(Received for publication, January 28, 1997, and in revised form, March 31, 1997)
From the School of Pharmacy and Pharmacology, University of
Bath, Claverton Down, Bath, BA2 7AY, United Kingdom and § The
Biomedical Research Centre and
Department of Medicine,
University of British Columbia, Vancouver, British Columbia, V6T
1Z3, Canada
The cytoplasmic tyrosine phosphatases, SHP1 and
SHP2, are implicated in the control of cellular proliferation and
survival. Here we demonstrate that both SHP1 and SHP2 associate with
the c subunit of the human interleukin-3 (IL-3) receptor following IL-3 stimulation and that the src homology region 2 (SH2)
domains of these phosphatases mediate this interaction. Sequential
immunoprecipitation analyses suggest this interaction is direct.
Competition studies, using phosphotyrosine-containing peptides based on
sequences surrounding key tyrosine residues within
c, suggest that
phosphorylation of tyrosine 612 is the key event mediating the
association of
c with SHP1 and SHP2. However, inhibition of SHP2
binding to
c, did not prevent tyrosine phosphorylation of SHP2.
Interestingly, this same phosphopeptide served as a substrate for the
tyrosine phosphatase activity of both SHP1 and SHP2. Binding of these
protein-tyrosine phosphatases to the IL-3 receptor may regulate IL-3
signal transduction pathways, both through their catalytic activity and
through the recruitment of other molecules to the receptor complex.
Interleukin-3 (IL-3)1 stimulates the
growth, survival, and differentiation of a broad range of hemopoietic
cells, including pluripotent stem cells and progenitors, mast cells,
megakaryocytes, macrophages, neutrophils, and basophils (1). The high
affinity human IL-3 receptor is composed of a specific subunit of
60-70 kDa and a
subunit of 130-140 kDa (2), which is also a
component of the high affinity human GM-CSF and IL-5 receptors (3, 4) and is referred to as
c. Although lacking intrinsic tyrosine kinase
activity, upon IL-3 binding,
c becomes phosphorylated on multiple
tyrosine residues, some of which serve as selective binding sites for
molecules containing SH2 domains. A number of cellular proteins
including JAK-2 (5), STAT-5 (6), SHP2 (7), Shc (8), Erk-1 and Erk-2 (9)
are also inducibly tyrosine-phosphorylated in response to IL-3
stimulation.
A subgroup of cytoplasmic protein-tyrosine phosphatases (PTPases),
characterized by containing two SH2 domains NH2-terminal to
their catalytic phosphatase domain, are now referred to as SHP, for
SH2 domain containing phosphatases. SHP1, also
referred to as PTP-1C (10), HCP (11), SH-PTP1 (12), and SHP (13), is
expressed almost exclusively in lymphohemopoietic cells, but has also
been detected in epithelial cells (12). Mutations within SHP1 are
responsible for the phenotype of the two moth-eaten mouse strains (me/me and
mev/mev; Refs. 14 and
15). SHP1 appears to act as a negative regulator of growth factor
signaling, as these moth-eaten mice die soon after birth due
to overproliferation and accumulation of macrophages in the lungs (16).
In addition, increased SHP1 levels have been shown to supress cell
growth in response to IL-3 (17). Activation of certain hemopoietic
growth factor receptors by their ligands results in the association of
SHP1 with the EpoR, via tyrosine 429 (18, 19), c-kit (20),
and the murine IL-3 receptor subunit, Aic2A (17), the sites of
interaction with which are not currently known.
SHP2 has been referred to as SH-PTP2 (21), Syp (22), SH-PTP3 (23), PTP-1D (24), and PTP-2C (25) and appears to be expressed ubiquitously. SHP2 is likely to be the mammalian homologue of the Drosophila csw gene, which transmits positive signals downstream of torso (26). SHP2 becomes phosphorylated on tyrosine and threonine residues following stimulation with a number of growth factors, including EGF, PDGF (22, 24, 27), SCF (28), Epo (29), and IL-3 (7). In addition, SHP2 directly binds, via its SH2 domains, to the activated PDGFR, at tyrosine 1009 (30, 31), the EpoR, at tyrosine 425 (32), and to the EGFR (22, 24). Both tyrosine phosphorylation and engagement of the SH2 domains contribute to the catalytic activation of SHP2. Although not tyrosine-phosphorylated in response to insulin, SHP2 binds to tyrosine-phosphorylated IRS-1 (33) and acts as a positive mediator of insulin and prolactin signals (34-37). SHP2 may have an additional function as an adaptor molecule, because following treatment of responsive cells with PDGF, SCF, Epo, or IL-3, Grb2, via its SH2 domain, associates with tyrosine-phosphorylated SHP2 (7, 28, 29, 38). SHP2 itself binds to the PDGFR, c-kit, and EpoR, resulting in the localization of Grb2 to the membrane and thus may influence the ras pathway.
