(Received for publication, March 20, 1995; and in revised form, June 27, 1995)
From the
We recently reported that phosphatidylinositol (PI) 3-kinase
becomes associated with the activated erythropoietin receptor (EpR),
most likely through the Src homology 2 (SH2) domains within the p85
subunit of PI-3 kinase and one or more phosphorylated tyrosines within
the EpR. We have now investigated this interaction in more detail and
have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these
fusion proteins to SDS-denatured EpRs, that this binding is direct.
Moreover, both in vitro competition studies, involving
phosphorylated peptides corresponding to the amino acid sequences
flanking the eight tyrosines within the intracellular domain of the
EpR, and in vivo studies with mutant EpRs bearing tyrosine to
phenylalanine substitutions, indicate that phosphorylation of
Tyr within the EpR is essential for the binding of PI
3-kinase. The presence of PI 3-kinase activity in EpR
immunoprecipitates from DA-3 cells infected with wild-type but not
Y503F EpRs confirms this finding. Our results demonstrate that the SH2
domains of p85 can bind, in addition to their well established
Tyr-Met/Val-X-Met consensus binding sequence, a
Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of
erythropoietin-induced tyrosine phosphorylations and proliferation of
wild-type and Y503F EpR-infected DA-3 cells revealed no differences.
However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the
erythropoietin-induced proliferation of both cell types, suggesting
that PI 3-kinase is activated in Y503F EpR expressing cells. This was
confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine
immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected
DA-3 cells and suggested that PI 3-kinase has a role in regulating
erythropoietin-induced proliferation, but at a site distinct from the
EpR.
Erythropoietin is the principal in vivo stimulator of
mammalian erythropoiesis (1) and exerts its action by binding
to receptors on the surface of relatively mature erythroid progenitors (2, 3, 4) . These erythropoietin receptors
(EpRs) ()belong to a family of hematopoietin receptors whose
members are characterized by the presence of conserved cysteines and
Trp-Ser-X-Trp-Ser motifs in their extracellular domains and
the absence of any known catalytic activity in their intracellular
regions(5, 6) . Nonetheless, although the EpR lacks
tyrosine kinase activity, it, along with a number of other cellular
proteins, becomes transiently phosphorylated on tyrosine residues
within minutes of binding
erythropoietin(7, 8, 9, 10, 11, 12, 13) .
Recent studies suggest that this rapid phosphorylation is carried out,
at least in part, by the cytoplasmic tyrosine kinase, Jak
2(14, 15) . Following this activation of the EpR, a
number of intracellular proteins apparently become physically
associated with
it(8, 16, 17, 18, 19) .
These proteins include Grb2(8, 18) ,
Shc(8, 18) , and the enzyme, phosphatidylinositol (PI)
3-kinase(16, 17, 19) . All three of these
proteins contain at least one Src homology 2 (SH2) domain, consisting
of approximately 100 amino acids (20) that may direct their
physical association with specific tyrosine phosphorylated regions
within the activated EpR. In the case of PI 3-kinase, a heterodimeric
enzyme complex that phosphorylates PI, PI-4 phosphate, and
PI-4,5-bisphosphate at the D-3 position of the inositol
ring(21) , it is now generally believed that its
85-kDa subunit, which contains two SH2 domains(22) ,
functions as an adaptor molecule that targets its associated catalytic
110-kDa component (23) to activated growth factor
receptors(24) .
In a previous report we demonstrated that
erythropoietin stimulates the association of PI 3-kinase with the
activated EpR, most likely through the SH2 domains of p85 and either
the activated EpR itself or a phosphorylated protein
intermediate(16) . In the current study we have further
investigated the nature of the interaction between p85 and the
activated EpR. Our results suggest that this interaction is direct and
that p85 binds to the phosphorylated COOH-terminal tyrosine within the
cytoplasmic region of the EpR, i.e. Tyr. This
finding reveals a new recognition motif for both the NH
-
and COOH-terminal SH2 domains of p85, i.e. Tyr-Val-Ala-Cys.
