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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3746-3755
SHP-1 Phosphatase C-Terminus Interacts With Novel Substrates
p32/p30 During Erythropoietin and Interleukin-3 Mitogenic Responses
By
Wentian Yang,
Mina Tabrizi,
Karim Berrada, and
Taolin Yi
From the Department of Cancer Biology, The Lerner Research Institute
of the Cleveland Clinic Foundation, Cleveland, OH.
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ABSTRACT |
SHP-1 protein tyrosine phosphatase is a critical negative regulator
of mitogenic signaling, as demonstrated by the heightened growth
responses to hematopoietic growth factors in hematopoietic cells of
motheaten mice, which lack functional SHP-1 expression due to mutations
in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated
by SHP-1 have not been fully identified. We detected two proteins
(p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor
(EpoR) cell line that expresses a mutant containing the SHP-1
C-terminus that suppresses the function of the endogenous phosphatase
and induces hyperproliferative responses to interleukin-3 (IL-3) and
Epo. Hyperphosphorylated p32/p30 are also detected in motheaten
hematopoietic cells, demonstrating an association of p32/p30
hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with
the phosphatase and the C-terminal mutant and they bind in vitro to a
synthetic peptide of the mutant but not the GST fusion proteins of
SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo
occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell
line, indicating p32/p30 as novel signaling molecules during cell cycle
progression. These data demonstrate a function for the SHP-1 C-terminus
in recruiting potential substrates p32/p30 and suggest that SHP-1 may
regulates mitogenic signaling by dephosphorylating p32/p30.
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INTRODUCTION |
HEMATOPOIETIC growth factors, such as
interleukin-3 (IL-3) and erythropoietin (Epo), play critical roles in
hematopoiesis. Signal transduction for both IL-3 and Epo depends on the
induction of tyrosine phosphorylation to activate the Ras and the Jak2
signaling pathways.1 The receptors for IL-3
(IL-3R) and Epo (EpoR) have no intrinsic kinase activity. Ligand
binding activates the receptor-coupled Jak2 protein tyrosine
kinase.2,3 As shown by mutational analysis, the IL-3R and
EpoR have distinct functional domains mediating the two signaling
pathways.4,5 The membrane proximal part of the receptor
cytoplasmic region is required for the activation of the Jak2 tyrosine
kinase, STAT5 phosphorylation, and c-myc expression. The carboxyl
terminal region of the receptor is responsible for activating the Ras
pathway, leading to MAPK phosphorylation and c-fos expression. Whereas
the activation of the Ras pathway is not required for mitogenic
signaling, it is important for cell viability by preventing
apoptosis6 and may also promote cell proliferation.7 On the other hand, activation of the Jak2
pathway and the induction of c-myc expression correlate with signaling of cell proliferation.5 However, the molecule(s) that
mediates mitogenic signals downstream from the Jak/Ras pathways to
apparatus controlling cell cycle progression has not been unequivocally identified. Although STAT5 activation enhances the cell proliferative response,8 it is not required for mitogenic
signaling.5 Importantly, the signal transduction for IL-3
and Epo is also regulated by protein tyrosine phosphatases (PTPases),
because the tyrosine phosphorylation induced by either of the growth
factors is transient and returns to basal levels shortly after ligand stimulation.9,10
SHP-1 (previously called PTP1C/HCP/SHPTP1/SHP) is a cytoplasmic protein
tyrosine phosphatase that contains two SH2 domains and is expressed
predominantly in hematopoietic cells.11-15 Previous studies
from our laboratory and others demonstrate that SHP-1 is a critical
negative regulator of the mitogenic signaling of IL-3 and Epo.
Hematopoietic cells from the motheaten mice, which lack functional
SHP-1 due to mutations in the SHP-1 gene,16,17 are
hyperproliferative in response to Epo,18 macrophage
colony-stimulating factor (M-CSF),19 and
granulocyte-macrophage colony-stimulating factor
(GM-CSF).20 This hyperproliferation is likely one of the
factors responsible for the elevated myelopoiesis of erythroid, monocytic, and granulocytic lineages in motheaten mice and for the
early death of the mice from massive accumulation of myeloid cells in
vital organs.21 The observation that overexpression of
SHP-1 suppresses IL-3-induced cell proliferation in a murine IL-3-dependent cell line provides additional support for a negative role for the phosphatase in mitogenic signaling.22
Furthermore, we and others have shown that SHP-1 associates with the
receptors for hematopoietic growth factors, such as IL-3,22
Epo,23,24 and stem cell factor,25 through its
SH2n domain that bind to phosphotyrosine sites in the cytoplasmic
regions of the receptors. In addition, SHP-1 also associates with and
regulates signals from membrane molecules of other receptor complexes,
such as Fc RIIB1,26 CD22,27 the nature killer
cell inhibitory receptor (KIR),28 the B-cell antigen
receptor,29 the receptor of interferon
 / ,30 and the member of the signal-inhibition
regulatory proteins (SRP).31,32
SHP-1 regulates mitogenic signaling by dephosphorylating key substrates
essential for cell proliferation. The Jak family kinases have been
implicated as SHP-1 substrates in recent studies. It was
shown23 that an EpoR mutant, which has two of the tyrosines in the cytoplasmic domain substituted with phenylalanines and was not
recognized by SHP-1 SH2 domains, caused marked and prolonged Jak2
hyperphosphorylation in response to Epo stimulation. This suggests that inhibition of SHP-1 binding to the receptor prevented it
from dephosphorylating Jak2 and may have enhanced mitogenic signaling.
