From the Section of Neonatal-Perinatal Medicine, Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received for publication, July 15, 2002, and in revised form, December 9, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Kit receptor tyrosine kinase and
erythropoietin receptor (Epo-R) cooperate in regulating blood cell
development. Mice that lack the expression of Kit or Epo-R die in
utero of severe anemia. Stimulation of Kit by its ligand, stem
cell factor activates several distinct early signaling pathways,
including phospholipase C Receptor tyrosine kinases
(RTKs)1 trigger multitude of
cellular events, including proliferation, survival, differentiation, and migration. In response to ligand-induced stimulation, RTKs undergo
dimerization and autophosphorylation on several distinct cytoplasmic
tyrosine residues (1-4). These phosphorylated tyrosine residues become
binding sites for a variety of Src homology 2 domain-containing enzymes
and adaptor proteins such as phospholipase C In hematopoietic cells, the RTK Kit plays an essential role in
regulating proliferation, survival, differentiation, and migration of
stem and progenitor cells (9-11). The proto-oncogene Kit encodes the
receptor for stem cell factor (SCF) and belongs to the type III
receptor tyrosine kinase subfamily (9-11). This family of cytokine
receptor includes the macrophage colony-stimulating factor (M-CSF)
receptor, the PDGF receptor, and the Flk-2/Flk-3 receptor (1).
The structure of these receptors includes an extracellular domain with
five Ig-like motifs, a single short membrane-spanning domain, and a
cytoplasmic domain with tyrosine kinase activity (9-11). The kinase
domain is separated by a kinase insert sequence that divides the kinase
domain into an ATP binding region and phosphotransferase region
(9-11). The product of the Kit gene is a transmembrane receptor
composed of 976 amino acids (aa) with 519 extracellular aa, a
transmembrane domain of 23 aa, and an intracellular tail of 433 aa
(9-11). In addition to being expressed on hematopoietic cells, Kit is
also expressed on cells of nonhematopoietic origin, including
melanocytes, primordial germ cells, and interstitial cells of Cajal
(9). In hematopoietic cells, Kit can synergize with other growth factor
receptors to promote survival, proliferation, and differentiation of
multiple hematopoietic lineages (9-11).
Intriguingly, the most profound phenotype due to the lack of Kit
expression in mice is manifested in erythroid cells. Mutant mice that
lack the expression of Kit (dominant white spotting, or W, mutants)
demonstrate severe deficiencies in erythroid cell development (9, 12).
Kit-deficient mice exhibit a severe reduction of colony-forming
unit-erythroid (CFU-E) progenitors in the fetal liver and die of anemia
around day 16 of gestation (9, 12). Epo-R-deficient mice also
demonstrate a similar decrease in CFU-E progenitors and die of anemia
between days 13 and 15 of gestation (13), suggesting that erythroid
progenitors cannot survive, proliferate, or differentiate unless both
Kit and the Epo-R signal transduction pathways are functional. Recent studies have suggested that Epo and Epo-R interactions may contribute to this process by preventing erythroid progenitors from undergoing apoptosis by activating Stat5 and subsequently inducing the expression of an antiapoptotic protein, Bcl-xL (14). Consistent with these studies, mice deficient in the expression of Stat5 or Bcl-xL manifest a
decrease in the number of erythroid progenitors due to enhanced apoptosis (14-17). However, the role of Kit in erythroid cell
development alone or in combination with Epo-R is poorly understood. To
this end, we have recently demonstrated an essential role for Kit in proliferation of erythroid progenitors (18). Further, we and others
have also demonstrated that Kit synergizes with Epo-R in enhancing
proliferation and survival of erythroid progenitors (18-25). However,
the role of early activating signal transduction pathways downstream
from Kit in erythroid cell growth, proliferation, and cooperation with
Epo-R is not known.
Activated Kit binds signaling molecules at specific tyrosine residues:
PLC- In this study, we took advantage of the ability of Tyr Cell Line--
G1E-ER2 cells have been described previously
(15). G1E-ER2 cells were grown in Iscove's modified Dulbecco's medium
(Invitrogen) with 15% heat-inactivated fetal bovine serum
(Fisher), recombinant Epo (2 units/ml) (Amgen, Thousand Oaks, CA), and
recombinant rat SCF (50 ng/ml) (Amgen).
Construction of Wild Type (WT) and Mutant Chimeric Kit Receptors
(CHRs)--
The CHR gene was constructed from DNA encoding aa 1-513
of the human M-CSF receptor and aa 528-977 of the murine Kit receptor joined at an EcoRI site. Plasmid containing the human
full-length M-CSF receptor cDNA (a kind gift of Dr. Sherr, St.
Judes, Memphis, TN) was utilized. Forward (NotI-containing)
and reverse (EcoRI-containing) primers corresponding to the
start site and transmembrane region of the M-CSF receptor were utilized
to perform PCR on the extracellular domain of the M-CSF
receptor. Forward (EcoRI-containing) and reverse (XhoI-containing) primers corresponding to the transmembrane
and the stop site were used to perform PCR on the transmembrane
and the cytoplasmic domain of the murine Kit receptor. The PCR product was digested and ligated into the NotI and XhoI
sites of MIEG3 bicistronic retroviral expression vector (40). The
sequence of the CHR was verified. To generate mutant CHRs, the
NotI-XhoI WT CHR DNA fragment (2.9 kb) spanning
the sites to be mutated was subcloned into Bluescript. The QuikChange
site-directed mutagenesis kit (Stratagene) and primers containing the
appropriate mutations were used to mutate tyrosine residues 567, 569, 702, 719, 728, and 745 to Phe. The NotI-XhoI
fragment containing mutations at tyrosine residues 567, 569, 702, 719, 728, and 745 in murine Kit receptor was verified by sequencing released
from Bluescript and religated into the NotI-XhoI
site of MIEG3 retroviral vector. In some experiments,
mutant CHR lacking all six tyrosine residues was used as a
template to restore phenylalanine mutations at positions 567 and 569 back to tyrosine. Utilizing this bicistronic retroviral vector, we
inserted the WT and the mutant CHR cDNAs upstream of the internal
ribosome entry site (IRES) and the enhanced green fluorescence protein
(EGFP) gene (see Fig. 1).
Expression of WT and Mutant CHRs in G1E-ER2 Cells--
To
produce WT and mutant CHR viral supernatants for infection of G1E-ER2
cells, Phoenix ecotropic cells were transiently transfected with WT or
the mutant CHR retroviral constructs using LipofectAMINE Plus reagent
(Invitrogen). Supernatants were collected 48 h after transfection,
filtered through 0.45-µm membranes, and used. Cells were
infected with 2 ml of virus supernatant in the presence of 8 µg/ml
polybrene. Virus-infected cells were harvested 48 h later, sorted
by a fluorescence-activated cell sorter, and expanded in culture.
G1E-ER2 cells expressing similar levels of EGFP and M-CSF receptors
were utilized to perform all of the experiments described in these studies.
