(Received for publication, September 7, 1995; and in revised form, March 11, 1996)
From the
To clarify the role of protein-tyrosine phosphatase (PTPase)
containing Src homology 2 regions (SHPTP2) in insulin signaling, either
wild-type or mutant SHPTP2 (PTP; lacking full PTPase domain) was
expressed in Rat 1 fibroblasts overexpressing human insulin receptors.
In response to insulin, phosphorylation of insulin receptor substrate 1
(IRS-1), IRS-1-associated PTPase activities and phosphatidylinositol
(PI) 3`-kinase activities were slightly enhanced in wild-type cells
when compared with those in the parent cells transfected with
hygromycin-resistant gene alone. In contrast, introduction of
PTP
inhibited insulin-induced association of IRS-1 with endogenous SHPTP2
and impaired both insulin-stimulated phosphorylation of IRS-1 and
activation of PI 3`-kinase. Furthermore, decreased content of p85
subunit of PI 3`-kinase was also found in mutant cells. Consistently,
the insulin-stimulated mitogen-activated protein kinase activities and
DNA synthesis were also enhanced in wild-type cells, but impaired in
mutant cells. Thus, the interaction of SHPTP2 with IRS-1 may be
associated with modulation of phosphorylation levels of IRS-1,
resulting in the changes of PI 3`-kinase and mitogen-activated protein
kinase activity. Furthermore, an impaired insulin signaling in mutant
cells may be partly reflected in a decreased content of p85 protein of
PI 3`-kinase.
The phosphorylation state of tyrosine residue is regulated by
both protein-tyrosine kinase (PTK) ()and protein-tyrosine
phosphatase (PTPase) and is critical for cell growth, differentiation
and metabolism(1, 2) . Insulin binding to its receptor
results in the sequential autophosphorylation of tyrosine residues in
the cytoplasmic region of the receptor, stimulation of receptor PTK,
then the phosphorylation of insulin receptor substrate-1 (IRS-1). IRS-1
contains several potential phosphorylation sites at tyrosine residues (3) , and tyrosine-phosphorylated IRS-1 binds to
phosphatidylinositol (PI) 3`-kinase and Grb2 through the association of
their Src homology 2 (SH2) regions(4) . SH2 domains, at first
identified in Src family PTK, are regions of about 100 amino acids, and
they can directly interact with proteins containing phosphotyrosine.
Thus, SH2 domains are thought to play important roles in signal
transduction via tyrosine phosphorylation(5, 6) .
The novel non-transmembrane PTPase, SHPTP2 (7, 8) (also known as Syp (9) ,
PTP1D(10) , and PTP2C(11) ), which contains a single
phosphatase domain and two adjacent copies of SH2 regions at its amino
terminus, has been cloned in mammalian cells. SH-PTP2 is widely
expressed and is probably the mammalian homologue of Drosophila Corkscrew (12) . The Corkscrew gene product potentiates
the Drosophila homologue of mammalian c-Raf to positively
transmit signals downstream of the Torso receptor PTK. These data
suggest an important role for SHPTP2 downstream of the receptor PTK in
mammalian cells. In fact, activated platelet-derived growth factor and
epidermal growth factor receptors bind directly to the SH2 domains of
SHPTP2, leading to the phosphorylation of the PTPase on tyrosine
residues, which further stimulates its catalytic activity and
positively regulates mitogenic signaling of these growth factor
receptors via SHPTP2Grb2
Sos complex
formation(9, 10, 13, 14, 15, 16) .
Although it has been reported that SHPTP2 also binds to
tyrosine-phosphorylated IRS-1 in vitro and in
vivo(13, 17, 18, 19) , the
precise role of SHPTP2 in insulin signal transduction remains unclear.
