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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 132-138
HEMATOPOIESIS
From the Programme in Molecular Biology and Cancer, Samuel Lunenfeld
Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;
Department of Chemistry and Biochemistry, University of California, San
Diego, La Jolla, CA; PE SCIEX, Concord, Ontario, Canada; and the
Department of Molecular and Medical Genetics, University of Toronto,
Toronto, Ontario, Canada.
The Shc adaptor protein possesses 2 distinct phosphotyrosine (pTyr)
recognition modules
Cytokines play an important role in hematopoiesis by
regulating the proliferation, differentiation, and cellular functions of various hematopoietic cell lineages at different stages of development.1 Among these cytokines, interleukin 3 (IL-3)
can stimulate the growth, survival, and differentiation of multipotent progenitors and more mature cells, including those of erythroid, mast,
and granulocyte cell lineages. The high-affinity IL-3 receptor is
composed of a specific The The adaptor protein Shc has been implicated in linking growth factor,
cytokine, and antigen receptors to Ras signaling.7 Shc
contains an amino-terminal PTB domain, a central region (CH1), and a
carboxyl-terminal SH2 domain.8-11 The Shc SH2
and PTB domains have quite distinct binding specificities for
pTyr-containing motifs. The PTB domain requires residues amino-terminal
to the pTyr with the consensus sequence In addition to interacting with activated receptors and Grb2, Shc also
associates with the SH2-containing 5' inositol phosphatase (SHIP).21-23 This 145-kd protein contains an SH2 domain at
its amino-terminal, a central catalytic domain containing 2 motifs highly conserved among inositol polyphosphate 5 phosphatases, and a
carboxy-terminal tail with 2 potential PTB-binding motifs (NXXY
sequences) and several proline-rich sequences with the potential to
bind SH3 domains.21,22 SHIP becomes Tyr phosphorylated on activation of several receptors in hematopoietic cells and subsequently forms a complex with Shc.24-27 SHIP has been implicated in
inhibitory signaling by means of Fc Structure-function analyses of the The precise role of Shc in mediating IL-3-specific pathways and
biologic events has remained unclear. In this study, we used the
immature murine IC2 premast cell line, which depends on IL-3 for its
proliferation and viability, to examine the role of the different
modular domains and phosphorylation sites of Shc in IL-3 signaling by
analyzing wild-type and mutant forms of Shc. We found both in vitro and
in vivo that the PTB domain plays a central role in Shc phosphorylation
and consequently in Grb2 binding and association with SHIP. Analysis of
the binding properties of the Y239/240 and Y317 Shc phosphorylation
sites indicated that the pY317 site interacts preferentially with
Grb2-Sos1 and SHIP, whereas the pY239 site bound more weakly to Grb2.
Our data suggest that Shc may be involved in multiple signaling
pathways leading to the different biologic responses stimulated by
IL-3.
Cell lines and antibodies
Generation of IC2 cells expressing FLAG-tagged mutant forms of Shc
In vitro binding assays pGEX glutathione-S-transferase (GST)-Shc fusion constructs were generated and prepared as described previously.11 IC2 cells (4 × 107) were starved in RPMI-1640 medium containing 0.5% FBS and 1% bovine serum albumin (pH 7) (Sigma, St Louis, MO) for 6 hours and subsequently treated with 100 ng/mL recombinant mouse IL-3 (PharMingen, San Diego, CA) at 37°C for 5 minutes. Cells were washed once with cold phosphate-buffered saline (PBS) and then solubilized on ice with lysis buffer (0.1% Triton X-100, 150 mmol/L sodium chloride [NaCl], 50 mmol/L Tris [pH 7.6], 10% glycerol, 0.1 mmol/L sodium vanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin) for 30 minutes. Lysates were cleared by centrifugation at 12 000 rpm for 5 minutes and incubated for 2 hours at 4°C on a rocker with 10 µL glutathione-Sepharose beads containing 5 µg fusion protein. Beads were washed 3 times with lysis buffer and once with PBS buffer. Bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.Immunoprecipitations and Western blotting Cell lysates of IC2 or IC2 infected with Shc were prepared as described above. The supernatants were then incubated in the presence of the designated serum or monoclonal antibody and 50% Protein A- or antimouse IgG-Sepharose beads (Sigma) for 2 hours at 4°C. Immune complexes were washed as described above and boiled for 3 minutes with sample buffer for SDS-PAGE. Eluted proteins were separated by electrophoresis and transferred to supported nitrocellulose (Schleicher & Schuell, Dassel, Germany). For immunoblotting, membranes were blocked for 1 hour at room temperature in PBS-0.2% Tween 20 (PBST) buffer containing 1% gelatin and then incubated with the designated antibodies in PBST with 0.1% gelatin for 1 hour at 4°C. Blots were washed 3 times for 10 minutes each time with PBST. Membranes were incubated for 1 hour with horseradish peroxidase conjugated either to Protein A or goat antimouse antibodies (Bio-Rad, Hercules, CA) in PBST with 0.5% gelatin. Reactive proteins were visualized with an enhanced chemiluminescence detection system (Amersham, England) as directed by the manufacturer. Densitometric scanning was performed with a high-resolution charged coupled device (CCD) camera (ImageQuant, Molecular Dynamics, Sunnyvale, CA).Phosphopeptide inhibition assays The phosphopeptides used for the inhibition studies were the 11-mers EMINPNpYIGMG (p145-1), MFENPLpYGSVS (p145-2), and IISDPEpYLLDQ (p145-3), corresponding to the sequence flanking Y917, Y1020, and Y798, respectively, within SHIP, and the 16-mer QLPSFDFNGPpYLGPPQ (pIL-3R )
corresponding to the sequence flanking Y577 within the c chain of
the IL-3 receptor. Unstimulated IC2 cell lysates or IC2 lysates
stimulated with IL-3 for 5 minutes were incubated with GST-Shc-PTB
fusion protein and the appropriate concentration of phosphopeptide and
rotated at 4°C for 2 hours. GST beads were then washed as described
above, and the bound proteins were eluted by boiling for 3 minutes in
SDS-sample buffer and subjected to Western blot analysis with anti-pTyr
4G10 monoclonal antibodies.
