(Received for publication, January 30, 1995; and in revised form, May 24, 1995)
From the
We constructed mutant receptors by mutating transmembrane
Val of the human insulin-like growth factor I receptor
(IGF-IR). Assays of receptor kinase and autophosphorylation revealed
constitutively augmented tyrosine kinase activity of V922E IGF-IR in
both transient and stable expression. The constitutively active
tyrosine kinase of this mutant was verified by promoted tyrosine
phosphorylation of insulin receptor substrate-1 (IRS-1) in the absence
of IGF-I. In CHO cells stably expressing V922E IGF-IR, both IRS-1
phosphorylation and the IRS-1-associated phosphoinositide 3-kinase
activity were stimulated in the absence of IGF-I to the level attained
by 1 nM IGF-I stimulation of wild-type IGF-IR, whereas the
Ras-mitogen-activated protein kinase pathway was not activated under
the same condition. In these CHO cells, V922E IGF-IR significantly
stimulated glucose uptake but did not promote mitogenesis in the
absence of IGF-I. We thus conclude that the V922E mutation of IGF-IR
switches on the intrinsic tyrosine kinase and differentially activates
the downstream pathways. This mutant is extremely useful in clarifying
the turning-on mechanism of IGF-IR as well as the differential roles of
individual downstream pathways of receptor tyrosine kinases.
Insulin-like growth factor I (IGF-I) ()is a regulator
of cell growth and metabolism(1) . IGF-IR, a receptor tyrosine
kinase belonging to the IR superfamily, mediates most actions of
IGF-I(2, 3, 4) . It consists of a 135-kDa
subunit and a 95-kDa
subunit and forms an
heterodimer on the cell
surface(5) . The binding of IGF-I to the
subunit turns on
the tyrosine kinase in the
subunit(6) . IRS-1 is the
major protein that undergoes tyrosine phosphorylation in response to
IGF-I(7, 8) . Phosphorylated IRS-1 then switches on
the downstream pathways by recruiting PI
3-kinase(8, 9) , tyrosine-specific protein phosphatase
Syp(10) , and probably others. As ligand-activated IGF-IR also
autophosphorylates its
subunit, phosphorylation of both the
subunit and IRS-1 thus provides the best index of the activation of
IGF-IR.
A basic question is what physiological roles IGF-IR plays in living cells. IGF-I produces a variety of cellular responses, both mitogenic and metabolic. Which action of IGF-I is mediated by IGF-IR is, however, less defined. This is mainly because IGF-I also acts on IR, which mediates similar cellular responses(11) . Another question is how the differences in the cellular outputs between IGF-IR and IR are created in vivo. IGF-I and insulin preferentially activate mitogenesis and metabolism via IGF-IR and IR, respectively(1) . Nevertheless, these two receptors are highly homologous structurally and biochemically(1) . The downstream pathways of IGF-IR so far clarified are all shared by IR(11) . Thus, it remains unknown if each receptor has its unique signaling pathway or if such differences result from the different activation profiles of the same downstream pathways.
To address these
questions, it is necessary to turn on IGF-IR selectively. One of the
best ways is to construct and use a constitutively active mutant of
IGF-IR. The expression of such an autoactive mutant allows the precise
analysis of the downstream pathways and functional roles of IGF-IR.
There are mutants of growth factor receptors whose tyrosine kinase
activities are constitutively turned on in the absence of their
ligands(12, 13, 14, 15) . In the
present study, based on analogy with mutation-dependent activation of
these receptors, we have constructed mutant IGF-IRs by substituting Glu
or Ile for the transmembrane Val residue and have found
that the former mutant retains increased tyrosine kinase activity and
constitutively and differentially activates the downstream cascade of
IGF-IR. Differential roles of IGF-IR signaling pathways in biological
outputs are also discussed by comparing the metabolic and mitogenic
effects of this activated mutant to its intracellular signals.