Recently, Xiao et al., have reported that microinjection of
either anti-Syp antibody or the GST-SH2 fusion protein of Syp into Rat
1 fibroblasts over expressing human insulin receptors (HIRc) blocks
insulin-stimulated DNA synthesis(20) . Furthermore, Milarski et al.(21) have reported that the introduction of a
catalytically inactive mutant Syp (Cys-459 Ser) in NIH-3T3 cells
leads to impairment of insulin-stimulated mitogen-activated protein
(MAP) kinase activity and thymidine uptake. Milarski's results
have been confirmed by Noguchi (22) and Yamauchi (23) ,
who showed that the introduction of either catalytically inactive
(Cys-459
Ser) or a SH2 mutant of SHPTP2 into Chinese hamster
ovary cells overexpressing the human insulin receptor (CHO-IR)
attenuates insulin-stimulated MAP kinase
activity(22, 23) , but not insulin-stimulated PI
3`-kinase activity(22) . Furthermore, overexpressing wild-type
SHPTP2 provides no additional increment in insulin-stimulated MAP
kinase (22, 23) and PI 3`-kinase activities in CHO-IR
cells(22) .
However, in our preliminary study, the
introduction of mutant SHPTP2, which completely lacked the catalytic
domain (PTP), attenuated insulin-stimulated PI 3`-kinase activity
in HIRc cells. Furthermore, overexpression of wild-type SHPTP2 enhanced
MAP kinase activity. The contrast of this stimulation to the inhibition
observed in CHO-IR cells suggests that the influence of introduction of
wild-type or mutant SHPTP2 differs between CHO cells and Rat 1
fibroblasts. Therefore, we examined insulin signaling in cells
overexpressing either wild-type (WT) or
PTP (MT) to clarify the
role of SHPTP2 in insulin signal transduction. We found that
insulin-stimulated PI 3`-kinase activities were enhanced in WT cells,
but attenuated in MT cells, resulting in modulation of DNA synthesis
via the regulation of IRS-1 phosphorylation. This is the first direct
evidence that SHPTP2 positively regulates both the insulin-stimulation
of MAP kinase cascade and PI 3`-kinase pathway.
Figure 1:
Screening for positive clones
expressing wild-type (A) or mutant SHPTP2; PTP (B). A, screening for positive clones expressing
wild-type SHPTP2. The cell lysate (40 µg of protein) was resolved
by SDS-PAGE and transferred to an Immobilon membrane using the standard
procedures. Immunoblotting proceeded using
Syp, and the blots were
visualized using anti-rabbit antiserum and enhanced chemiluminescence.
Several clones expressed wild-type SHPTP2 (68 kDa). Clone WT11 was used
for the present study. B, screening for positive clones
expressing mutant SHPTP2.
PTP (24 kDa) were expressed in some
clones, and MT15 was used for the present
study.
Figure 2:
Association of GST-SHPTP2 fusion proteins
with phosphorylated proteins in response to insulin. After being
starved for 24 h, cells were stimulated with 100 nM insulin
for 1 min at 37 °C. Following insulin stimulation, whole cell
lysates (500 µg of protein) were incubated with either GST-SHPTP2 (right) or GST-SH2 (left) fusion protein coupled to
glutathione-Sepharose beads for 90 min at 4 °C. After extensive
washing, bound proteins to either GST-SHPTP2 or GST-SH2 fusion protein
were resolved by SDS-PAGE, electrotransferred to membrane, and
immunoblotted with PY20.
Figure 3:
Insulin-stimulated phosphorylation of
insulin receptor and IRS-1 in WT11, MT15, and pHyg cells. A,
tyrosine-phosphorylated proteins in the cell lysate (200 µg of
protein) from the basal and 10 nM insulin-stimulated cells
were analyzed by Western blotting using PY20. B, after
immunoprecipitated with
IRS-1 antibody, bound proteins were
analyzed by Western blotting using
PY20. Both basal and 10 nM insulin-stimulated phosphorylation levels of IRS-1 were presented. C, to quantify IRS-1 protein content, equal amount of cell
lysates (40 µg of protein/line) was analyzed by Western blotting
using
IRS-1 antibody.