For
proliferation assays, the various EpR-infected DA-3 cells were grown to
near confluence with interleukin-3, washed once with RPMI, and
resuspended in RPMI containing 10% fetal calf serum. The cells were
then aliquoted into 96-well U-bottom microtiter plates (Linbro, ICN,
Mississauga, Ontario) to give a final volume of 0.1 ml/well and the
plates incubated for 20 h at 37 °C in a humidified atmosphere of 5%
CO, 95% air. Twenty µl of a 50 µCi/ml solution of
[
H]thymidine in RPMI was then added to each well
to give a final concentration of 1 µCi/well. After another 2 h at
37 °C, the contents of the wells were harvested onto filtermats and
counted using an LKB Betaplate Harvester and Liquid Scintillation
Counter (LKB Wallac, Turku, Finland).
Studies from both our laboratory (16) and that of
Miura et al.(19) recently established that the
activated EpR is capable of binding in vitro to the SH2
domains of the p85 subunit of PI 3-kinase. However, one issue that
remained unresolved at the completion of these studies was whether this
binding was direct or through an intermediary protein. This was an
important question since none of the eight tyrosines within the
intracellular domain of the EpR possess the canonical
Tyr-Met/Val-X-Met binding motif for the SH2 domains of
p85(27, 28) . To address this, DA-ER cells (i.e. DA-3 cells expressing approximately 12,000 EpRs/cell(8) )
were incubated in the presence and absence of erythropoietin for 5 min
at 37 °C, and anti-EpR or anti-p85 immunoprecipitations carried out
with the cell lysates. An anti-EpR immunoblot of the anti-EpR
immunoprecipitates demonstrated equal loading of EpRs and revealed, as
has been shown previously(9) , that erythropoietin stimulates
an increase in the apparent molecular mass of a fraction of the total
EpRs from approximately 66 to 72 kDa (Fig. 1A). An
anti-PY immunoblot of identical aliquots of anti-EpR immunoprecipitates
demonstrated, as has been shown
previously(9, 10, 12) , that this 72 kDa
species represents the tyrosine phosphorylated form of the EpR (Fig. 1B). An anti-EpR immunoblot of anti-p85
immunoprecipitates from these same cells revealed that p85 associates
only with this 72 kDa form of the EpR (Fig. 1C) and
blotting studies carried out with anti-EpR immunoprecipitates (using a
mixture of P-labeled GST fusion proteins containing the
NH
- and COOH-terminal SH2 domains of p85) showed that the
SH2 domains of p85 bound directly to the 72-kDa tyrosine-phosphorylated
EpR (Fig. 1D). Direct binding of each individual p85
SH2 domain was confirmed by incubating SDS-denatured EpRs with
bead-bound NH
- and COOH-terminal p85 SH2-GST fusion
proteins. As can be seen in Fig. 2, both the NH
- and
COOH-terminal SH2 of p85 recognized SDS-denatured,
tyrosine-phosphorylated EpRs.
Figure 1:
p85 SH2 domains
bind directly to the phosphorylated EpR. Lysates of control(-)
and erythropoietin-stimulated (+) DA-ER cells were subjected to
immunoprecipitation with anti-EpR antibodies (panels A, B, and D) or anti-p85 antibodies (panel C)
and then electrophoresed on SDS gels and analyzed by Western blotting
with anti-EpR (A and C) or anti-PY antibodies (B). The blot shown in panel D was probed with P-labeled NH
- and COOH-terminal p85 SH2
domains and visualized by autoradiography.
Figure 2:
SDS denatured EpRs bind to the
NH- and COOH-terminal SH2 domains of p85. Lysates from
DA-ER cells stimulated (lanes 2 and 3) or not (lane 1) with erythropoietin were either added directly to
agarose bound GST-SH2 fusion proteins containing the
NH
-terminal and COOH-terminal SH2 domains of p85 (lanes
1 and 2) or first immunoprecipitated with anti-EpR
antibodies, eluted with 100 µl of 100 °C 1% SDS in PSB, diluted
10-fold with PSB containing 0.5% Nonidet P-40 (lane 3), and
then added to the same amount of agarose-bound GST-SH2 fusion proteins.
Following elution with SDS-sample buffer, the samples were subjected to
SDS-PAGE and Western analysis with anti-PY
antibodies.
To determine which of the eight
tyrosines within the intracellular region of the
tyrosine-phosphorylated EpR was responsible for p85 binding, synthetic
phosphopeptides corresponding to these eight regions of the EpR were
tested for their ability to inhibit, in vitro, the binding of
the activated EpR to agarose-bound GST fusion proteins containing
either the NH- or COOH-terminal SH2 of p85. As can be seen
in Fig. 3, A and B, only one phosphopeptide
was capable of inhibiting this binding. This phosphopeptide
corresponded to the region flanking the most COOH-terminal tyrosine
within the EpR, i.e. Tyr
.