However, the hypothesis is complicated by the observation that
comparable Jak2 phosphorylation was detected from engagement of the
wild-type EpoR or an EpoR null mutant with all of the tyrosines in the
receptor cytoplasmic domain substituted by phenylalanines.8 Moreover, it was shown recently that SHP-1 directly binds to and dephosphorylates Jak family kinase.33 Importantly, we found that GM-CSF stimulation of motheaten macrophages, which are
hyperproliferative in response to the growth factor, induces only a
rather modest and transient Jak2 hyperphosphorylation, whereas the
cells contain several yet unidentified proteins that are markedly
hyperphosphorylated.24 This suggests that SHP-1 may play a
limited role in Jak2 dephosphorylation and that SHP-1 may regulate
mitogenic signaling by dephosphorylating additional substrates.
To identify key substrates of SHP-1 and elucidate mitogenic signaling
pathways, we sought to suppress the endogenous SHP-1 activity in a
murine myeloid cell line (DA3EpoR) that depends on IL-3 or Epo for
proliferation by introducing dominant negative SHP-1 mutants into the
cells and to characterize hyperphosphorylated proteins induced by the
mutant. Previous studies showed that deletion of the C-terminus of
SHP-1 increased the SHP-1 PTPase catalytic activity,34-36
suggesting that the C-terminus is inhibitory to the phosphatase. We
examined Epo/IL-3 growth responses and phosphotyrosine proteins of
DA3EpoR cells expressing an SHP-1 mutant containing the C-terminus of
phosphatase. Two SHP-1-associated proteins, p32/p30, were detected
whose hyperphosphorylation correlates with defective SHP-1 activity and
cell hyperproliferation. Our data indicate that p32/p30 are novel SHP-1
substrates that function understream of Jak/Ras pathways and during
cell cycle progression in mitogenic signaling of Epo/IL-3.
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MATERIALS AND METHODS |
Cells and cell culture.
PA317 cells37 were maintained in Dulbecco's modified
Eagle's medium (DMEM) with 10% fetal calf serum (FCS). The growth
factor-dependent murine myeloid cell line DA3EpoR10 and
DA3EpoR-H/Y343F5 were maintained in RPMI 1640 medium
supplemented with 10% FCS and 10% WEHI-3 conditioned medium (WCM) as
a source of IL-3 (~30 U/mL). Spleen cells and bone marrow-derived
macrophages from normal and viable motheaten mice were prepared as
previously described.20,33,38
Cell proliferation responses were determined by an MTT assay, as
described.20 For growth factor stimulation, cells were washed in 10% CSF RPMI 1640 and incubated in the medium without IL-3
for 16 hours at 37°C. Recombinant murine IL-3 (200 U/mL) or
recombinant Epo (100 U/mL) was added to the cell cultures, which were
then incubated at 37°C for various times, as indicated. Stimulation
reactions were terminated by lysing the cells in cold lysis buffer (50 mmol/L Tris, pH 7.4, 50 mmol/L NaCl, 0.5% sodium deoxycholate, 0.2 mmol/L Na3VO4, 20 mmol/L NaF, 1% NP-40, 2 mmol/L phenylmethyl sulfonyl fluoride, 20 µg/mL of
aprotinin, and 10% glycerol).
SHP-1-C mutant, transfection, and retroviral infection of DA3EpoR
cells.
The SHP-1 C-terminus cDNA was generated by polymerase chain
reaction39 from a murine SHP-1 cDNA clone15
with synthetic oligonucleotide primers
(5 -GGAATTCAGGATGAAGGCCTCGCGTACTTCC and 5-GGTCGACCTTCCTCTTGAGAGA). The resulting cDNA
fragment encodes SHP-1 C-terminus from amino acid 551 to 595, with a
translation initiation codon incoporated into the 5 end. The
cDNA fragment was then ligated via its 3 end to a double-strand
DNA fragment encoding KT3 epitope (VDKPPTPPPEPET) of the SV40 T
antigen40 to derive the cDNA clone of the KT3-tagged SHP-1
C mutant. The cDNA encoding this mutant was sequenced by the chain
termination method,41 cloned into the pBabe/puro vector
containing the puromycin resistant gene,42 and
transfected24 into the retroviral packaging cell line
PA317.37 DA3EpoR cells were infected by coculturing with
the transfected PA317 cells for 48 hours, followed by selection in the
presence of puromycin (2 µg/mL; Sigma, St Louis, MO) for 2 weeks in medium supplemented with 10% FCS and 10% WCM. DA3EpoR cells infected with the pBABE/puro vector alone were also generated under comparable conditions as a control.
RNA isolation and Northern hybridization.
Total cellular RNA was isolated from cells following the procedures
previously described.20,33,38,39,43 The RNA samples (~20
µg/well) were separated in 1.2% agarose formaldehyde gel by
electrophoresis. The RNA samples were then blotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH), probed with a
32P-labeled random-primed cDNA fragment encoding the
SHP-1-C mutant, and detected by autoradiography.
Antibodies, immunoprecipitation, binding assays, and Western
blotting.