We have shown previously that treatment of responsive cells with IL-3
or GM-CSF results in the tyrosine phosphorylation of SHP2, creating a
docking site (Tyr304 or Tyr542) for the SH2
domain of Grb2 (7). In addition, we found that IL-3 treatment of cells
resulted in co-precipitation of phosphatidylinositol 3-kinase with
SHP2, as well as increasing the phosphatase activity of SHP2 (7). We
were interested in determining other interactions mediated by SHP2 and
SHP1 in response to IL-3, to investigate the possible roles for these
PTPases in hemopoietic cells. Here we provide evidence that in
vitro and in vivo, both SHP1 and SHP2 bind directly to
the phosphorylated form of the IL-3 receptor. Using synthetic
phosphopeptides we have identified potential sites for this binding and
shown that these peptides can also be utilized as substrates by the
SHP1 and SHP2 catalytic domains in in vitro assays.
TF-1 is a human erythroleukemic cell line (39) that responds to IL-3, IL-5 GM-CSF, Epo, IL-4, IL-13, and insulin (39, 40). These cells were cultured as described previously (41).
Cell StimulationGibbon IL-3 expressed in Ag×63 cells is fully bioactive on human cells and was used as a source of IL-3 for stimulation of TF-1 cells. No effect on TF-1 cells has been observed with conditioned medium from untransfected cells. Cells were washed three times with Hanks' buffered saline solution containing 20 mM HEPES, resuspended at 2 × 107 cells/ml in serum-free RPMI, 20 mM HEPES, incubated for 30 min at 37 °C, and then stimulated with IL-3-conditioned medium (33% (v/v) final concentration) for 10 min. Cell pellets were lysed at 2 × 107 cell equivalents/ml in immunoprecipitation buffer (IP buffer: 50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 40 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 0.7 µg/ml pepstatin).
GST Fusion ProteinsThe construction, expression, and purification of SHP1SH2-GST and SHP2SH2-GST fusion proteins have been described previously (42).
PrecipitationsPrecipitations with antibodies and GST
fusion proteins were carried out as described previously (7, 41). The
following antibodies were used: 10 µg of a monoclonal antibody (3D7)
raised against human c (a kind gift of Prof. A. Lopez, Hansen
Center, Adelaide, Australia); 1 µg of anti-SHP1 (SH-PTP1 (SC-287),
Santa Cruz Biotechnology Inc.); and 0.1 µg of anti-SHP2 (SH-PTP2
(SC-280), Santa Cruz Biotechnology Inc.) antibodies. 5 µg of
SHP2SH2-GST or 15 µg of SHP1SH2-GST were used per precipitation.
The synthesis,
purification, and mass spectroscopic analysis of
phosphotyrosine-containing peptides has been described (43, 44). Table
I lists the phosphopeptides used in this study. Throughout the text the
phosphotyrosine-containing peptides are referred to by the relative
position of the tyrosine residue in c, lacking the 14-amino acid
signal peptide.
|
SHP1SH2-GST and SHP2SH2-GST fusion proteins were preincubated with the phosphopeptides at 100 µM or 50 µM and glutathione-Sepharose by rotating at 4 °C for 60 min. Cell extracts, from 1 × 107 cells, also containing the appropriate concentration of phosphopeptide, were added to the preincubated mixture and rotated at 4 °C for a further 60 min. For the anti-SHP2 phosphopeptide competition analyses, 1 × 107 cells were lysed in the presence of 500 µM phosphopeptide 612 or 750 or 100 µM 1009 and immunoprecipitated with the anti-SHP2 antibody as described above.
SDS-Polyacrylamide Gel Electrophoresis and ImmunoblottingSDS-polyacrylamide gel electrophoresis and
immunoblotting were carried out as described previously (8, 45).
