From the
Insulin activates hexose transport via at least two mechanisms:
a p21
Activation of the insulin receptor results in receptor
autophosphorylation and subsequent phosphorylation of its substrate,
insulin receptor substrate 1
(IRS-1)
Recent observations indicate that the phosphatase (PTP)
domain of SHPTP2 functions to send a positive signal independent of
Grb2/SOS. Overexpression of catalytically inactive SHPTP2 inhibits
insulin-induced activation of MAP
kinase
(9, 10, 11) , stimulation of c-fos reporter gene expression
(11) and GTP-loading onto
p21
Insulin signaling proceeds via at
least two divergent pathways. To date, all of the studies examining the
role of SHPTP2 in insulin-stimulated signaling have utilized cells in
which insulin induces mitogenesis but does not activate physiologically
important metabolic pathways. However, the primary function of insulin
is to regulate metabolic processes, most strikingly an increase in
hexose uptake in adipose tissue and skeletal muscle. In these tissues,
insulin rapidly and reversibly augments sugar uptake by promoting the
translocation of the ``insulin-responsive glucose
transporter'', GLUT4, from an intracellular compartment to the
cell surface
(16) . In a number of tissue culture cell lines,
insulin also increases glucose transport via an alternative pathway,
which shows similarity to that used for mitogenesis in most cell types.
Stimulation of this pathway results in a modest translocation of the
basal transporter, GLUT1, and a larger increase in its expression,
mediated primarily by transcriptional
activation
(17, 18) .
The mouse cell line 3T3-L1 has
been used as a model system for the study of insulin-stimulated
processes
(19) . They express GLUT1 as well as GLUT4 and respond
to insulin by increasing both GLUT1 expression and GLUT4 translocation.
We have shown previously that p21
For mitogenesis
assays, NIH-3T3 fibroblasts expressing the insulin receptor were
serum-starved in DMEM + 0.1% FBS for 24 h. GST-NC-SHPTP2 at a
concentration of 0.5 mg/ml and neutralizing antibodies to SHPTP2 at a
concentration of 10 mg/ml were microinjected using a Narishige injector
model IM200 and a Leitz manual micromanipulator. After approximately 2
h, the cells were stimulated with 1 µM insulin or 10% FBS
and BrdUrd was added to a final concentration of 10 µM.
The cells were incubated for an additional 18 h, fixed in methanol for
10 min, washed three times with PBS, permeabilized in 0.1% Nonidet
P-40, and incubated with a primary antibody to BrdUrd and a
fluorescent-labeled secondary antibody. Cells injected with SH2 domains
were detected by coinjection of M
We investigated the role of SHPTP2 in the pathways through
which insulin stimulates hexose uptake in cultured adipose cells. For
these experiments, two reagents were generated for microinjection into
3T3-L1 adipocytes: a glutathione S-transferase (GST) fusion
protein encoding the N- and C-terminal SH2 domains of SHPTP2
(GST-NC-SH2), and antibodies against full-length SHPTP2. We first
confirmed that GST-NC-SH2 could associate with IRS-1 following insulin
stimulation as previously reported
(2, 3) . Agarose beads
bound to GST-NC-SH2 were incubated with lysates prepared from 3T3-L1
adipocytes either left untreated or exposed to insulin for 5 min. The
GST-NC-SH2 beads were centrifuged and washed, and bound proteins were
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted
for the presence of phosphotyrosine (Fig. 1). A single pY protein
of 180 kDa corresponding to IRS-1 was specifically precipitated with
GST-NC-SH2 but not with GST alone. This result indicates that
GST-NC-SH2 associates with IRS-1 and confirms earlier results that
binding of the SH2 domains competes with endogenous
SHPTP2
(2, 3) . Thus, we hypothesized that upon
microinjection of GST-NC-SH2 into fibroblasts or adipocytes, GST-NC-SH2
would block association of endogenous SHPTP2 with IRS-1 and prevent
propagation of downstream signaling. Moreover, preincubation of
GST-NC-SH2 domains with anti-SHPTP2 antibodies
(986) inhibited
(by approximately 50%) the association of the PDGF receptor with
GST-NC-SH2 domains, while preincubation of the GST-NC-SH2 domains with
preimmune SHPTP2 antibodies was without effect on PDGF Receptor
association.(
Nonetheless, studies in which
catalytically inactive SHPTP2 has been overexpressed in fibroblasts
support the idea that the PTP domain is critical for signaling in some
insulin stimulated pathways
(9, 10, 11) .
