From the
Epidermal growth factor (EGF) stimulation of HepG2 and NIH 3T3
cells expressing high levels of the human EGF receptor (3T3/ER)
resulted in the tyrosine phosphorylation of a 115-kDa protein that was
co-immunoprecipitated with the Src homology 2 (SH2) domain containing
protein tyrosine phosphatase, SHPTP2. In contrast, activation of the
EGF receptor resulted in a relatively low level (<1%) of the total
SHPTP2 pool associated with the tyrosine-autophosphorylated EGF
receptor itself. Similarly, quantitative immunoprecipitations also
demonstrated that only trace amounts of the total EGF receptor pool
were associated with SHPTP2. Further, activation of the EGF receptor
did not result in any significant tyrosine phosphorylation of SHPTP2
and/or the association of the 115-kDa protein with Grb2. In comparison,
activation of Jurkat cells with a T cell receptor agonist monoclonal
antibody resulted in the co-immunoprecipitation of a 120-kDa
tyrosine-phosphorylated protein with Grb2 and a 105-kDa protein with
SHPTP2. Thus, these data have identified the 115- and 105-kDa proteins
as the predominant SHPTP2-associated phosphotyrosine proteins in EGF-
and T cell receptor-activated cells, respectively.
Protein phosphatases are typically thought to function as the
inactivation arms of protein kinase-mediated signaling pathways.
However, there are several recent examples whereby protein
tyrosine-specific phosphatases play an essential positive signaling
role. For example, in T cell receptor signaling, the CD45 protein
tyrosine-specific phosphatase dephosphorylates the inhibitory
carboxyl-terminal tyrosine phosphorylation site on Fyn and/or Lck
allowing for activation of tyrosine protein kinase
activity(1, 2, 3) . Similarly, recent studies
have demonstrated that SHPTP2 protein tyrosine-specific phosphatase
plays an important positive role in tyrosine kinase downstream
signaling. Microinjection of SHPTP2-specific antibodies was observed to
block insulin-stimulated DNA synthesis and expression of dominant
interfering SHPTP2 mutants inhibited activation of mitogen-activated
protein kinase, c-fos transcription, DNA synthesis,
and fibroblast growth factor-stimulated Xenopus oocyte
mesoderm
induction(4, 5, 6, 7, 8) .
These data have provided substantial evidence demonstrating a
positive signaling role for SHPTP2 in mediating tyrosine kinase growth
factor receptor action. To identify potential physiological targets for
SHPTP2, previous studies have observed that the SH2 domains of SHPTP2
result in the targeting to the tyrosine-phosphorylated epidermal growth
factor (EGF)
We and others have recently observed that SHPTP2 functions as
a positive mediator in the regulation of tyrosine kinase receptor
downstream
signaling(4, 5, 6, 7, 8) . To
identify potential targets for this phosphatase, we assessed the
presence of phosphotyrosine-containing proteins that associated with
SHPTP2 (Fig. 1). Stimulation of NIH 3T3/ER cells with 100 ng/ml
EGF for 5 min resulted in a marked increase in the tyrosine
phosphorylation of the EGF receptor (ER) following immunoprecipitation
with an ER-specific antibody (Fig. 1A, lanes1 and 2). In contrast, immunoprecipitation of
SHPTP2 primarily resulted in the co-immunoprecipitation of a 115-kDa
protein and another band migrating at approximately 55 kDa (Fig. 1A, lanes3 and 4).
Surprisingly, at this exposure level there was no detectable
co-immunoprecipitation of the tyrosine-phosphorylated ER. To assess the
presence of the ER in the SHPTP2 immunoprecipitates, panelA was overexposed (Fig. 1B). At this
exposure level, we were able to detect a small amount of an
EGF-stimulated phosphotyrosine band that co-migrated with the ER in the
SHPTP2 immunoprecipitates (Fig. 1B, lanes3 and 4). In addition to the predominant EGF-stimulated
115-kDa protein, several other lower molecular weight bands were
detected. These additional SHPTP2-associated phosphotyrosine-containing
bands probably represent degradation products of the 115-kDa protein.
In a complementary approach, control and
EGF-stimulated NIH 3T3/ER cells were initially immunoprecipitated with
an ER antibody and subsequently subjected to Western blotting with the
ER antibody (Fig. 2B, lanes1 and 2). Under these conditions the ER antibody was able to
immunoprecipitate greater than 95% of the total pool of ER protein
present in the whole cell detergent extracts (data not shown). However,
there was no detectable ER co-immunoprecipitated SHPTP2 protein (Fig. 2B, lanes5 and 6).