Primary antibodies were used at 0.1 µg/ml for the monoclonal
anti-phosphotyrosine antibody 4G10 and the polyclonal antibodies raised
against c, SHP1 and SHP2 were used at 0.5 µg/ml. Goat anti-mouse
or anti-rabbit horseradish peroxidase-conjugated secondary antibodies
(Dako) were used at 0.1 µg/ml. Immunoblots were developed using the
ECL system (Amersham Corp.) and Kodak X-AR5 film. Blots were stripped as described previously (45).
SHP2 and SHP1 GST fusion proteins were
expressed, purified, and thrombin-cleaved as described previously (42,
43). To assay the dephosphorylation of the c subunit
phosphopeptides, the release of Pi was measured by a
Malachite Green assay in half-volume 96-well plates (Costar) at
23 °C (44). SHP1(
SH2/
CT; Ref. 42) assays were performed in 25 mM Bistris-propane/HCl, pH 6.5, 50 mM NaCl, and
10 mM 2-mercaptoethanol. The assay buffer for SHP2(
SH2; Ref. 42) was the same except for 25 mM Bistris-propane/HCl, pH 6.25. Reaction mixtures containing assay buffer, 1 mM
phosphopeptides, and 45 nM SHP1(
SH2/
CT) or 20 nM SHP2(
SH2) were incubated at room temperature with
occasional agitation. After several time points, 25-µl samples were
removed and added to 50 µl of Malachite Green reagent to detect the
release of inorganic phosphate. The absorbance at 620 nm was measured
and compared with a standard Pi curve.
It has been demonstrated previously that
SHP1 can associate with the murine IL-3 receptor subunit, Aic2A,
via an unmapped site (17). No such association has been demonstrated in
human cells for SHP1 or SHP2. However, we had observed previously that SHP2 was inducibly tyrosine-phosphorylated in response to IL-3, at a
site that enabled binding of Grb2. In addition we also observed co-precipitation of SHP2 with phosphatidylinositol 3
-kinase and activation of SHP2, raising the possibility that SHP2 has multiple functions in IL-3 signaling (7). Therefore, we were interested in
determining whether SHP1 and SHP2 were able to act as adaptor molecules
by binding to the human IL-3 receptor
subunit (
c). TF-1 cells
were left untreated as a control or stimulated with IL-3, lysed, and
immunoprecipitates prepared with either monoclonal anti-
c or
polyclonal anti-SHP1 or anti-SHP2 antibodies. Immunoblotting was
performed on the precipitates with the 4G10 anti-phosphotyrosine antibody, the results are shown in Fig. 1.
A tyrosine-phosphorylated 130-kDa protein was immunoprecipitated with
the anti-c monoclonal antibody after IL-3 stimulation (Fig.
1A, lane 4). Reprobing this same blot with a polyclonal anti-
c antibody confirmed this to be
c (Fig. 1B, lanes
3 and 4). The anti-SHP1 antibodies precipitated three
tyrosine-phosphorylated proteins of 130, 105, and 60 kDa (Fig.
1A, lanes 5 and 6). The 60-kDa phosphoprotein was
present in SHP1 precipitates from both control and IL-3-stimulated
cells (Fig. 1A, lanes 5 and 6), and blotting with
anti-SHP1 antibodies (Fig. 1C, lanes 5 and 6)
confirmed that this protein was SHP1. SHP1 is constitutively
phosphorylated in other hemopoietic cells (17), and this also appears
to be the case in TF-1 cells. The 130- and 105-kDa species were only observed in the SHP1 precipitates from IL-3-treated cells. The identity
of the 105-kDa protein is unknown; however, the 130-kDa species
co-migrated with the tyrosine-phosphorylated
c precipitated by the
anti-
c antibody (Fig. 1A, lane 4), suggesting that
tyrosine-phosphorylated
c co-precipitates with SHP1. We could not
confirm this by reprobing this blot with polyclonal anti-
c
antibodies (Fig. 1B, lane 6), probably because the amount of
tyrosine-phosphorylated 130-kDa species precipitated by the anti-SHP1
antibodies (Fig. 1A, lane 6) was considerably less than that
precipitated by the anti-
c antibodies (Fig. 1A, lane 4)
and so below the limits of detection.
The SHP2 antibodies precipitated four tyrosine-phosphorylated
proteins of 70, 72, 90, and 130 kDa from IL-3-stimulated TF-1 cells
(Fig. 1A, lane 8). The doublet at 70 and 72 kDa corresponds to SHP2, as confirmed by immunoblotting with the SHP2 antibody (Fig.