Figure 3:
Phosphopeptide Y503 competes with the
phosphorylated EpR for binding to p85 SH2 domains. Lysates from DA-ER
cells stimulated with erythropoietin (+) were added to
agarose-bound GST-SH2 fusion proteins containing the
NH-terminal (A) and COOH-terminal (B) SH2
domains of p85 after preincubation of the beads for 20 min with 50
µM phosphopeptides corresponding to the sequences flanking
the eight tyrosines within the EpR. Following SDS-PAGE and transfer to
Immobilon, activated EpRs were visualized by blotting with the anti-PY
antibody 4G10. The positive control lane is a mixture of
unphosphorylated 11 mer peptides (see ``Materials and
Methods'').
From our in
vitro data to this point, it appeared that the phosphorylated
Tyr within the EpR might be playing a role in the binding
of PI 3-kinase. However, to test if this was actually the case in
vivo, we converted each of the eight tyrosines within the
cytoplasmic domain of the EpR to phenylalanines, using site-directed
mutagenesis. Clones bearing similar cell surface EpRs were selected
using biotinylated erythropoietin and FACS analysis as described
previously(4) . Western blot analysis of these selected clones,
using anti-EpR antibodies, revealed less than a 4-fold variation in EpR
numbers among the nine clones (Fig. 4A). To address the
more relevant question of the EpR level at the cell surface,
I-erythropoietin binding studies were carried out with
the nine clones and the relative levels were found to be similar to
those observed when total EpR numbers were assessed (data not shown).
These clones were treated with erythropoietin for 5 min at 37 °C
and half of the cell lysates incubated with agarose beads containing
the NH
- or COOH-terminal SH2 domain of p85. Bound proteins
were eluted and subjected to Western analysis with anti-PY antibodies
to monitor the level of activated EpRs. As can be seen in Fig. 4B, the only EpR mutant that did not appear to
bind to either the NH
- or COOH-terminal SH2 domain of p85
was Y503F. However, this conclusion would not be valid if Tyr
was the only tyrosine normally phosphorylated following
erythropoietin stimulation. To test this, one-quarter of the cell
lysates were immunoprecipitated with anti-EpR antibodies and subjected
to Western analysis with anti-PY antibodies. As can be seen in Fig. 4B (lower panel), this immunoblot clearly
shows that the Y503F EpR mutant is tyrosine-phosphorylated. The reduced
level of tyrosine phosphorylation of this mutant, compared, for
example, to a mutant with similar EpR surface expression (e.g. Y453F and Y455F) also indicates, as our results to this point
would suggest, that Tyr
is a site of tyrosine
phosphorylation.
Figure 4:
A, EpR levels in biotinylated Ep/FACS
selected DA-3 cells expressing wild and mutant EpRs. Lysates from 2
10
unstimulated DA-3 cells expressing wild-type (WT) EpRs or the eight tyrosine to phenylalanine points were
subjected to SDS-PAGE and Western analysis using anti-EpR antibodies. Numbers above the autoradiogram refer to relative intensities
of the EpR bands, determined by densitometry. B, a Y503F EpR
point mutant does not bind to p85 SH2 domains in vitro. Upper panels, lysates from erythropoietin-stimulated DA-3
cells expressing tyrosine to phenylalanine point mutants of the EpR
were incubated with bead-bound GST-SH2 fusion proteins containing the
NH
- or COOH-terminal SH2 domain of p85 and tightly bound
proteins were eluted by boiling in SDS-sample buffer and subjected to
Western analysis with the anti-PY antibody 4G10. Lower panel,
identical lysates were subjected to immunoprecipitation with anti-EpR
antibodies and the precipitates analyzed by Western blotting with 4G10
in order to detect activated EpRs.
To examine if p85 actually binds the activated EpR in vivo at position Tyr, the remaining
one-quarter of the cell lysates were subjected to immunoprecipitation
with anti-p85. As can be seen from the anti-PY immunoblot shown in Fig. 5, the only mutant EpR that did not co-precipitate with p85
was Y503F. The anti-PY immunoblot of the anti-EpR immunoprecipitates
shown in Fig. 4B (lower panel) demonstrates
that this mutant was tyrosine phosphorylated in response to
erythropoietin at residues other than Tyr
and that these
phosphorylated tyrosines were not involved in binding p85 in
vivo.