Antibodies against SHP-1,15 Jak2,3
EpoR,24 and IL-3R22 have been described
previously. The antiserum against SHP-1 C-terminus was developed in
rabbits with a synthetic peptide (KVKKQRSADKEKNKGSLKRK), whereas the
anti-SHP-1-M antibody recognizes a peptide sequence from the middle
section of the phosphatase (QKQEVKNLHQRLEGQRPENK). The hybridoma cell
line of anti-KT3 monoclonal antibody was a gift from Dr G. Walter (San
Diego, CA) and the antibody was purified from mouse ascites following
established procedures.40 Antibodies against
phosphotyrosine (anti-ptyr; UBI, Lake Placid, NY), STAT5 (Santa Cruz,
Santa Cruz, CA), phospho-MAPK (Promega, Madison, WI), c-myc (UBI), and c-fos (Santa Cruz) were purchased
from commercial sources.
Immunoprecipitation and Western blotting were performed as described
previously.24 Immune complexes were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels,
blotted onto nitrocellulose membrane (Schleicher & Schuell), probed
with specific antibodies, and detected using an enhanced chemiluminescence kit (ECL; Amersham, Arlington Heights,
IL). For the detection of SHP-1-C protein, which has a
calculated size of 6 kD and thus was difficult to quantitate by
SDS-PAGE/Western blotting, nitrocellulose membrane in a dot blotting
apparatus was coated with anti-KT3 antibody (20 mg/well) for 1 hour,
washed three times, blocked with 5% milk solution, and incubated with various amounts of cell lysates for 2 hours. The membrane was probed
with an anti-SHP-1-C antiserum (1:5,000 dilution) and detected by ECL
as described above.
For binding assays,28 cell lysates were incubated with a
GST-fusion protein of SHP-1 SH2 domains or with a synthetic peptide of
SHP-1-C (SSKHKEEVYENVHSKSQKEEKVKKQRSADKEKNKGSLKRK; Quality Controlled
Biochemicals, Inc) that was conjugated to Affi-gel 10 beads (BioRad Laboratories, Richmond, CA). Cellular
proteins associated with the fusion proteins (2 µg/reaction) and
peptides (1 µg/reaction) after washing in lysis buffer were analyzed
by SDS-PAGE and Western blotting.
Phosphatase assays.
The preparation of GST fusion protein of SHP-1 has been described
previously.28 The phosphatase (PTPase) activity of the GST-SHP-1 fusion protein was determined using pNPP (Sigma) as a
substrate. The PTPase assay was performed in the absence or presence of
synthetic peptide SHP-1-C (20 to 40 µmol/L) at 22°C for 30 minutes in 50 mL of reaction mixture (100 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10 mmol/L pNPP, and 20 nmol/L GST-SHP-1). The reaction was terminated by
adding 950 mL of 1 N NaOH. The reaction product p-nitrophenolate was
quantified by measuring absorbance at 405 nm.
| |
RESULTS |
An SHP-1 mutant (SHP-1-C) containing the C-terminus of the phosphatase
induces heightened growth response to IL-3 and Epo in DA3EpoR cells.
An SHP-1 mutant (SHP-1-C) containing the C-terminal 45 amino acids of
the phosphatase was tagged with the KT3 epitope
(Fig 1A) and introduced, by retroviral
infection in pBabe vector carrying the puromycin-resistant gene, into
the murine myeloid cell line DA3EpoR that depends on IL-3 or Epo for
proliferation. Puromycin-resistant populations of DA3EpoR cells
infected with the mutant (D/C) or the control vector (D/V) were further
characterized. SHP-1-C transcript was detected by Northern
hybridization in D/C but not D/V cells (Fig 1B, lanes 1 and 2). The
SHP-1-C transcript in D/C cells was expressed at about twofold to
threefold higher than that of the endogenous SHP-1. SHP-1-C protein
expression was detected in D/C (Fig 1C, upper panel, lanes 2 and 3) but
not in D/V cells (Fig 1C, lower panel, lanes 2 and 3), as expected.
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| Fig 1.
The SHP-1-C mutant induced heightened growth responses to
Epo/IL-3 in DA3EpoR cells. (A) The SHP-1-C mutant containing the C-terminus of the SHP-1 was tagged with the KT3 epitope and introduced into DA3EpoR cells by retroviral infection. The antigenic epitopes recognized by antibodies specific for SHP-1 (a-SHP-1-M) or SHP-1-C (a-KT3) or for both are indicated by the arrows. (B) The expression of
SHP-1-C transcript in cells infected with the SHP-1-C construct (D/C)
or the vector control (D/V) was determined by Northern hybridization with a SHP-1-C probe, which also detected the endogenous SHP-1. (C) The
expression of SHP-1-C protein was determined by dot blotting. A
membrane coated with the KT3 antibody was incubated without (lane 1) or
with lysates from D/V or D/C cells (lanes 2 and 3) and probed with an
anti-SHP-1-C antibody as indicated. (D) The growth responses of D/V
and D/C cells to Epo or IL-3 were determined by cell proliferation
assays using an MTT method and the values are the mean ± SD of three
replicates.
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Whereas both D/V and D/C populations displayed a significant growth
response to IL-3 or Epo in cell proliferation assays, the D/C cells
showed heightened growth response to IL-3 (Fig 1D, left) and Epo (Fig
1D, right) in comparison to the D/V cells. D/C cells also showed an
increased cell proliferation to different doses of Epo or IL-3 (data
not shown). This increased cell growth of D/C cells was not due to
changes in cell survival or caused by growth factor independent
proliferation, because both populations underwent apoptosis in a
similar manner after growth factor deprivation and could not be
maintained in the absence of IL-3 or Epo (data not shown). A similarly
increased cell growth was also detected in two other SHP-1C-expressing
cell populations derived from independent transfection experiments
(data not shown).