Since SHPTP2 was associated with IRS-1
both in vitro and in vivo as reported
previously(13, 17, 18, 19) , we
tested whether this association could be observed in HIRc cells. Cells
were stimulated with insulin, and then the cell lysates were
immunoprecipitated with IRS-1 and immunoblotted with
Syp as
shown in Fig. 4. In WT11 cells, a significant amount of SHPTP2
associated with IRS-1 was found in the basal state and was remarkably
increased in response to insulin. In contrast, when the
immunoprecipitate of
Syp was immunoblotted with
PY20, a
185-kDa tyrosine-phosphorylated IRS-1 protein was co-immunoprecipitated
with SHPTP2 only after insulin stimulation, but not in the basal state
(data not shown). Thus, this association of IRS-1 with SHPTP2 in the
basal state was not consistent by different means. In contrast to WT11
cells, basal and insulin-stimulated association of IRS-1 with rat
endogenous SHPTP2 was not detected in MT15 cells.
Figure 4:
Association of IRS-1 with SHPTP2 in WT11,
MT15, and pHyg cells. After cells were incubated with or without 100
nM insulin for 5 min, lysed, and immunoprecipitated with
IRS-1. Bound proteins were resolved by SDS-PAGE. The
immunoprecipitate was analyzed by Western blotting using
Syp.
To assess how much
SHPTP2 was associated with IRS-1 after insulin stimulation, we measured
total cellular SHPTP2-specific PTPase activity and studied the effects
of IRS-1 immunoabsorption on the PTPase activity in these cells.
The total cellular PTPase activity of SHPTP2 was not stimulated by
insulin (data not shown). In response to insulin, PTPase activity was
decreased due to immunoabsorption by
IRS-1 antibody by 0.005 and
0.169
A
/15 min/10
cells in
pHyg cells and WT11 cells, respectively, but was not significantly
affected in MT15 cells as shown in Table 1. The activity
reductions are explained by a portion of SHPTP2 binding to IRS-1 in
response to insulin. Even with more than 5-fold greater SHPTP2 activity
in WT11 cells than in pHyg cells, a 6-fold higher proportion of this
elevated PTPase activity was removed. These results strongly suggest
that overexpression of native SHPTP2 acts to enhance
insulin-facilitated IRS-1 binding of the SHPTP2 and that overexpression
of the SH2 region blocks this enhancement.
Figure 5: Insulin-stimulated PI 3`-kinase activity in WT11, MT15, and pHyg cells using HIRc cells as parent cells. A, PI 3`-kinase activities immunoprecipitated with anti-IRS-1 antiserum were measured as described under ``Experimental Procedures.'' B, the radioactivity of phosphatidylinositol phosphate was measured by liquid scintillation counter. PI 3`-kinase activity in each cell line was presented as mean ± S.E. of four separate experiments (*, p < 0.025 compared to the corresponding activity in pHyg cells). C, PI 3`-kinase activities immunoprecipitated with anti-p85 antibody were measured as described under ``Experimental Procedures.'' PI 3`-kinase activity in each cell line was presented as mean ± S.E. of four separate experiments (D) (*, p < 0.01 compared to the corresponding activity in pHyg cells).
It is
possible that PTP mutant, which consists only SH2 domains,
nonspecifically inhibits p85 binding to IRS-1, resulting in the
impaired PI 3`-kinase activation in MT cells. To rule out this
possibility, we have performed the stoichiometric analysis to determine
relationships between the degree of phosphorylation of IRS-1 and degree
of its association of p85 in pHyg and MT cells. In response to insulin,
IRS-1 was less phosphorylated in MT cells compared to that in pHyg
cells as shown in Fig. 6A. However, when association of
IRS-1 with p85 in MT cells was adjusted to the value of pHyg cells,
degree of tyrosine-phosphorylation of IRS-1 in MT cells was only 20% of
pHyg cells, suggesting that IRS-1 more efficiently binds p85 in MT
cells (Fig. 6B). These results clearly indicate that
impairment of insulin-stimulated PI 3`-kinase activity in MT cells may
be mainly due to decreased levels of IRS-1 phosphorylation, but not due
to nonspecifically inhibition of p85 binding to IRS-1 by mutant protein
expression.