Similarly, SHPTP2 immunoprecipitation of the ER-cleared supernatants
did not result in the co-immunoprecipitation of the ER (Fig. 2B, lanes3 and 4) but
did immunoprecipitate the SHPTP2 protein (Fig. 2B, lanes7 and 8). Thus, these data indicated
that essentially an undetectable level of the total SHPTP2 protein pool
was associated with the ER. Similarly, the total amount of the ER
associated with SHPTP2 was also relatively low. The fact that a
detectable, albeit small amount, of tyrosine-phosphorylated ER was
co-immunoprecipitated with SHPTP2 (Fig. 1) probably reflects a
greater sensitivity of the PY20-HRP antibody compared with the SHPTP2
and ER antibody used in the Western blots (Fig. 2). Nevertheless,
all these data were consistent with the ER playing a minor role in the
association of SHPTP2 whereas the predominant SHPTP2 binding
phosphotyrosine protein in EGF-stimulated 3T3/ER cells was apparently
the 115-kDa species.
To determine if the EGF-stimulated association
of SHPTP2 with the tyrosine-phosphorylated 115-kDa protein was unique
to NIH 3T3 cells genetically engineered to express high levels of the
human ER, we directly compared the 3T3/ER cells with the human
hepatoblastoma cell line, HepG2 (Fig. 3). Phosphotyrosine
immunoblotting of whole cell detergent extracts demonstrated the
EGF-stimulated tyrosine phosphorylation of the ER and proteins of
approximately 55 and 42 kDa in the 3T3/ER cells (Fig. 3A, lanes1 and 2). The
55- and 42-kDa proteins most likely reflect the tyrosine
phosphorylation of Shc and ERK2. In any case, EGF treatment of HepG2
cells resulted in a significantly weaker tyrosine phosphorylation of
the ER, consistent with the lower levels of the ER in the HepG2 cells
compared with the 3T3/ER cell line (Fig. 3A, lanes3 and 4). As previously observed, SHPTP2
immunoprecipitation of extracts from EGF-stimulated 3T3/ER cells
resulted in the predominant co-immunoprecipitation of the 115-kDa
phosphotyrosine-containing protein with a relatively low amount of the
tyrosine phosphorylated ER (Fig. 3B, lanes1 and 2). Similarly, EGF stimulation of the
HepG2 cells also demonstrated that the predominant SHPTP2
co-immunoprecipitated phosphotyrosine-containing protein was pp115 (Fig. 3B, lanes3 and 4). As
expected, a small amount of tyrosine-phosphorylated ER was detected in
the SHPTP2 immunoprecipitate of HepG2 cell extracts (Fig. 2B, lane4) when the gel was
overexposed (data not shown). These data demonstrate that the
EGF-stimulated tyrosine phosphorylation and association of pp115 with
SHPTP2 do not result from high level of ER expression and also occur in
a typical EGF-responsive cell line.
We thank Dr. John Koland for providing the human EGF
receptor expressing NIH 3T3 cells. We also thank Dr. Gary Koretzky for
providing the Jurkat T cells and the OKT3 monoclonal antibody.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)receptor, platelet-derived growth
factor receptor, and insulin receptor substrate-1
(IRS1)(9, 10, 11, 12, 13) . In
addition, it has been reported that tyrosine-phosphorylated IRS1
functions as a substrate for SHPTP2 in vitro(14) .
However, the tyrosine dephosphorylation of IRS1 is difficult to
reconcile with a positive effector role for SHPTP2 tyrosine phosphatase
activity, and a quantitative assessment of these associations has not
yet been determined. In this paper, we have determined that only a
small fraction of SHPTP2 associates with the EGF receptor and that a
115-kDa tyrosine-phosphorylated protein (pp115) is the major SHPTP2
binding protein in EGF-stimulated cells.
Cell Culture
HepG2 cells were obtained from the
American Type Tissue Culture collection and were maintained in
Dulbecco's modified Eagle's medium plus 10% fetal bovine
serum. NIH 3T3 cells overexpressing human EGF receptor (3T3/ER) were
obtained from Dr. John Koland (University of Iowa). 3T3/ER cells were
maintained in Dulbecco's modified Eagle's medium plus 10%
fetal bovine serum. The human Jurkat T cell line and TCR monoclonal
antibody (OKT3) were provided by Dr. Gary Koretzky (University of
Iowa). The Jurkat T cells were maintained in RPMI 1640 containing 10%
fetal bovine serum.
