From the Dipartimento di Biologia e Patologia
Cellulare e Molecolare & Centro di Endocrinologia ed Oncologia
Sperimentale del Consiglio Nazionale delle Ricerche, Federico II
University of Naples, 80131 Naples, Italy and ¶ INSERM U145,
06107 Nice Cedex 2, France
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ABSTRACT |
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In L6 muscle cells expressing the
Arg1152 Insulin induces proliferative and metabolic responses in several
different tissues (1). The biological effects of insulin in its target
cells are initiated by binding and activating tyrosine kinase receptors
(2), followed by phosphorylation of intracellular protein substrates
(2, 3). The phosphorylated substrates, in turn, bind
SH21 domain-containing
proteins (2, 3) further propagating receptor signal into at least two
major transduction routes. These pathways include the
Ras/mitogen-activated protein kinase (MAP kinase) cascade and the PI
3-kinase system (2, 3), and convey insulin signal to the final
cytoplasmic and nuclear effectors.
Insulin receptor substrate-1 (IRS-1) is an important intracellular
substrate for the insulin receptor kinase (3). IRS-1 features at least
7 tyrosine residues undergoing rapid phosphorylation upon insulin
receptor activation (4) and providing binding sites for at least six
distinct SH2 proteins (2-5). Tyrosine-phosphorylated IRS-1 binds the
SH2 domain in the p85 regulatory subunit of PI 3-kinase (6, 7) inducing
several metabolic responses (8, 9). Phosphorylated IRS-1 also interact
with the Grb2·SOS complex, activating p21ras,
the MAP kinase cascade and mitogenesis (10-12). Similar to IRS-1, the
oncoprotein Shc is tyrosine phosphorylated by the insulin receptor
followed by binding to Grb2·SOS and MAP kinase activation (13, 14).
Therefore, IRS-1 and Shc represent distinct links conveying insulin
signal through the MAP kinase machinery and evoking proliferative
responses. In Rat1 fibroblasts expressing human insulin receptors (15),
as well as in other cells, most SOS guanylnucleotide exchange activity
co-precipitated with Shc rather than IRS-1, suggesting a major role of
Shc in Ras activation by insulin. However, in 32-D cells expressing
insulin receptors, MAP kinase activation by insulin requires Grb2
binding to IRS-1 (16). Thus, the relative role of IRS-1 and Shc in
mediating proliferative response through the Ras·MAP kinase cascade
remains controversial and may be cell- and tissue-specific.
IRS-2 is another cellular substrate for the insulin receptor kinase,
which has been more recently identified in liver and skeletal muscle
cells (17). IRS-2 shares many structural features with IRS-1 (2, 3,
18). Like IRS-1, insulin receptor-phosphorylated IRS-2 binds to both PI
3-kinase and Grb2 (2, 3, 19). In IRS-1 knock-out mice, IRS-2
phosphorylation is substantially increased as compared with the
wild-type animals, suggesting that this increase may compensate for the
lack of IRS-1 thus improving insulin action on glucose metabolism (20).
Evidence has also been reported that IRS-2 mediates insulin-stimulated
translocation of GLUT4 in a fashion similar to IRS-1(2, 3). However,
whether each of these two substrates specializes in mediating certain
insulin bioeffects or whether they are largely redundant into the cells has not been conclusively established. Additionally, it remains unclear
whether variability exists in the relative role of IRS-1 and IRS-2 in
mediating insulin action in the different target tissues.
In the present report, we have studied insulin signaling in L6 skeletal
muscle cells expressing the IR1152 insulin receptor. This
mutant receptor maximally activates metabolic responses, preventing
further insulin stimulation, but normally transduces insulin mitogenic
signals. In addition, as we show in this work, IR1152
differentially phosphorylates IRS-1, IRS-2, and Shc, enabling us to
address their relative function in mediating proliferative and
metabolic signals in skeletal muscle, a major insulin-responsive tissue.
General Procedures--
L6 cell clones expressing 3 × 103 insulin receptors were selected and transfected with
either the mutant IR1152 or the wild-type insulin receptors
and have been previously characterized and described (21). In the
present study, two clones of transfected cells expressing 3.2 × 104 or 9 × 103 wild-type IRs/cell and
3.1 × 104 or 9.5 × 103 mutant
IRs/cell were used. At these low levels, overexpression of wild-type
receptors in L6 as in other cells (22) is accompanied by little change
in maximal insulin effects on most signaling events and cell responses.
However, a 3-10-fold decrease in the ED50 for insulin
effect on IRS-1 and IRS-2 phosphorylation and on glycogen synthase
activity and thymidine incorporation could be consistently detected
(Table I). These cells express fully functional GLUT4 transporters (21, 23-27). The antibodies against phosphotyrosine, IRS-1, IRS-2, Shc, Grb2, MAP kinase, and p85 PI
3-kinase were purchased from either Upstate Biotechnology Inc. (Lake
Placid, NY) or Santa Cruz Biotechnology (Santa Cruz, CA). Media and
sera for tissue culture were from Life Technologies, Inc.,
electrophoresis and Western blot reagents from Bio-Rad, and protein A
beads (Trisacryl) from Pierce. All other chemicals were from Sigma. The
yeast strain L40 (MAT a, trp1, leu2, his3, LYS2::lexA-His3, URA3::lexA-lacZ) and the
yeast expression plasmids pBTM116 were obtained from A. Vojtek (Fred
Hutchinson Cancer Research Center, Seattle, WA). The plasmid pACTII was
provided by Dr. S. Elledge (Baylor College of Medicine, Houston, TX)
and the human insulin receptor cDNA by Dr. S. Gammeltoft
(Bispebjerg Hospital, Copenhagen, Denmark). The two-hybrid
plasmids, the L40 strains, and the cDNA constructs used in this
study have been described previously (28, 29).
