From the Veterans Affairs Medical Center Research
Service and the § Department of Medicine, University of
Colorado Health Sciences Center, Denver, Colorado 80220, the ** Veterans
Affairs Medical Center Research Service, La Jolla, California 92161, and the
Department of Medicine, University of California at San
Diego, and the
Whittier Diabetes Institute,
La Jolla, California 92093
Received for publication, October 16, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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We assessed the roles of insulin
receptor substrate-1 (IRS-1) and Shc in insulin action on
farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I)
using Chinese hamster ovary (CHO) cells that overexpress wild-type
human insulin receptors (CHO-hIR-WT) or mutant insulin receptors
lacking the NPEY domain (CHO- Although insulin is a weaker mitogen than many other growth
factors, it is nevertheless essential for growth and differentiation of
many, if not all, tissues and cell types. The mechanism of the
mitogenic influence of insulin remains incompletely understood. It
appears that activation of the
Ras/MAPK1 and
phosphatidylinositol (PI) 3-kinase pathways is necessary for the
nuclear effects of insulin (1-6). Both DNA synthesis and transcription
regulation have been shown to involve the MAPK and PI 3-kinase pathways
(1-6).
We have recently identified another aspect of the mitogenic influence
of insulin: its ability to stimulate the prenylation of the Ras family
of GTPases (7, 8). Insulin promotes the phosphorylation and activation
of farnesyltransferase (FTase) and geranylgeranyltransferases (GGTases)
I and II (9-11). Activation of these enzymes results in increases in
the amounts of prenylated Ras, Rho, and Rab proteins available for
activation by other growth factors (9, 12). Cells grown in the presence
of high concentrations of insulin and tissues of hyperinsulinemic
animals contain increased amounts of farnesylated p21ras
and geranylgeranylated RhoA (9-12). We have also shown that ambient hyperinsulinemia potentiates the mitogenic influence of insulin-like growth factor-1, epidermal growth factor, platelet-derived growth factor, and lysophosphatidic acid in a variety of tissues (9-13). These findings indicate that hyperinsulinemia creates a certain background for cellular responses to the mitogenic influence of other
growth-promoting agents.
FTase and GGTase I are ubiquitous heterodimers, each consisting of an
Because IRS and Shc proteins lie upstream of Ras in the relay of
insulin signaling, we investigated the role of IRS-1 and Shc proteins
in insulin signaling to the prenyltransferases. Since phosphorylation
of IRS and Shc proteins requires the presence of the NPEY domain
of the insulin receptor (16, 17), we utilized a mutant insulin receptor
with a deletion of the NPEY domain ( Materials--
All standard chemicals were from Sigma. Anti-Ras
monoclonal antibody was from Transduction Laboratories (Lexington, KY);
anti-RhoA antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-phosphotyrosine antibody was from Sigma; and
anti-ACTIVE MAPK was from Promega (Madison, WI). All supplies
and reagents for SDS-polyacrylamide gel electrophoresis were from
Bio-Rad, and the enhanced chemiluminescence kit was from Amersham
Pharmacia Biotech. Recombinant Ras (Ras-CVLS) and Rho (Ras-CVLL)
proteins were from Calbiochem. Insulin was from Lilly, and
[32P]orthophosphate was from PerkinElmer Life Sciences.
CHO cells transfected with a wild-type human insulin receptor
(CHO-hIR-WT), a mutant insulin receptor lacking the NPEY motif
( Phosphorylation Assay of IRS-1 and Shc Proteins in CHO-hIR-WT,
Phosphorylation Assay of the FTase Phosphorylation of MAPK--
3T3-L1 fibroblasts were transduced
with control recombinant adenovirus or recombinant adenovirus
expressing the Shc SH2 domain-blocking peptide and subsequently
incubated in the absence or presence of insulin (100 nM)
for 5 min. Cell lysates were normalized for protein and resolved by
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a
polyvinylidene difluoride membrane and immunoblotted with
anti-phospho-MAPK antibodies. Phosphorylated MAPK was determined by chemiluminescence.