Affinity chromatography and protein identification by quadrupole/time-of-flight mass spectrometry The synthetic phosphopeptides used to identify associated proteins were biotin- -aminocaproic acid (Aca)-DHQpYYNDFPGKE,
biotin-Aca-DHQYpYNDFPGKE, biotin-Aca-DHQpYpYNDFPGKE, and
biotin-Aca-DPSpYVNIQNLD, corresponding to the sequences flanking Y239,
Y240, Y239/240, and Y317, respectively, within Shc. For each affinity
reagent, a 100-µL bed volume of streptavidin agarose resin (Sigma)
was washed with PBS and then resuspended in an excess of biotinylated
peptide. After mixing at room temperature for 15 minutes, the resins
were poured into a 1.5-mL chromatography column (Bio-Spin disposable
chromatography column; Bio-Rad) and washed extensively with cold Tris
buffer (50 mmol/L Tris-hydrochloric acid [pH 7.5], 50 mmol/L NaCl, 1 mmol/L EDTA, 2 mmol/L benzamidine, and 4 mmol/L dithiothreitol). IC2
cell lysate from 1.3 × 109 cells was prepared as
described above, diluted with 2 parts volume Tris buffer, and applied
to each column. The columns were washed with 3 mL Tris buffer and
eluted with 1 mL of 50 mmol/L phenylphosphate in Tris buffer. Eluted
proteins were precipitated by adding trichloroacetic acid to a final
concentration of 12%, and the resulting pellets were washed with
anhydrous ether and ethanol (80/20 vol/vol). Proteins were resolved by
using 9% SDS-PAGE and visualized with use of silver staining as
described by Shevchenko et al.40 Bands selected for mass
spectrometry (MS) analysis were excised from the gel and subjected to
tryptic digestion.40 MS measurements were carried out on a
Q-TOF apparatus (MDS-Sciex, Concord, ON, Canada).41
The PTB domain of Shc interacts with specific pTyr-containing proteins in mast cells stimulated with IL-3 The Shc adaptor protein can potentially interact with Tyr-phosphorylated proteins through its amino-terminal PTB domain, its carboxy-terminal SH2 domain, or both. To examine whether these distinct pTyr-recognition modules of Shc associate with different proteins in IL-3-stimulated cells, we incubated lysates from IC2 premast cells treated with IL-3 with GST fusion proteins containing the Shc PTB or SH2 domains (Figure 1A). Several pTyr-containing proteins of approximately 150 to 120, 115, 98, 56, 52, and 41 kd associated with the GST-PTB-domain fusion protein after IL-3 treatment, whereas neither GST alone nor the GST-SH2-domain fusion protein formed detectable complexes with phosphorylated proteins (Figure 1B). In addition, a mutant form of the PTB domain in which arginine 175, which is critical for pTyr recognition, is substituted with methionine,42,43 failed to recognize IL-3-induced pTyr-containing proteins. Two of the proteins known to coimmunoprecipitate with Shc after IL-3 stimulation are the IL-3c
subunit and the p145 inositol phosphatase SHIP21 (Figure
1B, C). Interestingly, a prominent phosphoprotein of approximate
150-120 kd was present in the GST-PTB precipitate (Figure 1B). To
investigate the identity of this band, PTB-associated proteins
from IL-3-stimulated cells were immunoblotted with anti-SHIP or
anti-c antiserum. Both SHIP and the c proteins were identified in
the GST-PTB complex (Figure 1D).
