Insulin Receptor Substrate (IRS) Proteins IRS-1 and IRS-2 Differential Signaling in the Insulin/Insulin-Like Growth Factor-I Pathways in Fetal Brown Adipocytes
Angela M. Valverde,
Margarita Lorenzo,
Sebastian Pons,
Morris F. White and
Manuel Benito
Departamento de Bioquimica y Biologia Molecular II
(A.M.V., M.L., M.B.) Instituto de Bioquimica Centro Mixto
Consejo Superior de Investigaciones Cientificas/Universidad
Complutense Facultad de Farmacia Universidad Complutense
28240-Madrid, Spain
Joslin Diabetes Center (S.P., M.F.W.,
M.B.) Harvard Medical School Boston, Massachusetts 02215
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ABSTRACT
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In the present study we have investigated the
contribution of the insulin receptor substrate proteins (IRS-1 and
IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling
pathways in fetal rat brown adipocytes, a model that expresses both
insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and
IRS-2 tyrosine phosphorylation, their association with p85
, and
IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase
activation to the same extent, the effect of insulin being stronger
than the effect of IGF-I at the same physiological dose (10
nM). Furthermore, insulin/IGF-I stimulated
IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2
phosphorylation was barely detected. Pull-down experiments with
glutathione-S-transferase-fusion proteins containing
SH2-domains of p85
revealed a strong association between IRS-1 and
IRS-2 with p85
in response to insulin/IGF-I, the insulin effect
being stronger than IGF-I. However, the Grb-2-SH2 domain showed
functional differences. While a strong association between IRS-1/Grb-2
was found, IRS-2/Grb-2 association was virtually absent in response to
insulin/IGF-I, as also demonstrated in competition studies with a
phosphopeptide containing the phosphotyrosine 895 residue within the
putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated
tyrosine phosphorylation of the three SHC proteins (46, 52, and 66
kDa). Moreover, insulin/IGF-I markedly increased the amount of
Grb-2-associated SHC proteins by the same extent. Our results suggest
that both IRS-1 and IRS-2 are required for phosphatidylinositol
3-kinase activation that leads to adipogenic and thermogenic
differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and
SHC, but not IRS-2, associate with Grb-2 leading to the
ras-mitogen-activated protein kinase-signaling pathway required for
fetal brown adipocyte proliferation.
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INTRODUCTION
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The pleiotropic effects of insulin and insulin-like growth
factor-I (IGF-I) on metabolic and mitogenic processes are mediated by a
complex network of intracellular signaling pathways (reviewed in Refs.
1 and 2). The biological effects of both molecules are mediated by the
activation of their cognate cell surface receptors, consisting of two
extracellular
-subunits and two membrane-spanning ß-subunits that
possess tyrosine kinase activity (3, 4). These receptors are believed
to transduce signals, at least in part, by phosphorylation of cellular
proteins. It is well known that phosphorylation of the insulin receptor
substrate-1 (IRS-1) upon multiple tyrosine residues results in the
interaction with SH2 domain-containing proteins including the growth
factor receptor-bound-2 (Grb-2/Sem5), the protein tyrosine phosphatase
SHPTP2, and the p85 subunit of phosphatidylinositol 3-kinase (PI
3-kinase) that leads to activation of various signaling pathways,
including the stimulation of the p21.ras-MAPK cascade (5, 6, 7, 8).
To elucidate the role of IRS-1 in insulin/IGF-I action, two
laboratories (9, 10) created IRS-1-deficient mice by targeted gene
mutation. These mice showed an alternative high molecular weight
substrate of the insulin receptor named IRS-2, which has been recently
purified and cloned (11, 12). IRS-2 has been found to be the major
substrate of tyrosine kinases activated by interleukin (IL)-4 in murine
lymphohemopoietic cells (13). Furthermore, the expression of IRS-2 was
detected in a variety of cell types, including fibroblasts, liver,
skeletal muscle, and brain (12). However, the exact role of both IRS
proteins in mediating physiological functions in the different tissues
has not yet been clarified.
Fetal brown adipocyte primary cultures offer a nonfibroblastic
mesenchymal cell model that has proven to be an excellent system by
which to study the role of IGF-I/insulin in the proliferation (14, 15, 16)
and differentiation processes (15, 17, 18, 19, 20), as well as their balance in
the insulin/IGF-I signal transduction pathways (18, 21, 22). These
cells bear a large number of high-affinity insulin and IGF-I receptors
per cell (15, 20), allowing a variety of studies to be performed with
no overexpression of signaling molecules. We have recently shown that
IRS-1 is tyrosine phosphorylated in fetal brown adipocytes upon
insulin/IGF-I stimulation, resulting in the activation of
IRS-1-associated PI 3-kinase (18, 21, 22). Importantly, this enzyme is
a requirement for IGF-I-induced brown adipocyte adipogenic and
thermogenic differentiation, but not for mitogenesis (18). However, the
presence of IRS-2 in brown adipose tissue and its potential role in the
insulin/IGF-I signaling cascade throughout late fetal development has
not yet been established.
In the present study, we demonstrate that IRS-1 and IRS-2 proteins
equally contribute to the association and subsequent activation of PI
3-kinase by insulin/IGF-I that lead to adipogenic and thermogenic
differentiation of primary fetal brown adipocytes. However, the
association of IRS-2 with the adapter protein Grb-2 is very poor, IRS-1
and SHC being the main docking proteins involved in the activation of
the ras-MAPK pathway in brown adipose tissue before birth.