Flow Cytometric Analysis--
Phycoerythrin (PE)-conjugated
secondary monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) was used to detect the antibody to the extracellular domain
of the CHR (M-CSF receptor; 2-4A5; Santa Cruz Biotechnology). G1E-ER2
cells (1 × 106) expressing WT or mutant CHRs were
incubated at 4 °C for 30 min with 1 µg of the primary antibody.
Cells were washed three times with phosphate-buffered saline containing
0.1% bovine serum albumin (Sigma) and incubated with a secondary
antibody for 30 min at 4 °C, washed as above, and analyzed by a
fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, San
Jose, CA).
Effect of M-CSF and Epo on Proliferation and Survival of G1E-ER2
Cells--
The effect of M-CSF and Epo on proliferation of G1E-ER2
cells was assayed by thymidine incorporation. 96-Well tissue culture plates were utilized for these studies. G1E-ER2 cells expressing WT or
mutant CHRs were plated at 5 × 104 cells/well for
48 h, either in the absence or presence of M-CSF (50 ng/ml) and/or
Epo (2 units/ml). Subsequently, 1.0 µCi of
[3H]thymidine was added to each well for 6-8 h at
37 °C. Cells were then harvested using an automated cell harvester
(96-well harvester; Brandel, Gaithersburg, MD), and thymidine
incorporation was determined in a scintillation counter. The effect of
M-CSF and/or Epo on cell growth and viability was performed by plating
1 × 105 cells in a six-well tissue culture plate in
replicates of three for 24 and 48 h, after which cells were
subjected to trypan blue and counted under the microscope. Viable cells
were scored at various time points.
Erythroid Colony Analysis--
Fetal liver cells were harvested
from day 12.5 WT embryos. Single cell suspensions were prepared and
incubated with or without retrovirus expressing the WT CHR or CHR
mutants containing tyrosine to phenylalanine mutations at positions 567 and 569 as described above. After infection, cells were plated in
triplicate in Immunoprecipitation--
Immunoprecipitations (IPs) were
performed as previously described (41). Briefly, cells expressing
either the WT or the mutant CHR deficient in the activation of Src
kinases (CHR 567/569) or Src add-back (CHR 567/569B) CHR mutants were
stimulated for the indicated times. Thereafter, cells were lysed in
lysis buffer (10 mM K2HPO4, 1 mM EDTA, 50 mM EGTA, 10 mM
MgCl2, 1 mM Na2VO4, 50 mM Western Blot Analysis--
For Western blot analysis, 1-2 × 106 WT CHR and CHR 567/569 were plated in duplicate
six-well tissue culture plates for 12 and 24 h at 37 °C in the
presence or absence of M-CSF (100 ng/ml) and/or Epo (2 units/ml).
Thereafter, cells were harvested and lysed in lysis buffer as described
above. An equal amount of protein was fractionated on a 10% SDS-PAGE
gel and electrophoretically transferred to nitrocellulose membrane.
Expression of c-Myc was determined by using an anti-c-Myc antibody
(Santa Cruz Biotechnology). Expression of Bcl-xL was determined by
using an anti-Bcl-xL antibody (Invitrogen). Activation of Stat5 was
determined by utilizing a phosphospecific Stat5 antibody (Cell
Signaling, Beverly, MA).
Construction of Wild Type and Mutant Chimeric Kit
Receptors--
To analyze the role of diverse early signaling pathways
activated via Kit in response to SCF stimulation in erythroid cells, we
utilized an erythroid progenitor cell line G1E-ER2 (15). These cells
were utilized to specifically examine the role of Kit and Epo-R in the
context of a cell type that is most affected due to mutations in Kit
and Epo-R. We and others have previously shown that these cells mimic
primary proerythroblasts and respond to both SCF and Epo (15, 18).
Since G1E-ER2 cells express endogenous Kit receptors, we constructed
eight chimeric Kit receptors (CHRs) to bypass endogenous Kit receptors
(Fig. 1). The CHR approach to investigate
the role of a specific signaling pathway in nonhematopoietic cells
downstream from a RTK, such as the PDGF receptor, has been previously
described (6, 42). The M-CSF receptor and Kit belong to the same
subfamily of RTKs but possess distinct ligand binding specificity (1).
G1E-ER2 cells do not express endogenous M-CSFR (Fig.
2B, left
panel) and show no response to M-CSF stimulation (Fig.
2D, panel A). Based on these
observations, we cloned a cDNA encoding a protein consisting of the
extracellular domain of the human M-CSFR and the transmembrane and the
cytoplasmic domain of murine Kit (Fig. 1). This CHR is activated upon
binding M-CSF but signals in a fashion similar to endogenous Kit
receptor (Fig. 2, C and D (panel
B)). Eight mutant chimeric receptor cDNAs were constructed, encoding tyrosine to phenylalanine mutations in the cytoplasmic domain of Kit. These mutant receptors cannot bind and
activate signaling molecules (10, 11, 26-29, 31, 43). We mutated the
binding sites for Src kinases at positions 567 and 569 of the Kit
cytoplasmic domain, PI 3-kinase at position 719, PLC- Expression and Function of Wild Type Chimeric Receptor--
For
expression, biochemical and functional analysis of the WT CHR, we
cloned this receptor into a bicistronic retroviral vector MIEG3 (Fig.
2A) that expresses the EGFP via an internal ribosome entry
site and generated viral supernatants as described under "Experimental Procedures." We have previously reported the use of
this vector in generating high transduction of hematopoietic cells
(40). After infection of G1E-ER2 cells, EGFP-positive cells were sorted
to homogeneity and utilized to perform functional and biochemical
studies. Flow cytometry was utilized to examine the expression of
M-CSFR in G1E-ER2 cells expressing the WT CHR. Fig. 2B
(left panel) demonstrates complete lack of M-CSFR
expression in parental untransduced G1E-ER2 cells stained with a
PE-conjugated antibody against M-CSFR (y axis). Fig.
2B (right panel) demonstrates 100%
co-expression of both EGFP and M-CSFR in G1E-ER2 cells transduced with
the WT CHR.
To determine whether ligand-induced phosphorylation of the WT CHR is
similar to endogenous Kit receptor, we starved the cells for 6 h
of serum and growth factors, and stimulated them with either 100 ng/ml
recombinant rat SCF or human M-CSF for 10 min, after which cells were
lysed and subjected to immunoprecipitation using an anti-Kit antibody
or an anti-M-CSFR antibody and subjected to Western blot analysis using
an anti-phosphotyrosine antibody. As shown in Fig. 2C,
stimulation of G1E-ER2 cells expressing the WT CHR with SCF
(lane 2) or M-CSF (lane 4)
resulted in comparable tyrosine phosphorylation, suggesting that the WT
CHR biochemically behaves in a fashion similar to endogenous Kit
receptor. The phosphorylation of various CHR mutants examined in the
present study was also similar to WT CHR except for CHR mutants
impaired in the activation of Src and PI 3-kinase pathway. These two
mutant CHRs demonstrated a slight decrease in phosphorylation compared
with WT CHR (data not shown).