Specific phosphopeptides containing the NXXY motifs of SHIP and c chain of the IL-3 receptor contains the sequence PSFDFNGPYLGPP within its
membrane-proximal domain, which provides a potential Shc PTB binding
site.45 We investigated whether binding of the Shc PTB
domain to SHIP or the c subunit of the IL-3 receptor in mast cell
lysates stimulated with IL-3 is mediated specifically by these NXXpY
motifs. Lysates from IC2 cells treated with IL-3 for 5 minutes were
incubated with GST-PTB domain fusion protein in the presence of
increasing concentrations of phosphopeptides corresponding to the NXXpY
sequences of SHIP (p145-1 and p145-2) or of the IL-3 receptor c
chain (pIL-3R). All 3 NXXpY phosphopeptides, but not a control
phosphopeptide (p145-3), inhibited binding of the Shc PTB domain to
SHIP, c, and other pTyr proteins in the mast cell lysates (Figure
2A). Reprobing the blots with anti-SHIP and
anti-c sera confirmed these results (Figure 2B, C, and data not
shown). These results indicate that the Shc PTB domain is primarily
responsible for the recognition of pTyr-containing proteins by the Shc
adaptor observed in IL-3-stimulated IC2 cells. The data also suggest
that these associations depend on the presence of a phosphorylated NXXY
motif in the Shc binding partners.
Mutations in Shc affect its Tyr phosphorylation in vivo in response to IL-3 Previous studies showed that Shc becomes Tyr phosphorylated in vitro and in vivo after stimulation with either IL-3 or epidermal growth factor (EGF) at 3 major Tyr phosphorylation sites Y239, Y240, and
Y317.12,16,46,47 We examined the in vivo roles of the Shc
PTB and SH2 domains, as well as the functions of each Shc
phosphorylation site, in IL-3 signaling. For this purpose, IC2 mast
cells were infected with retroviral vectors encoding either wild-type
or mutant forms of p52 Shc in which either the PTB or SH2 modular
domains or the Tyr phosphorylation sites had been inactivated (Figure
3A). These proteins were all tagged with a
FLAG epitope at their amino-terminals. Stable cell lines were established from pools of infected cells, and the expression levels of
the different FLAG-tagged Shc proteins were assessed by immunoblotting with anti-FLAG antiserum (Figure 3B).
The Shc PTB domain and Tyr residues Y239 and Y317 are required for complex formation of Shc with SHIP and Grb2 in mast cells To examine whether the mutant forms of Shc can associate with SHIP, Grb2, or both, after IL-3 stimulation, we treated mast cells expressing the different Shc mutant proteins with IL-3. Cells were then lysed, immunoprecipitated with anti-FLAG antibodies, and immunoblotted with either anti-pTyr or anti-Grb2 antibodies. Densitometric analysis was performed on all blots (Table 1). Wild-type Shc coprecipitated with both SHIP and Grb2 after IL-3 stimulation. Substitution of either Y239 or Y240 had only a small effect on association with Grb2 and SHIP, whereas replacement of both Y239 and 240 had a more significant effect on association with SHIP than with Grb2 (Figure 4 and Table 1). Shc mutants possessing either an inactive PTB domain (R175M Shc and 2M Shc) or lacking all 3 major phosphorylation sites (3F Shc) were unable to associate with SHIP or Grb2. Furthermore, substitution of Y317 resulted in a markedly impaired association with SHIP and Grb2, although residual binding to both proteins was detected (Figure 4 and Table 1). Reprobing this blot with anti-Shc antibodies confirmed similar expression levels of the FLAG-tagged Shc proteins (Figure 4). Thus, these results indicate that in mast cells, an active PTB domain as well as IL-3-induced phosphorylation of Shc, is required for high-affinity interactions with SHIP and Grb2.
The aim of the current study was to analyze the function of
the modular domains and Tyr-phosphorylation sites of Shc in IL-3 signaling and its effects in IL-3 cellular responses. Data obtained using coprecipitation experiments and in vitro binding to GST-fusion proteins indicated that 2 of the main phosphoproteins interacting with
Shc after stimulation of IC2 mast cells with IL-3 were the receptor
We thank Dr G. Caruana and Dr T. Kubiseski for discussions and critical
review of this manuscript.
Submitted October 7, 1999; accepted February 24, 2000.
Supported by grants from the Medical Research Council of Canada (MRC),
the National Cancer Institute of Canada (NCIC), and MDS-Sciex. L.V. was
supported by an MRC fellowship and P.v.d.G. was a postdoctoral fellow
of the NCIC. T.P. is a distinguished MRC professor.
Reprints: Tony Pawson, Programme in Molecular Biology
and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
600 University Ave, Toronto, ON M5G 1X5, Canada; e-mail: pawson{at}mshri.on.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
subunit of 60 to 70 kd and a 
XNXXY (where
RIIB on mast and B
cells,
) or IC2 cells treated (+)
with IL-3 (100 ng/mL) for 5 minutes were precipitated with the
GST-PTB-domain fusion protein in the presence and absence of
phosphopeptides corresponding to the NXXpY motifs of SHIP (p145-1 and
p145-2) and the 