Figure 6:
Relationship between degree of
insulin-stimulated phosphorylation of IRS-1 and its association with
p85 in pHyg cell and MT cells. A, after insulin stimulation
(1-100 nM), whole cell lysates (1 mg of protein) were
immunoprecipitated with IRS-1. Bound proteins were resolved by
SDS-PAGE, electrotransferred to membrane, and immunoblotted with either
PY20 or
p85 antibody. B, stoichiometric analysis of
the relationships between the degree of phosphorylation of IRS-1 and
degree of its association with p85 in pHyg and MT cells using
densitometric scanning. Degree of IRS-1 phosphorylation and its
association with p85 stimulated by three different insulin
concentrations were corrected by those values in pHyg cells stimulated
by 1 nM insulin.
To further analyze the total PI 3`-kinase activity in
the cells, PI 3`-kinase activity immunoprecipitated with p85
antibody was measured, we found that total PI 3`-kinase activities
immunoprecipitated with
p85 subunits was decreased in MT cells,
but unchanged in WT11 cells (Fig. 5, C and D).
To rule out the possibility that the decreased PI 3`-kinase activity in
HIRc cells which expressed the
PTP originated from clonal
variation or from specific characteristics of the parent HIRc cells, we
tested the effects of
PTP expression on PI 3`-kinase activity in
another independent cell line, HIRY/F2 cells, where Y/F2 mutant insulin
receptors were expressed(28) . We have cloned HIRY/F2 cells,
which overexpress either wild-type (WT1) or
PTP (MT6) at
expression levels similar to those for HIRc cells (WT11 and MT15). In
HIRY/F2 cells, PI 3`-kinase activities immunoprecipitated with
IRS-1 antiserum were also decreased in MT6 cells but increased in
WT1 cells as shown in Fig. 7(A and B).
Furthermore, PI 3`-kinase activity immunoprecipitated with
p85
antibody was decreased in only MT6 cells but increased in WT1 cells. To
study the reason for decreased total PI 3`-kinase activity, we next
assessed the protein content of p85 subunit in these cell lines. As
shown in Fig. 8, Western blot analysis using polyclonal
p85
antibody showed that the content of p85 protein was decreased in MT
cells in both HIRc and HIRY/F2 cells. Using another monoclonal p85
antibody that recognizes a different epitope, we confirmed that the
protein content of the p85 subunit was decreased in MT cells,
suggesting that expression of
PTP might induce decreased protein
content of p85 subunits in both HIRc and HIRY/F2 cells. On the other
hand, p85 protein content in WT cells was comparable to that in pHyg
cells.
Figure 7: Insulin-stimulated PI 3`-kinase activity in WT1, MT6, and pHyg cells using HIRY/F2 cells as parent cells PI 3`-kinase activities immunoprecipitated with either anti-IRS-1 or anti-p85 antibody were measured as described under ``Experimental Procedures.'' A, insulin-stimulated PI 3`-kinase activity immunoprecipitated with IRS-1. B, PI 3`-kinase activity in each cell line was presented as means ± S.E. of four separate experiments. *, p < 0.05;**, p < 0.01 compared to the corresponding activity in pHyg cells. C, insulin-stimulated PI 3`-kinase activity associated with p85. D, PI 3`-kinase activity in each cell line was presented as means ± S.E. of four separate experiments. *, p < 0.05; **, p < 0.01 compared to the corresponding activity in pHyg cells.
Figure 8:
Quantification of content of p85 subunit
of PI 3`-kinase. Content of p85 subunit of PI 3`-kinase was assessed by
Western blotting using two p85 antibodies (polyclonal and monoclonal
p85 antibodies). Content of p85 subunit of PI 3`-kinase was decreased
in both HIRc and HIRY/F2 cells expressing PTP (MT15 and MT6 cells)
compared with cells expressing wild-type SHPTP2 and pHyg
cells.