Immunoprecipitations and Western Blot
Analysis
Whole cell extracts were prepared by detergent
solubilization in lysis buffer (20 mM Hepes, pH 7.4, 1% Triton
X-100, 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10
µg/ml aprotinin, and 1.5 mM pepstatin) for 1 h at 4
°C. The resultant cell extracts (500 µg) were diluted 5-fold
using the lysis buffer without Triton X-100 and incubated with 4 µg
of a carboxyl-terminal SHPTP2 polyclonal antibody (Santa Cruz) or an
EGF receptor monoclonal antibody, LA1 (Upstate Biotechnology), for 2 h
at 4 °C. The primary polyclonal and monoclonal antibodies were
incubated with Protein A-agarose or Protein G PLUS-agarose,
respectively, for 1 h at 4 °C. The resulting immunoprecipitates
were then subjected to SDS-polyacrylamide gel electrophoresis. The
whole cell extracts and the immunoprecipitates were then subjected to
Western blot analysis using an amino-terminal SHPTP2 antibody
(Transduction Laboratories), an EGF receptor antibody (Transduction
Laboratories), or a phosphotyrosine antibody (PY20-HRP, Santa Cruz) and
visualized with the enhanced chemical luminescence (ECL) detection
system (Amersham Corp.).
Figure 1:
Identification of pp115 as the
predominant tyrosine-phosphorylated protein associated with SHPTP2
following EGF stimulation of 3T3/ER cells. NIH 3T3/ER cells were
incubated in the absence (lanes1 and 3) or
presence (lanes2 and 4) of 100 ng/ml EGF
for 5 min at 37 °C. Whole cell detergent lysates were prepared and
immunoprecipitated with an ER-specific monoclonal antibody (ER, lanes1 and 2) or with a
SHPTP2-specific polyclonal antibody (lanes3 and 4) as described under ``Experimental Procedures.''
The immunoprecipitates were then subjected to phosphotyrosine
immunoblotting using the PY20-HRP antibody and visualized by the ECL
method for 5 s (A) or for 2 min (B). IB,
immunoblot; IP, immunoprecipitate.
Since previous studies have demonstrated that SHPTP2 can associate
with EGF-stimulated tyrosine-phosphorylated ER(10) , we next
determined the relative extent of SHPTP2 binding compared with the
total cellular pool of ER and SHPTP2 (Fig. 2). Control and
EGF-stimulated NIH 3T3/ER cells were initially immunoprecipitated with
the SHPTP2 antibody and subjected to Western blotting with the ER
antibody (Fig. 2A, lanes1 and 2). Under these conditions, we were unable to detect any
significant level of ER protein by ER Western blotting. In contrast,
when the supernatant from the initial SHPTP2 immunoprecipitate was
subjected to a second round of immunoprecipitation using the ER
antibody, a strong positive ER Western blotting signal was readily
observed (Fig. 2A, lanes3 and 4). It should be noted that the apparent increase in the
molecular weight of the ER following EGF stimulation (Fig. 2A, lane4) compared with
unstimulated cells (Fig. 2A, lane3)
reflects a decrease in electrophoretic mobility due to the
phosphorylation of the ER.
Figure 2:
Determination of the relative amount of
ER associated with SHPTP2. 3T3/ER cells were either left untreated (lanes 1, 3, 5, and 7) or incubated with 100 ng/ml
EGF (lanes 2, 4, 6, and 8) for 5 min at 37 °C.
Whole cell detergent lysates were prepared and subjected to
immunoprecipitation with the SHPTP2 polyclonal antibody (A) or
with the ER monoclonal antibody (B) as described under
``Experimental Procedures.'' The resulting supernatants from
the initial SHPTP2 immunoprecipitation in panelA were subjected to a second round of ER immunoprecipitation (lanes 3, 4, 7, and 8). The resulting supernatants
from the initial ER immunoprecipitation in panelB were subjected to a second round of SHPTP2 immunoprecipitation (lanes 3, 4, 7, and 8). The immunoprecipitates were
then Western blotted with an ER-specific antibody (lanes
1-4) or with a SHPTP2 specific antibody (lanes
5-8). IB, immunoblot; IP,
immunoprecipitate.
To demonstrate that the SHPTP2 antibody
was effective, the initial SHPTP2 immunoprecipitation was Western
blotted for the presence of SHPTP2 (Fig. 2A, lanes5 and 6). Under these conditions, the SHPTP2
antibody quantitatively immunoprecipitated 100% of the total SHPTP2
protein pool present in the whole cell detergent extracts (data not
shown). In addition, the supernatant from the initial SHPTP2
immunoprecipitation was then subjected to immunoprecipitation with the
ER antibody. As expected, the immunoprecipitation of ER from the
initial SHPTP2-cleared supernatant was unable to immunoprecipitate any
detectable SHPTP2 protein (Fig. 2A, lanes7 and 8).