Figure 5: Endogenous p85 does not associate with the activated Y503F EpR in vivo. Lysates from erythropoietin-stimulated DA-3 cells expressing the wild-type and each of the tyrosine to phenylalanine EpR point mutants were immunoprecipitated with anti-p85 (upper) or anti-EpR antibodies (see Fig. 4B, lower panel). Tightly bound proteins were eluted with SDS-sample buffer and subjected to Western analysis with 4G10 in order to detect activated EpRs. The(-) lane represents a lysate from unstimulated DA-3 cells expressing the wild-type EpR.
To ensure that our results with the selected EpR cell line containing Y503F were not due to a cloning artifact but were representative of this mutant, anti-p85 immunoprecipitates were carried out with lysates from two additional, independently isolated Y503F clones. As shown in Fig. 6, no co-precipitation of activated EpRs was observed with these clones either.
Figure 6: The lack of association between endogenous p85 and the EpR is representative of the Y503F EpR mutant. Lysates from DA-3 cells expressing a wild-type and two Y503F EpR mutant clones were immunoprecipitated with anti-p85 antibodies and subjected to Western analysis with 4G10 (A) or immunoprecipitated with anti-EpR antibodies and subjected to Western analysis with 4G10 (B).
We (16) and
others (17, 19) have shown previously that PI 3-kinase
activity becomes associated with the EpR following ligand binding. To
determine if this association occurs solely through the interaction of
its p85 subunit with the activated EpR, PI 3-kinase assays were carried
out with wild-type and Y503F EpR expressing DA-3 cells, using clones
expressing identical cell surface EpR levels. As expected, the
generation of PIP was observed in EpR immunoprecipitates
from erythropoietin-stimulated wild-type but not Y503F EpR containing
DA-3 cells (Fig. 7). On the basis of this correlation, we
conclude that the major binding site within the EpR for PI 3-kinase is
the phosphorylated sequence Tyr
-Val-Ala-Cys.
Figure 7: PI 3-kinase activity is not associated with the activated Y503F EpR in DA-3 cells. DA-3 cells expressing the wild-type or Y503F EpR were incubated for 5 min at 37 °C with erythropoietin and the lysates immunoprecipitated with anti-p85 or anti-EpR antibodies. Immunoprecipitated protein A beads were washed, PI 3-kinase assays carried out, and thin layer chromatography performed as described previously(16) .
Having
established that PI 3-kinase binds to the activated EpR via the SH2
domains of the former with the phosphorylated Tyr of the
latter, we set out to determine the biochemical and biological
consequences of this interaction by first comparing tyrosine
phosphorylation events stimulated by erythropoietin in DA-3 cells
expressing the same number of cell surface wild-type and Y503F EpRs.
Interestingly, anti-PY immunoblots of anti-PY immunoprecipitates
revealed that there were no obvious differences in the intensities of
the major phosphoproteins, aside from the expected difference in the
EpR itself (Fig. 8A). Anti-PY immunoblots of anti-Shc (Fig. 8B) and anti-Jak 2 (Fig. 8C)
immunoprecipitates confirmed this finding.
Figure 8: Erythropoietin stimulates the same tyrosine phosphorylation pattern in DA-3 cells expressing the wild-type and Y503F EpR. Lysates from unstimulated(-) or erythropoietin-stimulated (+) DA-3 cells expressing the wild-type (WT) or Y503F (503) EpR were subjected to anti-PY (A), anti-Shc (B), or anti-Jak2 (C) immunoprecipitation and tightly bound proteins eluted with SDS-sample buffer and resolved by SDS-PAGE. Following transfer to Immobilon, tyrosine-phosphorylated proteins were visualized by immunoblotting with anti-PY.