Detection of IRS-1, IRS-2, Shc, Grb2, and p85--
Cells were
stimulated with 100 nM insulin for 5 min at 37 °C as
indicated, and lysed in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM
EDTA, 10 mM Na4P2O7, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, 1 mM
phenylmethylsulfonyl fluoride (TAT buffer). Lysates were then incubated
with IRS-1, IRS-2, or Shc Abs for 18 h at 4 °C, and immunocomplexes precipitated with protein A beads, solubilized in
Laemmli buffer, separated by PAGE, and Western-blotted as described in
Ref. 30. Blots were probed with phosphotyrosine, Grb2, or p85 Abs as in
Ref. 30 and revealed with 125I-labeled protein A and
autoradiography. Quantitation was achieved by laser densitometry of the bands.
Determination of PI 3-Kinase and MAP Kinase Activities--
For
PI 3-kinase, cells were stimulated with 100 nM insulin for
15 min at 37 °C as indicated, and solubilized for 40 min at 4 °C
in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10%
glycerol, 1% Nonidet P-40, 10 mM EDTA, 10 mM
Na4P2O7, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 100 mM NaF, 1 mM
phenylmethylsulfonyl fluoride (TAN buffer). Aliquots of the lysates
were precipitated with IRS-1 or IRS-2 Abs coupled to protein A
Sepharose for 2 h at 4 °C. PI 3-kinase activity was determined
in pellets as described in Ref. 31.
For MAP kinase assays, cells were lysed in 50 mM
Glycogen Accumulation, Glycogen Synthase, and Thymidine
Incorporation Assays--
Cells were exposed to 50 nM
wortmannin for 30 min at 37 °C before stimulation with insulin (20 min at 37 °C), as indicated. Glycogen content of the cells was
determined as described previously (21). Glycogen synthase activity was
assayed by a modification of the method by Thomas et al.
(33). Briefly, cells were preincubated in Hepes buffer, pH 7.8, for
3 h at 37 °C before the assay. The cells were further incubated
with 50 nM wortmannin and then stimulated with 100 nM insulin (in the presence or the absence of wortmannin) as indicated, resuspended in ice-cold 100 nM NaF, 10 mM EDTA, and sonicated for 10 s at 300 watts. Cells
were then centrifuged for 10 min at 2,000 rpm, and 20-µl aliquots of
the supernatants (20 µg of cell proteins) incubated with 60 µl of a
reaction mixture containing 40 mM Tris-HCl, pH 7.8, 25 mM NaF, 20 mM EDTA, 10 mg/ml glycogen, 7.2 nM uridine 5'-diphosphate-glucose, in the absence or the
presence of 6.7 mM glucose 6-phosphate. The incubation was
prolonged for 20 min at 30 °C and terminated by spotting on filter
paper followed by precipitation with ice-cold ethanol. Precipitated
radioactivity was quantitated in a Beckman scintillation counter.
Enzyme activity was expressed as a percentage of the glucose
6-phosphate-independent form (21).
For thymidine incorporation, six-well plates were seeded with
105 cells/well. After 24 h, the medium was replaced
with Dulbecco's modified Eagle's medium supplemented with 0.05%
bovine serum albumin and no serum, incubated for 24 h, and than
further incubated for 16 h with the same medium supplemented with
50 mM wortmannin and 100 nM insulin as
indicated. [3H]Thymidine was then added at a specific
activity of 500 nCi/ml, and incorporation into DNA was quantitated as
described previously (34).
Transformation of Yeast Strains and Insulin Receptor Interaction with IRS-2 Fusion Proteins and
Phosphorylation of Immobilized Substrates--
Construction of the
IRS-2 fusion proteins, partial purification of insulin receptors, and
precipitation of autophosphorylated insulin receptors by IRS-2 fusion
proteins were performed as described in Ref. 37. To analyze the
phosphorylation of insulin receptor substrates in vitro,
parental L6 myotubes were lysed with TAT buffer and precipitated with
IRS-1, IRS-2, or Shc antibodies. Precipitated proteins were then
incubated with protein A-Sepharose and the immobilized proteins further
incubated with wild-type or mutant insulin receptors (250 fmol/assay).