SHP-1-C induced hyperphosphorylation of p32/p30 but had no marked
effect on the activation of Jak2 and Ras pathways.
Because suppression of endogenous SHP-1 by the SHP-1-C mutant should
cause hyperphosphorylation in the substrates of SHP-1, we examined
Epo-induced protein tyrosine phosphorylation in D/V and D/C cells. In
comparison with D/V cells, D/C cells contained several distinct
proteins that were hyperphosphorylated. Among these, two proteins of
approximately 32 and 30 kD (p32/p30) were prominent
(Fig 2A), with p32 hyperphosphorylation
more pronounced than p30. The phosphorylation of p32/p30 in D/V cells,
as in the parental DA3EpoR cells (data not shown), was modestly and
transiently induced by Epo at 10 minutes (Fig 2A, lane 3) and then
again at 120 minutes after stimulation (Fig 2A, lane 6). p32/p30
phosphorylation in D/C cells was generally onefold to fourfold higher
than that in D/V cells with or without Epo stimulation (Fig 2A, lanes 7 through 11; see also Figs 4 and 5) and were also further induced at 120 minutes after stimulation (Fig 2A, lane 12). In addition, increased
phosphorylation was also detected in yet unidentified proteins of
approximately 150, 104, 56, and 53 kD (Fig 2A, lanes 7 through 12). In
contrast, Epo-induced phosphorylation of EpoR (Fig 2A), Jak2 (Fig 2D),
and Stat5 (data not shown) were comparable in D/V and D/C cells, with a
minor increase of Jak2 phosphorylation in growth factor-deprived D/C
cells (compare Fig 3C and D, lanes 1 and
7). Consistent with the lack of effect of SHP-1-C mutant on Jak2/Stat5
phosphorylation, Epo induced similar expression levels of c-myc (Fig
2C) and c-fos (data not shown) in both cell populations. Similar
results were obtained when the cells were stimulated with IL-3 (data
not shown).
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| Fig 2.
SHP-1-C induced hyperphosphorylation of p32/p30 but had
no marked effect on the phosphorylation of EpoR and Jak2 and c-myc expression. Total cell lysates (TCL) were prepared from D/V and D/C
cells stimulated with Epo for various times. The lysates were analyzed
directly by SDS-PAGE/Western blotting or used in immunoprecipitation with antibodies as indicated. The positions of hyperphosphorylated proteins of approximately 30, 32, 53, 56, 104, and 150 kD and protein
size markers (in kilodaltons) are indicated.
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| Fig 4.
Hyperphosphorylated p32/p30 coimmunoprecipitated with
SHP-1 and SHP-1-C from D/C cells but were not recognized by SHP-1 SH2 domains in vitro. (A) TCLs were prepared from D/V and D/C cells and
were used for immunoprecipitation with antibodies specific for SHP-1
(lanes 5 and 6) or SHP-1-C (lanes 3 and 4). TCLs and the
immunocomplexes were analyzed by SDS-PAGE/Western blotting with
antibodies as indicated. (B) Cell lysates of D/V or D/C cells stimulated without ( ) or with (+) Epo for 5 minutes were used in
anti-EpoR immunoprecipitation (lanes 1 through 4) or in in vitro
binding assays with a GST fusion protein of SHP-1 SH2 domains containing amino acids 1-198 of the murine SHP-1 (lanes 5 through 8).
Phosphoproteins were analyzed by SDS-PAGE/Western blotting with an
anti-ptyr antibody. The higher level of EpoR phosphotyrosine signaling
from D/C cells (lane 8) was not reproducible and may have been caused
by variations in the amount of fusion proteins. The positions of
phosphoproteins and protein size markers are indicated.
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| Fig 5.
Synthetic peptide of SHP-1-C binds to p32/p30 from D/V
and D/C cells but has no marked effect on SHP-1 PTPase activity in vitro. (A) Total cell lysates (TCL) from D/V and D/C cells were incubated in binding assays with a synthetic peptide of SHP-1C conjugated to Affi-gel 10. TCL (lanes 1 and 2) and cellular proteins associated with peptide (lanes 3 and 4) were analyzed by SDS-PAGE and
Western blotting with antibodies as indicated. The positions of
phosphotyrosine proteins and protein size markers are indicated. (B)
The PTPase activity of a GST-SHP-1 fusion protein was determined in the
absence or presence of the synthetic SHP-1-C peptide (20 to 40 µmol/L). The data represent the mean ± SD values of duplicated samples.
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| Fig 3.
Reduced induction of p32/p30 phosphorylation from the
EpoR mutant H/Y343F that was defective in Ras but not Jak2 activation. DA3 cells expressing the EpoR mutant H/Y343F were deprived of growth
factors and then stimulated with IL-3 or Epo for various times. Cell
lysates were prepared and analyzed by SDS-PAGE/Western blotting with
antibodies as indicated.
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The data presented in Fig 2A were protein samples separated in a 7.5%
SDS-PAGE gel that failed to distinctively resolute p32/p30 proteins. We
used an 8.5% gel in the following experiments for p32/p30 analysis to
achieve better results.
Reduced induction of p32/p30 phosphorylation from the EpoR mutant
H/Y343F that was defective in Ras but not Jak2 activation.