1D, lanes 7 and 8). The identity of the 90-kDa
protein is unknown. The 130-kDa protein co-migrated with
tyrosine-phosphorylated c precipitated by the anti-
c antibodies
(Fig. 1A, lane 4), and its identity as
c was confirmed by
reprobing the blot with the polyclonal anti-
c antibodies (Fig.
1B, lane 8). In reciprocal experiments, in which blots of
material precipitated with anti-
c were reprobed with antibodies
specific for either SHP1 or SHP2, neither were detectable (Fig. 1,
C and D). The monoclonal anti-
c antibody
precipitates less than 10% of the
c expressed in the TF-1
cells,2 only a proportion of which is
likely to be phosphorylated at the appropriate sites and will hence
interact with downstream signaling molecules. Thus, our failure to
detect SHP1 and SHP2 in the anti-
c precipitates most likely reflects
the fact that the amounts present were below our limits of
detection.
We next investigated whether the SH2 domains of the
two PTPases mediated the observed interactions with c. Precipitates
from extracts of TF-1 were prepared using fusion proteins in which GST
was fused to either the SH2 domains of SHP1 (SHP1SH2-GST) or SHP2
(SHP2SH2-GST). Both SHP1SH2-GST and SHP2SH2-GST precipitated a
tyrosine-phosphorylated 130-kDa protein from extracts of cells stimulated with IL-3 (Fig. 2A, lanes 2 and
4) but not from control samples (Fig. 2A, lanes 1 and 3), which was recognized by the polyclonal anti-
c
antibody (Fig. 2A, lower panel). These results clearly
demonstrated that the SH2 domains of SHP1 and SHP2 can associate
in vitro with the tyrosine-phosphorylated
c following IL-3 stimulation. The additional proteins observed in the antibody immunoprecipitations were not observed in the SH2 domain-fusion protein
precipitates. This could be for a number of reasons: (i) they interact
with regions of the phosphatase other than the SH2 domains, (ii) they
bind the tyrosine-phosphorylated phosphatases themselves, or (iii) they
are bound to other proteins in the co-immunopreciptated complex.
To ascertain whether the interactions between c and the SH2 domains
of SHP1 and SHP2 were direct, we performed sequential immunoprecipitations. Extracts of TF-1 cells were immunoprecipitated with the monoclonal anti-
c antibody and the precipitated material boiled and denatured in the presence of SDS and 2-mercaptoethanol. The
denatured primary immunoprecipitates were diluted 1 in 10 (so the
concentration of SDS was <0.1%) and re-precipitated with either
SHP1SH2-GST or SHP2SH2-GST. The primary anti-
c immunoprecipitation precipitated tyrosine-phosphorylated
c from cells treated with IL-3
(Fig. 2B, lane 2), but not from control cells (Fig.
2B, lane 1). The secondary precipitations with SHP1SH2-GST
and SHP2SH2-GST re-precipitated tyrosine-phosphorylated
c from the
IL-3-stimulated cell extracts (Fig. 2B, lanes 4 and
6). Similar results were observed when the fusion proteins
were used as the primary precipitants and anti-
c as the secondary
precipitating agent (data not shown). These results demonstrate that
the SH2 domains of SHP1 and SHP2 can bind directly to
tyrosine-phosphorylated
c.
To confirm that the observed associations involved
interactions of the SH2 domains with phosphotyrosine and to provide an indication as to which of the potential tyrosines on c was
responsible for mediating the interactions with the SH2 domains of SHP1
and SHP2, we used a peptide competition assay. The tyrosine residues within the
c that become phosphorylated upon IL-3 stimulation have
not been biochemically mapped, so phosphopeptides corresponding to the
sequences surrounding five of the tyrosine residues within
c (see
Table I) were tested for their ability to block
precipitation of the tyrosine-phosphorylated
c by SHP1SH2-GST and
SHP2SH2-GST. Phosphopeptides corresponding to residues surrounding
tyrosines 806 and 856 were not tested, as mutants of
c that were
truncated at residue 763 or beyond retain normal functions in response
to IL-3 (46, 47). The results of the competition analyses are shown in
Fig. 3, A and B. The
phosphopeptide incorporating tyrosine 612 (612) inhibited the
precipitation of tyrosine-phosphorylated
c by SHP1SH2-GST (Fig.