Phosphorylation reactions were initiated by adding 2 mM CTP, 2 µM ATP, 10 mM HEPES, pH
7.4, 0.02% Triton X-100, 5 mM MnCl2, 7 mM MgCl2, 0.02 µM
[ IRS Phosphorylation in L61152 Myotubes--
Lysates
were prepared from L6 skeletal muscle cells expressing the
constitutively active Arg1152
Tyrosine-phosphorylated IRSs bind different SH2 proteins, including the
Grb2 adaptor and the p85 regulatory subunit of PI-3 kinase, which
propagate insulin signal (2, 3, 5, 19). In parallel with IRS-1
phosphorylation, Grb2 co-precipitation with IRS-1 was also slightly
increased in Mut as compared with WT cells (Fig. 1, middle
panel, p < 0.05). Likewise, recovery of PI
3-kinase activity in the IRS-1 immunoprecipitates was 20-30% increased in cells expressing IR1152 (Fig. 1,
bottom panel, p < 0.001). After
insulin addition to both WT and Mut cells, Grb2 association with IRS-1
and IRS-1-bound PI 3-kinase were stimulated by almost 3-fold.
At variance with IRS-1, basal phosphorylation of IRS-2 in Mut cells was
constitutively increased by almost 4-fold as compared with control
cells (Fig. 2, top
panel). These phosphorylation levels were comparable to
those of wild-type cells after maximal insulin stimulation. Exposure of
Mut cells to insulin concentrations up to 100 nM did not
further increase IRS-2 tyrosine phosphorylation, however, while
increasing that in control cells by 5-fold. Grb2 co-precipitation with
IRS-2 and recovery of PI 3-kinase activity in IRS-2 immunoprecipitates
also increased by 4- and 2-fold, respectively, in the
insulin-stimulated control cells, while exhibiting high basal levels
and no insulin sensitivity in the mutant cells (Fig. 2,
middle and bottom panels). It appeared
therefore that the IR1152 mutant phosphorylated IRS-1 and 2 and induced their binding to SH2 intracellular proteins differentially
both in the absence and in the presence of insulin. This effect could
not be ascribed to differences in IRS-1, IRS-2, PI 3-K (Fig.
3), or Grb2 (Fig. 7) levels since these
were comparable in all of the cell clones analyzed. The relative levels
of endogenous, wild-type, and mutant insulin receptors in the cell
clones are also shown in Fig. 3. Data similar to those in the L6 cells
were also obtained with NIH-3T3 fibroblasts expressing the mutant and
wild-type insulin receptors (data not shown). As in the intact cells,
in vitro phosphorylation of immobilized IRS-2 by
affinity-purified IRWT showed a 3-fold increase upon
insulin addition, while phosphorylation of immobilized IRS-2 by basal
IR1152 exhibited a 2.8-fold increase as compared with the
wild-type receptors and was not further stimulated by insulin (Fig.
4, top panel).
In vitro phosphorylation of immobilized IRS-1 by the mutant receptors showed a slight 30% basal increase compared with that by the
wild-type receptor (p < 0.05) and was fully
phosphorylated upon insulin stimulation, suggesting that the
IR1152 mutation mainly enhances the interaction of the
receptor with IRS-2. To examine this possibility, we analyzed the
ability of IR1152 to bind the kinase regulatory loop
binding (KRLB) domain of IRS-2. This insulin receptor binding region
has been previously reported to be unique to IRS-2 and not present in
IRS-1 (38). IRWT and IR1152 were incubated with
immobilized GST-IRS-2-KRLB and KRLB-bound receptors were then
immunoblotted with insulin receptor antibodies. As shown in Fig. 4
(middle panel), almost no wild-type receptors bound to the KRLB domain in basal conditions while insulin activation of these receptors determined a 4-fold increase in KRLB binding. At
variance, with the IR1152 mutant, binding to the KRLB
domain was already maximal in the absence of insulin and did not
further increase upon insulin addition. In this same assay, there was
no difference in IR1152 and IRWT binding to the
IRS-2 PTB domain (Fig. 4, bottom panel). Thus, the constitutive IR1152 kinase activity toward IRS-2 is
accompanied by an enhanced binding of the mutant receptor to the KRLB
domain of IRS-2, independent of insulin.
Induction of Metabolic and Proliferative Responses through the
IR1152 Receptor--
While tyrosine phosphorylation is
known to represent a prerequisite for enabling IRS-1 and IRS-2 to
transduce insulin signal downstream the receptor, their relative role
in inducing metabolic responses in target tissues remains unsettled
(20). As we previously reported in the L6 myotubes (21), expression of
IR1152 constitutively activated glycogen synthase blocking
insulin stimulation of the enzyme. Glycogen content in these cells was
also maximal in the basal state and not further stimulated by insulin
(Fig. 5). In Mut cells, both glycogen
synthase and glycogen accumulation were returned to levels similar to
those detected in basal control cells after incubation with the PI
3-kinase inhibitor wortmannin. Wortmannin also blocked
insulin-stimulated activation of glycogen synthase and glycogen
accumulation in WT cells, indicating that PI 3-kinase mediated both the
induction of these responses through the wild-type IR and through the
constitutively active mutant (Fig. 5).
Activation of the MAP kinase system by the Grb2·SOS complex
represents a major mechanism conveying IR mitogenic signals to the
nucleus (11). In the Mut cells, MAP kinase activity exhibited a modest
basal increase (30%, p < 0.001) as compared with the control cells (Fig. 6, top
panel). [3H]Thymidine incorporation into DNA
also exhibited a slight basal increase in the mutant-expressing cells
(p < 0.05; Fig. 6, bottom panel). Insulin addition rapidly stimulated MAP kinase
activity in the Mut and control cells by 2.5- and 3-fold, respectively, and increased [3H]thymidine incorporation into DNA by
more than 6-fold in both cell lines. At variance from glycogen
synthesis and glycogen synthase activity, insulin-stimulated thymidine
incorporation was not affected by preincubation of the cells with
wortmannin. Thus, in the Mut cells, glucose storage reflected IRS-2 but
not IRS-1 phosphorylation and was blocked by PI 3-kinase inhibition. In
contrast, proliferative responses better correlated with IRS-1 than
with IRS-2 phosphorylation levels and are independent of PI 3-kinase
activity.