Figure 9: Insulin-stimulated BrdUrd incorporation in HIRc and HIRY/F2 cells. After starvation, the cells were stimulated with insulin, pulsed with BrdUrd, and stained with anti-BrdUrd as described under ``Experimental Procedures.'' A, percentage of BrdUrd-labeled cells in WT11, MT15, and pHyg cells were determined in the presence or absence of 10 nM insulin. B, percentage of BrdUrd-labeled cells in WT1, MT6, and pHyg cells were determined in the presence or absence of 10 nM insulin. Each column is presented as mean ± S.E. of 4-6 separate experiments. *, p < 0.05;**, p < 0.01 compared to pHyg cells.
As shown in Fig. 10, we found that insulin-stimulated PI 3`-kinase activity was well correlated with IRS-1 phosphorylation levels in WT and MT cells (r = 0.953, p < 0.05). Furthermore, changes in PI 3`-kinase activity were well correlated with those in DNA synthesis (r = 0.986, p < 0.01), as well as in the case of MAP kinase (r = 0.91, p < 0.05) in HIRc cells.
Figure 10:
Relationship between phosphorylation of
IRS-1 and PI 3`-kinase activity (A) and relationship between
PI 3`-kinase activity and DNA synthesis among pHyg, MT11, and WT15
cells (B). A, magnitude of increment in
phosphorylation of IRS-1 and PI 3`-kinase activity in response to
insulin in WT11 and MT15 cells were divided by those in pHyg cells,
respectively. B, magnitude of increment in PI 3`-kinase
activity and DNA synthesis in WT11 and MT15 cells in response to
insulin were divided by those in pHyg cells. Similar relationships were
observed in HIRY/F2 cells expressing wild-type and PTP. Data
represent means ± S.E. of 4-5 separate
experiments.
We have established cell lines overexpressing either
wild-type or mutant SHPTP2 (PTP) originated from HIRc and HIRY/F2
cells. In response to insulin, IRS-1-associated SHPTP2 activity in WT11
cells was significantly greater than that in pHyg cells. On the other
hand, SHPTP2 activities bound to IRS-1 in MT15 cells was not affected
by insulin treatment. Thus, introduction of
PTP inhibited the
association of IRS-1 with native endogenous SHPTP2 and overexpression
of wild-type SHPTP2 enhanced its association with IRS-1 compared to
that in pHyg cells in response to insulin.
Does the change in
association between SHPTP2 and IRS-1 modulate insulin signaling? In our
current study, we found that both insulin-stimulated levels of tyrosine
phosphorylation of insulin receptors and IRS-1 were increased in WT
cells and decreased in MT cells. Thus, we speculate that SHPTP2
potentiates the phosphorylation levels of IRS-1 and regulates the
insulin signal through either the inhibition of an undefined PTPase(s)
or the activation of undefined PTK(s). As shown in Fig. 2, in
addition to IRS-1 and insulin receptor subunit, two undefined
tyrosine-phosphorylated proteins bound to SH2 domains of SHPTP2 in
response to insulin. One was a 115-kDa tyrosine-phosphorylated protein
and thought to be a SHPTP2-binding protein as reported in several
studies(21, 22, 23, 35) . The other
high molecular weight tyrosine-phosphorylated protein (pp135) remains
undefined. Although the precise roles of these proteins for regulation
of insulin signaling are unclear, they may be substrate(s) for SHPTP2.
Further characterization of these proteins may have important
implications for clarifying the role of SHPTP2 in the signal
transduction of insulin.