Adenoviral Transduction--
3T3-L1 cells were transduced at a
multiplicity of infection of 1-20 plaque-forming units/cell for 1 h with stocks of either control recombinant adenovirus containing the
CMV promoter (Ad5-CMV/Ad5-LacZ viruses not expressing domains of the
signaling peptides) or recombinant adenoviruses Ad5-PTB, Ad5-SAIN,
Ad5-Shc-SH2, Ad5-p85-NSH2, and Ad5-p110-CAAX. All constructs
were developed in the laboratory of J. M. O. and have been
characterized and described in at least two studies (18, 19). To
achieve 90% transduction efficiency, the cells were incubated with the
virus for 12-16 h, with experiments being performed 60-90 h after
viral infection. The presence of the transduced domains was detected by
polymerase chain reaction and described in detail previously (18,
19)
PI 3-Kinase Activity--
3T3-L1 fibroblasts were
transduced with stocks of either control recombinant adenovirus
containing the CMV promoter (Ad5-CMV/Ad5-LacZ) or recombinant
adenoviruses expressing the IRS-1 PH or Shc SH2 domain. The incubation
solution was replaced with serum-free medium for 24 h.
Subsequently, cells were incubated without or with insulin (100 nM) for 10 min. Cell lysates were normalized for protein, and IRS-1 was immunoprecipitated and placed into a reaction mixture (100 mM MgCl2, 10 mM Tris, 0.55 mM ATP, and 1 mCi/ml [ FTase and GGTase I Activity Assay--
Cells were challenged
with 100 nM insulin for 1 or 24 h and then lysed in
lysis buffer. Prenyltransferase activity was assayed in
vitro using a modified method of Moores et al. (20).
Lysates containing endogenous FTase or GGTase I from control and
insulin-treated cells were normalized for protein. The in
vitro filtration assay was initiated by adding a 5-µl aliquot of
normalized extract to 45 µl of a reaction assay solution (5 mM MgCl2, 5 mM dithiothreitol, 100 nM Ras-CVLS (Ras) or Ras-CVLL (RhoA analog) protein, 100 nM tritiated farnesyl pyrophosphate (15 mCi/mmol) or
geranylgeranyl pyrophosphate (15 mCi/mmol), respectively, and 50 mM HEPES, pH 7.5) and incubated at 37 °C for 30 min. The
assay was stopped with 1 ml of 1 N HCl in ethanol, and the
reaction mixture was filtered through Whatman GF/C glass-fiber filters
and air-dried. Labeled protein (a measure of enzymatic activity) was
quantified by liquid scintillation spectrometry.
Assay for Amounts of Farnesylated p21ras and
Geranylgeranylated RhoA--
Cells were incubated with or without
insulin (100 nM) for 1 or 24 h and lysed. Normalized
lysates were mixed with an equal volume of Triton X-114 and incubated
at 37 °C for 3 min, and aqueous and detergent phases were allowed to
separate at room temperature as previously described (7-12).
Antibodies to p21ras or RhoA were used to immunoprecipitate
their respective GTPases from both the aqueous (unprenylated GTPase)
and detergent (prenylated GTPase) phases. Proteins were resolved by
SDS-polyacrylamide gel electrophoresis, determined by Western blotting,
and quantified by densitometry. The amount of prenylated p21ras
and RhoA proteins is expressed as a percentage of total cellular p21ras and RhoA immunoprecipitated from both phases.
Statistical Analysis--
All statistics were analyzed by
Student's t test, with p < 0.05 considered significant.
Role of the NPEY Domain of the Insulin Receptor--
Initially, we
assessed the effects of insulin on the phosphorylation and stimulation
of FTase and GGTase I in CHO cells overexpressing the wild-type insulin
receptor or a mutant lacking the NPEY domain (
Insulin (100 nM) promoted phosphorylation of IRS-1 and Shc
in the CHO-hIR-WT cells (Figs. 1,
A and B), but not in the
Since phosphorylation of Shc and IRS-1 is required for their
association with Grb2, we assessed co-immunoprecipitation of Grb2 with
either Shc or IRS-1 in cells expressing the wild-type and mutant
insulin receptors. Insulin stimulated an association of Grb2 with Shc
and IRS-1 in the CHO-hIR-WT cells, but not in the
In concert with these findings, insulin failed to promote
phosphorylation of the
Consistent with its stimulatory effect on the phosphorylation of the
Role of Shc Versus IRS-1--
Thus far, our experiments strongly
suggested the importance of either IRS-1 or Shc for insulin action on
the prenyltransferases, but did not distinguish between these two
signaling intermediates. To address this point, we used adenoviral
transduction of 3T3-L1 fibroblasts with proteins that block the
IRS-1/Shc PTB domain, IRS-1/Shc SAIN domain, IRS-1 PH domain, or Shc
SH2 domain. These constructs have been fully characterized and
described previously (18, 19). The roles of the PH, PTB, and SAIN
domains in mediating the interactions of the IRS and Shc proteins with
the insulin receptor have been thoroughly reviewed elsewhere (21-23).