Shc Phosphorylation in IR1152-expressing Cells--
In
most cells, phosphorylation of the IR substrate Shc and its association
to the Grb2·SOS complex is considered to represent a major and
wortmannin-independent pathway transducing insulin mitogenic signals
(15). Whether the Shc route is necessary or redundant for
insulin-induced mitogenesis is currently unknown. Based on
immunoprecipitation of Shc followed by blotting with Tyr(P) Ab,
phosphorylation of p52shc was detectable in both
the control and the Mut cells (Fig. 7, middle panel). However, Shc was not
constitutively phosphorylated in Mut cells. In addition, insulin
increased Shc phosphorylation by 3-fold in the wild-type cells, but had
almost no effect in several mutant clones. The levels of Shc and Grb2
were identical in control and Mut cells (Fig. 7, top
panel). Insulin had also no effect on Grb2 association with
Shc in Mut cells, although increasing that in the WT by 2-fold (Fig. 7,
bottom panel). Therefore, in the
IR1152-expressing cells, insulin-induced mitogenesis
occurred in the absence of Shc phosphorylation and its subsequent Grb2
association.
Shc interaction with the insulin receptor has been reported to depend
on a PTB domain homologous to that of IRS-1 and IRS-2 (39). Since the
IR1152 mutant normally binds to the PTB domain of IRS-2, we
sought to investigate further the ability of this mutant receptor to
bind Shc in a yeast two-hybrid analysis. Shc full-length cDNA was
fused to the Gal4 activation domain, whereas the catalytically active cytoplasmic portion of the insulin receptor (including the
juxtamembrane region) was fused to the LexA DNA binding domain.
Gal4-fused IRS-1 and IRS-2 full-lengths and Raf full-length were
included as positive and negative controls, respectively. As shown in
Fig. 8 (top panel), Shc interacted with IR1152 as well as with the wild-type
insulin receptor in the yeast two-hybrid assay. Consistent with the
data shown in the previous sections of this report, IRS-1 and IRS-2
also interacted with the mutant and the wild-type insulin receptors,
while Raf did not. In vitro, IR1152 elicited a
30% increase in the basal phosphorylation of Shc as compared with
IRWT (p < 0.05; Fig. 8,
bottom panel). Insulin addition induced a further 70% increase in Shc phosphorylation by the mutant receptor, similar to
that measured with receptors from control cells (difference not
statistically significant). Thus, the data indicated that the lack of
Shc phosphorylation in intact Mut cells did not directly result from an
effect of the IR1152 mutation on IR1152-Shc
binding or phosphorylation. Alternatively, we postulated that the lack
of in vivo phosphorylation might be caused by the abnormal
intracellular routing, which characterizes the IR1152
receptors (40). To test this hypothesis, we analyzed Shc
phosphorylation upon 24-h preincubation of the cells with TPA. This
treatment shifts the internalized insulin receptors toward the
retroendocytotic rather than the degradative compartment, thus
mimicking IR1152 routing in the L6 myotubes as well as in
the NIH-3T3 fibroblasts (41). As shown in Fig.
9 (top panel), TPA
preincubation of control cells, reduced the insulin-stimulated
phosphorylation of Shc to levels comparable to those measured in
untreated Mut cells with no change in the total Shc levels of the cells
(Fig. 9, bottom panel). TPA did not further
reduce the insulin-stimulated Shc phosphorylation in the Mut cells. At
variance with Shc phosphorylation, preincubation with TPA elicited no
change in IRS-1 phosphorylation by either the wild-type or the
IR1152 receptors (Fig. 9, middle
panel).
Insulin evokes a wide range of metabolic and mitogenic responses
by binding tyrosine kinase receptors and phosphorylating tyrosines on
several intracellular protein substrates (1-3). These include IRS-1,
IRS-2, and Shc. While the relevance of these IR substrates in
propagating insulin signal has been well established (2, 3), the
specific role of each of them as well as the extent to which they are
redundant or complementary is less clear (3). In addition, IRS-1,
IRS-2, and Shc may feature tissue specificity in the major targets for
insulin action, muscle, liver, and adipose tissues (3). In the present
work, we have addressed these issues by analyzing signaling through the
IR1152 mutant insulin receptor in cultured L6 skeletal
muscle cells. The IR1152 receptor maximally activates
metabolic responses in several cell types, preventing further
stimulation by insulin (21, 34). In contrast, insulin mitogenic signals
are normally mediated by this mutant (34), enabling us to investigate
which receptor substrates are involved in proliferative and metabolic
insulin effects. While the L6 muscle cells may not necessarily reflect all of the properties of skeletal muscle tissue in vivo,
they have been widely used for studies on insulin action since they possess several characteristics of this tissue (21, 23-27). In addition, cultured cells with preserved IR substrate function provide
an important tool for investigating the specific function of each
substrate, complementary to the in vivo/ex vivo
models. In fact, very recent data in knock-out animals have shown that disruption of IRS-1 gene results in compensatory mechanisms affecting the function of other substrates (20). These effects do not occur or
may be more easily controlled in cultured cells with unaffected
substrate expression. Furthermore, in the present study, L6 cell clones
have been chosen expressing only small numbers of exogenous receptors.