However, previous studies (21, 22, 23) reported that introduction of
dominant negative mutant SHPTP2 did not modulate the phosphorylation
level of IRS-1. Noguchi et al.(22) showed that
introduction of negative SHPTP2 inhibited Ras-MAP kinase pathway but
that the phosphorylation of IRS-1 was unchanged. In the present study,
our PTP mutant consists only SH2 domains of SHPTP2 and tertiary
structure of its SH2 domains may differ from those of native SHPTP2 and
their catalytically inactive mutant (Cys
Ser). Therefore, it may
be important to rule out the possibility that this SH2 mutant
nonspecifically interacts with other SH2 binding sites rather than
endogenous SHPTP2 binding sites, leading to inhibit functions of other
SH2-containing molecules. However, SH2 domains of SHPTP2 are reported
to behave differently compared with those of PTP1C in vitro(25) and in vivo(36) , even though they
have 60-70% of homology in SH2 regions of both PTPases. Moreover,
overexpression of wild-type SHPTP2 induces the opposite effects when
compared with MT cells. Furthermore, we could not find any qualitative
difference in the profile of binding proteins between GST fusion
proteins containing full-length SHPTP2 and
PTP as shown in Fig. 2. Thus, we speculate that
PTP mutant interfere with
native SHPTP2 to associate endogenous SHPTP2 binding sites including
IRS-1. In the present study, we confirmed that the introduction of
mutant SHPTP2 attenuated insulin-stimulated MAP kinase activity as
reported(21, 22, 23) . However, we also found
that
PTP impaired the insulin-stimulated activation of PI
3`-kinase associated with IRS-1 in MT cells. Furthermore, we found that
insulin-stimulated PI 3`-kinase activity was well correlated with IRS-1
phosphorylation levels that was evaluated using three cell lines, as
shown in Fig. 10A. Thus, we speculate that changes in
phosphorylation levels of IRS-1 might be associated with both changes
in PI 3`-kinase and MAP kinase activities in HIRc cells.
Although
PTP may act by nonspecifically inhibiting between IRS-1 and p85
subunit of PI 3`-kinase in MT cells, this seem unlikely based on the
following. First, our SH2 protein was not able to bind a GST-IRS-1
fusion protein that includes binding motif for p85 of PI 3`-kinase and
no SHPTP2 binding site(17) . Furthermore, p85 was more
efficiently bound to phosphorylated IRS-1 in MT cells, when compared
with that in pHyg cells (Fig. 6). Considering these data
together, we believe that decreased levels of phosphorylation of IRS-1
may cause impairment of its association with p85, resulting in decrease
in PI 3`-kinase activity rather than unphysiological inhibition of
IRS-1 association with p85 of PI 3`-kinase. Therefore, the reason for
this discrepancy between Noguchi's study and ours still remains
unclear. However, one can speculate that different cell lines (CHO-IR versus HIRc) may respond differently to SHPTP2 depending on
its concentration in the cells.
Interestingly, the content of the
p85 subunit of PI 3`-kinase was significantly decreased in MT cells,
and its content in WT cells was comparable with that of pHyg cells.
Since the protein content of IRS-1 (Fig. 3C) was not decreased
in MT cells compared with that in pHyg cells, the decrease in protein
content of p85 subunit might be specific. The decrease in p85 content
in MT cells was confirmed in two different parent cell lines.
Therefore, it may be possible that SHPTP2 regulates expression of a p85
subunit of PI 3`-kinase. Similarly, Hausdorff et al.(37) reported that microinjection of either GST-SH2 fusion
protein or anti-SHPTP2 antibody into 3T3-L1 adipocytes inhibited
insulin-stimulated expression of Glut1 and that SHPTP2 was necessary
for insulin-stimulated expression of Glut1 protein(37) . They
speculate that this may be caused by inhibition of p21 activation(38) . However, McGuire et al.(39) have reported that expression of p85 is not affected
in NIH3T3 cells after a 40-h treatment with lovastatin, which inhibits
farnesylation of p21
, suggesting that p21
does not modulate the expression of p85(39) . Although
the mechanism for regulation of p85 expression remains unclear, it has
been reported that differentiation of preadipocyes to adipocytes leads
to increased levels of the expression and phosphorylation of insulin
receptor and IRS-1, and resulted in increased expression of
p85(40) . Therefore, based upon these data, SHPTP2 may modulate
expression of p85 via activation of insulin signaling.
In
conclusion, overexpression of SHPTP2 enhanced the insulin-stimulated
activities of both MAP kinase and PI 3`-kinase through increased IRS-1
phosphorylation and introduction of dominant negative mutant SHPTP2
(PTP) attenuated these pathways through impaired IRS-1
phosphorylation. This is the first direct evidence that levels of
SHPTP2 activity positively regulate the stimulation of both MAP kinase
cascade and PI 3`-kinase pathway via modulation of phosphorylation of
IRS-1 in Rat 1 fibroblasts expressing human insulin receptors.