The consensus is that the PTB and SAIN domains of the IRS and Shc
proteins facilitate direct interaction of these proteins with the
insulin receptor (21-23). In contrast, the PH domain (68% homology
between IRS-1 and IRS-2) directs the IRS proteins to the insulin
receptor, but interacts with the plasma membrane lipid groups (24).
We first determined the utility of overexpression of the PH domain for
distinguishing between the IRS and Shc signaling pathways. Overexpression of the PH domain blocked the ability of insulin to
stimulate phosphorylation of IRS-1, association of p85 with IRS-1, and
activation of PI 3-kinase and Akt while not affecting the amounts of
the IRS-1 protein (Fig. 4). In contrast,
insulin-induced phosphorylation of Shc and activation of MAPK remained
unaffected in these cells (Fig. 5). Thus,
overexpression of the PH domain appears to be a useful tool in
examining the influence of IRS-1 versus Shc pathways on
prenyltransferases.
Overexpression of the PTB or SAIN domain of the IRS-1 and Shc proteins
prevented the interaction of the endogenous domains of these
intermediates with the juxtamembrane domain of the insulin receptor
(18, 19) and thereby blocked insulin's ability to promote
phosphorylation of the
In concert with these observations, we found that functional disruption
of IRS-1/Shc binding to the insulin receptor by adenoviral transduction
of the PTB and SAIN domain proteins also eliminated the ability of
insulin to increase the activities of prenyltransferases and the
amounts of prenylated p21ras and RhoA (Fig.
7, A-D). In contrast,
overexpression of the IRS-1 PH domain did not impair the effect of
insulin on either the FTase (Fig. 7A) or GGTase I (Fig.
7B) activity or on the amounts of farnesylated
p21ras (Fig. 7C) and geranylgeranylated RhoA (Fig.
7D), even though it completely blocked the IRS-1-related
signaling. These observations argue against the role of IRS proteins in
the mechanism of insulin action on prenyltransferases. Even though the
Shc SH2 domain is not involved in Shc-insulin receptor interactions
(25, 26), an interference with the Shc SH2 domain completely inhibited
the insulin effect on the amounts of farnesylated p21ras and
geranylgeranylated RhoA (Fig. 7, C and D).
Taken together, these findings indicated that interference with Shc
binding (overexpression of the PTB or SAIN domain) to the insulin
receptor blocked the insulin effect on prenyltransferases. In contrast,
interference with IRS-1 binding alone (overexpression of the PH domain)
did not block the insulin effect. Interestingly, interference with the
Shc SH2 domain (a domain not needed for the interaction of Shc with the
insulin receptor) (25) inhibited the effect of insulin on
prenyltransferases, even though Shc continued to mediate an insulin
effect on MAPK (Fig. 8).
Finally, we confirmed a lack of potential involvement of PI 3-kinase in
this process using adenoviral transduction of the blocking peptide of
either the p85 NSH2 domain or constitutively active p110.
Neither of these transductions had any effect on the ability of insulin
to stimulate FTase activity (Fig.
9A) or to increase the amounts
of farnesylated p21ras (Fig. 9B). Similar results
were obtained in experiments with GGTase I (data not shown).
Insulin-specific stimulation of prenylation of the Ras family of
small molecular mass GTPases has emerged as an important aspect of
insulin action that modulates cellular mitogenic responses to a variety
of growth factors (9, 11, 12). In previous studies, we identified
several steps in the mechanism of insulin action on prenylation. We
found that insulin promotes phosphorylation of the In this study, we first determined that disruption of the interaction
of IRS-1 and Shc with the insulin receptor abrogates insulin effects on
the prenyltransferases. Thus, an insulin effect was absent in cells
overexpressing a mutant insulin receptor lacking the NPEY domain
( A novel observation presented in this study is the ability of the
overexpressed IRS-1 PH domain to block insulin signaling along the
IRS-1 pathway without any effect on the Shc/MAPK pathway. In cells with
overexpression of the IRS-1 PH domain, insulin failed to stimulate
IRS-1 phosphorylation, association of p85 with IRS-1, and activation of
PI 3-kinase and Akt (Fig. 4). In contrast, insulin still promoted
phosphorylation of Shc and MAPK in these cells (Fig. 5). Thus,
overexpression of the PH domain allows one to study the
Shc/MAPK-mediated signaling independently of the IRS-1/PI 3-kinase
signaling branch. The mechanism whereby interference with the PH domain
blocks interactions of IRS-1 with the insulin receptor remains unknown.