3A, lane 7). Some inhibition was also consistently observed
with phosphopeptide 750 (Fig. 3A, lane 5), but not
consistently with the other phosphopeptides. Only phosphopeptide 612 consistently inhibited the precipitation of tyrosine-phosphorylated
c by SHP2SH2-GST (Fig. 3B, lane 4). Reprobing of this
same blot with polyclonal anti-
c antibodies confirmed that the
presence of phosphopeptide 612 had inhibited precipitation of
c by
SHP2SH2-GST (Fig. 3B, lower panel).
SHP1 and SHP2 each contain two SH2 domains, which appear to differ in
their functions (48). Therefore, we used combinations of phosphopeptide
612 with the other phosphopeptides to examine the possibility that any
of the latter might make a secondary contribution to the binding of
SHP2 to c. At a concentration of 100 µM (Fig.
3C, lane 3) phosphopeptide 612 almost completely inhibited
(>95%) precipitation of tyrosine-phosphorylated
c by SHP2SH2-GST.
At 50 µM 612, inhibition was approximately 80% (Fig. 3C, lane 4). Combinations of the other phosphopeptides (50 µM) with 612 (50 µM) did not result in
further reduction of the amount of tyrosine-phosphorylated
c
precipitated by SHP2SH2-GST (Fig. 3C, lanes 5-8).
Therefore, it appears that the SH2 domains of SHP2 interact solely with
residues surrounding phosphotyrosine 612 of
c. Technical
difficulties have prevented clearcut results from being obtained in
similar experiments with SHP1.
We also performed phosphopeptide competition analyses of anti-SHP2
immunoprecipitates from control and IL-3-treated TF-1 cells to
investigate whether endogenous SHP2 could also be inhibited from
binding to the tyrosine-phosphorylated c by 612. A phosphopeptide incorporating the residues surrounding tyrosine 1009 within the PDGFR
(1009; Ref. 42) has been shown to be the binding site for SHP2 (27, 31)
and was used as a control. Cells were lysed in the presence of 500 µM phosphopeptide 612 or 750 or 100 µM 1009 and precipitations prepared with the anti-SHP2 antibody. Phosphopeptides 612 and 1009 significantly reduced the co-precipitation of endogenous SHP2 with tyrosine-phosphorylated
c from
IL-3-stimulated cells (Fig. 3D, lanes 3 and 5),
whereas phosphopeptide 750 did not. It is interesting that although
SHP2 is inhibited from binding to
c by phosphopeptides 612 and 1009, the level of SHP2 tyrosine phosphorylation is not decreased and in this
experiment appears to have increased (72-kDa protein, Fig.
3D, compare lanes 3 and 5 with
lanes 2 and 4). Fig. 3D, lower panel,
shows the blot to be evenly loaded with respect to SHP2. This result
suggests that association of SHP2 with
c may not be required for its
tyrosine phosphorylation. Higher concentrations of phosphopeptide were required to inhibit co-precipitation of
c by anti-SHP2 antibodies compared with the fusion proteins, perhaps reflecting a high affinity complex between SHP2 and
c. Competition experiments were attempted using the anti-SHP1 antibody, but proved technically challenging. The
anti-SHP1 antibody appears to be much less efficient in
immunoprecipitation (even though 10 times more was used than for the
SHP2 antibody), and the amount of the tyrosine-phosphorylated
c
precipitated was often low (see Fig. 1A, lane 6), making
interpretation of the results difficult. This could be explained if the
association between SHP1 and the receptor is of low affinity or
transient in nature.
To assess
whether the synthetic phosphopeptides tested in the competition
experiments could also serve as substrates for SHP1 and SHP2,
phosphatase assays were performed. GST-fusion proteins of SHP1 and SHP2
lacking the SH2 domains were cleaved with thrombin to release the
GST-protein part. However, due to a cryptic thrombin cleavage site
within the carboxyl terminus of SHP1 a 5-kDa fragment was also released
after complete thrombin digestion (42). The activity of both PTPases,
SHP1(SH2/
CT), and SHP2 (
SH2) toward the five different
phosphopeptides was assessed, and the results are shown in Fig.
4. With both enzymes, phosphopeptide 612 was by far the
best substrate as reflected in the highest release of Pi.
Phosphopeptide 750 showed a much diminished, but significant, release
of Pi, whereas with phosphopeptides 695, 577, and 452, either no or minimal dephosphorylation was seen. Thus, a phosphopeptide corresponding to a potential binding site for the SH2 domains of both
enzymes serves as a good substrate for the PTPase domain of both SHP1
and SHP2.