The late induction of p32/p30 phosphorylation at 2 hours after Epo
stimulation indicates that it is a signaling event downstream of the
Jak/Ras signaling pathways that are activated transiently (1 to 60 minutes) after Epo/IL-3 stimulation. Using an IL-3-responsive cell
line (DA3EpoR-H/Y343F) that expresses an EpoR mutant competent in
activating the Jak2 but not the Ras pathway,5 we examined the relative roles of the Jak2 and Ras activations in the induction of
p32/p30 phosphorylation.
As reported previously, stimulating DA3EpoR-H/Y343F cells with IL-3 or
Epo induced rapid Jak2 phosphorylation (data not shown). We found that
IL-3 induced p32/p30 phosphorylation in DA3EpoR-H/Y343F cells at 2 to 4 hours after stimulation (compare Fig 3, lanes 3 and 1), similar to the
induction of p32/p30 phosphorylation via the wild-type EpoR in D/V and
D/C cells (Fig 2A). However, p32/p30 phosphorylation in DA3EpoR-H/Y343F
cells stimulated with Epo for 2 to 4 hours (Fig 3, lanes 7 and 8) was
barely above the levels of growth factor-deprived cells (Fig 3, lane
5), indicating that the EpoR-H/Y343F mutant was less effective in
inducing p32/p30 phosphorylation. Curiously, there were reduced p32/p30
phosphorylation signals in these cells at 30 minutes after stimulation
by Epo (Fig 3, lane 7) or IL-3 (Fig 3, lane 2), which was also visible in D/V cells at 30 to 60 minutes (Fig 2A, lanes 4 and 5).
p32/p30 phosphoproteins associate with SHP-1-C and SHP-1 in D/C
cells.
To detect phosphotyrosine proteins that interact with SHP-1-C or the
endogenous SHP-1, we immunoprecipitated the mutant and SHP-1 from D/V
and D/C cells using antibodies specific for the mutant (anti-KT3) or
SHP-1 (anti-SHP-1-M; Fig 1A). p32 and p30 phosphoproteins were detected
in the SHP-1-C and SHP-1 immunocomplexes from the D/C
(Fig 4A, lanes 4 and 6), demonstrating that
p32/p30 associated with SHP-1-C and SHP-1 in vivo in D/C cells. This
association was specific, because the hyperphosphorylated p56 and p53
in D/C cells were not detected in the immunocomplexes under comparable conditions. The result also showed that SHP-1 and SHP-1-C associated with the phosphoproteins independently because SHP-1 was not detected in the SHP-1-C immunocomplexes (Fig 4A, lane 4). Because the GST fusion
protein of SHP-1 SH2 domains bound to phosphorylated EpoR but not
p32/p30 in vitro (Fig 4B, lane 8), it indicated that the association of
p32/p30 with SHP-1 was mediated via the C-terminus of the phosphatase
and that the SHP-1-C mutant competes against SHP-1 for p32/p30
association. In addition, phosphotyrosine proteins of approximately 48 and 26 kD (p48 and p26) were also detected in the immunocomplexes (Fig
4A, lanes 4 and 6) with weaker and less consistent signals, suggesting
that their association with SHP-1 and SHP-1-C was unstable or indirect.
Interestingly, phosphorylated p32/p30 were not detected in SHP-1
immunocomplexes from D/V cells (Fig 4A, lane 3). Whether
unphosphorylated forms of p32/p30 associate with SHP-1 in D/V cells
remains to be determined.
Synthetic peptide of SHP-1-C bind to p32/p30 in D/V and D/C cells and
is inactive against SHP-1 PTPase in vitro.
To further define the interactions of SHP-1 with p32/p30, we examined
the binding of a synthetic peptide of the SHP-1-C to the
phosphoproteins in vitro. The peptide bound to p32/p30 in D/V and D/C
cells (Fig 5A, lanes 3 and 4 of the upper
panel). This association of p32/p30 with the SHP-1-C peptide did not
require the SHP-1 phosphatase (Fig 5A, lanes 3 and 4 of the lower
panel), consistent with the independent association of p32/p30 with
SHP-1-C and SHP-1 in vivo (Fig 4A). Interestingly, phosphorylated
p32/p30 in association with the peptide from D/V were twofold to
threefold higher than those from D/C cells. This indicated that
pre-existing complexes of SHP-1-C mutant and p32/p30 in D/C cells (Fig
4A, lane 6) reduced the amount of p32/p30 available for binding to the
SHP-1-C peptide. It also demonstrated that p32/p30 in D/V cells had
SHP-1-C binding activity. Thus, the failure of p32/p30 to
coimmunoprecipitate with SHP-1 in D/V cells (Fig 4A, lane 3) may result
from failure of the C-terminus of SHP-1 to interact with p32/p30.
To examine the effect of the SHP-1-C peptide on SHP-1 PTPase activity,
we performed in vitro SHP-1 PTPase assays. We found that the peptide
had no marked effect on the PTPase activity of a GST fusion protein of
SHP-1 in the absence (Fig 5B) or presence of phosphotyrosine peptides
that bind to the SH2 domains of SHP-1 (data not shown). Similarly, we
failed to detect marked differences in the PTPase activities of the
SHP-1 proteins immunoprecipitated from D/V and D/C cells (data not
shown).
Induction of p32/p30 phosphorylation at 2 to 18 hours after Epo
stimulation.