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RESULTS
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Both IRS-1 and IRS-2 Are Tyrosine Phosphorylated by Insulin/IGF-I
in Fetal Brown Adipocytes
Insulin and IGF-I stimulate tyrosine phosphorylation of 180- to
190-kDa proteins that are composed of IRS-1 and the recently reported
IRS-2. Our first purpose was to establish the balance of tyrosine
phosphorylation of the IRS proteins in fetal brown adipocyte primary
cultures. Quiescent cells (20 h serum-deprived) were incubated with 10
nM insulin or 10 nM IGF-I, which has been shown
previously to give maximal phosphorylation (21, 22), for 5 min at 37 C.
After incubation, cell lysates were prepared, and equal amounts of
protein (600800 µg) were immunoprecipitated either with the
anti-IRS-1 or with the anti-IRS-2 glutathione-S-transferase
(GST)-fusion protein antibodies. The immune complexes were analyzed by
SDS-PAGE, followed by Western blotting with the anti-Tyr(P) antibody
(Py20). Figure 1
shows the tyrosine
phosphorylation of the IRS proteins induced by insulin or IGF-I
treatment. As shown in Fig. 1
, both insulin/IGF-I induce a significant
increase in tyrosine phosphorylation of IRS-1, as compared with control
cells, the insulin effect being stronger than the effect of IGF-I at
the same dose (10 nM). When the anti-IRS-2
immunoprecipitates were analyzed by anti-Tyr(P) Western blotting, we
found a strong phosphorylation band showing a slightly higher molecular
mass (approximately 190 kDa) in insulin/IGF-I-treated cells. However,
the level of tyrosine phosphorylation of this band was similar to the
level that we previously found in the IRS-1 immunoprecipitates, the
effect of insulin being also stronger than the effect of IGF-I at the
same dose. These results suggest that both IRS-1 and IRS-2 are involved
in the insulin/IGF-I signaling in fetal brown adipocytes.

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Figure 1. Effect of Insulin/IGF-I on IRS-1 and IRS-2 Tyrosine
Phosphorylation in Fetal Brown Adipocytes
Fetal brown adipocytes serum starved for 20 h were incubated for 5
min at 37 C with 10 nM insulin or 10 nM IGF-I.
Control cells were cultured in serum-free medium. At the end of the
culture time, cells were lysed and immunoprecipitated with the
anti-IRS-1 and anti-IRS-2 polyclonal antibodies. The immune complexes
were washed and analyzed by Western blotting with the anti-Tyr(P)
antibody (Py20). The positions of IRS-1 and IRS-2 are indicated by an
arrowhead. The positions of mol wt markers
(x10-3) are shown on the left. The results
shown are representative of at least three independent experiments.
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Association of IRS Proteins with the p85
Subunit of
Phosphatidylinositol 3-Kinase by Insulin/IGF-I
The next step was to investigate whether tyrosine-phosphorylated
IRS-2 also associates with the p85
subunit of PI 3-kinase as
compared with IRS-1. Serum-deprived cells were incubated with 10
nM insulin or 10 nM IGF-I for 5 min at 37 C.
After incubation, cell lysates were prepared, and equal amounts of
protein were immunoprecipitated either with the anti-IRS-1 or
anti-IRS-2 GST-fusion protein antibodies. The immune complexes were
analyzed by Western blotting with the anti-
p85 polyclonal antibody.
As shown in Fig. 2A
, control cells did
not show any p85
bound to either IRS-1 or IRS-2. However, treatment
of brown adipocytes with 10 nM insulin or 10 nM
IGF-I resulted in a marked increase in the amount of p85
bound to
either IRS-1 or IRS-2, insulin stimulating more binding of p85
to
either IRS-1 or IRS-2 than IGF-I did (Fig. 2A
).
To further demonstrate the balance of the association IRS-1/2-p85
subunit of PI 3-kinase in fetal brown adipocytes, quiescent cells were
stimulated with 10 nM insulin or 10 nM IGF-I
for 5 min at 37 C. The cells were lysed, and 600 µg of protein were
immunoprecipitated with the anti-
p85 polyclonal antibody as
described in Materials and Methods. The immune complexes
were subjected to Western blotting analysis with the anti-IRS-1 and
anti-IRS-2 polyclonal antibodies. As shown in Fig. 2B
, we found a huge
increase in the amount of both IRS-1 and IRS-2 in the anti-
p85
immunoprecipitates upon treatment with insulin/IGF-I, insulin
stimulating more binding of either IRS-1 or IRS-2 to p85
than IGF-I
did.
Both IRS Proteins Induce PI 3-Kinase Activity in Fetal Brown
Adipocytes
We have recently demonstrated the stimulation of IRS-1-associated
PI 3-kinase enzymatic activity in fetal brown adipocyte primary
cultures treated with insulin or IGF-I (21, 22). Our next step was to
investigate whether PI 3-kinase activity was also associated with IRS-2
as it was to IRS-1, under the same experimental conditions. Upon
stimulation with 10 nM insulin or 10 nM IGF-I,
cell lysates were subjected to immunoprecipitation with the anti-IRS-1
or IRS-2 antibodies and assayed for PI 3-kinase activity as described
in Materials and Methods. As shown in Fig. 3
, insulin/IGF-I markedly increased IRS-1
and also IRS-2-associated PI 3-kinase activity, the effect of insulin
on both activities being stronger than that induced by IGF-I. Similar
PI 3-kinase activity associated with IRS-1/IRS-2 induced by insulin was
found after two successive immunoprecipitations with anti-IRS-1 and
anti-IRS-2 antibodies of the same lysate (results not shown).