Figure 3:
Comparison of EGF-stimulated tyrosine
phosphorylation and association of SHPTP2 with pp115 in 3T3/ER and
HepG2 cells. NIH 3T3/ER and HepG2 cells were incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100 ng/ml EGF for 5 min at 37
°C. A, whole cell detergent lysates were prepared and
directly subjected to Western blotting using the phosphotyrosine
antibody PY20-HRP. B, the detergent lysates prepared in A were immunoprecipitated with the SHPTP2 polyclonal antibody and
subjected to Western blotting using the phosphotyrosine antibody
PY20-HRP. IB, immunoblot; IP,
immunoprecipitate.
Recently it was reported that
activation of the T cell antigen receptor (TCR) resulted in the
tyrosine phosphorylation of proteins in the 116-kDa range that were
associated with the small adapter proteins Crk and
Grb2(15, 16) . To determine if the SHPTP2-associated
115-kDa protein was potentially related to these proteins, we compared
the effect of EGF on HepG2 cells with that of TCR activation on Jurkat
T cells (Fig. 4). As expected, EGF stimulation of HepG2 cells
resulted in the co-immunoprecipitation of the 115-kDa
tyrosine-phosphorylated protein with the SHPTP2 antibody (Fig. 4A, lanes1 and 2).
Incubation of Jurkat cells with an activating TCR antibody (OKT3)
resulted in the predominant tyrosine phosphorylation of a 105-kDa
protein that was co-immunoprecipitated with the SHPTP2 antibody (Fig. 4A, lanes3 and 4). The
other major band observed at approximately 25 kDa following TCR
activation represents the light chain of the OKT3 monoclonal antibody.
We next assessed whether the SHPTP2-associated proteins were also bound
to Grb2 by co-immunoprecipitation with a Grb2 antibody (Fig. 4B). Immunoprecipitation of Grb2 from
EGF-stimulated HepG2 cells demonstrated the presence of
tyrosine-phosphorylated 52- and 46-kDa proteins, which probably
represent the Shc proteins (Fig. 4B, lanes1 and 2). Due to the relatively low level of ER
in these cells, the tyrosine-phosphorylated ER was only observed when
the gel was overexposed (data not shown). Importantly, the
SHPTP2-associated 115-kDa protein was not co-immunoprecipitated with
Grb2. However, as previously reported(15, 16) ,
activation of the TCR resulted in the Grb2 co-immunoprecipitation of a
120-kDa tyrosine-phosphorylated band (Fig. 4B, lanes3 and 4). This band did not co-migrate with the
SHPTP2-associated 105-kDa protein observed in these cells. Thus, the
115- and 105-kDa SHPTP2-associated proteins were unrelated to the
previously described Grb2-associated tyrosine-phosphorylated protein.
Furthermore, we have attempted to identify these components by
immunoprecipitation with known antibodies against
tyrosine-phosphorylated proteins in this molecular weight range.
However, we have been unable to co-immunoprecipitate SHPTP2 with any
EGF-stimulated tyrosine-phosphorylated proteins using antibodies
directed against Jak1, Jak2, Jak3, Tyk2, STAT2, FAK, rasGAP, c-Dbl,
c-Cbl, or the p120 Src substrate (data not shown).
Figure 4:
Comparison of SHPTP2- and Grb2-associated
phosphotyrosine proteins in HepG2 and Jurkat T cells. HepG2 cells were
incubated in the absence (lane1) or presence (lane2) of 100 ng/ml EGF for 5 min at 37 °C.
Jurkat T cells (JKT) were incubated in the absence (lane3) or presence of the TCR agonist antibody OKT3 (lane4). Whole cell detergent lysates were prepared and were
either immunoprecipitated with a SHPTP2 antibody (A) or with a
Grb2 antibody (B). The immunoprecipitates were then subjected
to Western blotting using the phosphotyrosine antibody PY20-HRP. IB, immunoblot; IP,
immunoprecipitate.
In summary, the
data presented in this paper demonstrate that a protein of 115 kDa is a
significant EGF-stimulated SHPTP2-associated tyrosine-phosphorylated
protein in NIH 3T3/ER and HepG2 cells. Although SHPTP2 can bind to the
autophosphorylated ER, the extent of this association is relatively
minor and only accounts for a small fraction of the total cellular ER
and SHPTP2 pool. Since we have not yet identified the nature of the
115-kDa protein and currently no antibodies are available, we have not
been able to determine the relative extent of SHPTP2 association with
pp115. However, based upon the tyrosine phosphorylation signal, we
speculate that this protein is both a substrate for the ER and is the
major SHPTP2 binding protein. In addition, an apparently related
105-kDa protein was detected in Jurkat T cells, and neither the 115-
nor 105-kDa proteins were found to associate with Grb2. Clearly, the
identification of the physiological function of pp115 is an important
issue necessary to determine the molecular role of SHPTP2 in ER
signaling.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.