Because the PH domain of IRS-1 interacts with the plasma membrane, one
can assume that the inability of IRS-1 to anchor at the plasma membrane
precludes the interactions of its PTB domain with the insulin receptor.
Further studies are needed to detail this process. For the purpose of
our investigation, we demonstrated that the blockade of insulin
signaling via the IRS-1 pathway with overexpression of the IRS-1 PH
domain does not interfere with the ability of insulin to activate the
prenyltransferases and to augment prenylation of Ras and Rho proteins.
Another novel and unexpected observation was made when we blocked the
Shc SH2 domain. A functional knockout of this domain prevented insulin
action on the prenyltransferases. In cells transduced with the anti-Shc
SH2 protein antibody, insulin failed to stimulate phosphorylation of
the We have previously demonstrated that the insulin-induced signaling to
the prenyltransferases involves activation of the Ras/MAPK pathway
(10). Cells with a dominant-negative mutant of Ras and cells treated
with PD98059, an inhibitor of MEK, fail to respond to insulin in terms
of stimulation of the prenyltransferases (10). These observations
suggest that insulin stimulates the prenyltransferases in a positive
feedback fashion. Insulin activates p21ras and promotes the
phosphorylation and activation of MAPK. The latter appears to
phosphorylate and activate FTase, which, in turn, farnesylates more
p21ras, allowing farnesylated p21ras to anchor at the
plasma membrane in preparation for subsequent activation (12). What
remains unresolved is an important question as to why other growth
factors that activate MAPK fail to mimic the insulin effect on the prenyltransferases.
One possibility is that activation of MAPK is a necessary (but not
sufficient) step for activation of the prenyltransferases by insulin.
Conceivably, the putative additional steps that complement the
influence of MAPK are exclusively under insulin's control. In addition
to the MAPK pathway, signaling via the PI 3-kinase pathway is equally
important for mitogenesis (1-3). These two pathways may interact in
mediating the insulin effect on the prenyltransferases. However, using
wortmannin, an inhibitor of PI 3-kinase, we have previously
demonstrated that inhibition of PI 3-kinase has no effect on the
ability of insulin to activate the prenyltransferases (10). The present
study is in agreement and demonstrates that neither a functional
knockout of PI 3-kinase (overexpression of either the IRS-1 PH domain
or the p85 NSH2 domain) nor constitutively active p110 has any effect
on the insulin-stimulated prenylation.
The present findings also indicate that activation of MAPK is not
sufficient to phosphorylate and activate the prenyltransferases. Thus,
overexpression of the Shc SH2 domain resulted in full activation of
MAPK in response to insulin, but a complete block in activation of the
prenyltransferases. In summary, these experiments emphasize the
importance of the Shc-mediated signaling from the insulin receptor to
FTase and GGTase I. Downstream from Shc, this signal appears to proceed
along two pathways. It involves activation of the MAPK pathway (29-31)
and a yet unidentified pathway that involves the Shc SH2 domain. Both
branches are necessary, but neither one alone is sufficient to promote
the phosphorylation and activation of the prenyltransferases by
insulin. The Shc SH2 domain may recruit additional proteins (possibly
kinases) into this process. Further studies are needed to identify
these putative intermediates.
NPEY) or 3T3-L1 fibroblasts
transfected with adenoviruses that express the PTB or SAIN
domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1,
or the Src homology 2 (SH2) domain of Shc. Insulin promoted
phosphorylation of the
-subunit of FTase and GGTase I in CHO-hIR-WT
cells, but was without effect in CHO-
NPEY cells. Insulin increased
FTase and GGTase I activities and the amounts of prenylated Ras and
RhoA proteins in CHO-hIR-WT (but not CHO-
NPEY) cells. Overexpression
of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc
signaling) prevented insulin-stimulated phosphorylation of the FTase
and GGTase I
-subunit activation of FTase and GGTase I and
subsequent increases in prenylated Ras and RhoA proteins. In contrast,
overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not
Shc) signaling, did not alter insulin action on the prenyltransferases,
but completely inhibited the insulin effect on the phosphorylation of
IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt.