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Figure 3. PI 3-Kinase Activity Associated with IRS-1 and
IRS-2 during Insulin/IGF-I Stimulation in Fetal Brown Adipocytes
A, Fetal brown adipocytes serum starved for 20 h were incubated
for 5 min at 37 C with 10 nM insulin or 10 nM
IGF-I. Control cells were cultured in serum-free medium. Cells were
lysed and immunoprecipitated with the anti-IRS-1 and anti-IRS-2
antibodies. The immune complexes were washed and immediately used for
an in vitro phosphatidylinositol kinase assay as
described in Materials and Methods. The conversion of
phosphatidylinositol to phosphatidylinositol phosphate in the presence
of [ -32P]ATP was analyzed by TLC. A representative
experiment is shown. B, The corresponding autoradiograms were
quantitated by scanning densitometry. Results are expressed as
arbitrary units of PI 3-kinase activity and are means ±
SEM from three independent experiments.
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Association of IRS Proteins with the Adapter Protein Grb-2 in Brown
Adipocytes
Alignment of the sequences of IRS-2 and IRS-1 revealed a poorly
conserved carboxyl terminus containing several tyrosine phosphorylation
motifs (11). To further explore possible signaling diversity downstream
IRS-1/2 in brown adipocytes, we proceeded to investigate the balance of
the association of both IRS proteins with the adapter protein Grb-2. To
study this, quiescent cells were incubated with 10 nM
insulin or 10 nM IGF-I for 5 min at 37 C. After incubation,
cell lysates were prepared, and equal amounts of protein were
immunoprecipitated either with the anti-IRS-1 or anti-IRS-2 GST-fusion
protein polyclonal antibodies. The resulting immunoprecipitates were
analyzed by Western blotting with the anti-Grb-2 polyclonal antibody.
As shown in Fig. 4A
, control cells showed
a considerable amount of IRS-1-associated Grb-2. However,
IRS-2-associated Grb-2 was barely detected. Insulin/IGF-I induced a
marked increase in the amount of IRS-1-associated Grb-2 but a very poor
increase in the amount of IRS-2-associated Grb-2. To confirm these
data, we analyzed the presence of IRS-1 and IRS-2 in anti-Grb-2
immunoprecipitates. Cells were stimulated with 10 nM
insulin or 10 nM IGF-I for 5 min at 37 C, and lysates were
immunoprecipitated with the anti-Grb-2 polyclonal antibody as described
in Materials and Methods. The immune complexes were
subjected to Western blotting analysis with the anti-IRS-1 and
anti-IRS-2 GST-fusion protein polyclonal antibodies. As shown in Fig. 4B
, insulin/IGF-I induced a marked increase in the amount of
Grb-2-associated IRS-1. However, the amount of Grb-2-associated IRS-2
in response to insulin/IGF-I was virtually undetectable.

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Figure 4. Effect of Insulin and IGF-I on IRS-1 and IRS-2
Association with the Adapter Protein Grb-2 in Fetal Brown Adipocytes
A, Quiescent fetal brown adipocytes were cultured as described in Fig. 2 . At the end of the culture time, cells were lysed and
immunoprecipitated with the anti-IRS-1 and anti-IRS-2 polyclonal
antibodies. Immunoprecipitates were analyzed by SDS-PAGE followed by
transfer of proteins to Immobilon and Western blotting with the
anti-Grb-2 antibody. The position of Grb-2 is indicated by an
arrowhead. The positions of mol wt markers
(x10-3) are shown on the left. The results
shown are representative of at least three independent experiments. B,
Cells were stimulated as described in panel A, immunoprecipitated with
the anti-Grb-2 antibody, and analyzed by Western blotting with the
anti-IRS-1 and anti-IRS-2 antibodies. The positions of IRS-1 and IRS-2
are indicated by arrowheads. The results shown are
representative of at least three independent experiments.
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Association of IRS-1 and IRS-2 with SH2-Containing Proteins
To substantiate the results shown above, we investigated the
ability of GST-fusion proteins containing SH2-domains of the p85
subunit of PI 3-kinase, or full-length Grb-2, to bind
tyrosine-phosphorylated IRS-1 and IRS-2 in fetal brown adipocytes. Cell
lysates were incubated with 10 µg GST fusion proteins containing SH2
domains as indicated in Materials and Methods and
precipitated with glutathione Sepharose beads. Samples were subjected
to SDS-PAGE followed by Western blotting with the anti-IRS-1 and
anti-IRS-2 polyclonal antibodies.
As shown in Fig. 5
, the
NH2-terminal SH2 domain of p85
bound strongly to both
IRS-1 and IRS-2 upon stimulation with 10 nM insulin or 10
nM IGF-I, insulin stimulating more binding than IGF-I did.
However, the SH2 domains of Grb-2 revealed functional differences.
Thus, control cells showed a basal amount of Grb-2-associated IRS-1,
but not Grb-2-associated IRS-2. Insulin/IGF-I markedly increased the
amount of Grb-2-associated IRS-1. However, the amount of
Grb-2-associated IRS-2 in response to insulin/IGF-I was virtually
undetectable (Fig. 5
).

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Figure 5. Differential Binding of p85 and Grb-2 SH2
Domains to IRS-1 and IRS-2 in Insulin/IGF-I-Stimulated Fetal Brown
Adipocytes
Quiescent brown adipocytes were incubated in the absence or presence of
insulin (10 nM) (ins) or IGF-I (10 nM) (I) for
5 min at 37 C. Cell lysates were precipitated with the indicated GST
fusion proteins as described in Materials and Methods,
separated by SDS-PAGE, and immunoblotted with the anti-IRS-1 and
anti-IRS-2 antibodies. The positions of IRS-1 and IRS-2 are indicated
on the right. The data are representative of two similar
experiments.