In this study we have demonstrated that the tyrosine phosphatases
SHP1 and SHP2 can both bind inducibly to c following IL-3 stimulation. This association appears to be directly mediated by
interactions between the SH2 domains of SHP1 and SHP2 and
phosphotyrosine residues within
c. A phosphotyrosine-containing
peptide based on the sequence surrounding tyrosine 612 of
c was able
to compete the binding of both SHP1SH2-GST and SHP2SH2-GST fusion
proteins to tyrosine-phosphorylated
c in in vitro assays
and also the binding of endogenous SHP2 to
c in immunoprecipitation
studies. These results strongly suggest that the SH2 domains of both
SHP1 and SHP2 bind to tyrosine 612 of
c.
Tyrosine 612 of c is located within the motif LEYLCLP, which has
similarities to motifs previously identified for SHP1 and SHP2 SH2
interactions. The NH2-terminal SH2 domain of SHP1 showed a
broad selectivity for pY-hydrophobic-X-hydrophobic motifs
from a phosphopeptide library (49). In addition, tyrosine 429 of the
EpoR, which lies in the pYLYL motif, has been show to be essential for
the SHP1 binding (18). Pei et al. (50) have previously shown
that a phosphopeptide based on the sequence surrounding Tyr612 of
c (referred to as Tyr628, which
includes the 14-amino acid signal peptide) bound the
NH2-terminal SH2 domain of SHP1, activated the phosphatase,
and acted as a substrate for SHP1. They suggested this tyrosine may be
the binding site for SHP1 to
c (50), and our results show a similar
peptide does compete with SHP1 for binding to
c. We observed weaker
inhibition of precipitation of
c with SHP1SH2-GST by phosphopeptide
750. Tyrosine 750 is located in the sequence pYVEL, which also conforms to the predicted SHP1 SH2 binding motif (49).
The selectivity of the NH2-terminal SH2 domain of SHP2,
determined using a degenerate peptide library, was shown to be
pY-V/I/T-X-V/L/I (51). Experiments using both mutant
receptors (31) and peptide competition assays (27) demonstrate that
tyrosine 1009 of the PDGFR, in the motif pYTAV, is required for SHP2
binding. Recently, using EpoR mutant receptors, it has been shown that
SHP2 binds, via its SH2 domains, to the activated EpoR at tyrosine 425, in the motif pYTIL (32). The residues surrounding tyrosine 612 (YLCL)
of c are similar to these previously described binding motifs for
SHP2.
The effects of mutagenesis of tyrosines 612 and 750 of c on tyrosine
phosphorylation of substrates in response to GM-CSF have been reported
(52, 53). In these transfectants, normal levels of SHP2 tyrosine
phosphorylation were observed (53). However, the association of SHP2
with
c was not examined in these mutant
c-expressing cells. An
interesting point relating to this is whether stable association of
SHP2 with
c is required for its tyrosine phosphorylation. Our data
suggest that in the presence of phosphopeptide 612, which competes for
the binding of SHP2 to
c, the levels of SHP2 tyrosine
phosphorylation are not diminished and may actually increase (Fig.
3D), supporting the notion that SHP2 does not need to be
bound to
c to become phosphorylated. However, we cannot rule out the
possibility that SHP2 associates transiently with
c and that this is
all that is required for its phosphorylation or that another mechanism
is leading to increased SHP2 tyrosine phosphorylation in the presence
of the phosphopeptide, e.g. a kinase is activated.
Additional mutational analyses of
c have implicated two regions of
c, which appear to influence SHP2 tyrosine phosphorylation (54).
Mutation of Tyr577 in conjunction with a truncation up to
residue 589 resulted in greatly reduced levels of SHP2 tyrosine
phosphorylation in response to GM-CSF, although either mutation alone
had no effect (54). Our results suggest that Tyr577 is not
involved in SHP2 binding to
c and that Tyr612 is the
major site of interaction. Since Tyr612 is removed in the
589
c truncation mutant, but SHP2 is still tyrosine-phosphorylated,
it may be that association of SHP2 is not required for its tyrosine
phosphorylation, and our data are consistent with this possibility.