Although in vivo substrates of SHPTP2 have yet to be
identified, current data strongly suggest that SHPTP2 can function as
an upstream mediator of p21
-dependent pathway, leading to an increase
in the amount of cell surface GLUT1; and a metabolic,
p21
-independent pathway, leading to
translocation of the insulin-responsive transporter GLUT4 to the cell
surface. Following insulin stimulation, SHPTP2, a non-transmembrane
protein-tyrosine phosphatase, associates with insulin receptor
substrate 1 via its Src homology 2 (SH2) domains. Microinjection of a
glutathione S-transferase fusion protein encoding the N- and
C-terminal SH2 domains of SHPTP2 (GST-NC-SH2) or anti-SHPTP2 antibodies
into NIH-3T3 fibroblasts overexpressing the insulin receptor blocks
insulin-induced DNA synthesis. Microinjection of either GST-NC-SH2 or
anti-SHPTP2 antibodies into 3T3-L1 adipocytes inhibited the
insulin-stimulated increase in expression of GLUT1. In contrast,
translocation of GLUT4 to the cell surface was unaffected by either
GST-NC-SH2 or anti-SHPTP2 antibodies. These data confirm a role for
SHPTP2 in insulin-stimulated mitogenesis and indicate that whereas
SHPTP2 is necessary for insulin-stimulated expression of GLUT1, it is
not required for activation of the metabolic pathway leading to GLUT4
translocation.
(
)(1) . Tyrosine-phosphorylated
IRS-1 then serves to recruit Src homology 2 (SH2) domain-containing
proteins, which regulate downstream signaling pathways. These signaling
molecules include the p21
activator complex
Grb2/SOS, the p85 subunit of phosphatidylinositol 3-kinase, and the
protein tyrosine phosphatase SHPTP2
(1) . SHPTP2 associates with
IRS-1
(2, 3) , as well as with the EGF
receptor
(3, 4, 5) and PDGF
receptor
(3, 4, 5) , following stimulation by
insulin, EGF, or PDGF, respectively. Upon stimulation of cells with
either EGF or PDGF, SHPTP2 becomes
tyrosine-phosphorylated
(2, 4, 5) . In the case
of PDGF stimulation, the C-terminal phosphotyrosine residue on SHPTP2
binds Grb2 and may couple the PDGF receptor to the p21
pathway
(6, 7) . Microinjection of either the SH2
domains of SHPTP2 or antibodies to SHPTP2 blocks insulin-induced
mitogenesis
(8) . SHPTP2 is not, however, tyrosine-phosphorylated
in response to insulin
(3) , suggesting that the Grb2/SOS
association of SHPTP2 is not operative in the insulin signaling
pathway.
(9), suggesting that SHPTP2 is an upstream
mediator of p21
activation in insulin signaling.
The role of the PTP domain of SHPTP2 also extends beyond signaling
pathways related to mitogenesis. Injection of catalytically inactive
SHPTP2 blocks fibroblast growth factor-induced mesoderm induction in
Xenopus embryos and prevents completion of gastrulation (12).
Association of SHPTP2 through its N-terminal SH2 domain with a
phosphotyrosyl (pY) peptide corresponding to its binding site on either
the PDGF receptor
(13) or IRS-1
(14, 15) leads to
a substantial increase in phosphatase activity. This might well provide
a signaling mechanism utilized by SHPTP2 that does not rely on
recruitment of a Grb2/SOS complex.
is necessary
for increased GLUT1 expression but is not required for translocation of
GLUT4 to the cell surface
(20) . Since SHPTP2 is required in both
mitogenic and non-mitogenic signaling pathways, we asked whether SHPTP2
is required for the insulin-stimulated increase in cell surface
expression of glucose transporters.
Materials
Crystalline porcine insulin was a gift
of Lilly Laboratories. Rhodamine-conjugated donkey anti-rabbit
antibodies and fluorescein dichlorotriazine (DTAF)-conjugated donkey
anti-rat antibodies were purchased from Jackson Immunologicals (West
Grove, PA). Bovine serum albumin used in translocation assays was from
Calbiochem. 5-Bromo-2-deoxyuridine (BrdUrd, RPN 201) and
mouse-anti-BrdUrd (RPN 202) were purchased from Amersham. MBPras for
microinjection studies was purified as described
(20) .