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IRS-1 and IRS-2 Displayed Differential Affinity for Grb-2 in Fetal
Brown Adipocytes
The functional difference between IRS-1 and IRS-2 with respect to
the ability to bind Grb-2 and subsequently activate the ras-MAPK
pathway could be explained by differential affinities of both IRS
proteins for the Grb-2 adapter protein. To examine this possibility, a
synthethic phosphopeptide containing a single tyrosine-phosphorylated
motif, based on the established Grb-2-binding site in IRS-1 and IRS-2
(11), was tested for the ability to displace IRS-1 or IRS-2 from Grb-2
immune complexes. Serum-deprived brown adipocytes were stimulated for 5
min with 10 nM insulin, and immune complexes, prepared with
the anti-Grb-2 antibody, were incubated with increasing doses of
phosphopeptide as described in Materials and Methods. The
remaining endogenous IRS-1/IRS-2 bound to Grb-2 was analyzed by Western
blotting with the anti-IRS-1 and anti-IRS-2 antibodies. Representative
phosphopeptide competition experiments are shown in Fig. 6
, A and B. Dissociation of IRS-2 from
Grb-2 was much more sensitive (ED50 = 0.01
µM) (Fig. 6D
) to the phosphopeptide than dissociation of
IRS-1 from Grb-2 (ED50 > 1 µM) (Fig. 6C
),
suggesting that the Grb-2-binding site of IRS-1 displays a much higher
affinity for Grb-2 than that of IRS-2.

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Figure 6. IRS-1 and IRS-2 Associate to Grb-2 with Different
Affinity in Fetal Brown Adipocytes
Quiescent brown adipocytes were incubated with 10 nM
insulin for 5 min at 37 C. Control cells were cultured in serum-free
medium. Cell lysates were incubated for 16 h at 4 C with
anti-Grb-2 antibody either in the absence or in the presence of
increasing doses of the synthetic phosphopeptide PPEPKSPGEY(P)VNIEFG.
The immune complexes were collected, resolved by SDS-PAGE, and detected
by immunoblotting with the anti-IRS-1 (panel A) or anti-IRS-2 (panel B)
antibodies. The resulting immunoreactive proteins were detected by
enhanced chemiluminescence, longer exposure being necessary for
visualizing the IRS-2 bands than for IRS-1 bands. The migration of the
IRS-1 and IRS-2 proteins is indicated on the left.
Representative experiments are shown. The amounts of IRS-1 (panel C)
and IRS-2 (panel D) associated with the immune complexes were
quantitated by scanning densitometry. Results are expressed as
arbitrary units and are means ± SEM from three
independent experiments.
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Insulin/IGF-I-Induced SHC Tyrosine Phosphorylation and Its
Association with Grb-2
Finally, we have investigated the contribution of SHC
proteins to the Grb-2-association, which could be relevant to the
proliferation induced by IGF-I in fetal brown adipocyte primary
cultures. Serum-detk;2prived cells were stimulated either with
10 nM insulin or 10 nM IGF-I for 5 min at 37 C.
Cell lysates were prepared, and equal amounts of protein were
immunoprecipitated with the Py72 anti-Tyr(P) antibody. The
resulting immunoprecipitates were analyzed by Western blotting with the
anti-SHC polyclonal antibody. As shown in Fig. 7A
, control cells showed basal
phosphorylation bands of the three SHC proteins. However, 10
nM insulin/IGF-I increased the 46- and 52-kDa SHC
phosphotyrosine bands by 2-fold and the 66-kDa SHC phosphotyrosine band
by 3-fold.

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Figure 7. SHC Is Tyrosine Phosphorylated and Associated with
Grb-2 during Insulin/IGF-I Stimulation in Fetal Brown Adipocytes
A, Quiescent brown adipocytes were incubated in the absence or presence
of insulin (10 nM) or IGF-I (10 nM) for 5 min
at 37 C. Cell lysates were immunoprecipitated with the anti-Tyr(P) Py72
antibody and analyzed by Western blotting with the anti-SHC antibody.
The migration of the SHC proteins is indicated on the
right. The positions of mol wt markers
(x10-3) are shown on the left. B,
Quiescent brown adipocytes were stimulated as described in panel A.
Cell lysates were precipitated with the SH2Grb-2 fusion
protein as described in Materials and Methods, separated
by SDS-PAGE, and immunoblotted with the anti-SHC antibody. The position
of the SHC proteins is indicated on the right. The data
are representative of two similar experiments.
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To further investigate the importance of the association of SHC-Grb-2
in fetal brown adipocytes, we determined the ability of the
SH2Grb-2 GST-fusion protein to bind tyrosine-phosphorylated
SHC following insulin/IGF-I treatment in brown adipocytes. Cell lysates
were incubated with 10 µg GST fusion protein as indicated in
Materials and Methods and precipitated with glutathione
Sepharose beads. Samples were subjected to SDS-PAGE followed by Western
blotting with the anti-SHC antibody. As shown in Fig. 7B
, there is an
important association of SHC with Grb-2 before stimulation of brown
adipocytes with insulin/IGF-I. However, insulin/IGF-I markedly
increased by the same extent the amount of Grb-2-associated SHC
proteins.
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DISCUSSION
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The recent identification and cloning of IRS-2 as an IRS-1-like
molecule indicate that a family of docking proteins may be used for
insulin/IGF-I and cytokine signaling. Our laboratory has established
that quiescent fetal brown adipocytes in primary culture constitute a
suitable cell model in which to study the insulin/IGF-I
signaling network, under physiological conditions (21, 22). These cells
bear a large number of high-affinity insulin- and IGF-I-binding sites
per cell (15, 20), which allow the unhampered investigation of the
early events in the insulin/IGF-I action. In previous reports we have
shown that IRS-1 tyrosine phosphorylation is maximally induced at
physiological concentrations of both molecules (21, 22). In the present
paper, we have shown that in fetal brown adipocytes IRS-2 is tyrosine
phosphorylated, to the same extent as IRS-1, in insulin/IGF-I-treated
cells. These results prompted us to study, in our cell model, the
contribution of both IRS-1 and IRS-2 docking proteins to the
association with two molecules that propagate two distinct pathways
downstream of IRSs: the association with the lipid/serine-threonine
kinase PI 3-kinase and the association with the adapter protein
Grb-2.