We next compared the
erythropoietin-responsive proliferation of DA-3 cells expressing
wild-type and Y503F EpRs. Because of previous studies in which we found
that erythropoietin-responsive proliferation was highly dependent on
cell surface EpR levels(9) , clones expressing identical
numbers of cell surface EpRs, based on I-erythropoietin
binding studies, were again used. As can be seen in Fig. 9A, erythropoietin-induced proliferation of the
two cell types was very similar, suggesting that EpR-associated PI
3-kinase activity does not play a role in erythropoietin-induced
proliferation. However, since PI 3-kinase has recently been shown to be
directly activated by Ras
in COS cells (29) we
investigated whether PI 3-kinase might play a role in
erythropoietin-induced proliferation, but at a locus other than the
EpR. Specifically, we examined the effect of the PI-3 kinase inhibitor,
wortmannin(30) , on erythropoietin-induced proliferation of
DA-3 cells expressing wild-type and Y503F EpRs. Interestingly,
wortmannin reduced the erythropoietin responsiveness of both wild-type (Fig. 9B) and Y503F EpR (Fig. 9C)
expressing DA-3 cells to approximately the same degree, i.e. between one and two logs, suggesting, perhaps, that PI 3-kinase is
activated by erythropoietin in Y503F EpR expressing DA-3 cells and that
PI 3-kinase plays a significant role in erythropoietin-induced
proliferation but at a site distinct from the EpR.
Figure 9:
The
effect of erythropoietin on the proliferation of DA-3 cells expressing
wild-type and Y503F EpRs. A, erythropoietin dose-response
curves generated with DA-3 cells expressing similar cell surface levels
of wild-type () and Y503F (
) EpRs. B,
erythropoietin dose-response curves with DA-3 cells expressing
wild-type EpRs, carried out in the presence (
) and absence
(
) of 10
M wortmannin. C,
erythropoietin dose-response curves with DA-3 cells expressing Y503F
EpRs carried out in the presence (
) and absence (
) of
10
M wortmannin (for B and C wortmannin was added every 5 h). Each point represents the mean of
three replicates ± S.E.
To confirm this,
PI 3-kinase assays were carried out with anti-PY immunoprecipitates
from both wild-type and Y503F EpR expressing DA-3 cells, treated with
or without erythropoietin. As can be seen in Fig. 10, almost as
much PIP was generated in response to erythropoietin in the
Y503F EpR as in the wild-type EpR expressing DA-3 cells, consistent
with the notion that PI 3-kinase is activated by erythropoietin both at
the level of the EpR and also at a locus distinct from the EpR.
Figure 10: PI 3-kinase activity is stimulated by erythropoietin in Y503F EpR expressing DA-3 cells. DA-3 cells expressing the wild-type or Y503F EpR were incubated for 5 min at 37 °C with or without erythropoietin and the lysates immunoprecipitated with anti-PY antibodies. Immunoprecipitated protein A beads were washed, PI 3-kinase assays carried out, and thin layer chromatography performed as described previously(16) . An anti-p85 immunoprecipitate was also assayed as a positive control (first lane).
Within minutes of binding erythropoietin, the EpR becomes
phosphorylated on tyrosine residues and attracts a number of
intracellular proteins to its intracellular
domain(8, 16, 18, 31) . As we (16) and others (17, 19, 31) have
shown, one of these proteins is PI 3-kinase. In a previous report we
demonstrated that PI 3-kinase becomes rapidly associated with the EpR
following erythropoietin stimulation of Ba/F3 cells expressing high
levels of the murine EpR and that this interaction might be mediated by
the SH2 domains of p85 and tyrosine-phosphorylated motifs within the
EpR(16) . This was subsequently confirmed by both Miura et
al.(19) and He et al.(17) . Miura et
al.(19) went on to show that PI 3-kinase did not
associate with a mitogenically active EpR which lacked the
COOH-terminal 108 amino acids and therefore concluded that p85 binds to
the carboxyl terminus of the EpR and that this association is not
required for transducing a mitogenic signal. He et
al.(17) , on the other hand, using various EpR mutants
with cytosolic truncations and deletions concluded that p85 binds to a
more membrane proximal region of the EpR, i.e. between
Pro and Glu
in the extended box-2 region.
In the current study we have tried to resolve this controversy and
further explore the nature of this interaction. For this study we used
DA-3 cells since they do not express any endogenous EpRs(12) .