Sheep-anti-MBPras antibodies were purchased from Elmira Biologicals
(Iowa City, Iowa). Anti-phosphotyrosine antibody 4G10 was a gift of Dr.
Tom Roberts (Dana Farber Cancer Institute, Boston, MA).
Cell Culture
3T3-L1 fibroblasts were grown and
differentiated upon confluence as described
(21) . Adipocytes
were maintained in DMEM containing 10% fetal bovine serum and used at
10-20 days post-differentiation. NIH-3T3 fibroblasts stably
expressing the human insulin receptor (3T3-HIR) were maintained in DMEM
containing 10% calf serum and 200 µg/ml Geneticin (G418).
Protein and Antibody Preparation
A glutathione
S-transferase fusion protein encoding amino acids 1-251
of SHPTP2 was generated by polymerase chain reaction from the
full-length SHPTP2 cDNA using the primers
3`-TCACTATAGGGCGAATTGGGTACC-5` and 3`-GTTGTCCTAAGGTTTGAA-5`. The
resulting PCR product was digested with EcoRI and ligated into
pGEX-2T to yield GST-NC-SH2. GST-NC-SH2 protein was produced from
Escherichia coli as described previously
(2) . In
preparation of GST-NC-SH2 protein for microinjection experiments, the
fusion protein was eluted from glutathione-Sepharose with 8 M
urea. The eluted protein was dialyzed against phosphate-buffered saline
using an Amicon microconcentrator and concentrated to 0.5 mg/ml.
Antibodies to SHPTP2 were generated against full-length GST fusion
protein and purified to a final concentration of 10 mg/ml by Protein G
affinity purification. To establish whether GST-NC-SH2 domains bind
activated IRS-1, 3T3-L1 adipocytes were serum-starved for 24 h in DMEM
+ 0.1% FBS, and cells were either stimulated with 500 nM
insulin for 5 min or left untreated. Cell lysates were prepared as
described previously
(2) and incubated with either GST alone or
GST-NC-SH2 (4 µg) at 4 °C for 2 h. Glutathione-agarose beads
were collected by centrifugation and washed four times in 1%-Nonidet
P-40 plus 1 mM sodium orthovanadate. Bound proteins were
separated on 8% SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose. Phosphotyrosine-containing proteins were detected
with an anti-phosphotyrosine antibody (4G10) and a donkey anti-mouse
horseradish peroxidase-conjugated secondary antibody using Enhanced
Chemiluminescence (Amersham Corp.).
Microinjection
3T3-L1 adipocytes were
microinjected using an Eppendorf model 5242 injector and a Narishige
hydraulic manual micromanipulator. Tips were pulled from
filament-containing borosilicate glass (World Precision Instruments) to
diameters of 0.2-0.5 mm using a Sachs-Flaming micropipette puller
(Sutter, model PC-84). Anti-SHPTP2 antibodies were mixed with MBPras to
yield final concentrations of 5 mg/ml antibody and 1 mg/ml marker
protein in phosphate-buffered saline. In all experiments, proteins were
injected into the cytoplasm of 50-100 cells.
70,000
fluorescein isothiocyanate-dextran at 5 mg/ml. Antibody-injected cells
were identified by staining for the presence of rabbit IgG using a
tetramethyl rhodamine isothiocyanate-coupled goat anti-rabbit antibody.
Plasma Membrane Sheet Assay and
Immunofluorescence
For GLUT4 translocation sheet assays, cells
were injected with either GST-NC-SH2 proteins or polyclonal antibodies
to SHPTP2 and were allowed to recover from microinjection for 30 min.
They were then incubated in Leibovitz L-15 medium containing
0.2% bovine serum albumin for 2 h and treated with 100 nM
insulin for 15 min. Plasma membrane ``sheets'' were prepared
essentially as described
(22) . Cells to be assayed for GLUT1
expression were incubated for 4 h in DMEM containing 0.5% calf serum
prior to injection with GST-NC-SH2 or anti-SHPTP2 antibodies. They were
then incubated in the absence or presence of 500 nM insulin
for an additional 20 h before preparation of sheets. All plasma
membrane sheets were processed for immunofluorescence as described
(21) using affinity-purified antibodies to the C terminus of
GLUT4 or serum containing antibodies to the C terminus of GLUT1.
Antibodies to GLUT1 were a gift of Miles Pharmaceuticals (West Haven,
CT). Injected cells were identified by staining with antibodies to
MBPras and DTAF-conjugated secondary antibodies. The amount of glucose
transporter on the plasma membrane was quantitated by digital image
processing as described previously
(20) .