At these low levels of expression, abnormal cellular events that do not
occur in the untransfected cells are unlikely to complicate the
interpretation of our findings.
We report that IRS-2 was constitutively tyrosine-phosphorylated in L6
cells expressing the IR1152 receptor and did not undergo
further phosphorylation following insulin exposure. At variance, in
cells expressing the mutant receptor, IRS-1 phosphorylation exhibited
little increase under basal conditions, but featured comparable insulin
phosphorylation in cells expressing the wild-type and the
IR1152 receptors. The differential phosphorylation of the
two IRSs likely reflects an enhanced capability of IR1152
to bind the KRLB domain of IRS-2, independent of insulin. In fact, we
have shown that (i) in vitro, IR1152 shows
increased binding to the KRLB domain, which is only present in IRS-2;
(ii) IR1152 normally binds to IRS-2 PTB domain and exhibits
normal phosphorylation of the juxtamembrane NPEY motif (42), which is
crucial for binding IRS-2 as well as IRS-1 and Shc PTB domains (39);
and (iii) the differential phosphorylation of IRS-1 and IRS-2 by
IR1152 occurs similarly in vitro and in intact
cells, suggesting that it is not caused by discrete effects of the
mutant receptor on the cellular mechanisms controlling phosphorylation
of these substrates. Previous work has shown that binding of the KRLB
domain requires phosphorylation of the tyrosine triplet in the insulin
receptor regulatory domain (38), while phosphorylation of these
residues is depressed in the IR1152 mutant (42). In the
IR1152 receptor, however, we showed that the mutation
mimics the effect of phosphorylation of the regulatory tyrosines (21),
activating transduction of several biological effects in the absence of
insulin. Thus, the constitutive IRS-2 binding to the regulatory loop of IR1152 may contribute to its unique signaling.
At variance with IRS phosphorylation, IR1152 was unable to
phosphorylate Shc in intact cells, either in the absence or the
presence of insulin. The absence of Shc phosphorylation in the Mut
cells upon insulin exposure is consistent with the previously reported dominant activity of IR1152 over the small complement of
endogenous IRs (34). However, the mechanism responsible for this defect
does not seem to involve the inability of IR1152 to
interact with Shc. In fact IR1152 binds Shc in a yeast
two-hybrid system and phosphorylates it in vitro.
Alternatively, we postulated that the abnormal cellular routing of the
mutant receptor (40) may impair its ability to bind Shc. Consistent
with this hypothesis, in the present paper, we show that TPA treatment
alters the cellular routing of wild-type insulin receptors, mimicking
that of IR1152, and, simultaneously, impairs Shc, although
not IRS, phosphorylation. Previous studies also showed that IRS
phosphorylation is unaffected by the inhibition of insulin and IGF-I
receptor endocytosis (43, 44) while phosphorylation of Shc by the
insulin and IGF-I receptors does appear to require receptor endocytosis
(43, 44). Thus, the possibility exists that Shc phosphorylation occurs
at an intracellular site distant from the plasma membrane and away from
the IR1152 route.
In IR1152-expressing clones, basal
[3H]thymidine incorporation is only slightly increased
compared with that of cells expressing wild-type receptors, despite
maximal IRS-2 phosphorylation and Grb2 association with IRS-2. On the
other hand, insulin exposure simultaneously increased IRS-1
phosphorylation and Grb2 association, MAP kinase activity and
[3H]thymidine incorporation almost identically in mutant
and wild-type cells. At variance with other cell types (8, 45),
inhibition of PI 3-kinase activity with wortmannin did not impair at
all insulin-induced DNA synthesis in L6 cells. It appears therefore that induction of the IRS-2·Grb2 complex is less efficient than that
of IRS-1·Grb2 complex in transducing mitogenic responses and thus
IRS-2 cannot substitute for IRS-1 in mediating the mitogenic action of
insulin in Mut cells. In addition, these data indicate that the
Grb2·SOS-activated MAP kinase cascade is a major pathway conveying
insulin proliferative signals in these muscle cells via IRS-1
phosphorylation. Hence, preliminary experiments in our laboratory show
that ribozyme suppression of IRS-1 in L6 cells and pre-exposure of the
cells to the MAP kinase inhibitor PD98059 blocked insulin effect on
thymidine incorporation. Consistent with our findings, Sharma et
al. (46) have shown that adenovirus-mediated overexpression of
IRS-1 interacting domains abolishes insulin-stimulated mitogenesis in
3T3-L1 adipocytes. Also, IRS-1-deficient mice exhibit growth
retardation despite supranormal levels of IRS-2 phosphorylation in
tissues (20) while IRS-2-deficient mice show progressive deterioration
of glucose homeostasis with only small differences in growth (47).