Finally, overexpression of the Shc SH2 domain completely blocked the
insulin effect on FTase and GGTase I activities without interfering
with insulin signaling to MAPK. These data suggest that insulin
signaling from its receptor to the prenyltransferases FTase and GGTase
I is mediated by the Shc pathway, but not the
IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin
signaling to MAPK may be necessary (but not sufficient) for activation
of prenyltransferase activity. An additional pathway involving the Shc
SH2 domain may be necessary to mediate the insulin effect on FTase and
GGTase I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and a
-subunit (14). Although the
-subunit of each enzyme
confers substrate specificity, these two prenyltransferases share a
common
-subunit (15). We have previously shown that insulin promotes
phosphorylation of the
-subunit and that this phosphorylation
correlates with increased enzymatic activity of these enzymes (10).
Insulin-induced phosphorylation of the
-subunit of FTase and GGTase
I and subsequent activation of these enzymes require the presence of
the intact C-terminal domain of the insulin receptor, is dependent on
the activation of the Ras/MAPK pathway, and does not involve the PI
3-kinase pathway (10).
NPEY) to address this question.
In addition, we employed adenoviral transduction of proteins (the
PTB and SAIN domains of IRS-1 and Shc, the PH domain of IRS-1,
and the Shc SH2 domain) that, when overexpressed, functionally
eliminate the influence of endogenous IRS and/or Shc proteins. This
approach allowed us to assess the roles of IRS and Shc proteins in the
mechanism of insulin action on FTase and GGTase I in 3T3-L1 fibroblasts
and Chinese hamster ovary (CHO) cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NPEY), or a neomycin resistance-containing plasmid vector (CHO-neo)
were generated in the laboratory of P. B, and the adenoviral
constructs were developed in the laboratory of J. M. O.
NPEY, and CHO-neo Cells--
Cells were preincubated in serum-free
medium for 24 h and then incubated without or with 100 nM insulin for 5 min. Cell were homogenized in lysis buffer
(150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 1 mM sodium vanadate, 1 mM
sodium phosphate, 1% Triton X-100, 0.05% SDS, 10 µg/ml aprotinin,
10 µg/ml leupeptin, and 50 mM HEPES, pH 7.5) and
normalized for protein. IRS-1 and Shc were immunoprecipitated from cell
lysates and resolved on 12% polyacrylamide gels. Amounts of
phosphorylation or protein were determined by Western blotting using
anti-phosphotyrosine antibodies. To assess an association of Shc with
Grb2, cell lysates were immunoprecipitated with anti-Shc antibodies and
immunoblotted with anti-Grb2 antibodies.
-Subunit in CHO-hIR-WT,
NPEY, and CHO-neo Cells and 3T3-L1 Fibroblasts--
Cells were
incubated for 6 h at 37 °C in serum- and phosphate-free medium
and preincubated overnight with 250 µCi of
[32P]orthophosphate (10 mCi/mmol). Cells were then
incubated for 60 min with or without insulin (100 nM),
lysed as previously described (10), sonicated, and centrifuged, and
protein concentrations were diluted to 0.5 mg/ml. The FTase
-subunit
was immunoprecipitated with antiserum to the
-subunit and analyzed
by 12% SDS-polyacrylamide gel electrophoresis. Amounts of
phosphorylation were visualized by autoradiography, whereas amounts of
protein were determined by Western blotting; both were quantified by densitometry.
-32P]ATP (0.42 µM), pH 7.5) for 10 min at room temperature. Reactions were stopped by the addition of 8 N HCl followed by the
addition of CHCl3/methanol (1:1, v/v). Solutions were
centrifuged at 14,000 rpm for 1 min. The organic layer was lyophilized
and resuspended in methanol. Samples were loaded onto Silica Gel 60 TLC
plates that had been previously coated with 1% potassium oxalate. The samples were resolved in a solution of
CHCl3/methanol/H2O/NH4OH (60:47:11:2). The TLC plates were dried and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NPEY). The NPEY domain
is the site of IRS-1 and Shc binding to the cytoplasmic portion of the
insulin receptor. The
NPEY cells contain 9 × 105
mutant human insulin receptors/cell, similar to the cells transfected with the wild-type human insulin receptor (CHO-hIR-WT). The control CHO-neo cells (transfected with a plasmid for neomycin resistance) possess ~3000 rodent insulin receptors/cell. All three cell lines have been characterized and described previously (16).