Next, we analyzed the function of WT CHR by examining proliferation in
response to M-CSF stimulation. Consistent with the lack of M-CSFR
expression in G1E-ER2 cells shown earlier (Fig. 2B,
left panel), the addition of M-CSF to parental
untransduced G1E-ER2 cells did not induce proliferation and did not
cooperate with Epo-R to enhance proliferation (Fig. 2D,
panel A). In contrast, and as expected and
previously shown (18), stimulation of G1E-ER2 cells with SCF resulted
in significant proliferation, which was further augmented in the
presence of Epo (Fig. 2D, panel A).
Consistent with the expression and the biochemical observations noted
above (Fig. 2, B and C), G1E-ER2 cells expressing
the WT CHR demonstrated a similar increase in proliferation in response
to M-CSF stimulation, as seen with SCF stimulation of endogenous Kit
receptors (Fig. 2D, panel B). This
increase in proliferation was further augmented in the presence of Epo,
to levels observed in response to endogenous Kit activation with SCF
and Epo (Fig. 2D, panel B).
Collectively, these data demonstrate that the WT CHR appears to
function in a fashion similar to the endogenous Kit receptor in G1E-ER2 cells.
Expression and Function of Mutant Chimeric Receptors--
To
determine the effect of abrogating the activation of diverse early
signaling pathways in Kit-induced proliferation/growth and cooperation
with Epo-R, we expressed all eight CHR mutants in G1E-ER2 cells and
examined M-CSF induced growth and proliferation over a 2-day culture
period by trypan blue exclusion. Fig. 3
demonstrates similar co-expression of all eight CHR mutants
(y axis) and EGFP (x axis) in G1E-ER2 cells as
determined by flow cytometry using a PE-conjugated antibody against the
M-CSFR. G1E-ER2 cells expressing a similar level of CHR mutants were
utilized in subsequent studies. As shown in Fig.
4, Kit mutants lacking the binding sites
for Src kinases (Y567F/Y569F) (Fig. 4, panel C)
completely lacked the ability to sustain the growth over a 2-day
culture period in the presence of M-CSF. In contrast, Kit mutants
lacking the binding sites for Grb2 (Y702F) (Fig. 4, panel
D), PLC-
We and others have previously shown that cooperation between Kit and
Epo-R is essential for regulating proliferation of erythroid progenitors (18-25). To determine the role of Kit-induced early signaling pathways in mediating cooperation with Epo-R, we examined various Kit mutants for their ability to induce growth in the presence
of both M-CSF and Epo. As shown in Fig.
5, and consistent with a role for Epo in
maintaining the survival of erythroid progenitors, costimulation of
G1E-ER2 cells expressing various Kit mutants with Epo and M-CSF
maintained their growth and survival over a 2-day culture period.
However, mutants of Kit impaired in the activation of Grb2 (Fig. 5,
panel D), PI 3-kinase (Fig. 5, panel E), and the
Y745F (Fig. 5, panel G) demonstrated an enhanced ability to
cooperate with Epo-R in inducing growth over a 2-day culture period. A
Kit mutant impaired in the activation of Src kinases demonstrated a
complete lack of cooperation with Epo-R in inducing growth (Fig. 5,
panel C). Similar results were obtained using a thymidine
incorporation assay (Fig. 6A).
As shown in Fig. 6A, Kit mutants defective in the activation
of Src and PI 3-kinase showed the most profound reduction in thymidine
incorporation over 48 h in the presence of M-CSF alone (Fig.
6A, panels C and E). In contrast, and
similar to the results shown in Fig. 4, panels D,
F, and G, mutants of Kit defective in the
activation of Grb2 (Fig. 6A, panel D), PLC-
To further substantiate a role for Src kinases in Kit and Epo-R-induced
cooperation, we treated G1E-ER2 cells with a Src kinase-specific inhibitor (PP1) and measured proliferation by thymidine incorporation in the presence of SCF and/or Epo over 48 h. As shown in Fig. 6C, a dose-dependent decrease in proliferation
of G1E-ER2 cells was observed in PP1-treated cells both in the presence
of SCF as well as in combination with Epo. Collectively, these results strongly implicate the Src kinase pathway in regulating both Kit- and
Epo-R-mediated cooperation in erythroid cells.
Several previously described studies have demonstrated a physical
association between Kit and Epo-R (19, 22, 41). Further, stimulation of
Kit- and Epo-R-expressing cells with SCF alone has been shown to
transphosphorylate Epo-R (19, 22). Wu et al. (21) have
argued that Kit-induced phosphorylation of Epo-R is essential for the
generation of CFU-Es. We hypothesized that the lack of cooperation
between Src kinase-defective Kit mutant and Epo-R may in part be due to
reduced transphosphorylation of the Epo-R in response to M-CSF
stimulation. To test this, WT or 567/569 CHR-expressing G1E-ER2 cells
were starved and stimulated for 10 min with M-CSF, subsequently the
lysates were prepared and subjected to IP with an anti-Epo-R antibody,
followed by Western blot analysis using an anti-phosphotyrosine
antibody. As shown in Fig. 7A,
and consistent with previously published results in HCD57 cells (19,
22), stimulation of G1E-ER2 cells expressing the WT CHR with M-CSF
resulted in a slight increase in the transphosphorylation of Epo-R.
However, only a modest increase in the phosphorylation of Epo-R was
noted in G1E-ER2 cells expressing the Src kinase-defective Kit mutant
(lane 4), suggesting that the reduced
proliferation of G1E-ER2 cells expressing the 567/569 Kit receptor may
in part be due to reduced transphosphorylation of the Epo-R in response to M-CSF stimulation. The level of Epo-induced Epo-R phosphorylation in
WT and 567/569-expressing G1E-ER2 cells was comparable (data not
shown).
Previous studies in nonhematopoietic cells have implicated Src kinases
in inducing growth/proliferation by regulating the expression of c-Myc
(44, 45). To determine whether reduced proliferation and cooperation
between Src kinase-defective Kit mutant and Epo-R is associated with
reduced expression of c-Myc, we cultured WT and 567/569 Kit
mutant-expressing G1E-ER2 cells in the presence of M-CSF or M-CSF and
Epo and examined the expression of c-Myc. As shown in Fig.
7B, compared with WT CHR, stimulation of Src
kinase-defective Kit mutant-expressing cells showed a significant decrease in the expression of c-Myc in response to M-CSF stimulation alone (lane 4) as well as in the presence of Epo
(lane 6), although expression of c-Myc in
response to Epo alone was comparable between WT and the 567/569 Kit
mutant (lanes 2 and 5). Further,
Epo-induced Stat5 activation (Fig. 7C) and Bcl-xL expression
(Fig. 7D) was also comparable between WT and the Src
kinase-defective Kit mutant. Interestingly, c-Myc expression was also
reduced in a PI 3-kinase-defective Kit mutant but not in other mutants
of Kit (data not shown).