Previous studies performed in IRS-1-deficient mice showed that the
residual insulin/IGF-I action of these animals correlated with the
appearance of IRS-2 bound to the p85
subunit of PI 3-kinase in a
tissue-dependent manner, providing evidence of an alternative and
independent signaling pathway in the insulin/IGF-I action (9, 10, 23, 24, 25). Furthermore, IRS-2 has been found to be the major high
molecular mass protein phosphorylated on tyrosine in hematopoietic
cells (13). However, in fetal brown adipocytes, IRS-1 and IRS-2
strongly associate with the p85
subunit, indicating that both
docking proteins are contributing to the PI 3-kinase activation
throughout late fetal development. In a recent work, we have shown that
PI 3-kinase is an essential requirement for the IGF-I-induced
adipogenic and thermogenic differentiation of fetal brown adipocytes
(18). These results, together with the data presented here, suggest
that IRS-2 signaling may be as necessary as IRS-1 for the complete
differentiation of brown adipose tissue before birth, in contrast to
other adipose cells such as primary white adipocytes and differentiated
3T3-L1 fibroblasts in which phosphorylated IRS-2 was not found in
response to insulin (12). The relative contribution of IRS-1/IRS-2 to
the adipogenic and thermogenic differentiation is difficult to
determine in primary cultures and deserves further experimental work in
these cells from IRS-1-deficient mice. Actually, PI 3-kinase activation
in response to insulin is severely impaired in IRS-1-deficient
fibroblasts, although a strong association between IRS-2/p85
was
found (26). The fact that insulin turned out to be a stronger signal in
activating IRS-1 and IRS-2-associated PI 3-kinase activity, as compared
with IGF-I, further supports our previous data showing that insulin was
a more potent signal than IGF-I in adipogenic and thermogenic
differentiation (20).
The adapter protein Grb-2 has been shown to link IRS-1 to the
activation of ras and the MAPK cascade (6, 27). Activation of p21.ras
to its GTP active form has been shown to occur in fetal brown
adipocytes upon insulin or IGF-I stimulation (21, 22). Importantly,
serum-starved brown adipocytes showed a considerable percentage of ras
in its active GTP form (21), as a consequence of their intrinsic
mitogenic competence (15). Results presented in this paper show that
there is a significant IRS-1/Grb-2 association in control cells in the
absence of external stimuli, which could explain the intrinsic ras.GTP
activation that we found in fetal brown adipocyte primary cultures.
Furthermore, there is a marked increase in the amount of
Grb-2-associated IRS-1 in response to insulin/IGF-I. However, despite
the fact that the amino acid sequence of IRS-2 predicts a tyrosine
phosphorylation motif that could serve as a Grb-2 binding site (11, 12, 28), our data show that the association between IRS-2/Grb-2 in response
to insulin/IGF-I is virtually absent. In addition, the phosphopeptide
competition assay revealed that IRS-2 binds to Grb-2 with 2 orders of
magnitude lower affinity than IRS-1. Therefore, a molecular mechanism
that may account for the differential signaling between IRS-1 and IRS-2
underlies the differential affinities for Grb-2 of their conserved
Grb-2 binding domains, probably due to the different environment
surrounding this domain in both molecules. These data are consistent
with the severe inhibition of the DNA synthesis found in
IRS-1-deficient fibroblasts, in which the presence of IRS-2 could not
compensate for the absence of IRS-1 in maintaining the mitogenic
response of IGF-I (26).
SHC has recently been established as another early phosphotyrosine
substrate, which has been proposed as the predominant signaling
molecule coupling insulin receptors to p21 ras.GTP formation in Rat-1
fibroblasts (29, 30). More recently, SHC signaling, rather than IRS-1,
has been proposed to account for the proliferation of Rat-1 fibroblasts
in response to insulin (31). In fetal brown adipocyte primary cultures,
the three SHC proteins are tyrosine phosphorylated in response to
insulin/IGF-I. Furthermore, we have found a marked increase in the
amount of SH2Grb-2 fusion protein associated with SHC in
response to insulin/IGF-I. The results presented here indicate that in
fetal brown adipocytes IRS-1 and SHC, but not IRS-2, are the docking
proteins that lead to the ras-MAPK cascade after insulin and IGF-I
stimulation. The fact that IGF-I is as potent as insulin in inducing
Grb-2-SHC association is in agreement with the role of IGF-I as a
potent mitogen for fetal brown adipocytes, in a p21.ras-dependent
manner (16).
In summary, the findings described in this paper strongly suggest that
IRS-1 and IRS-2 contribute equally to the association with the p85
that leads to the PI 3-kinase activation in response to insulin/IGF-I,
a signaling required for the onset of brown adipose tissue adipogenic
and thermogenic differentiation before birth. Moreover, IRS-1 and SHC,
but not IRS-2, associate with the adapter protein Grb-2 that leads to
the ras-MAPK cascade, a signaling required for fetal brown adipocyte
proliferation.
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MATERIALS AND METHODS
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Materials
FCS and culture media were from Imperial Laboratories
(Hampshire, U.K.). IGF-I was purchased from Calbiochem
(Calbiochem-Novabiochem, La Jolla, CA). Insulin,
L-
-phosphatidylinositol, and
L-
-phosphatidyl-L-serine were purchased from
Sigma Chemical Co. (St. Louis, MO). Collagenase and Protein A-agarose
were purchased from Boehringer Mannheim (Mannheim, Germany).
Glutathione Sepharose beads were purchased from Pharmacia (Upsala,
Sweden). [
32P]ATP (3000 Ci/mmol) was purchased from
Amersham (Aylesbury, U.K.). All other reagents used were of the purest
grade available.