The induction of p32/p30 phosphorylation at 2 hours after stimulation
(Fig 2A) prompted us to examine p32/p30 phosphorylation in D/V and D/C
cells at later stages after stimulation. Consistent with data shown in
Fig 2, p32/p30 phosphorylation was induced about twofold to threefold
at 2 hours after Epo stimulation in both cell populations
(Fig 6A, lanes 2 and 8). The
phosphorylation of p32/p30 was further induced twofold to fourfold from
6 to 18 hours after stimulation, with those in the D/C cells threefold to sixfold higher than those in the D/V cells at each of the time points (lanes 3 through 6 and 9 through 12). Increased phosphorylation in proteins of 48 and 26 kD (p48 and p26) in D/C cells was also detected (Fig 6A). Phosphorylated p32/p30 coimmunoprecipitated with
SHP-1 (Fig 6B, lanes 1 through 6) and the SHP-1-C mutant (data not
shown) from D/C but not D/V cells (Fig 6B, lanes 1 through 6), despite
their existence in D/V cell lysates (Fig 6A, lanes 1 through 6).
Similar results were derived from D/V and D/C cells stimulated with
IL-3 (data not shown).
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| Fig 6.
p32/p30 phosphorylation in D/V and D/C cells was mainly
induced at 2 to 18 hours after Epo stimulation. Growth factor-deprived D/V and D/C cells were stimulated with Epo for various times. Cell
lysates were prepared and used for immunoprecipitation with an
anti-SHP-1 antibody. The cell lysates and immunocomplexes were analyzed by SDS-PAGE/Western blotting with antibodies as indicated. The
positions of phosphoproteins and protein size markers are indicated.
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Hyperphosphorylated p32/p30 are major novel proteins in SHP-1-C
immunocomplexes.
To determine the relative amounts of p32/p30 in SHP-1-C
immunocomplexes, anti-KT3 immunocomplexes from D/V or D/C cells were separated in an SDS-PAGE gel in duplicates and analyzed by either direct Coomassie Blue staining or by Western blotting with an anti-ptyr
antibody. As expected, hyperphosphorylated p32/p30 were detected by
Western blotting in the immunocomplex from D/C cells (Fig 7, lane 3) but not from D/V cells (Fig
7, lane 4). Two proteins that comigrate with the hyperphosphorylated
p32/p30 were detected uniquely in the immunocomplex from D/C cells (Fig
7, lane 1), indicating that these two proteins were likely to be the
hyperphosphorylated p32/p30 and were among the major proteins in
association with SHP-1-C. In addition, tyrosine-phosphorylated p48 and
p26 were also detected in complexes with p32/p30 (Fig 7, lane 3). Using a similar approach, we found that proteins of 32 and 30 kD were also
among the major cellular proteins associated with the SHP-1-C peptide
in binding assays (data not shown). However, it was not clear whether
SHP-1-C interacted with p32/p30 directly or indirectly, because
additional cellular proteins were also presented in the immunoprecipitation and peptide binding complexes.
View larger version (80K):
[in this window]
[in a new window]
| Fig 7.
Hyperphosphorylated p32/p30 are major novel proteins in
SHP-1-C immunocomplexes. Cell lysates were prepared from approximately 1 × 108 D/V or D/C cells and incubated with anti-KT3
antibody. Immunocomplexes were separated in a 10% SDS-PAGE gel in
duplicates with 90% of the immunocomplexes loaded in lanes 1 and 2 and
about 0.1% in lanes 3 and 4. Samples in lanes 1 and 2 were detected by
direct staining with Coomassie Blue R-250 and those in lanes 3 and 4 were detected by Western blotting with an anti-ptyr
antibody. The positions of p32/p30, p48, p26, IgH/IgL, and protein size markers (M) are indicated.
|
|
A number of cellular proteins have similar sizes as p32/p30 and are
tyrosine phosphorylated or regulated during cell cycle progression.
These include p34Cdc2, p33Cdk2, p38Cdk2, p33Cdk4, p40Cdk6, p36cyclinD1,
p37/42CAK, p33cyclinD3, p36PCNA, and p27Kip1. Antibodies against these
proteins failed to react with p32/p30 (data not shown).
Hyperphosphorylation and SHP-1 association of p32/p30 in motheaten
hematopoietic cells.
The hyperphosphorylation of p32/p30 in D/C cells indicated that p32/p30
are potential novel SHP-1 substrates. In support of this, we found that
hyperphosphorylated proteins that comigrated with the p32/p30 of D/C
cells in SDS-PAGE were also detectable in the spleen cells
(Fig 8A) and bone marrow macrophages (Fig 8C) of viable motheaten mouse, which express two forms of catalytically inactive SHP-1 proteins.16 Reminiscent of the results from
D/V and D/C cells, we also found that the hyperphosphorylated p32/30 coimmunoprecipitated with the inactive SHP-1 proteins from viable motheaten spleen cells (Fig 8B, lane 2) and macrophages (data not
shown), whereas the SHP-1-C peptide bound to p32/p30 from normal as
well as motheaten cells. In consistence with previous studies, we
detected a phosphoprotein of 120 to 130 kD20 and several
yet-unidentified proteins in the SHP-1 immunocomplexes from the
motheaten cells (Fig 8B, lane 2). The larger number of phosphoproteins
in association with the catalytically inactive SHP-1 in motheaten cells
than with the SHP-1 in D/C cells is consistent with the notion that
SHP-1-C only block the C-terminal function of the phosphatase. As in
D/V cells, GM-CSF induced an early and modest p32/p30 phosphorylation
in normal mouse macrophages (Fig 8C, lane 2). But we failed to detect
marked induction of p32/p30 phosphorylation in normal or motheaten
macrophages after CSF-1 or GM-CSF stimulation at later stages (data not
shown). It is known that the primary macrophages express lower levels
of hematopoietic receptors and proliferate at a much slower rate than
the established DA3 cell lines. Whether the lack of marked late
induction of p32/p30 in these primary cells was due to these
differences or whether it was a cell lineage-specific character has not
been determined.