To determine whether restoring the Src kinase pathway in a Kit mutant
lacking the activation of all five early signaling pathways (naked CHR)
could restore Kit-induced proliferation and cooperation with Epo-R,
including its transphosphorylation, we constructed and expressed a CHR
mutant in which only the Src kinase pathway was restored by replacing
the phenylalanines at positions 567 and 569 back to tyrosines
(567/569B). Fig. 8A
(top panel) demonstrates the expression of WT CHR
(left panel), naked CHR (right
panel) and CHR mutant in which the Src binding sites at
positions 567 and 569 were restored to tyrosines (Src add-back mutant;
middle panel). As shown in Fig. 8A
(bottom left panel), stimulation of the Src kinase add-back mutant receptor with M-CSF alone, demonstrated a 50% restoration in proliferation, and completely restored the cooperation between Kit and Epo-R (Fig. 8A,
bottom right panel). Further,
restoration of the Src binding sites in the naked mutant Kit receptor
also restored the transphosphorylation of the Epo-R in response to
M-CSF stimulation (Fig. 8B). These observations were further
confirmed by expressing the WT, 567/569B and naked CHRs in 32D myeloid
cell line that lack the expression of endogenous Kit
receptors.2 Collectively,
these results demonstrate that early activation of Src kinase pathway
in Kit is necessary and sufficient to restore the cooperation between
Kit and Epo-R in G1E-ER2 cells.
Kit is crucial for the development of erythroid progenitors, since
mice that lack Kit or its ligand SCF exhibit significant reduction of
fetal liver erythroid progenitors and die of anemia (9, 12). The
survival and proliferation of erythroid progenitors also depends on
Epo, suggesting that erythroid progenitors cannot proliferate and/or
survive unless both Kit and the Epo-R signal transduction pathways are
functional. We have recently demonstrated an essential role for SCF/Kit
interactions in proliferation of proerythroblasts (18). Further, we
have shown that Epo-R, in addition to cooperating with Kit in further
augmenting the expansion of proerythroblasts, also maintains their
survival by inducing the activation of Stat5 and the expression of
Bcl-xL, a function Kit is incapable of performing (18). These results
are in agreement with a previously described role for Epo and
consistent with the manifestation of severe anemia in mice lacking the
expression of Stat5 or Bcl-xL (14-16). Although a role for
Epo-R/Stat5/Bcl-xL pathway has been well documented, and it is thought
to be the major pathway in regulating the survival of erythroid
progenitors, the nature of Kit-induced early signaling pathways in
regulating erythroid cell proliferation alone or with Epo-R is not known.
Activation of Kit by its ligand, SCF, activates at least five early
signaling pathways via binding to specific tyrosine residues in the
intracellular domain of Kit. Whether these pathways play a redundant or
a nonredundant role in erythroid cell expansion alone or in combination
with Epo-R is not known. Utilizing a CHR approach and tyrosine to
phenylalanine mutations in Kit, we have explored the importance of
these early signaling pathways in Kit-induced expansion alone and in
cooperation with Epo-R. The major conclusions of our study are as
follows. 1) Tyrosine to phenylalanine mutations in the Kit
intracellular domain responsible for activating the PI 3-kinase
(position 719), PLC- The lack of a profound effect on proliferation in erythroid cells
expressing Kit mutants defective in the activation of PLC- The mechanism(s) by which Kit and Epo-R cooperate to regulate the
expansion of committed erythroid progenitors is poorly understood (19-25). Thus far, two mechanisms of cooperation between Kit and Epo-R
have been described in cell line models. Studies by Pircher et
al. (24) and Sui et al. (23) have provided evidence
that mitogen-activated protein kinase extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 at least in
part may function as downstream integrators of Epo-R and Kit signals
(23, 24). Wu et al. (19) have shown that activation of Kit
induces tyrosine phosphorylation of the Epo-R, and that Kit interacts
with the Epo-R by physically associating with its cytoplasmic domain.
Further, the ability of SCF to support proliferation of 32D cells
expressing Kit requires co-expression of the Epo-R, demonstrating that
at least one proliferative signal generated by Kit involves the Epo-R
as a downstream signal transduction protein (19). Consistent with these
findings, a functional interaction of activated Kit with the Epo-R was
shown to be crucial for CFU-E generation (21). Specifically, erythroid
progenitors from Epo-R In fibroblasts, Src kinases play an essential role in regulating DNA
synthesis by inducing the expression of c-Myc in response to PDGF
stimulation (44, 45, 47). Studies have shown that neutralizing
antibodies for Src kinase and dominant negative mutants for c-Src and
Fyn inhibit DNA synthesis in response to PDGF, indicating that these
kinases can act downstream from RTKs in a pathway that is required for
proliferation. Remarkably, the expression of c-Myc was found to be
sufficient to overcome the requirement for induction of DNA synthesis
by RTKs (44, 45). Consistent with these observations, we demonstrate
that Kit-induced activation of Src kinases also regulates the
expression of c-Myc, which is associated with reduced proliferation in
erythroid cells expressing Kit mutants defective in the activation of
Src kinases. A similar decrease in the expression of c-Myc was also
observed when G1E-ER2 cells were treated with the Src kinase inhibitor
PP1 in the presence of SCF and
Epo.3 Collectively, these
results demonstrate that in addition to the above described mechanisms
of cooperation between Kit and Epo-R, c-Myc at least in part may
function as a downstream integrator of Epo-R and Kit signaling in
erythroid cells.
The observation that only 50% correction in proliferation of erythroid
cells was observed in cells expressing the Src add-back Kit mutant in
response to M-CSF stimulation is intriguing and requires further
investigation. It is possible that the Src kinase pathway alone is
unable to activate all of the necessary signaling proteins essential
for efficient Kit-induced proliferation, although Src kinases have been
shown to regulate multiple signaling pathways, including the activation
of the Ras pathway via Shc, the PI 3-kinase/Akt pathway via Cbl,
and the Rac/c-Jun N-terminal kinase pathway via guanine exchange
factors, such as Vav (29, 47, 48). Whether all of these signaling
molecules are activated by Src kinases in response to Kit activation is
currently being investigated. It is possible that only a few of these
pathways are activated in Src add-back Kit mutants and that other
pathways are necessary for complete rescue in proliferation.
Alternatively, it is conceivable that all of the above pathways are
activated in Src add-back Kit mutants, however at a significantly
reduced level compared with wild type Kit receptor. Thus, it possible
that quantitative rather than qualitative differences in the activation
of various signaling pathways exist between wild type and Src
add-back Kit receptor. These possibilities are currently being investigated.
, phosphatidylinositol 3-kinase, Src
kinase, Grb2, and Grb7. The role of these pathways in Kit-induced
growth, proliferation, or cooperation with Epo-R is not known. We
demonstrate that inactivation of any one of these early signaling
pathways in Kit significantly impairs growth and proliferation.