)
This result suggested that
anti-SHPTP2 antibodies had a neutralizing effect on the binding of
SHPTP2 SH2 domains to their corresponding phosphotyrosines. We then
assessed whether either anti-SHPTP2 antibodies or GST-NC-SH2 had a
biological effect on insulin-induced mitogenesis. GST-NC-SH2 and
anti-SHPTP2 antibodies were injected into quiescent NIH-3T3 cells
overexpressing the insulin receptor (3T3-HIR). As shown in
Fig. 2
,
45% of the insulin-stimulated and
80% of the
serum-stimulated cells were in S-phase, as compared to
15% of the
unstimulated cells. Microinjection of anti-SHPTP2 antibodies
(986) into 3T3-HIR cells blocked their entry into S-phase in
response to insulin (p < 0.05), but had no effect on
serum-induced DNA synthesis (Fig. 2A). Similarly,
3T3-HIR cells that were microinjected with GST-NC-SH2 also failed to
enter S-phase in response to insulin (p < 0.05), whereas
the serum-stimulated increase was unaffected (Fig. 2B).
Neither GST alone nor preimmune SHPTP2 antibodies had an effect on
insulin-stimulated mitogenesis. These results indicate that both
GST-NC-SH2 and the anti-SHPTP2 antibodies specifically block
insulin-induced mitogenesis and that these reagents are not toxic to
cell growth. The data confirm the efficacy of these reagents and
support previously published studies showing that SHPTP2 is necessary
for insulin-stimulated DNA synthesis but is not required for mitogenic
induction by serum
(8) .
Figure 1:
SH2 domains of SHPTP2
bind to IRS-1. GST fusion proteins encoding the N- and C-terminal
domains of SHPTP2 (GST-NC-SH2) were bound to glutathione-agarose beads.
Either GST alone or GST-NC-SH2 beads were incubated with lysates
prepared from 3T3-L1 adipocytes treated with (+) or without
(-) insulin for 5 min. Tyrosine-phosphorylated proteins were
detected by immunoblotting using 4G10 monoclonal antibody.
TCL, total cell lysate.
Figure 2:
GST-NC-SH2 and anti-SHPTP2 antibodies
block insulin-stimulated DNA synthesis. 3T3-HIR cells were
serum-starved for 24 h prior to microinjection with GST-NC-SH2
(A) or antibodies against SHPTP2 (B). The cells were
stimulated with 1 µM insulin or 10% FBS, incubated in the
presence of BrdUrd for 18 h and fixed in methanol. The fixed cells were
stained with a primary antibody to BrdUrd and a fluorescent-labeled
secondary antibody, and cells showing nuclear BrdUrd staining were
scored positive for entry into S-phase. Results represent the mean
± S.E. of 200 injected cells obtained from three separate
experiments (*, p < 0.05).
Exposure of 3T3-L1 adipocytes to
insulin for several hours increases total GLUT1 mRNA and protein as
well as the fraction of the hexose carrier on the cell
surface
(17, 23, 24) . We therefore chose to
examine the effect of SHPTP2 on the level of cell surface GLUT1 in the
absence and presence of chronic insulin exposure. As shown in
Fig. 3A, treatment of 3T3-L1 adipocytes with insulin for
20 h increases the amount of cell surface GLUT1 by 5-fold.
Microinjection of GST-NC-SH2 inhibited expression of GLUT1 in the
presence of insulin (Fig. 3A) whereas GST or MBPras
alone had no effect (data not shown). The relative brightness of
injected versus uninjected cells was quantified by image
processing; the abundance of GLUT1 on plasma membrane sheets is
summarized in Fig. 3B. GLUT1 expression was also
inhibited by microinjection of antibody to SHPTP2
(Fig. 3B), while preimmune serum had no effect on GLUT1
distribution (data not shown).
Figure 3:
SHPTP2 is required for expression of GLUT1
in 3T3-L1 adipocytes. 3T3-L1 adipocytes were serum-starved for 4 h
prior to microinjection with GST-NC-SH2 or antibodies to SHPTP2. The
adipocytes were then stimulated with 500 nM insulin and
incubated for an additional 20 h before preparation of plasma membrane
sheets. The sheets were stained with an antibody to MBPras and a
fluorescein-coupled anti-sheep antibody to detect injected cells, and
with serum containing antibody to GLUT1 and rhodamine-coupled
anti-rabbit antibody for quantitation of GLUT1. PanelA shows a representative field of cells injected with GST-NC-SH2 and
stained for GLUT1, with arrowheads denoting injected cells.