NPEY cells. There was
a minimal but consistent effect of insulin in the CHO-neo cells,
reflecting the presence of the endogenous insulin receptors.
Overexpression of the
NPEY receptors had a dominant-negative influence on the small number of endogenous insulin receptors, as
reflected by the absence of insulin-stimulated phosphorylation of IRS-1
and Shc in the
NPEY cells.
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Fig. 1.
Effect of insulin on the phosphorylation of
IRS-1 and Shc and on the association of Grb2 with IRS-1 and Shc.
CHO-hIR-WT, NPEY, and CHO-neo cells were preincubated in
serum-free medium for 24 h and then incubated without or with
insulin (100 nM) for 1 or 5 min. IRS-1 and Shc were
immunoprecipitated (IP) from cell lysates and resolved on
12% polyacrylamide gels. Phosphorylation of IRS-1 (A) and
Shc (B) was determined by Western blotting (WB)
using anti-phosphotyrosine antibodies. The association of Grb2 with Shc
and IRS-1 is depicted in C and D. Cell lysates
were immunoprecipitated with anti-Shc or anti-IRS-1 antibodies and
immunoblotted with anti-Grb2 antibodies.
NPEY cells (Fig.
1, C and D), confirming the lack of Shc- and
IRS-1-mediated downstream signaling in these cells.
-subunit of FTase/GGTase I in the
NPEY cells (Fig. 2A), indicating
that an interaction of the insulin receptor with either IRS-1 or Shc is
required for insulin signaling to the prenyltransferases. Insulin (100 nM) strongly promoted phosphorylation of the
-subunit in
both the CHO-hIR-WT and CHO-neo cells (Fig. 2A), suggesting
that even a minimal number of the intact insulin receptors in CHO-neo
cells is sufficient for this aspect of insulin action in response to
maximally effective insulin concentrations. A
dose-dependent effect of insulin on the phosphorylation of
the
-subunit revealed significantly reduced sensitivity of the
CHO-neo cells to insulin (Fig. 2, B and C),
consistent with a lower number of insulin receptors. In subsequent
experiments, the maximally effective dose of insulin was used to
compare insulin effects on the different cell lines.
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Fig. 2.
Effect of insulin on the phosphorylation of
the FTase -subunit. A,
CHO-hIR-WT (lanes 1 and 4),
NPEY (lanes
2 and 5), and CHO-neo (lanes 3 and
6) cells were preincubated with
[32P]orthophosphate (250 µCi/plate) for 24 h and
then incubated in medium without (lanes 1-3) or with
(lanes 4-6) 100 nM insulin for 1 h. The
FTase
-subunit was immunoprecipitated from lysates, normalized for
protein, resolved on 12% polyacrylamide gels, and determined by
autoradiography. B and C, CHO-hIR-WT
(WT) and CHO-neo (Neo) cells were preincubated in
medium containing [32P]orthophosphate (250 µCi/plate)
for 24 h and then challenged with insulin for 1 h at the
indicated concentrations. The FTase
-subunit was immunoprecipitated
from lysates, resolved on 12% polyacrylamide gels, determined by
autoradiography, and quantified by densitometry. The upper
panel in B depicts an autoradiogram from a
representative experiment. In C, the results are expressed
as a percentage of the maximal response to insulin and are presented as
the mean ± S.E. *, p < 0.05 versus
Neo (n = 3-4/group).
-subunit, insulin increased the activities of FTase (Fig.
3A) and GGTase I (Fig.
3B) and the amounts of farnesylated p21ras (Fig.
3C) and geranylgeranylated RhoA (Fig. 3D),
respectively, in the CHO-hIR-WT cells and, to a lesser extent, in
the CHO-neo cells. Insulin was ineffective in the
NPEY cells.
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Fig. 3.