However, inactivation of the Src pathway demonstrated the most profound
defect. Combined stimulation with Epo also resulted in impaired
cooperation between Src-defective Kit mutant and Epo-R and, to a lesser
extent, with Kit mutants defective in the activation of
phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between
the Src-defective Kit mutant and Epo-R was associated with reduced
transphosphorylation of Epo-R and expression of c-Myc. Remarkably,
restoration of only the Src pathway in a Kit receptor defective in the
activation of all early signaling pathways demonstrated a 50%
correction in proliferation in response to Kit stimulation and
completely restored the cooperation with Epo-R. These data demonstrate
an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PLC-
),
phosphatidylinositol 3-kinase p85 subunit (PI 3-kinase), Ras
GTPase-activating protein, SHP2 phosphatase, Src kinases, Grb2, Grb7,
and Shc (5). In this manner, the phosphorylated tyrosine residues
initiate signal transduction via several distinct early signaling
pathways. A major unresolved question in the field of RTK signaling is
whether these diverse signaling pathways result in redundant or
nonredundant biological functions. Recent studies utilizing the
platelet-derived growth factor (PDGF) RTK have begun to address some of
these issues in nonhematopoietic cells (6-8). However, relatively
little is known about the biological consequence(s) of activation of
diverse signaling pathways by RTKs in hematopoietic cells.
at tyrosine 728 (26), PI 3-kinase at tyrosine 719 (28), Src
class kinases at positions 567/569 (27, 29-31), Grb2 at tyrosine 702 (32), and Grb7 at tyrosine 934 (32). Other classes of signaling
proteins have also been reported to bind activated Kit with unknown
sequence specificity (33-37). Studies in multiple cell types have
shown that Kit carrying tyrosine to phenylalanine mutations at the
critical residues fail to bind the associated signaling molecules and
consequently fail to activate these signaling pathways (10, 11).
Phe
mutations in Kit to block the activation of downstream signaling molecules to comprehensively investigate the effect of lack of activation of five early signaling pathways in Kit-induced growth, proliferation, and cooperation with Epo-R in a relevant cell type. Utilizing an erythroid progenitor cell line (G1E-ER2) that closely mimics primary proerythroblasts (15, 18, 38, 39) and primary fetal
liver cells, we demonstrate that inactivation of any one of these five
early signaling pathways in Kit significantly impairs growth and
proliferation in response to Kit activation. The most profound defect
was observed in Kit mutants impaired in the activation of the Src
kinase pathway. Interestingly, when stimulated in combination with
erythropoietin (Epo), the mutant of Kit deficient in the activation of
Src kinase demonstrated a profound reduction in proliferation, which
was associated with impaired transphosphorylation of Epo-R and
expression of c-Myc. Remarkably, restoration of the Src kinase pathway
in Kit, alone, in the absence of remaining four early signaling
pathways restored the cooperation and transphosphorylation of Epo-R in
response to Kit activation. These results demonstrate an essential role
for the Src signaling pathway in regulating growth, proliferation, and
cooperation with Epo-R downstream from Kit.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methylcellulose (Stemcell Technologies, Vancouver,
Canada) with Epo (2 units/ml) or Epo plus M-CSF (2 units/ml Epo and 100 ng/ml M-CSF). Benzidine-positive colonies were counted 2-3 days after plating.
-glycerol phosphate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 µg/ml pepstatin A (pH 7.2)). Lysates were clarified by
centrifugation at 10,000 × g, 4 °C for 30 min. IP
was performed by incubating equivalent amounts of cell lysates with
either anti-Epo-R or an anti-Kit or anti-M-CSF receptor antibody
overnight at 4 °C (all from Santa Cruz Biotechnology). Protein
A- or protein G-Sepharose beads (Amersham Biosciences) were used to
collect the antigen-antibody complexes. IPs were separated by
SDS-PAGE), and proteins were electrophoretically transferred onto
nitrocellulose membranes (Bio-Rad). After blocking residual binding
sites on the transfer membrane by incubating the membrane with 5% milk
overnight, Western blot analysis using an anti-phosphotyrosine antibody
(Upstate Biotechnology, Inc., Lake Placid, NY) and Supersignal West
Dura extended duration detection system (Pierce) was utilized
according to the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
at position
728, Grb2 at position 702, an additional mutation at position 745, a
double mutant encoding tyrosine to phenylalanine mutations at positions
567 and 569, and a "naked" receptor that encodes tyrosine to
phenylalanine mutations at all six positions (Fig. 1).
View larger version (39K):
[in a new window]
Fig. 1.
Schematic structure of WT and mutant CHRs.
A, the Kit CHR includes the ligand-binding domain of the
human M-CSF receptor, the transmembrane, and the cytoplasmic tail of
the Kit receptor. K, the kinase domain of the receptors. In
the mutant Kit CHRs, tyrosines (Y) at the indicated
positions have been changed to phenylalanine (F). In the
naked CHR, tyrosines at all six positions have been changed to
phenylalanine. B, schematic representation of the Kit
cytoplasmic domain, indicating signaling molecules that bind to the
phosphorylated tyrosines.
View larger version (29K):
[in a new window]
Fig. 2.
Expression, biochemical, and functional
analysis of WT CHR. A, schematic of retroviral vectors
MIEG3 and MIEG3-CHR. An improved Moloney leukemia stem cell virus-based
bicistronic retroviral vector, MIEG3, expressing EGFP was utilized. WT
and mutant CHRs were cloned in front of the internal ribosome entry
site (IRES). LTR, long terminal repeat.
B, expression of the WT CHR by flow cytometry. The
left panel (y axis) demonstrates lack
of M-CSF receptor expression in the parental (untransduced) G1E-ER2
cells stained with a PE-conjugated antibody against M-CSF receptor. The
right panel demonstrates co-expression of both
EGFP (x axis) and the M-CSF receptor (y axis) in
G1E-ER2 cells transduced with the WT CHR. C, phosphorylation
of WT CHR in response to M-CSF stimulation. Cells expressing endogenous
Kit or WT CHR were stimulated for 10 min with either SCF
(lane 2) or M-CSF (lane 4)
or left unstimulated (lanes 1 and 3)
and subjected to IP with an anti-Kit (lanes 1 and
2) or an anti-M-CSF (lanes 3 and
4) receptor antibody and subjected to Western blot analysis
with an anti-phosphotyrosine antibody (top
panel). D, analysis of M-CSF-induced
proliferation in the absence and presence of Epo in G1E-ER2 cells
expressing the WT CHR. Parental (A) or WT CHR (B)
expressing G1E-ER2 cells were cultured in the presence of M-CSF or Epo
or SCF or a combination of SCF, M-CSF, and Epo for 48 h.
Proliferation was measured by thymidine incorporation assay.
Bars denote the mean thymidine incorporation (cpm ± S.D.) of at least two independent experiments performed in replicates
of six. *, p < 0.05 for Epo and M-CSF + Epo
versus M-CSF and no growth factor (D,
panel A). **, p < 0.05 for SCF
versus Epo and M-CSF + Epo (D, panel
A). ***, p < 0.05 for SCF + Epo
versus SCF (D, panel A). *,
p < 0.05 for M-CSF and SCF versus no growth
factor (D, panel B). **,
p < 0.05 for M- CSF + Epo and SCF + Epo
versus SCF (D, panel
B).
(Y728F) (Fig. 4, panel F), and Y745F (Fig.