Finally, our recent work indicates that IRS-1 but not IRS-2 mediates
IGF-I mitogenic responses (48), suggesting functional specialization in
the IRS system. In most cells, including skeletal muscle cells, Shc
also has an important function in transducing insulin effect on cell
proliferation (15). However, our data show that the
Shc-dependent component of the insulin mitogenic signal may
be largely redundant in the L6 muscle cells, at least in those
expressing the IR1152 mutant receptor. Hence, in these
cells, full insulin activation of mitogenesis occurs in the absence of
any detectable phosphorylation of Shc.
Different from thymidine incorporation, glycogen synthase activity and
glycogen accumulation in L61152 cells were constitutively
increased by the mutant receptor preventing further increase upon
insulin exposure. A severely impaired increase in glucose disposal in
response to insulin was also measured by us in vivo, in the
skeletal muscle of diabetic individuals expressing the mutant receptor
(49). It is possible therefore that insulin resistance in these
patients is also contributed by a constitutive increase in muscle
glycogen synthase activity and glycogen content due to the mutant
receptor. The inhibition of PI 3-kinase activity blocked both the
constitutively active glycogen synthesis in
IR1152-expressing cells and the
insulin-dependent glycogen synthesis in cells expressing
IRWT. These data suggest that the same mechanism is
involved in the control of the glycogen synthetic machinery by both the
constitutively active IR1152 and the insulin-activated
wild-type receptors. In the mutant cells, the glycogen synthetic
process is fully activated concomitantly with the constitutive
phosphorylation of IRS-2 but with almost no IRS-1 phosphorylation. In
addition, it did not further increase upon insulin addition despite a
12-fold increase in IRS-1 phosphorylation. It appears therefore that
IRS-2 mediates metabolic signaling in Mut cells. Consistent with this
interpretation, very recent data (47) show that disruption of IRS-2
gene in mice leads to increased PI-3 kinase activity in IRS-1
precipitates from basal muscle tissue and nevertheless the IRS-2
knock-out mice are severely insulin-resistant. An alternative
interpretation of our data is that dose responses for DNA synthesis and
glycogen synthesis are quantitatively different in the L6 cells. Thus,
only a small amount of phosphorylated IRS-1 (such as that present in
the Mut cells) is sufficient to fully activate glycogen synthesis. In
this event, however, one would expect that at submaximally effective
receptor expression, IRS-1 should be rate-limiting for glycogen
accumulation. In contrast, our data show that the expression of small
numbers of IR1152 receptors are accompanied by undetectable
levels of IRS-1 phosphorylation but still induce maximal IRS-2
phosphorylation and glycogen synthesis. Previous work in 3T3-L1
adipocytes showed that interference with the IRS-1-IR interaction did
not cause inhibition of insulin-stimulated glucose transport,
suggesting that alternate pathways exist in these cells (46). More
recently, Zhou et al. (50) have shown that, in rat
adipocytes, overexpression of high levels of IRS-1 as well as of IRS-2
increased basal and insulin-stimulated Glut4 levels in the plasma
membranes indicating that both IRSs may signal Glut4 translocation when
overexpressed in cells. Supramaximal IRS-2 phosphorylation or IRS-2
overexpression caused by IRS-1 deficiency may also be responsible for
the residual insulin-stimulated glucose transport in soleus muscles of
IRS-1 knock out mice (20). Here, we show that, even in the absence of
absolute increases in phosphorylation or of overexpression, IRS-2 can
mediate insulin metabolic effects in L6 cells.
In conclusion, we have provided evidence that IRS-2 mediates insulin
regulation of glucose storage in the L6 cells expressing IR1152 receptors. In addition, IRS-1 and Shc activation of
the MAP kinase cascade may be largely redundant in mediating
proliferative responses in these cells.
Gln insulin receptor (Mut), basal
tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was
increased by 35% compared with wild-type cells (WT). Upon exposure to
insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and
WT cells. IRS-2 was constitutively phosphorylated in Mut cells and not
further phosphorylated by insulin. The maximal phosphorylation of IRS-2
in basal Mut cells was paralleled by a 4-fold increased binding of the
kinase regulatory loop binding domain of IRS-2 to the
Arg1152
Gln receptor. Grb2 and phosphatidylinositol
3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation
levels of the two IRSs. Mitogen-activated protein kinase activation and
[3H]thymidine incorporation closely correlated with IRS-1
phosphorylation in Mut and WT cells, while glycogen synthesis and
synthase activity correlated with IRS-2 phosphorylation. The
Arg1152
Gln mutant did not signal Shc phosphorylation
or Shc-Grb2 association in intact L6 cells, while binding Shc in a
yeast two-hybrid system and phosphorylating Shc in vitro.
Thus, IRS-2 appears to mediate insulin regulation of glucose storage in
Mut cells, while insulin-stimulated mitogenesis correlates with the
activation of the IRS-1/mitogen-activated protein kinase pathway in
these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in
muscle cells.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
Basal and insulin-stimulated IRS phosphorylation and glycogen synthase
activity in L6 expressing endogenous and wild-type human insulin
receptor
-glycerol phosphate, 10 mM Hepes, pH 8.0, 70 mM NaCl, 1 mM Na3VO4,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride. Determination of MAP
kinase activity in the lysates was performed as described in Ref. 32.
