower panels) proteins were then added to
immunoprecipitates as substrates and incubated at room temperature for
30 minutes in in vitro kinase buffer with or without 1 mmol/L
ATP, as indicated. Reaction products were then subjected to
immunoblotting with antiphosphotyrosine followed by reprobing with
anti-EpoR or anti-Stat5, as indicated.
|
|
| |
DISCUSSION |
The present study has demonstrated that Lyn physically associates with
the EpoR in Epo-responsive hematopoietic cells. Transient expression
experiments in COS7 cells further showed that both LynA and LynB binds
to the membrane-proximal 91-amino acid residues of the EpoR cytoplasmic
domain. However, in contrast to the binding of Jak2 to the EpoR, the
membrane-proximal amino acid residues 281 through 300 of the EpoR were
dispensable for the binding of Lyn. In vitro binding studies
using GST-Lyn fusion proteins showed that the tyrosine kinase domain of
Lyn binds to the EpoR and further indicated that the binding of Lyn to
the EpoR may be additionally mediated through interaction between the
Lyn SH2 domain and phosphorylated Y-464 and Y-479 residues in the
carboxy-terminal region of the EpoR. In vitro binding studies also
raised a possibility that the Lyn SH2 may bind directly to
tyrosine-phosphorylated Jak2. When coexpressed in COS7 cells, Lyn
induced tyrosine phosphorylation of the EpoR. Furthermore, in vitro
reconstitution experiments in COS7 cells as well as overexpression
experiments in 32D/EpoR-Wt cells indicated that Lyn has the ability to
induce tyrosine phosphorylation of Stat5 to activate its DNA-binding
and transcription-activating activities. Lyn was also shown to
phosphorylate the EpoR and Stat5 on tyrosines in vitro. Together, these
results strongly suggest that Lyn may play a role in activation of the
EpoR-mediated signaling pathways including the Jak2/Stat5 pathway.
The coexpression experiments in COS7 cells demonstrated that Lyn binds
to the membrane-proximal 91-amino acid region of the EpoR cytoplasmic
domain, which is required for growth signaling from the
EpoR.4,5,7,36 However, the 20-amino acid region between the
Box1 and Box2 motifs, required for the binding of Jak2, was dispensable
for that of Lyn, thus suggesting that the two tyrosine kinases bind to
the EpoR membrane-proximal region in different manners. Because the
membrane-proximal 91-amino acid region does not contain any tyrosine
residues, it is expected that the binding to this region is mediated
through the Lyn tyrosine kinase domain, which was shown to bind to the
EpoR irrespective of its tyrosine phosphorylation status in the in
vitro binding study. The physical association between the Src family
tyrosine kinase domain and the cytokine receptor cytoplasmic domain has previously been reported in the case of binding of Lck to the IL-2
receptor subunit (IL-2R).38 Intriguingly, when
expressed in cells not expressing Lck, IL-2R has been shown to
couple with Lyn instead of Lck,39,40 which implies that the
Lyn tyrosine kinase domain may also have the ability to bind IL-2R.
The in vitro binding studies using GST-Lyn fusion proteins further
showed that the Lyn SH2 domain specifically bound to the tyrosine-phosphorylated 72-kD form of the EpoR in cell lysate from
Epo-stimulated cells. In fact, anti-EpoR immunoblotting showed that the
amount of the EpoR that was bound to the SH2 domain of Lyn was much
higher than that bound to the tyrosine kinase domain (Fig 4B). It is
thus suggested that the SH2 domain of Lyn binds to the EpoR with an
affinity that is substantially higher than that of the tyrosine kinase
domain. The difference in binding affinities may be quite remarkable,
because the amount of the tyrosine-phosphorylated EpoR in cells is much
smaller than that of the unphosphorylated EpoR; only a small portion of
the EpoR synthesized in cells as the 62- to 66-kD forms is expressed on the cell surface and becomes tyrosine phosphorylated as the 72-kD form
upon Epo binding.4,37,41 Consistent with this idea, we
could detect the EpoR binding of the Lyn SH2 domain but not that of the
tyrosine kinase domain by Far Western blotting. It is thus possible
that, unlike when overexpressed in COS7 cells, the Lyn binding to the
EpoR in hematopoietic cells may be mediated mainly through the
interaction between the Lyn SH2 domain and phosphotyrosines in the
activated EpoR and thus induced mainly after Epo stimulation. However,
this possibility remains to be proved, because the small quantity of
the surface expressed EpoR prohibited the direct detection of the EpoR
that is associated with Lyn in hematopoietic cells by anti-EpoR
blotting of Lyn immunoprecipitates (data not shown).