4, panel G) continued to grow in the presence of M-CSF,
although at a slightly reduced rate compared with cells stimulated via
the endogenous Kit receptor. Interestingly, G1E-ER2 cells expressing
the PI 3-kinase-defective Kit mutant (Fig. 4, panel E)
showed an initial burst in growth, after which these cells maintained
survival without undergoing proliferation. The Kit receptor impaired in
the activation of all five early signaling pathways (naked
receptor; Fig. 4, panel H), behaved in a fashion similar to
the Src kinase-defective Kit mutant (Fig. 4, panel C). These
results suggest that Kit-induced activation of the Src pathway may play
an essential role in regulating the growth of erythroid cells.
View larger version (46K):
[in a new window]
Fig. 3.
Expression of mutant CHRs in G1E-ER2
cells. G1E-ER2 cells expressing the indicated CHR mutants were
stained with a PE-conjugated antibody against M-CSF receptor and
analyzed by flow cytometry. Shown is the co-expression of EGFP
(x axis) and the M-CSF receptor (y axis) for all
eight CHR mutants.
View larger version (36K):
[in a new window]
Fig. 4.
Impaired Kit-induced growth
of G1E-ER2 cells deficient in the activation of indicated
early signaling pathways. G1E-ER2 cells expressing various CHR
mutants were plated at a concentration of 1 × 105
cells/well in the presence of 50 ng/ml SCF (solid
lines) or 50 ng/ml M-CSF (dashed
lines) in replicates of three for 24 and 48 h. Cells
were harvested at the indicated times and subjected to trypan blue and
counted under the microscope. Viable cells were scored at various time
points. Shown is the mean ± S.D. of an independent experiment. *,
p < 0.05 for SCF versus M-CSF
stimulation.
(Fig. 6A, panel F), and the Y745F (Fig. 6A, panel G) demonstrated only a modest decrease
in proliferation in response to M-CSF stimulation. The naked receptor
behaved in a fashion similar to the mutant of Kit defective in the
activation of Src kinase (Fig. 6A, panel H).
Further, similar to the results shown in Fig. 5, panel C,
only the Src kinase-defective Kit mutants showed a profound decrease in
thymidine incorporation in the presence of both Epo and M-CSF (Fig.
6A, panel C). Consistent with a role for Src
kinases in mediating cooperation between Epo and SCF in G1E-ER2 cells,
expression of the Src kinase-defective Kit mutants in primary fetal
liver cells also resulted in a significant decrease in the number of
CFU-E colonies in vitro in the presence of M-CSF and Epo
(Fig. 6B).
View larger version (34K):
[in a new window]
Fig. 5.
Impaired cooperation between Kit and Epo-R in
G1E-ER2 cells deficient in the activation of Src kinases. G1E-ER2
cells expressing various CHR mutants were plated at a concentration of
1 × 105 cells/well in the presence of 50 ng/ml SCF
and 2 units/ml Epo (solid lines) or 50 ng/ml of
M-CSF and 2 units/ml of Epo (dashed lines) or 2 units/ml Epo (dotted lines) in replicates of
three for 24 and 48 h. Cells were harvested at indicated times and
subjected to trypan blue and counted under the microscope. Viable cells
were scored at various time points. Shown are the mean ± S.D. of
an independent experiment. *, p < 0.05 for SCF + Epo
versus M-CSF + Epo.
View larger version (53K):
[in a new window]
Fig. 6.
A, analysis of M-CSF and M-CSF plus Epo
induced thymidine incorporation in G1E-ER2 cells expressing various CHR
mutants. G1E-ER2 cells expressing the indicated mutants were cultured
in the presence of M-CSF or M-CSF plus Epo for 48 h. Proliferation
was measured by thymidine incorporation assay. Bars denote
the mean thymidine incorporation (cpm ± S.E.) of two independent
experiments performed in replicates of six. *, p < 0.05 CHR mutants versus WT CHR. B, stimulation of
Kit receptors impaired in the activation of Src kinases results in
reduced CFU-Es. Fetal liver cells were harvested from day 12.5 WT
embryos. Single cell suspensions were prepared and incubated with or
without retrovirus expressing the WT CHR or CHR mutants containing
tyrosine to phenylalanine mutations at positions 567 and 569 or 567 and
569 (double mutant). After infection, cells were plated in triplicate
in -methylcellulose with Epo (2 units/ml) or Epo plus M-CSF (2 units/ml Epo plus 100 ng/ml M-CSF). Benzidine-positive colonies were
counted 2-3 days after plating. C, impaired proliferation
of G1E-ER2 cells in the presence of Src kinase inhibitor PP1. Parental
G1E-ER2 cells were treated with the indicated concentrations of the Src
kinase inhibitor PP1 and cultured in the presence of SCF or SCF plus
Epo for 48 h. Bars denote the mean thymidine
incorporation (cpm ± S.D.) of an independent experiment performed
in replicates of six. *, p < 0.05 for SCF and SCF + Epo (dimethyl sulfoxide (DMSO) or untreated versus SCF + SCF
and Epo PP1 inhibitor).
View larger version (21K):
[in a new window]
Fig. 7.
A, impaired phosphorylation of Epo-R by a mutant of
Kit deficient in the activation of Src kinases. G1E-ER2 cells
expressing WT or 567/569 CHR were starved and left unstimulated
(lanes 1 and 3) or stimulated with 100 ng/ml M-CSF (lanes 2 and 4) for the
indicated times. Lysates were subjected to IP with an anti-Epo-R
antibody and Western blot analysis with an anti-phosphotyrosine
antibody. The position of Epo-R is indicated. The bottom
panel demonstrates protein loading in each lane.
B, expression of c-Myc in G1E-ER2 cells expressing the WT or
the 567/569 CHR. G1E-ER2 cells expressing WT or the 567/569 CHR were
cultured in the presence of M-CSF (lanes 1 and
4) or Epo (lanes 2 and 5)
or with a combination of both (lanes 3 and
6). The position of c-Myc is indicated. C,
activation of Stat5 in G1E-ER2 cells expressing the WT or the 567/569
CHR. G1E-ER2 cells expressing the WT or the 567/569 CHR were starved
and left unstimulated (lanes 1 and 4)
or stimulated with M-CSF (lanes 2 and
5) or Epo (lanes 3 and 6).
Cell lysates were collected and subjected to Western blot analysis with
an anti-phospho-Stat5 antibody. The position of the phosphorylated
Stat5 is indicated in the top panel. The
bottom panel shows total Stat5 protein in each
lane. D, expression of Bcl-xL in G1E-ER2 cells expressing
the WT (lanes 1 and 2) or the 567/569
CHR (lanes 3 and 4) in the presence of
M-CSF (lanes 1 and 3) or Epo
(lanes 2 and 4) for 24 h. The
position of Bcl-xL is indicated.
View larger version (39K):
[in a new window]
Fig. 8.
Restoration of the Src binding sites in the
naked CHR restores cooperation between Kit and Epo-R.