View larger version (64K):
[in this window]
[in a new window]
| Fig 8.
Hyperphosphorylation and SHP-1 association of p32/p30 in
motheaten macrophages. (A) Total cell lysates were prepared from D/V or
D/C cells and from the spleen cells of normal (+/+) or viable
motheaten mice (mev/mev). The lysates were analyzed by SDS-PAGE/Western
blotting with antibodies as indicated. (B) Cell lysates from the spleen
cells of normal (+/+) or viable motheaten mice (mev/mev) were
incubated with an anti-SHP-1 antibody for immunoprecipitation assays
or with the synthetic SHP-1-C peptide in binding assays. SHP-1
immunocomplexes (lanes 1 and 2) and SHP-1-C peptide-binding proteins
(lanes 3 and 4) were analyzed by SDS-PAGE/Western blotting with
antibodies as indicated. (C) Bone marrow-derived macrophages from
normal (+/+) or viable motheaten mice (mev/mev) were deprived of
growth factors and then stimulated with GM-CSF for various times.
Lysates from these cells were analyzed by SDS-PAGE/Western blotting
with antibodies as indicated. Positions of phosphotyrosine proteins and
protein size markers are indicated.
|
|
| |
DISCUSSION |
DA3EpoR cells expressing the SHP-1-C mutant (D/C cells) show heightened
cell proliferation in response to Epo/IL-3 and contain hyperphosphorylated proteins similar to those detected in the motheaten
cells that lack functional SHP-1. This indicates that the mutant blocks
the function of the endogenous SHP-1 in D/C cells and causes
hyperphosphorylation in SHP-1 substrates involved in mitogenic
signaling of Epo/IL-3. Identification of these substrates is essential
in understanding SHP-1 function and in elucidating mitogenic signaling
pathways of the hematopoietic growth factors.
p32/p30 are likely major SHP-1 substrates, because they are among the
prominently hyperphosphorylated proteins in D/C cells as well as in
motheaten cells. The observation that p32/p30 coimmunoprecipitate with
SHP-1 in D/C and the motheaten cells demonstrates their interaction with the phosphatase in vivo and supports a role for SHP-1 in directly
dephosphorylating the two proteins. Indeed, we found that the
phosphorylation of p32/p30 was sensitive to SHP-1 PTPase activity in
vitro (unpublished data). Moreover, the marked and constitutive hyperphosphorylation of p32/p30 in these SHP-1-deficient cells demonstrates that their dephosphorylation is regulated mainly by
SHP-1. This is in contrast to Jak2 dephosphorylation, in which SHP-1
appears to play a limited role as indicated by the transient and modest
nature of Jak2 hyperphosphorylation after GM-CSF stimulation in
motheaten macrophages20 and the lack of major effect of
SHP-1-C mutant on Jak2 phosphorylation in D/C cells (Fig 2). It is
expected that the Jak family kinases are regulated by additional
phosphatase(s), because they are ubiquitously expressed, whereas SHP-1
expression is restricted predominantly in hematopoietic cells. Because
of their late induction and small sizes, p32/30 escaped detection in
previous studies focused on the relatively large Jak/Stat proteins and
on signaling events induced immediately after Epo/IL-3 stimulation.
Several lines of evidence indicate that p32/p30 are likely to be
involved in the mitogenic signaling of Epo and IL-3. Because they are
hyperphosphorylated in D/C cells and motheaten cells, which are
hyperproliferative in response to Epo/IL-3 and GM-CSF, respectively, a
correlation exists between p32/p30 hyperphosphorylation and a
heightened growth response to hematopoietic growth
factors. Moreover, we found that SHP-1 deficiency in D/C cells (Fig 2) and motheaten cells20 has little or limited effect on the
phosphorylation of Jak2/Stat5 and Epo/IL-3 receptors. On the other
hand, the other hyperphosphorylated proteins in D/C cells (eg, p56/53
in Fig 2) were not detected in association with the phosphatase and
thus may be not directly dephosphorylated by SHP-1. Thus, SHP-1 may downregulate mitogenic signaling of IL-3/Epo in large part by dephosphorylating p32/p30. SHP-1 regulates additional signaling pathways in hematopoietic cells, as indicated by its association with
various membrane receptors and the multiple hematopoietic abnormalities
in motheaten mice. The involvement of p32/p30 in these signaling
pathways remain to be determined.
p32/30 is clearly involved in late signaling events, as indicated by
the marked induction of p32/p30 phosphorylation at 2 to 18 hours. Their
potential involvement in early signaling is suggested by the modest,
but reproducible, induction immediately after ligand stimulation. They
appear to function downstream of the early Jak2/Ras pathways, because
Epo stimulation of the EpoR-H/Y343F mutant that activates the Jak2, but
not the Ras, pathway results in reduced induction of p32/p30
phosphorylation. Because p32/p30 phosphorylation is still modulated
modestly by the mutant that activates the Jak2 pathways, it is likely
that both Ras and Jak2 pathways are required for optimal induction of
p32/p30 phosphorylation. On the other hand, the marked induction of
p32/p30 phosphorylation at 2 to 18 hours indicates them as signaling
molecules in cell cycle progression. Consistent with this, we found
that D/C cells showed a 16% increase in G2/M population and a
corresponding decrease in G0/G1 population in comparison to D/V cells
when maintained in the presence of IL-3 (unpublished
data), indicating that D/C cells progress faster during
cell cycle than D/V cells. Because p32/p30 phosphorylation may be cell
cycle related, the differential cell cycle distributions of D/V and D/C
cells may contribute to the differences of p32/p30 phosphorylation in
these cells. Further studies to identify p32/p30 and characterize their
function are clearly needed to define their role in mitogenic signaling
and cell cycle progression. In this regard, the stable association of
p32/p30 with SHP-1-C mutant in D/C cells allows us, from a unique
position, to identify the proteins by micropeptide sequencing and to
define their roles in mitogenic signaling in hematopoietic cells.