Cell Culture
Brown adipocytes were obtained from interscapular brown adipose
tissue of 20-day Wistar rat fetuses and isolated by collagenase
dispersion as previously described (15). Cells were plated at 5 x
106 cells/100 mm or 11.2 x 106
cells/60-mm tissue culture plates in MEM supplemented with 10% FCS to
allow cell attachment to the plastic surface of the plates. After 46
h of culture at 37 C, cells were rinsed twice with PBS, and 80% of the
initial cells were attached. Cells were maintained for 20 h in a
serum-free medium supplemented with 0.2% (wt/vol) BSA. At this time
cells were treated for 5 min with insulin or IGF-I at the doses
indicated in the text and in the figure legends. Control cells were
cultured in the absence of factors in the medium.
Immunoprecipitations
Quiescent fetal brown adipocytes (after 20 h of serum
deprivation) were treated with insulin or IGF-I for 5 min and
subsequently lysed at 4 C in 1 ml of a solution containing 10
mM Tris-HCl, 5 mM EDTA, 50 mM NaCl,
30 mM sodium pyrophosphate, 50 mM NaF, 100
µM Na3VO4, 1% Triton X-100, and
1 mM phenylmethylsulfonyl fluoride, pH 7.6 (lysis
buffer). Lysates were clarified by centrifugation at 15,000 x
g for 10 min, and the supernatants were transferred to a
fresh tube. After protein content determination, equal amounts of
protein were immunoprecipitated at 4 C with the corresponding
antibodies, and the immune complexes were collected on Protein
A-agarose or anti-mouse Ig-agarose beads. Immunoprecipitates were
washed three times with lysis buffer and extracted for 10 min at 95 C
in 2 x SDS-PAGE sample buffer (200 mM Tris-HCl, 6%
SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol, pH
6.8) and analyzed by SDS-PAGE and as described in Results
and in the figure legends. Anti-
p85 and anti-Grb-2 polyclonal
antibodies were from Santa Cruz (Santa Cruz Biotechnology, Palo Alto,
CA). Anti-SHC polyclonal antibody was from Upstate Biotechnology (Lake
Placid, NY). For IRS-1/IRS-2 immunoprecipitations, polyclonal
antibodies were obtained as described (12). For anti-Tyr(P)
immunoprecipitations, the Py72 monoclonal antibody was the generous
gift of Dr. E. Rozengurt and J. Sinnett-Smith (Imperial Cancer Research
Foundation, London). The monoclonal anti-Tyr(P) antibody used for
Western blotting (Py20) was purchased from Santa Cruz.
Western Blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes
and were blocked using 5% nonfat dried milk in 10 mM
Tris-HCl and 150 mM NaCl, pH 7.5, and incubated overnight
with several antibodies as indicated in 0.05% Tween-20, 1% nonfat
dried milk in 10 mM Tris-HCl and 150 mM NaCl,
pH 7.5. Immunoreactive bands were visualized using the enhanced
chemiluminescence Western blotting protocol (Amersham, Arlington
Heights, IL).
PI 3-Kinase Activity
PI 3-kinase activity was measured by in vitro
phosphorylation of phosphatidylinositol as described (32). Fetal brown
adipocytes were incubated with IGF-I in the absence or presence of PI
3-kinase inhibitors as indicated in the figure legends. After washing
with ice-cold PBS, cells were solubilized in lysis buffer containing
leupeptin (10 µg/ml), aprotinin (10 µg/ml), and 1 mM
phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation
at 15,000 x g for 10 min at 4 C, and proteins were
immunoprecipitated with the anti-IRS-1/IRS-2 polyclonal antibodies. The
immunoprecipitates were washed successively in PBS containing 1%
Triton X-100 and 100 µM Na3VO4
(twice), 100 mM Tris (pH 7.5) containing 0.5 M
LiCl, 1 mM EDTA and 100 µM
Na3VO4 (two times), and 25 mM Tris
(pH 7.5) containing 100 mM NaCl and 1 mM EDTA
(twice). To each pellet were added 25 µl of 1 mg/ml
L-
-phospha-tidylinositol/L-
-phosphatidyl-L-serine
sonicated in 25 mM HEPES (pH 7.5) and 1 mM
EDTA.
The PI 3-kinase reaction was started by the addition of 100
nM [
32P]ATP (10 µCi) and 300
µM ATP in 25 µl of 25 mM HEPES, pH 7.4, 10
mM MgCl2, and 0.5 mM EGTA. After 15
min at room temperature, the reaction was stopped by the addition of
500 µl CHCl3-methanol (1:2) in a 1% concentration HCl
plus 125 µl chloroform and 125 µl HCl (10 mM). The
samples were centrifuged, and the lower organic phase was removed and
washed once with 480 µl methanol-100 mM HCl plus 2
mM EDTA (1:1). The organic phase was extracted, dried
in vacuo, and resuspended in chloroform. Samples were
applied to a silica gel TLC plate. TLC plates were developed in 2
N propanol-1-acetic acid (65:35, vol/vol), dried,
visualized by autoradiography, and quantitated by scanning laser
densitometry (Molecular Dynamics personal densitometer, Sunnyvale,
CA).