The Lyn SH2 domain was shown to bind directly with the EpoR by
Far-Western blotting (Fig 4D). Competition binding assays using synthetic phosphopeptides further indicated that the binding may involve phosphorylated Y-464 and Y-479 residues at the EpoR
carboxy-terminal region (Fig 4E and F). Notably, the amino acids
following Y-464 (Y-464 ENS) are similar to the optimal binding
sequences for the SH2 domains of the Src family kinases with the
general motif pY-hydrophilic-hydrophilic-I/P,42 although
those following Y-479 (Y-479 VAC) are quite different. Previously, we
showed that the p85 regulatory subunit of PI3K binds through its SH2
domain to the tyrosine-phosphorylated EpoR carboxy-terminal region,
which is, however, not required for growth signaling.10
Recently, Damen et al43 have shown that Y-479 of the EpoR,
one of the Lyn SH2 binding sites identified in the present study, is
involved in binding to the p85 SH2 domain. Intriguingly, a very recent
study by Klingmüller et al44 has further
shown that Y-479 may be involved in transducing signals for
proliferation and differentiation of the erythroid progenitor cells. It
is thus possible that Lyn may play a physiologically significant role by binding to this potentially important tyrosine residue or to Y-464
in the vicinity of Y-479. In this regard, it should be noted that Lyn
has been shown to bind directly or indirectly with p85 and to activate
the PI3K activity.20,45-48 It is also conceivable that Lyn
may compete with p85 for binding to Y-479 and thus inhibit the
EpoR-mediated activation of PI3K. The effect of Lyn on the EpoR-mediated activation of the PI3K pathway is currently under investigation in our laboratory using cells stably overexpressing Lyn.
As the present study was being completed, Tilbrook et al26
reported that Lyn physically associates with the EpoR and is essential
for Epo-induced differentiation of the J2E cell line, which was
generated by transforming immature erythroid cells with the
raf/myc-containing J2 retrovirus. Using the yeast
two-hybrid analysis, Tilbrook et al26 assigned the region
of Lyn involved in binding to the EpoR to the N-terminal 162-amino acid
residues, which contains the unique and SH3 binding domains and only a
portion of the SH2 domain. This is in contrast to the present study,
which demonstrated that the SH2 and catalytic domains of Lyn are
involved in in vitro binding studies. However, because Tilbrook
et al26 examined only this N-terminal region of Lyn in the
yeast two-hybrid analysis, it has remained to be determined whether the
other domains of Lyn may also mediate binding with the EpoR in the
yeast cells. Furthermore, it should be noted that the binding that
requires the tyrosine phosphorylation of the EpoR may not be examined
by the method used by Tilbrook et al.26 Further studies are
thus required to determined which domain of Lyn mainly mediates binding to the EpoR in hematopoietic cells.
Previous studies have implicated Lyn in signaling from the receptors
for IL-3, GM-CSF, and IL-5,17-22 which share the common signal-transducing subunit c, because Lyn physically associates with
c and its kinase activity shows a moderate increase after activation
of these receptors in several cell lines, including 32D
cells.19 In contrast, we have not observed any persistent and significant increase in the autokinase or exokinase activity of Lyn
by Epo stimulation of 32D/EpoR-Wt cells in the present study (negative
data not shown). This may be due to the high constitutive activity of
Lyn in cells we used, because a moderate increase in activity of a
fraction of Lyn that is associated with the EpoR could not be then
detectable by the in vitro kinase assay of Lyn immunoprecipitated from the whole cell. Alternatively, because we have
not observed any significant increase in Lyn activity even after IL-3
stimulation of 32D/EpoR-Wt cells (data not shown), the differences in
reagents, cells, or method we used as compared with those used in
previous studies might have caused our inability to demonstrate an
increase in Lyn activity after cytokine stimulation. In this regard, it
should be noted that Tilbrook et al26 have shown that Epo
induces a marginal increase in the Lyn kinase activity in the J2E
cells. Another possibility is that the binding of Lyn to the EpoR may
not modulate its kinase activity but may allow the kinase access to
substrates for phosphorylation, which include the EpoR itself and
signaling molecules recruited to the tyrosine-phosphorylated EpoR.