p32/p30 phosphorylation is regulated by SHP-1 via its C-terminus. We
demonstrate that a synthetic peptide containing the SHP-1 C-terminal 40 amino acids has no marked effect on the PTPase activity of recombinant
SHP-1 in vitro (Fig 5B). Moreover, the peptide forms stable complexes
with p32/p30 but not SHP-1 in vitro in binding assays (Fig 5A) and in
vivo when it was expressed as the SHP-1-C mutant (Fig 4A). p32/p30
binding activity is also detectable in intact SHP-1, as indicated by
the association of p32/p30 with SHP-1 in D/C and motheaten cells.
Because expression of the peptide and its association with p32/p30 in
vivo correlates with an increase in p32/p30 phosphorylation, it
indicates that the SHP-1-C mutant competes against the phosphatase for
binding to p32/p30, resulting in p32/p30 hyperphosphorylation. We also
found that p32/p30 in D/V and normal mouse cells are capable of binding
to the SHP-1-C peptide in vitro, although they do not associate with
the endogenous SHP-1 in vivo. This suggests that the p32/p30 binding
activity in the endogenous SHP-1 is regulated and that SHP-1-C mutant
may interfere with the regulation to cause SHP-1/p32/p30 interactions in D/C cells. In this regard, it is interesting to note that a tyrosine
phosphorylation site (Y564) resides in the SHP-1-C
mutant.44 The demonstrated binding of the synthetic peptide
of SHP-1 C-terminus to p32/p30 suggests the possibility that
phosphorylation of C-terminal tyrosines Y564 may affect SHP-1
configurations and its interactions with p32/p30. Alternatively, it may
regulate SHP-1 phosphatase activity and SHP-1 dephosphorylation of
p32/p30. Additional studies are clearly needed to define this
regulation and the role of Y564 phosphorylation in p32/p30 association.
An intramolecular inhibitory function was proposed previously for the
C-terminus of SHP-1, because truncation of the last 35 to 41 amino
acids of SHP-1 activates the PTPase.34-36 However, direct
interactions of C-terminus of SHP-1 with the SH2 domains or the PTPase
catalytic domain have not been demonstrated.35 Furthermore,
the enzyme with truncation to the C-terminal 60 amino acids35 or the C-terminal 20 amino acids (our unpublished
data) behaves like the wild-type SHP-1. Thus, the
activation of SHP-1 caused by C-terminal truncation of amino acid 35-41 is complicated. Although our data indicate that the SHP-1-C mutant
affects the function of SHP-1 PTPase and causes p32/p30
hyperphosphorylation primarily by a competition mechanism, it remains
possible that SHP-1-C may inhibit SHP-1 through unstable and/or
transient interactions with the catalytic domain of SHP-1 in vivo and
affect SHP-1 substrates. Nevertheless, the small size of the SHP-1-C
and its potent effect in suppressing SHP-1 function make it an
appealing target for further development of biochemical and
pharmaceutical reagents to block SHP-1 activities and manipulate
hematopoiesis and immunity. The approach described in this study could
also be useful for analyzing the functions of other enzymes and for
identification of their substrates.
Previous studies from our group and others showed that SHP-1 binds, via
its SH2 domains in N-terminal region, to receptors and kinases in
hematopoietic cells. The association leads to activation of SHP-1
PTPase and dephosphorylation of potential substrates, including the Jak
kinases and ZAP-70 kinases. We demonstrate here that SHP-1 regulates
p32/p30 phosphorylation through an alternative mechanism involving the
C-terminus of the phosphatase and that p32/p30 may be key molecules
through which SHP-1 regulates mitogenic signals. Despite marked
advances in our understanding of mitogenic pathways, critical elements
that link signals from the early Ras/Jak pathways and the later cell
cycle progression remain to be fully characterized. p32/p30
phosphorylation is induced at early and, more significantly, late
stages of Epo/IL-3 mitogenic signaling and thus may be involved in
linking the early and late signaling events. The cloning of p32/p30 and
characterization of their functions will lead to the elucidation of
their role in mitogenic signaling pathways in hematopoietic cells.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted January 12, 1998.
Supported by grants from the American Cancer Society (DB-74554) and the
American Heart Association (NEO-94 074-GIA ) to T.Y.
Address reprint requests to Taolin Yi, PhD, Department of Cancer
Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave, NN1-25, Cleveland, OH 44195; e-mail: yit{at}cesmtp.ccf.org.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr G. Walter for the anti-KT3 hybridoma cell line, Dr
J. Ihle for the DA3EpoR-H/Y343F cell line, and Drs Christine Campbell
and Robert Silverman for critical reading of the manuscript.
| |
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Abstract
Introduction
Abstract