Briefly, 10 µl of the lysates (10 µg of cell protein) were
incubated with 5 µg of myelin basic protein for 15 min at 25 °C in
a final volume of 25 µl of 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 10 mM MgCl2, 40 mM [
-32P]ATP. The reaction was terminated
by addition of 4× Laemli buffer and myelin basic protein
phosphorylation determined by PAGE separation, followed by quantitation
of radioactivity in the excised bands.
-Galactosidase
Assay--
Plasmid DNA transformations were performed using the
lithium acetate method of Gietz et al. (35). Cotransformants
were selected on Trp
, Leu
plates. The
transformants were tested for
-galactosidase activity by liquid
culture assays using the substrate
o-nitrophenyl-
-D-galactopyranoside as
described by Miller (36).
-32P]ATP (final concentrations), and prolonged for 30 min at 22 °C. Phosphorylated proteins were separated by SDS-PAGE and
analyzed by autoradiography.
RESULTS
Gln insulin receptors
(Mut cells) (21). Immunoprecipitation of these lysates
(Mut1 cell clone, 3.1 × 104 IR/cells)
with IRS-1 Abs followed by blotting with phosphotyrosine antibodies
(Tyr(P) Abs) revealed a slight increase (35%, p < 0.001) in basal IRS-1 tyrosine phosphorylation as compared with lysates from control cells, either those expressing a comparable number of
wild-type hIRs or those from parental cells (WT, L6 cells, respectively; Fig. 1, top
panel). A 20% basal increase in IRS-1 phosphorylation was
also detectable in cells expressing smaller number of mutant receptors
(Mut3; 9 × 103 receptors/cell;
p < 0.05). Exposure to insulin produced a
similar 10-12-fold increase in IRS-1 phosphorylation in all of the
cell lines.
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Fig. 1.
IRS-1 phosphorylation and association to SH2
proteins in Mut cells. Different clones of L6 myotubes, either
parental cells (L6; 3.1 × 103 IR/cell) or cells
expressing human wild-type (WT1 and WT3;
3.2 × 104 and 9 × 103 IR/cell,
respectively) or mutant insulin receptors (Mut1 and
Mut3; 3.1 × 104 and 9.5 × 103 IR/cell, respectively) were incubated in the absence or
the presence of 100 nM insulin as described under
"Materials and Methods." Cell lysates were
precipitated with IRS-1 Abs and then immunoblotted with phosphotyrosine
(top) or Grb2 (middle) Abs. Blotted proteins were
revealed with 125I-protein A and autoradiography. PI
3-kinase activity in IRS-1 precipitates (bottom) was assayed
as described under "Materials and Methods."
Quantitation of both the IRS-1·Grb2 bands and PIP spots was achieved
by laser densitometry. In each panel, the bars represent the
mean values ± S.D. from at least three independent experiments. A
representative experiment is shown in each inset. Based on
t test analysis, the differences in basal IRS-1
phosphorylation in Mut1 and Mut3
versus control cells were significant at the
p < 0.001 and p < 0.05 levels,
respectively. The increased basal IRS-1 association with Grb2 and PI
3-K activity in Mut1 and Mut3 cells was
significant at the p < 0.05 (Grb2 association) and
p < 0.001 (PI 3-K activity) levels.
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Fig. 2.
IRS-2 phosphorylation and association to SH2
proteins in Mut cells. WT and Mut cells were incubated with
insulin as described in the legend to Fig. 1, immunoprecipitated with
IRS-2 Abs, and then immunoblotted with phosphotyrosine (top
panel) or Grb2 (middle panel) Abs or
assayed for PI 3-kinase activity (bottom panel).
Detection and quantitation was achieved as in the experiment shown in
Fig. 1. In each panel, the bars represent the mean
values ± S.D. from at least three independent experiments. The
difference in basal values between Mut and control cells are
statistically significant (p < 0.001). A
representative experiment is shown in each inset.
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Fig. 3.
IR, IRS, and PI 3-kinase levels in Mut
cells. The myotubes were lysed and cell proteins separated by
SDS-PAGE as described under "Materials and Methods." Proteins were
then blotted using specific IR, IRS-1, IRS-2, or p85 antibodies and
revealed by 125I-protein A and autoradiography. The
autoradiographs shown are representative of at least three independent
experiment for each of the proteins analyzed.
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Fig. 4.
In vitro interaction of
IR1152 with IRSs. Top, substrates
phosphorylation by IR1152. IRS-1, and IRS-2
from L6 parental myotubes were immobilized on Sepharose beads and
phosphorylated in vitro as described under "Materials and
Methods," using insulin receptors purified from parental (L6), WT, or
Mut cells as indicated. Phosphorylated proteins were then separated and
analyzed by SDS-PAGE. Middle and bottom
panels, precipitation of IR1152 by IRS-2 fusion
proteins. Purified wild-type and mutant insulin receptors were
incubated with immobilized KRLB or PTB domains of IRS-2 as described
under "Materials and Methods," immunoblotted with specific receptor
antibodies, and revealed by ECL and autoradiography. Each
bar represents the mean ± S.D. of values from three
independent experiments. A representative autoradiograph is shown in
each inset.
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Fig. 5.