The data presented herein suggest that the binding between p85 and the
activated EpR is direct and not through an intermediate protein, such
as Syp, which has recently been shown to bind Grb2 to the
platelet-derived growth factor receptor(32, 33) . The
data also suggest that the NH
- and COOH-terminal SH2
domains of p85 bind specifically to the phosphorylated form of the EpR
at Tyr
. This not only demonstrates for the first time
that Tyr
within the EpR is phosphorylated in vivo in response to erythropoietin binding (and that at least one other
tyrosine within the EpR is phosphorylated as well) but reveals a
previously unreported recognition motif for the SH2 domains of p85, i.e. Y-V-A-C. This is not inconsistent with the exhaustive
synthetic phosphopeptide studies carried out by Songyang et al.(28) since they did not test cysteine at position 3+
and since cysteine and the bulkier methionine, which is the established
amino acid at position 3+, both provide a hydrophobic side chain
with sulfer atoms positioned in a similar orientation. We also found
that p85 COOH-terminal SH2 beads bound more activated EpRs, in repeated
experiments, than an equivalent amount of bead-bound
NH
-terminal SH2 (as shown in Fig. 2). This was
consistent with peptide inhibition studies where we found that
approximately 10-fold less phosphopeptide Y503 was needed to completely
block activated EpR binding to COOH-terminal SH2 (data not shown). This
is in keeping with the difference in affinity reported for the
NH
- and COOH-terminal SH2 domains for the phosphorylated,
established consensus sequence,
Tyr-Met/Val-X-Met(28) .
As far as the ramifications
of PI 3-kinase binding to the activated EpR are concerned, our studies
with the Y503F EpR mutant suggest that neither erythropoietin induced
proliferation nor tyrosine phosphorylation events are affected by the
absence of an EpR-associated PI 3-kinase. This is interesting, given
that in many cases there is a good correlation between growth factor
stimulation and a rapid increase in the levels of the lipid products of
PI-3 kinase, particularly PI 3,4-bisphosphate and PI
3,4,5-trisphosphate(34, 35) . Since these products are
poor substrates for any known phospholipase
C(36, 37) , it has been postulated that they may
represent a new class of second messengers involved in mitogenic signal
transduction, perhaps, in part, via activation of S6
kinase(38, 39) . Very recent studies, however, suggest
that not all growth factors require PI 3-kinase for cell growth (40) and that this enzyme might function in some cells to
stimulate events unrelated to mitogenesis, such as glucose
transport(41, 42) , membrane ruffling(43) ,
differentiation(44, 45) , cell survival(46) ,
or endocytic trafficking of activated receptors(47) . This last
finding prompted us to investigate whether the activated Y503F EpR was
internalized at a slower rate than the wild-type EpR in DA-3 cells,
using I-erythropoietin, but no difference was observed
(data not shown). An extra complication in studies designed to
investigate the role of the lipid products of PI 3-kinase is that this
enzyme has both lipid and serine kinase
activity(48, 49) , and inhibitors like wortmannin
inhibit both activities. Thus the substantial reduction in
responsiveness to erythropoietin that we observe with both our
wild-type and Y503F EpR expressing DA-3 cells could be attributed to
the loss of serine kinase activity. Consistent with this possibility,
Lam et al.(50) recently reported that PI 3-kinase
serine phosphorylates IRS-1 in response to insulin in rat adipocytes.
From our studies with wortmannin and our PI 3-kinase assays
demonstrating activation of PI 3-kinase in response to erythropoietin
in Y503F EpR expressing cells, we tentatively conclude that PI 3-kinase
is involved in erythropoietin-induced proliferation at a site other
than the EpR. However, the greater than 10-fold drop in erythropoietin
responsiveness we detect with wortmannin might represent an
underestimate of the role of PI 3-kinase involvement in
erythropoietin-induced mitogenesis since eight PI 3-kinases have been
found in mammals to date and it is not yet known how many of these are
wortmannin-sensitive(51) . On the other hand, the observed
inhibition could be an overestimate since wortmannin, even at the low
concentrations used in our studies, might be exerting its effects
through molecules other than PI 3-kinase(51, 52) .
Based on both our previous observations that erythropoietin stimulates
the tyrosine phosphorylation of Shc and its association with Grb2, (8) and recent reports that Ras can activate PI 3-kinase by
directly interacting with its catalytic p110 subunit(29) , we
hypothesize that this site could be immediately downstream from Ras.
In summary, we have demonstrated that PI 3-kinase becomes directly
associated with the EpR, following ligand binding, at Tyr within the EpR and that this binding is not critical for
erythropoietin-induced mitogensis. We are currently exploring whether
this association may play a role in erythropoietin-induced glucose
uptake or erythroid differentiation.