Effect of insulin on the activities of FTase
and GGTase I and the amounts of farnesylated p21ras and
geranylgeranylated RhoA in CHO cells. CHO-hIR-WT, NPEY,
and CHO-neo cells were incubated in the absence (open bars)
or presence of 100 nM insulin for 1 h (closed
bars) or 24 h (hatched bars). Cell lysates were
normalized for protein, and a 5-µl aliquot of lysate was used to
determine endogenous FTase (A) and GGTase I (B)
activities as described under "Experimental Procedures." Parallel
studies were performed to determine the amounts of farnesylated
p21ras (C) and geranylgeranylated RhoA
(D) as percentages of total cellular p21ras and
RhoA, respectively (as described under "Experimental Procedures").
Results represent the mean ± S.E. *, p < 0.05 versus controls (no insulin; n = 4-5/group).
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Fig. 4.
Effect of overexpression of the PH domain
peptide on insulin signaling via the IRS-1/PI 3-kinase branch.
Cells were transduced with adenoviruses expressing the IRS-1 PH
domain-blocking peptide or vector only (control) as
described under "Experimental Procedures." Cells were then treated
with 100 nM insulin for either 10 min or 1 h as
indicated and lysed. A, IRS-1 immunoprecipitates
(IP) were immunoblotted (IB) with the PY20
antibody to determine phosphorylation of IRS-1. B, IRS-1
immunoprecipitates were blotted with the anti-p85 antibody to determine
an association of IRS-1 with p85. C, IRS-1
immunoprecipitates were blotted with the anti-IRS-1 antibody to
determine the amounts of IRS-1 protein in these samples. D,
PI 3-kinase was activated by insulin (10 min) in the IRS-1
immunoprecipitates. Cells transduced with the Shc SH2 domain-blocking
peptide were used as additional controls. E, shown are the
results of the insulin-induced phosphorylation of Akt in cells
transduced with the PH domain (a 10-min incubation with insulin).
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Fig. 5.
Effect of overexpression of the PH domain
peptide on insulin signaling via the Shc/MAPK branch. Cells were
transduced with adenoviruses expressing the IRS-1 PH domain-blocking
peptide or vector only (control) as described under
"Experimental Procedures." Cells were then treated with 100 nM insulin for 10 min or 1 h as indicated and lysed.
A, immunoprecipitates (IP) of Shc were blotted
with the PY20 antibody. B, the cell lysate was immunoblotted
(IB) with the anti-phospho-MAPK antibody (a 10-min
incubation with insulin). Erk, extracellular
signal-regulated kinase.
-subunit of FTase/GGTase I (Fig. 6). In contrast, interference with the
IRS-1 PH domain had no effect on this aspect of insulin action,
suggesting that IRS-1 does not play a role in this process.
Unexpectedly, we found that blocking the Shc SH2 domain completely
inhibited the insulin effect on the phosphorylation of the
-subunit
of FTase/GGTase I (Fig. 6).
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Fig. 6.
Effect of insulin on the phosphorylation of
the FTase -subunit in 3T3-L1 fibroblasts
transduced with adenoviruses expressing PH, PTB, SAIN, or Shc SH2
domain-blocking peptides. 3T3-L1 fibroblasts were transduced with
vector only; Ad5-CMV (C); or adenoviruses expressing
the IRS-1 PH, IRS-1/Shc PTB, IRS-1/Shc SAIN, and Shc SH2
domain-blocking peptides. [32P]Orthophosphate (250 µCi/plate) was added to the medium for 24 h, followed by
incubation in medium without or with insulin (100 nM) for
1 h. Lysates were normalized for protein, and the FTase
-subunit was immunoprecipitated, resolved on a 12% polyacrylamide
gel, and determined by autoradiography.
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Fig. 7.
Effect of insulin on the activities of FTase
and GGTase I and the amounts of prenylated p21ras and RhoA in
3T3-L1 fibroblasts. Cells were transduced (as described in the
legend to Fig. 6) and then incubated in medium without (open
bars) or with (closed bars) insulin (100 nM) for 1 h. Endogenous FTase (A) and
GGTase I (B) activities and the amounts of farnesylated
p21ras (C) and geranylgeranylated RhoA
(D) were assayed in cell lysates as described under
"Experimental Procedures." Prenyltransferase activity is expressed
as the mean ± S.E. of four experiments/group. *,
p < 0.05 versus controls (CNT;
no insulin) (A and B). The amounts of prenylated
p21ras and RhoA are expressed as percentages of total cellular
p21ras and RhoA, respectively, and are plotted as the
means ± S.E. of four experiments/group. *, p < 0.05 versus controls (no insulin) (C and
D).
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Fig. 8.