Glycogen synthase activity and glycogen
content in Mut cells. The cells were preincubated with 50 nM wortmannin for 30 min and then further exposed to 100 nM insulin for 30 min (glycogen synthase) or 12 h
(glycogen content). Glycogen synthase activity (top
panel) and glycogen content (bottom
panel) in cell extracts were determined as described
under "Materials and Methods." Each value is
the mean ± S.D. of duplicate determinations in four
(glycogen synthase) and three (glycogen content) experiments. The
differences in basal values between Mut and control cells are
statistically significant (p < 0.001).
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Fig. 6.
MAP kinase and thymidine incorporation in Mut
cells. Different clones of L6 myoblasts expressing either human
wild-type (WT1 and WT3) or mutant insulin
receptors (Mut1 and Mut3) were preincubated
with 50 nM wortmannin and then exposed to 100 nM insulin. MAP kinase activity in cell lysates and
[3H]thymidine incorporation into DNA were determined as
described under "Materials and Methods." Each
value is the mean ± S.D. of duplicate determinations in six (MAP
kinase) and five (thymidine incorporation) experiments.
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Fig. 7.
Shc phosphorylation and Grb2 association in
Mut cells. The L6 myoblasts were incubated in the absence or the
presence of 100 nM insulin as indicated. Cell lysates were
precipitated with Shc Abs and then immunoblotted with phosphotyrosine
(middle panel) or Grb2 (bottom
panel) Abs. Blotted proteins were revealed with
[125I]protein A and autoradiography. Quantitation of the
bands was achieved by laser densitometry of the autoradiographs. In
each panel, the bars represent the mean values ± S.D.
from at least three independent experiments. A representative
experiment is shown in each inset. For detection of total
cellular levels of Shc and Grb2 (top panel),
equal amounts of solubilized cell proteins were separated by SDS-PAGE
and blotted with Shc or Grb2 Abs. A representative experiment is
shown.
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Fig. 8.
Analysis of IR1152 interaction
with Shc and other insulin receptor substrates. Top
panel, measurement of the -galactosidase activity in
transformed yeast is shown. The yeast reporter strain L40 was
co-transformed with a plasmid encoding the LexA DNA binding
domain-IR
in combination with a plasmid encoding GAD-Shc, GAD-IRS-1,
or GAD-IRS-2. Transformants were isolated on selective plates.
Activation of the LexA-LacZ reporter gene was monitored by measuring
-galactosidase activity in cell lysates using
o-nitrophenyl-
-D-galactopyranoside as
substrate. The activities are expressed in Miller's units (36) and are
the average ± S.D. of values obtained with samples prepared from
three independent transformants. Bottom, Shc phosphorylation by
IR1152. Shc from L6 parental myotubes were
immobilized on Sepharose beads and phosphorylated in vitro
as described under "Materials and Methods," using insulin receptors
purified from either the parental cells or the WT and Mut cells as
indicated. Phosphorylated proteins were then separated by SDS-PAGE and
revealed by autoradiography. Each bar represents the
mean ± S.D. of at least three independent experiments. A
representative autoradiograph is shown in the inset.
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Fig. 9.
Effect of TPA on insulin-stimulated Shc
phosphorylation in L6 cells. Top, L6 parental myotubes
and cells expressing WT and IR1152 mutant receptors were
incubated with 1 µM TPA for 24 h and further
stimulated with 100 nM insulin for 10 min at 37 °C as
indicated. Shc phosphorylation in the intact cells was then analyzed as
described in the legend to Fig. 7. Each bar represents the
mean value ± S.D. of at least three independent determinations. A
representative autoradiograph is shown in the inset.
Bottom, to verify the total levels of Shc and IRS-1 upon TPA
treatment, aliquots of the cell lysates were subjected to SDS-PAGE and
blotted with Shc or IRS-1 specific antibodies as indicated.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. E. Consiglio and G. Vecchio for continuous support and advice during the course of this work, and to Drs. P. Gorden and D. Accili (National Institutes of Health IH, Bethesda, MD) for generously donating anti-insulin receptor antibodies. We also like to thank Drs. L. Beguinot (DIBIT, H. S. Raffaele, Milan, Italy) and F. Andreozzi for advice and critical reading of the manuscript and Dr. D. Liguoro for technical help.
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FOOTNOTES |
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* This work was supported in part by the Biomed2 Program of the European Community (Grant BMH4-CT-0751 to F. B.), Telethon Grant E.554 (to F. B.), grants from the Associazione Italiana per la Ricerca sul Cancro (to F. B and P. F.), the Ministero dell' Università e della Ricerca Scientifica, and the Consiglio Nazionale delle Ricerche Target Project on Biotechnology.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.
§ Recipient of a fellowship from the Federazione Italiana per la Ricerca sul Cancro.
To whom all correspondence should be addressed: Dipartimento
di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Via S. Pansini 5, 80131 Naples, Italy. Tel.: 39-081-7463248; Fax: 39-081-7701016; E-mail: beguino{at}unina.it.
The abbreviations used are: SH2, Src homology 2; IR, insulin receptor; IRS, insulin receptor substrate; PAGE, polyacrylamide gel electrophoresis; Ab, antibody; WT, wild-type; MAP, mitogen-activated protein; PI, phosphatidylinositol; IGF-I, insulin-like growth factor-I; KRLB, kinase regulatory loop binding; PTB, phosphotyrosine binding; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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REFERENCES |
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