Interestingly, phosphotyrosine peptide 612, which competed the binding
of both SHP1 and SHP2 to c, was also the best substrate for SHP1 and
SHP2 catalytic activities. It has been shown previously that SHP1 and
SHP2 prefer substrates that have acidic residues to the NH2
terminus of the phosphotyrosine (42, 55), and this is the case for
Tyr612. Whether this site is an in vivo
substrate remains to be determined, although Yi et al. (17)
reported the SHP1-catalyzed dephosphorylation Aic2A. Therefore, the
same site that appears to be recognized by the SH2 domains of these
phosphatases can also be utilized as a substrate. In experiments using
full-length recombinant SHP2 and peptides for substrates, Sugimoto
et al. (55) found SHP2 to have a preference for tyrosine
1009 (to which the SH2 domain of SHP2 also binds, leading to its
activation (30)) and tyrosine 1021 of the PDGFR, suggesting that
tyrosine 1009 may both regulate and act as a substrate for the PTPase
activity of SHP2. We have observed similar substrate specificities with
PDGF receptor peptides (42), and the results presented here with SHP2
and
c suggest a similar mechanism may be used in IL-3 signaling.
However, using immunoprecipitated phosphorylated PDGF
receptor and
recombinant full-length SHP2, phosphotyrosines Tyr771 and
Tyr751, followed by Tyr740, were used
preferentially, while Tyr1021 and Tyr1009 were
very poor substrates (56). These discrepancies could arise from
differences in using the intact PDGF
receptor, which contains multiple potential phosphorylation sites, which may bind other proteins
and mask potential sites, instead of using peptides as substrates. In
our in vitro assay system, the SHP1 and SHP2 recombinant enzymes used lacked their SH2 domains, thus removing any potential "activating" effects of the various phosphopeptides.
It is likely that SHP1 and SHP2 have different roles when localized to
the IL-3 receptor. SHP1 is thought to be a negative regulator of growth
and functions to terminate signals. Overexpression of SHP1 in DA3 cells
leads to a decrease in IL-3-dependent proliferation and a
decrease in Aic2A tyrosine phosphorylation (17). The binding of SHP1 to
the EpoR activates the PTPase, which dephosphorylates Jak-2, leading to
the termination of proliferative signals (18). SHP1 has been shown
recently to interact directly with Jak-2, leading to its
dephosphorylation (57). IL-3 also induces activation of Jak-2 (5), so
SHP1 may function in a similar manner in IL-3 signal transduction. SHP1
may also function to dephosphorylate other phosphorylated tyrosine
residues on the IL-3 receptor, again leading to down-regulation of IL-3
responses. However, also competing for the same binding site on c is
SHP2, which is thought to act as a positive mediator of growth factor
signals. SHP2 associates with Jak-1 and Jak-2, and tyrosine 304 of SHP2
is phosphorylated by these kinases, leading to the creation of a Grb2
recognition motif (58). We have shown previously that
tyrosine-phosphorylated SHP2 associates with the adaptor molecule Grb2
(7), and the studies presented here suggest that SHP2 could act as an
adaptor between the activated
c and Grb2, thus leading to activation of the ras/mitogen-activated protein kinase pathway, known
to be activated by IL-3 (9, 59). This has been suggested in other
systems, where SHP2 acts as an adaptor between Grb2 and the EpoR (28)
and the PDGFR (38). Expression of a catalytically inactive version of
SHP2 results in reduced activation of the mitogen-activated protein
kinases Erk-1 and Erk-2 in response to insulin (60), which fits with
the proposed role of SHP2 in transducing positive growth-promoting
signals. Additionally, we have shown previously that SHP2 associates
with the p85 subunit of phosphatidylinositol 3
-kinase (7), so, SHP2
may also regulate this pathway. Although both SHP1 and SHP2 associate
with tyrosine 612 of
c, it is likely that only a portion of the
receptors are associated with SHP1 and SHP2 at any one time. The
kinetics of activation/deactivation of each phosphatase would have to
be individually assessed to determine the relative sequence of events.
Any shifting of the equilibrium between the two phosphatases would
result in either a positive or negative effect on IL-3-induced signals. Further detailed molecular analyses are required to dissect these pathways.
In summary, we have demonstrated that SHP1 and SHP2 bind through their
SH2 domains to tyrosine 612 of c. Both SHP1 and SHP2 appear to be
able to regulate their binding to the receptor as phosphopeptide 612 also served as a substrate for the catalytic domain of both the
PTPases, possibly enabling them to modulate signaling pathways,
regulated by tyrosine phosphorylation/dephosphorylation events.
We thank Phil Owen, Peter Borowski, and Ian Clark-Lewis for peptide synthesis and purification and Bridget Craddock for helpful comments and discussions.