Differential Binding of SH2 Domains from p85
and Grb-2 with
the IRS-Proteins
GST-fusion proteins containing nSH2
p85 and
SH2Grb-2 were prepared as described (7). Cell lysates were
prepared from unstimulated, insulin-stimulated, or IGF-I-stimulated
fetal brown adipocytes in lysis buffer. The extracts were clarified by
centrifugation at 12,000 x g for 15 min at 4 C. The
supernatants were incubated with 10 µg of the GST fusion proteins
containing SH2 domains as indicated at 4 C for 1 h and
precipitated with glutathione Sepharose at 4 C for 1 h, washed
twice in 50 mM Tris/HCl (pH 7.4) containing 100
mM NaCl, 250 µg/ml BSA, 0.2 mM
Na3VO4, and 0.4 mM
phenylmethylsulfonyl fluoride, and boiled for 5 min in Laemmli sample
buffer. Samples were separated on 7% SDS-PAGE and analyzed by
immunoblotting.
Phosphopeptide Competition Studies
Phosphopeptide binding competition assay was performed as
described (33). Lysates from insulin-stimulated cells (600800 µg of
protein) prepared as described above were incubated with the anti-Grb-2
antibody for 30 min at 4 C, divided into 1-ml aliquots, and then
incubated at 4 C for 14 h without or with various doses
(0.001100 µM) of a synthetic phosphopeptide
(PPEPKSPGEY(P)VNIEFG) corresponding to the IRS-1/IRS-2 consensus Grb-2
binding domain (11). The immune complexes were collected on 40 µl
protein A-agarose, resolved on a 7% gel, and analyzed by
immunoblotting with the anti-IRS-1 and anti-IRS-2 antibodies.
Protein Determination
Protein determination was performed by the Bradford dye method
(34), using the Bio-Rad (Richmond, CA) reagent and BSA as the
standard.
Experimental Animals
The animals used for the required experiments in this report
were treated in accord with the "Guidelines for Care and Use of
Experimental Animals."
 |
FOOTNOTES
|
---|
Address requests for reprints to: Manuel Benito, Departament de Bioquimica y Biologia Molecular II, Instituto de Bioquimica (C.S.I.C./U.C.M.), Facultad de Farmacia, Universidad Complutense, Madrid, Spain 28040.
This work was supported by Grant SAF96/0115 from the Comision
Interministerial de Ciencia y Tecnologia, Spain.
Received for publication May 27, 1997.
Revision received October 29, 1997.
Accepted for publication January 27, 1998.
 |
REFERENCES
|
---|
-
Cheatham B, Kahn CR 1995 Insulin action and the insulin
signaling network. Endocr Rev 16:117142[Medline]
-
Jones JI, Clemmons DR 1995 Insulin-like growth factors and
their binding proteins: biological actions. Endocr Rev 16:334[Medline]
-
Ullrich A, Bell JR, Chen EY, Herrera R, Petruzzelli LM, Dull
TJ, Gray A, Coussens L, Liao Y-C, Tsubokawa M, Mason A, Seeburg PH,
Grunfeld C, Rosen OM, Ramachandran J 1985 Human insulin receptor and
its relationship to the tyrosine kinase family of oncogenes. Nature 313:756761[Medline]
-
Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins
C, Henzel W, Bon TL, Kathuria S, Chen E, Jacobs S, Francke U,
Ramachandran J, Fujita-Yamaguchi Y 1986 Insulin-like growth factor I
receptor primary structure: comparison with insulin receptor suggests
structural determinants that define functional specificity. EMBO J 5:25032512[Abstract]
-
Backer JM, Myers Jr MG, Schoelson SE, Chin DJ, Sun XJ,
Miralpeix M, Hu P, Margolis B, Skolnik EY, Schlessinger J, White MF 1992 The phosphatidylinositol 3-kinase is activated by association with
IRS-1 during insulin stimulation. EMBO J 11:34693479[Abstract]
-
Skolnik EY, Batzer AG, Li N, Lee CH, Lowenstein E, Mohammadi
M, Margolis B, Schlessinger J 1993 The function of GRB2 in linking the
insulin receptor to Ras signaling pathways. Science 260:19531955[Medline]
-
Sun XJ, Crimmins DL, Myers Jr MG, Miralpeix M, White MF 1993 Pleiotropic insulin signals are engaged by multisite
phosphorylation of IRS-1. Mol Cell Biol 13:74187428[Abstract]
-
Khuné MR, Pawson T, Lienhard GE, Feng, G-S 1993 The
insulin receptor substrate 1 associates with the SH2-containing
phosphotyrosine phosphatase Syp. J Biol Chem 268:1147911481[Abstract/Free Full Text]
-
Araki E, Lipes MA, Patti ME, Bruning JC, Haag III BL, Johnson
RS, Kahn CR 1994 Alternative pathway of insulin signaling in mice with
targeted disruption of the IRS-1 gene. Nature 372:186190[CrossRef][Medline]
-
Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T,
Terauchi Y, Ueki K, Kaburagi Y, Satoh S, Sekihara H, Yoshioka S,
Horikoshi H, Furruta Y, Ikawa Y, Kasuga M, Yazaki Y, Aizawa S 1994 Insulin resistance and growth retardation in mice lacking insulin
receptor substrate-1. Nature 372:182186[CrossRef][Medline]
-
Sun XJ, Wang LM, Zhang Y, Yenush L, Myers Jr MG, Glasheen EM,
Lane WS, Pierce JH, White M 1995 Role of IRS-2 in insulin and
cytokine signaling. Nature 377:173177[CrossRef][Medline]
-
Sun XJ, Pons S, Wang L-M, Zhang Y, Yenush L, Burks D, Myers Jr
MG, Glasheen E, Copeland NG, Jenkins NA, Pierce JH, White MF 1997 The
IRS-2 gene on Murine chromosome 8 encodes a unique signaling adapter
for insulin and cytokine action. Mol Endocrinol 11:251262[Abstract/Free Full Text]