Effect of insulin on the phosphorylation of
MAPK in wild-type and transduced (Shc SH2 domain-blocking peptide)
3T3-L1 fibroblasts. Wild-type (WT) fibroblasts or
fibroblasts transduced with adenoviruses expressing the Shc SH2
domain-blocking peptide were incubated without or with insulin (100 nM) for 5 min. Cell lysates were normalized for protein and
resolved on 12% polyacrylamide gels. The amounts of phosphorylation
were determined by Western blotting using anti-phospho-MAPK antibodies.
CNT, control.
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Fig. 9.
Effect of insulin on FTase activity and the
amounts of farnesylated p21ras in 3T3-L1 fibroblasts.
Wild-type (WT) and 3T3-L1 cells transduced with adenoviruses
expressing the p85 NSH2 and constitutively active p110 (p110
CAAX) peptides of PI 3-kinase were incubated in medium
without (open bars) or with (closed bars) 100 nM insulin for 1 h. A, FTase activity is
expressed as the mean ± S.E. of three experiments/group. *,
p < 0.05 versus controls (no insulin).
B, the amounts of farnesylated p21ras are expressed
as a percentage of total cellular p21ras, and results represent
the mean ± S.E. of three experiments. *, p < 0.05 versus controls (no insulin).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of FTase
(which is shared with GGTase I) (9, 10) and the
-subunit of GGTase
II (27). This increase in phosphorylation of the corresponding
-subunits appears to correlate with the activities of all three
prenyltransferases (10, 27, 28). Augmented activities of FTase and
GGTase I increase the amounts of prenylated p21ras and RhoA,
respectively, in cells exposed to high concentrations of insulin
(7-13). Prenylation of p21ras and RhoA GTPases is a
prerequisite for their activation by GTP loading under the influence of
other growth factors (14). Thus, by providing greater amounts of
prenylated p21ras and RhoA, hyperinsulinemia increases the
mitogenic responsiveness of tissues to various growth-promoting agents.
We have proposed that the "priming effect" of hyperinsulinemia
plays a significant role in the cellular responsiveness to
growth-promoting agents.
NPEY) and in cells with a functional transient knockout of IRS-1
and Shc (adenoviral transduction of the PTB and SAIN domains). We
further demonstrated that interference with only IRS-1 binding by
blocking the IRS-1 PH domain inhibited insulin signaling via the
IRS-1/PI 3-kinase pathway, but did not affect insulin's ability to
signal to the prenyltransferases, suggesting that IRS-1-mediated
signaling is not involved in the latter process. On the other hand, our
results indicated an important role of the Shc-initiated pathway in
insulin signaling to the prenyltransferases.
-subunit (Fig. 6), activation of the prenyltransferases (Fig. 7,
A and B), and augmentation of the amounts of
prenylated GTPases (Fig. 7, C and D). In
contrast, insulin continued to activate MAPK in these cells (Fig. 8).
The Shc SH2 domain is not involved in binding of Shc to the insulin
receptor (25, 26); and in fact, its function in the mechanism of
insulin signaling remains obscure. We propose that a new role for this
domain is to mediate insulin signaling to the prenyltransferases. The
precise biochemical role of this domain in mediating insulin signaling
to prenyltransferases remains enigmatic. A possible unknown and yet to
be characterized docking intermediate(s) may be necessary for the
propagation of insulin signaling mediated through the Shc SH2 domain.
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FOOTNOTES |
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* This work was supported in part by the Medical Research Service of the Department of Veterans Affairs, National Institutes of Health Grant DK 54475 (to P. B.), the Foundation for Biomedical Education and Research, the Denver Research Institute, the HealthONE Foundation, the Diabetes and Endocrine Research Fund, and the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a fellowship from the American Heart Association and recipient of a Veterans Affairs Research Service career development award.
§§ To whom correspondence should be addressed: VA Medical Center (151), 1055 Clermont St., Denver, CO 80220. Tel.: 303-393-4619; Fax: 303-377-5686; E-mail: Boris.Draznin@med.va.gov.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009443200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; PI, phosphatidylinositol; FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; IRS, insulin receptor substrate; PH, pleckstrin homology; SH2, Src homology 2; CHO, Chinese hamster ovary; CMV, cytomegalovirus; Ad5, adenovirus type 5; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PTB, phosphotyrosine binding; SAIN, shc and IRS-1 NPXY.
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