Quantitation of GLUT1 in uninjected cells and GST-NC-SH2 or
antibody-injected cells from three separate experiments is shown in
panelB.
We then addressed whether SHPTP2 is
involved in the acute regulation of glucose uptake by insulin. As
visualized by the plasma membrane sheet assay, stimulation of 3T3-L1
adipocytes with insulin for 15 min significantly increases the amount
of GLUT4 on the plasma membrane
(Fig. 4A)
(22, 25) ; quantitation of
fluorescent staining by image processing indicated that cell surface
GLUT4 increased 15-fold in response to insulin
(Fig. 4B). Microinjection of either GST-NC-SH2
(Fig. 4A) or anti-SHPTP2 antibodies was without effect
on the distribution of GLUT4 in these cells in the presence or absence
of either 100 nM insulin (Fig. 4B) or
sub-saturating (10 nM) insulin (data not shown). GST, MBPras,
and preimmune serum also had no effect on localization of GLUT4 (data
not shown).
Figure 4:
SHPTP2 has no effect on translocation of
GLUT4 in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with
GST-NC-SH2 or antibodies to SHPTP2. The adipocytes were serum-starved
for 2 h, stimulated with 100 nM insulin, and incubated for an
additional 15 min before preparation of plasma membrane sheets. The
sheets were stained with an antibody to MBPras and a DTAF-coupled
anti-sheep antibody to detect injected cells, and with
affinity-purified antibody to GLUT4 and rhodamine-coupled anti-rabbit
antibody for quantitation of GLUT4. PanelA shows a
representative field of cells injected with GST-NC-SH2 and stained for
GLUT4, with arrowheads denoting injected cells. Quantitation
of GLUT4 in uninjected cells and GST-NC-SH2- or antibody-injected cells
from three separate experiments is shown in panelB.
One of the fundamental problems in understanding the
mechanism of insulin action is explaining its specificity in the
regulation of metabolism, in spite of significant commonality with
other peptide growth factors in terms of the signaling pathways
modulated. For example, EGF is a very poor activator of hexose uptake
in 3T3-L1 adipocytes, in spite of equivalent potency to insulin in
stimulating MAP kinase
(26) . Since SHPTP2 is affected quite
differently by insulin as compared to PDGF or EGF, in that the former
does not promote phosphorylation of the phosphatase, SHPTP2 seemed an
attractive target for transducing hormone-specific
signals
(2, 3, 4, 5) . Previous studies
evaluating the role of SHPTP2 in insulin action have utilized
non-differentiated cells in which the most prominent effects of the
hormone relate to the initiation of cell
growth
(8, 9, 10, 11) . The experiments
reported above exclude SHPTP2 as an obligate intermediate in the
signaling pathways by which insulin stimulates the translocation of
GLUT4, the most important means of rapidly activating hexose uptake
into muscle and adipose tissue. The requirement for SHPTP2 for the
increase in expression of GLUT1 is consistent both with the critical
role of p21 and Raf-1 in this response, as well
as the idea that the increase in transport mediated by GLUT1 represents
primarily a component of the mitogenic
response
(20, 27) .
.This notion is
supported by the observation that, when overexpressed, catalytically
inactive SHPTP2 functions as a dominant-inhibitory mutant and abrogates
insulin-induced p21
GTP-loading
(9) .
Moreover, signaling events distal to p21
such as
insulin-induced MAP kinase activation and GLUT1
expression
(9, 10) , fibroblast growth factor-induced
mesoderm induction in Xenopus embryos
(12) , c-fos transcription through SRE
(25) , and EGF-induced MAP kinase
transactivation of Elk-1(
)
are also inhibited by
overexpression of catalytically inactive SHPTP2. We have shown
previously that p21
mediates insulin-stimulated
GLUT1 expression but not GLUT4 translocation in 3T3-L1
adipocytes
(20) . Since SHPTP2 is also necessary for GLUT1
expression but is not required for GLUT4 translocation, our data
support published reports suggesting that SHPTP2 is required for
processes mediated by p21
. We conclude that
SHPTP2 plays a role in insulin-induced transcription of immediate early
genes such as GLUT1, but is not required for the metabolic increase in
hexose transport mediated by GLUT4 translocation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.