A, G1E-ER2 cells expressing the WT CHR (top
left panel), naked CHR (top
right panel), and 567/569 add-back CHR
(top middle panel) were stained with a
PE-conjugated antibody against the M-CSFR and analyzed by flow
cytometry. Shown is the co-expression of EGFP (x axis) and
the M-CSFR (y axis). G1E-ER2 cells expressing the WT CHR,
naked CHR, and 567/569 add-back CHR were cultured in the presence of
M-CSF or M-CSF plus Epo for 48 h. Proliferation was measured by
thymidine incorporation assay. Bars denote the mean
thymidine incorporation (cpm ± S.D.) of a representative
experiment performed in replicates of six. Similar results were
observed in two other experiments. B, analysis of
M-CSF-induced Epo-R transphosphorylation in G1E-ER2 cells expressing
the WT (lanes 1 and 2), 567/569
(lanes 3 and 4), or the 567/569 B CHR
(lanes 5 and 6). G1E-ER2 cells
expressing the WT or the 567/569 mutant or the 567/569 add-back CHR
were starved and stimulated with 100 ng/ml M-CSF (lanes
2, 4, and 6) for the indicated times.
Lysates were subjected to IP with an anti-Epo-R antibody and Western
blot analysis with an anti-phosphotyrosine antibody. The position of
Epo-R is indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(position 728), Grb2 (position 702), and
position 745 pathways show a substantial reduction in proliferation
upon Kit activation alone as well as in combination with Epo. However,
tyrosine to phenylalanine mutations in Kit at positions 567/569,
previously shown to bind Src kinase family members, are critical for
Kit as well as Epo-R-induced proliferation of erythroid progenitors. 2)
The lack of cooperation between a Kit mutant defective in the
activation of the Src kinase pathway and Epo-R is associated with
reduced transphosphorylation of the Epo-R and expression of c-Myc. 3)
Remarkably, restoration of the Src binding sites in a Kit mutant
stripped of all the other signaling pathways (naked receptor) restored
50% proliferation upon Kit activation alone and completely restored
the cooperation with Epo-R, including its transphosphorylation.
, PI
3-kinase, Grb2, and Y745F is intriguing. It is possible that in
erythroid cells, these pathways play only a minor role in proliferation downstream from Kit. Consistent with this interpretation, mice carrying
a mutation in the PI 3-kinase binding site (position 719) of Kit
in vivo also do not manifest an erythroid phenotype (43,
46), although these animals do show a defect in mast cell proliferation
and germ cell maturation in response to Kit activation (43, 46). Thus,
early signaling pathways downstream from Kit may be differentially
utilized in different cell lineages. Alternatively, since cooperation
between Kit and Epo-R is absolutely essential for normal erythroid
development in vivo, and since we demonstrate only a slight
reduction in the proliferation of erythroid cells in response to
co-stimulation with SCF and Epo in PI 3-kinase-defective Kit mutant, it
is conceivable that mice carrying a mutation in the PI 3-kinase binding
site of Kit in vivo may tolerate this mutation and hence a
lack of an erythroid phenotype. Based on this interpretation and our
results with the Src kinase Kit mutants, we hypothesize that other than
the Src kinase-defective mutants, disrupting any of the other early
signaling pathways in Kit, including the PLC-
, Grb2, Grb7, or
tyrosine Y745F would not result in an erythroid phenotype (anemia)
in vivo, since at least in vitro, all of these
mutations are well tolerated and do not manifest a profound phenotype
upon co-stimulation with SCF and Epo.
/
fetal livers, infected in vitro
with a retrovirus expressing the wild type Epo-R, require the addition
of both Epo and SCF to form CFU-E colonies (21). Therefore, it is
likely that Kit activates Epo-R by phosphorylating tyrosine residues in
its cytoplasmic domains, which allows for an efficient
recruitment/binding of Src homology 2-containing signaling proteins,
including the Src and the PI 3-kinases, resulting in further activation
of downstream signaling proteins. Our data demonstrate that tyrosine
residues 567 and 569 in Kit may play an essential role in regulating
this process. Since erythroid cells expressing mutants of
Kit lacking these two tyrosines demonstrate a significant reduction in
Kit-induced phosphorylation of the Epo-R, which is associated with
reduced proliferation and c-Myc expression. Remarkably, restoring these two residues back to tyrosines in the absence of other signaling pathways is sufficient to restore the phosphorylation of Epo-R and proliferation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Mervin Yoder, Wade Clapp, and Eddy Srour for critically reviewing the manuscript and members of our laboratories for useful discussions. We also thank Marsha Hippensteel for assistance in preparation of the manuscript and expert administrative assistance.
![]() |
FOOTNOTES |
---|
* 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.
Recipient of an American Society of Hematology Junior Faculty
Scholar Award. To whom correspondence should be addressed: Herman B
Wells Center for Pediatric Research, Cancer Research Bldg., 1044 W. Walnut St., Rm. 425, Indianapolis, IN 46202. Tel.: 317-274-4658; Fax:
317-274-8679; E-mail: rkapur@iupui.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M207068200
2 L. Hong and R. Kapur, unpublished observations.
3 B. L. Tan and R. Kapur, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RTK, receptor
tyrosine kinase;
PLC-, phospholipase C
;
PI 3-kinase, phosphatidylinositol 3-kinase;
PDGF, platelet-derived growth factor;
SCF, stem cell factor;
M-CSF, macrophage colony-stimulating factor;
CFU-E, colony-forming unit-erythroid;
Epo, erythropoietin;
Epo-R, Epo
receptor;
aa, amino acids;
WT, wild type;
CHR, chimeric kit
receptor;
EGFP, enhanced green fluorescent protein;
PE, phycoerythrin;
IP, immunoprecipitation;
PP1, pyrazolopyrimidine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. Rev. Cell Biol. 10, 251-337[CrossRef] |
2. | Pawson, T. (1995) Nature 373, 573-580[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Pawson, T.,
and Nash, P.
(2000)
Genes Dev.
14,
1027-1047 |
4. | Schlessinger, J. (2000) Cell 103, 211-225[Medline] [Order article via Infotrieve] |
5. | Pawson, T., and Saxton, T. M. (1999) Cell 97, 675-678[Medline] [Order article via Infotrieve] |
6. | Fambrough, D., McClure, K., Kazlauskas, A., and Lander, E. S. (1999) Cell 97, 727-741[Medline] [Order article via Infotrieve] |
7. |
Heuchel, R.,
Berg, A.,
Tallquist, M.,
Ahlen, K.,
Reed, R. K.,
Rubin, K.,
Claesson-Welsh, L.,
Heldin, C. H.,
and Soriano, P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11410-11415 |
8. | Klinghoffer, R. A., Mueting-Nelsen, P. F., Faerman, A., Shani, M., and Soriano, P. (2001) Mol. Cell. 7, 343-354[Medline] [Order article via Infotrieve] |
9. |
Broudy, V. C.