-
Welham MJ, Bone H, Levings M, Learmonth L, Wang LM, Leslie KB,
Pierce JH, Schrader JW 1997 Insulin receptor substrate-2 is the major
170-kDa protein phosphorylated on tyrosine in response to cytokines in
murine lymphohemopoietic cells. J Biol Chem 272:13771381[Abstract/Free Full Text]
-
Valverde AM, Benito M, Lorenzo M 1991 Proliferation of fetal
brown adipocyte primary cultures: relationship with the genetic
expression of glucose 6-phosphate dehydrogenase. Exp Cell Res 194:232237[Medline]
-
Lorenzo M, Valverde AM, Teruel T, Benito M 1993 IGF-I is a
mitogen also involved in differentiation-related gene expression in
fetal rat brown adipocytes. J Cell Biol 123:15671575[Abstract]
-
Valverde AM, Lorenzo M, Teruel T, Benito M 1995 cAMP inhibits
IGF-I-induced mitogenesis in fetal rat brown adipocytes: role of p21
ras. Exp Cell Res 218:305309[CrossRef][Medline]
-
Valverde AM, Benito M, Lorenzo M 1992 Hormonal regulation of
malic enzyme and glucose 6-phosphate dehydrogenase in fetal brown
adipocyte primary cultures. Eur J Biochem 203:313319[Abstract]
-
Valverde AM, Lorenzo M, Navarro P, Benito M 1997 Phosphatidylinositol 3-kinase is a requirement for insulin-like growth
factor I-induced differentiation, but not for mitogenesis, in fetal
brown adipocytes. Mol Endocrinol 11:595697[Abstract/Free Full Text]
-
Teruel T, Valverde AM, Alvarez A, Benito M, Lorenzo M 1995 Differentiation of rat brown adipocytes during late foetal development:
role of insulin-like growth factor I. Biochem J 310:771776[Medline]
-
Teruel T, Valverde AM, Benito M, Lorenzo M 1996 Insulin-like
growth factor I and insulin induce adipogenic-related gene expression
in foetal brown adipocyte primary cultures. Biochem J 319:627632[Medline]
-
Valverde AM, Teruel T, Lorenzo M, Benito M 1996 Involvement of Raf-1 kinase and protein kinase C
in insulin-like
growth factor I-induced brown adipocyte mitogenic signaling cascades:
inhibition by cyclic adenosine 3,5-monophosphate. Endocrinology 137:38323841[Abstract]
-
Valverde AM, Lorenzo M, Teruel T, Benito M 1997 Alterations in
the insulin signaling pathway induced by immortalization and H-ras
transformation of brown adipocytes. Endocrinology 138:31953206[Abstract/Free Full Text]
-
Patti ME, Sun XJ, Brunning JC, Araki E, Lipes MA, White MF,
Kahn CR 1995 4PS/Insulin receptor substrate-1 is the alternative
substrate of the insulin receptor in IRS-1 deficient mice. J Biol
Chem 270:2467024673[Abstract/Free Full Text]
-
Tobe K, Tamemoto H, Yamauchi T, Aizawa S, Yazaki Y, Kadowaki T 1995 Identification of a 190-kDa protein as a novel substrate for the
insulin receptor kinase functionally similar to insulin receptor
substrate-1. J Biol Chem 270:56985701[Abstract/Free Full Text]
-
Yamauchi T, Tobe K, Tamemoto H, Ueki K, Kaburagi Y,
Yamamoto-Honda R, Takahashi Y, Yoshizawa F, Aizawa S, Akanuma Y,
Sonenberg N, Yazaki, Y Kadowaki T 1996 Insulin signaling and insulin
actions in the muscles and livers of insulin-resistant, insulin
receptor substrate1-deficient mice. Mol Cell Biol 16:30743084[Abstract]
-
Brunning JC, Winnay J, Cheatham B, Kahn RC 1997 Differential
signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1
deficient cells. Mol Cell Biol 17:15131521[Abstract]
-
Skolnik EY, Lee CH, Batzer AG, Vicentini LM, Zhou M, Daly R,
Myers Jr MG, Baker JM, Ullrich A, White MF, Schlessinger J 1993 The
SH2/SH3 domain-containing protein GRB2 interacts with
tyrosine-phosphorylated IRS-1 and Shc: implications for insulin control
of ras signaling. EMBO J 12:19291936[Abstract]
-
White MF 1997 The insulin signalling system and the IRS
proteins. Diabetologia 40:S2S17
-
Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM 1994 Evidence for a functional role of Shc proteins in mitogenic
signaling induced by insulin, insulin-like growth factor-1, and
epidermal growth factor. J Biol Chem 269:1368913694[Abstract/Free Full Text]
-
Sasaoka T, Draznin B, Leitner JW, Langlois WJ, Olefsky JM 1994 Shc is the predominant signaling molecule coupling insulin receptors to
activation of guanine nucleotide releasing factor and p21 ras-GTP
formation. J Biol Chem 2691073410738
-
Giorgetti-Peraldi S, Ottinger E, Wolf G, Ye B, Burkee Jr TR,
Shoelson SE 1997 Cellular effects of phosphotyrosine-binding domain
inhibitors on insulin receptor signaling and trafficking. Mol Cell Biol 17:11801188[Abstract]
-
Reif K, Gout I, Waterfield MD, Cantrell DA 1993 Divergent
regulation of phosphatidylinositol 3-kinase P85
and P85ß isoforms
upon T cell activation. J Biol Chem 268:1078010788[Abstract/Free Full Text]
-
Smith-Hall J, Pons S, Patti ME, Burks DJ, Yenush L, Sun XJ,
Kahn CR, White MF 1997 The 60 kDa insulin receptor substrate functions
like an IRS protein (pp60IRS3) in adipose cells.
Biochemistry 36:83048310[CrossRef][Medline]
-
Bradford M 1976 A rapid and sensitive method for the
quantitation of microgram quantities utilizing the principle of protein
dye binding. Anal Biochem 72:248254[CrossRef][Medline]