Previously, v-Src has been shown to activate Stat3 in NIH3T3 and rat
embryo fibroblast cells.49,50 Furthermore, it has been
proposed that c-Src, which physically associates with growth factor
receptors with intrinsic tyrosine kinase activity,51 mediates the activation of Stat3 by these receptors.50 When expressed in 32D cells, v-Src was shown to activate Stat1, Stat3, and
Stat5 and to abrogate IL-3 dependence of this cell line.52 These observations led us to speculate that Lyn, the most abundantly expressed Src family kinase in 32D cells, may play a role in the EpoR-mediated activation of Stat5. In accordance with this speculation, the present study demonstrated that Stat5 as well as the EpoR is a
substrate for Lyn, because Lyn induced the tyrosine phosphorylation of
both the EpoR and Stat5 in COS7 cells (Figs 2 and 5A) and
phosphorylated these proteins on tyrosines in vitro (Fig 7). Although
the Lyn-induced tyrosine phosphorylation of the EpoR and Stat5 was
independent of Epo stimulation in COS7 cells, this could be due to the
high expression levels of Lyn and these substrates in these cells, because Jak2 also induced the constitutive tyrosine phosphorylation of
the EpoR and Stat5 when coexpressed in COS7 cells (Figs 2 and 5A). By
using a Stat5 mutant, it was further demonstrated that Lyn induced the
phosphorylation of Stat5 on Y-694, which is required for its activation
(Fig 5B).29 In accordance with this finding, Lyn stimulated
the DNA-binding and transactivation abilities of Stat5 in COS7 cells
(Figs 5C and 6A). The ability of Lyn to increase the Stat5-mediated
transcriptional activation was confirmed in 32D/EpoR-Wt cells by
transient overexpression of Lyn (Fig 6B). These data strongly support a
role for Lyn in enhancing the EpoR-mediated Stat5 activation.
Importantly, the Lyn SH2 domain was shown to bind to
tyrosine-phosphorylated Jak2 in vitro (Fig 4C and D), although
the in vivo binding could not be demonstrated by
coimmunoprecipitation (data not shown). Similarly, Uddin et
al53 have very recently reported that Fyn, another member
of the Src family, associates via its SH2 domain with Tyk2 or Jak2 in
cells stimulated with interferon (IFN) or IFN, respectively. In
addition, the Src family kinases have been shown to associate through
their SH2 domains with several receptor-type tyrosine kinases,
resulting in the mutual stimulation of catalytic activity and enhanced
phosphorylation of downstream targets of each of the tyrosine
kinases.51 It is thus tempting to speculate that the
Lyn-Jak2 interaction may similarly activate or stabilize the catalytic
activity of both tyrosine kinases. In vitro and in vivo binding studies
have previously shown that Lyn also binds to Cbl, Shc, PI3K,
phospholipase C, MAPK, and GAP,47 all of which have also
been implicated in signaling mediated through the EpoR. Therefore, Lyn
may also directly phosphorylate these signaling molecules involved in
EpoR-mediated signaling or indirectly potentiate their tyrosine
phosphorylation. Further studies are currently in progress to address
these possibilities and to investigate the effect of Lyn on
anti-apoptotic, growth-promoting, and differentiation-inducing
abilities of the EpoR.
In summary, this study suggests that Lyn constitutively binds to the
91-amino acid membrane-proximal EpoR region and, upon the activation
and tyrosine phosphorylation of the EpoR, further interacts through its
SH2 domain with the phosphorylated tyrosine residues, Y-464 and Y-479,
in the EpoR carboxy-terminal region. It is speculated that the binding
of these phosphotyrosines to the Lyn SH2 domain may increase the kinase
activity of Lyn by displacing the inhibitory phosphotyrosine at the
carboxy-terminal region of Lyn. Alternatively, Lyn may be newly
recruited to the activated EpoR through the phosphotyrosine-SH2
interaction, thus gaining access to substrates for phosphorylation,
which include the EpoR itself and signaling molecules recruited to the
tyrosine-phosphorylated EpoR. It is thus hypothesized that Lyn plays a
role in the EpoR-mediated signaling, which was supported by the finding
that Lyn enhanced the Epo-induced activation of Stat5.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted January 12, 1998.
Supported by grants from the Ministry of Education, Science and Culture
of Japan.
Address reprints requests to Osamu Miura, MD, First Department of
Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyoku, Tokyo 113, Japan.
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.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs James N. Ihle and Taolin Yi for
invaluable discussions and for the generous gifts of the murine Lyn
cDNAs and the EpoR-derived phosphotyrosine peptides. We thank Drs Yoji
Ikawa, Atsushi Miyajima, and Koh Yamamoto for helpful discussions and
Kaori Okada for excellent technical assistance.
| |
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A. Arai, E. Kanda, Y. Nosaka, N. Miyasaka, and O. Miura
CrkL Is Recruited through Its SH2 Domain to the Erythropoietin Receptor and Plays a Role in Lyn-mediated Receptor Signaling
J. Biol. Chem.,
August 24, 2001;
276(35):
33282 - 33290.
[Abstract]
[Full Text]
[PDF]
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Abstract
Introduction
Abstract