(1997)
Blood
90,
1345-1364 |
10. | Linnekin, D. (1999) Int. J. Biochem. Cell Biol. 31, 1053-1074[CrossRef][Medline] [Order article via Infotrieve] |
11. | Boissan, M., Feger, F., Guillosson, J. J., and Arock, M. (2000) J. Leukocyte Biol. 67, 135-148[Abstract] |
12. | Nocka, K., Majumder, S., Chabot, B., Ray, P., Cervone, M., Bernstein, A., and Besmer, P. (1989) Genes Dev. 3, 816-826[Abstract] |
13. | Wu, H., Liu, X., Jaenisch, R., and Lodish, H. F. (1995) Cell 83, 59-67[Medline] [Order article via Infotrieve] |
14. | Socolovsky, M., Fallon, A. E., Wang, S., Brugnara, C., and Lodish, H. F. (1999) Cell 98, 181-191[Medline] [Order article via Infotrieve] |
15. |
Gregory, T., Yu, C.,
Ma, A.,
Orkin, S. H.,
Blobel, G. A.,
and Weiss, M. J.
(1999)
Blood
94,
87-96 |
16. |
Motoyama, N.,
Kimura, T.,
Takahashi, T.,
Watanabe, T.,
and Nakano, T.
(1999)
J. Exp. Med.
189,
1691-1698 |
17. | Motoyama, N., Wang, F., Roth, K. A., Sawa, H., Nakayama, K., Negishi, I., Senju, S., Zhang, Q., Fujii, S., and Loh, D. Y. (1995) Science 267, 1506-1510[Medline] [Order article via Infotrieve] |
18. |
Kapur, R.,
and Zhang, L.
(2001)
J. Biol. Chem.
276,
1099-1106 |
19. | Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Joneja, B.,
Chen, H. C.,
Seshasayee, D.,
Wrentmore, A. L.,
and Wojchowski, D. M.
(1997)
Blood
90,
3533-3545 |
21. |
Wu, H.,
Klingmuller, U.,
Acurio, A.,
Hsiao, J. G.,
and Lodish, H. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
1806-1810 |
22. |
Jacobs-Helber, S. M.,
Penta, K.,
Sun, Z.,
Lawson, A.,
and Sawyer, S. T.
(1997)
J. Biol. Chem.
272,
6850-6853 |
23. |
Sui, X.,
Krantz, S. B.,
You, M.,
and Zhao, Z.
(1998)
Blood
92,
1142-1149 |
24. |
Pircher, T. J.,
Geiger, J. N.,
Zhang, D.,
Miller, C. P.,
Gaines, P.,
and Wojchowski, D. M.
(2001)
J. Biol. Chem.
276,
8995-9002 |
25. |
Miller, C. P.,
Heilman, D. W.,
and Wojchowski, D. M.
(2002)
Blood
99,
898-904 |
26. |
Gommerman, J. L.,
Sittaro, D.,
Klebasz, N. Z.,
Williams, D. A.,
and Berger, S. A.
(2000)
Blood
96,
3734-3742 |
27. |
Ueda, S.,
Mizuki, M.,
Ikeda, H.,
Tsujimura, T.,
Matsumura, I.,
Nakano, K.,
Daino, H.,
Honda, Zi. Z.,
Sonoyama, J.,
Shibayama, H.,
Sugahara, H.,
Machii, T.,
and Kanakura, Y.
(2002)
Blood
99,
3342-3349 |
28. | Serve, H., Yee, N. S., Stella, G., Sepp-Lorenzino, L., Tan, J. C., and Besmer, P. (1995) EMBO J. 14, 473-483[Abstract] |
29. |
Timokhina, I.,
Kissel, H.,
Stella, G.,
and Besmer, P.
(1998)
EMBO J.
17,
6250-6262 |
30. |
Linnekin, D.,
DeBerry, C. S.,
and Mou, S.
(1997)
J. Biol. Chem.
272,
27450-27455 |
31. | Lennartsson, J., Blume-Jensen, P., Hermanson, M., Ponten, E., Carlberg, M., and Ronnstrand, L. (1999) Oncogene 18, 5546-5553[CrossRef][Medline] [Order article via Infotrieve] |
32. | Thommes, K., Lennartsson, J., Carlberg, M., and Ronnstrand, L. (1999) Biochem. J. 341, 211-216[CrossRef][Medline] [Order article via Infotrieve] |
33. | Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1587-1591[Abstract] |
34. |
Cutler, R. L.,
Liu, L.,
Damen, J. E.,
and Krystal, G.
(1993)
J. Biol. Chem.
268,
21463-21465 |
35. |
Sattler, M.,
Salgia, R.,
Shrikhande, G.,
Verma, S.,
Pisick, E.,
Prasad, K. V.,
and Griffin, J. D.
(1997)
J. Biol. Chem.
272,
10248-10253 |
36. |
van Dijk, T. B.,
van Den Akker, E.,
Amelsvoort, M. P.,
Mano, H.,
Lowenberg, B.,
and von Lindern, M.
(2000)
Blood
96,
3406-3413 |
37. |
Jahn, T.,
Seipel, P.,
Urschel, S.,
Peschel, C.,
and Duyster, J.
(2002)
Mol. Cell. Biol.
22,
979-991 |
38. |
Kapur, R.,
Cooper, R.,
Xiao, X.,
Weiss, M. J.,
Donovan, P.,
and Williams, D. A.
(1999)
Blood
94,
1915-1925 |
39. |
Kapur, R.,
Cooper, R.,
Zhang, L.,
and Williams, D. A.
(2001)
Blood
97,
1975-1981 |
40. | Yang, F. C., Kapur, R., King, A. J., Tao, W., Kim, C., Borneo, J., Breese, R., Marshall, M., Dinauer, M. C., and Williams, D. A. (2000) Immunity 12, 557-568[Medline] [Order article via Infotrieve] |
41. |
Kapur, R.,
Majumdar, M.,
Xiao, X.,
McAndrews-Hill, M.,
Schindler, K.,
and Williams, D. A.
(1998)
Blood
91,
879-889 |
42. |
DeMali, K. A.,
and Kazlauskas, A.
(1998)
Mol. Cell. Biol.
18,
2014-2022 |
43. |
Kissel, H.,
Timokhina, I.,
Hardy, M. P.,
Rothschild, G.,
Tajima, Y.,
Soares, V.,
Angeles, M.,
Whitlow, S. R.,
Manova, K.,
and Besmer, P.
(2000)
EMBO J.
19,
1312-1326 |
44. |
Blake, R. A.,
Broome, M. A.,
Liu, X.,
Wu, J.,
Gishizky, M.,
Sun, L.,
and Courtneidge, S. A.
(2000)
Mol. Cell. Biol.
20,
9018-9027 |
45. | Barone, M. V., and Courtneidge, S. A. (1995) Nature 378, 509-512[CrossRef][Medline] [Order article via Infotrieve] |
46. | Blume-Jensen, P., Jiang, G., Hyman, R., Lee, K. F., O'Gorman, S., and Hunter, T. (2000) Nat. Genet. 24, 157-162[CrossRef][Medline] [Order article via Infotrieve] |
47. | Chiariello, M., Marinissen, M. J., and Gutkind, J. S. (2001) Nat. Cell Biol. 3, 580-586[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Corey, S. J.,
and Anderson, S. M.
(1999)
Blood
93,
1-14 |