An EcoRI fragment of human IGF-IR cDNA containing a
complete open reading frame was subcloned into pUC19. The mutagenized
cDNAs were generated by the unique site elimination method (Pharmacia
Biotech) using a selection primer in which an original SspI
site was replaced with an EcoRV site (5`-CTTCCTTTTTCGATATCATT
GAAGCATTT-3`: an EcoRV site is underlined). The sequences of
mutagenic target primers were 5`-GTACAGCATAATGATCAACCCTCC-3` for V922I
IGF-IR and 5`-GTACAGCATAATCTCCAACCCTCC-3` for V922E IGF-IR (the codon
for Val is underlined). These mutations were confirmed by
dideoxynucleotide sequencing using Sequenase (U. S. Biochemical
Corp.), and IGF-IR cDNAs were cloned into pcDNA1. COS-7 and
SV40-infected BALB c/3T3 cells (SV40-BALB cells, ATCC) were grown in
Dulbecco's modified Eagle's medium plus 10% calf serum. CHO
cells were grown in Ham F-12 plus 10% FCS. The antibodies used were:
IR3, monoclonal antibody against human IGF-IR (Oncogene Science);
1D6, monoclonal antibody against rat IRS-1(16) ; RC20H,
anti-Tyr(P) monoclonal antibody conjugated with horseradish peroxidase
(Transduction Laboratories); polyclonal antibody for the C terminus of
rat IRS-1 (UBI); and rabbit antisera against C terminus (residues
350-367) of rat ERK1.
Transient transfection was performed by
the lipofection method (10 µg of either WT, V922E, or V922I IGF-IR
cDNA and 20 µl of LipofectAMINE (Life Technologies, Inc.)) as
described(17) . After transfection, cells were treated with or
without IGF-I for 5 min at 37 °C. For stable expression of IGF-IRs,
CHO cells were transfected by the lipofection method using 1 µg of
pSV2neo and 10 µg of WT or mutant IGF-IR cDNA. Cells were split the
next day at 1:10, plated in Ham F-12 plus 10% FCS, and, 2 days later,
were selected with 450 µg/ml G418. After 2-3 weeks, colonies
were picked and tested for I-IGF-I binding as
described(11) . The colonies with high binding levels were
further subcloned by limiting dilution. Cells overexpressing WT, V922E,
or V922I IGF-IR were designated as CHO-IGF-IR, CHO-V922E, or CHO-V922I,
respectively.
For immunoprecipitation, cells were starved in Ham
F-12 for 24 h at 37 °C and treated with IGF-I at 37 °C. Cells
were lysed and precipitated with mouse IgG (Cappel), IR3, or 1D6,
as described (18) . The samples were electrophoresed on
SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride
sheet, probed with RC20H or anti-IRS-1 antibody/horseradish
peroxidase-conjugated anti-rabbit IgG, and detected by ECL. For
labeling studies, 45 h post-transfection cells were incubated with Met-
and Cys-free Dulbecco's modified Eagle's medium containing
[
S]Met and [
S]Cys (0.1
mCi/ml) with 10% dialyzed FCS for 3 h at 37 °C. Cells were then
incubated with IGF-I for 5 min and analyzed as described(19) .
The IGF-IR kinase activity was measured using Poly(Glu, Tyr) 4:1
(Sigma) as described(18) . The bands of poly(Glu, Tyr) and
IGF-IR subunit were excised and quantitated by Cerenkov counting.
The PI 3-kinase activity was measured directly in 1D6 precipitates in a
reaction mixture as described(18) . IGF-I-stimulated
2-deoxy-D-[1,2-
H]glucose uptake (20) and [
H]methylthymidine incorporation (21) were measured as described. The cell proliferation assay
using XTT (Boehringer Mannheim) was performed according to the
manufacturer's instructions. Analysis of Ras-bound guanine
nucleotides was performed as described(16) . The MAP kinase
activity was measured by immune-complex kinase assay in anti-ERK1
immunoprecipitates and in-gel kinase assay using cell lysates. ERK1 was
precipitated with anti-ERK1 rabbit sera (1:25 dilution), and its
activity was measured using myelin basic protein (MBP) as described (22) . In-gel kinase assay was performed using MBP as
described(23) . All radioisotopes were purchased from DuPont
NEN. All experiments were reproduced at least three times (only in
immune-complex MAP kinase assay, experiments were independently
repeated twice).
We first examined in vitro kinase activity of
Val mutants using an exogenous substrate. IGF-IRs
transiently expressed in COS-7 cells treated with or without 100 nM IGF-I were precipitated with
IR3. Tyrosine kinase activity
was assessed by measuring incorporation of radioactivity into poly(Glu,
Tyr) 4:1. As shown in Fig.1A, poly(Glu, Tyr)
phosphorylation markedly increased in the IGF-I-untreated precipitate
of V922E IGF-IR but not in that of WT or V922I IGF-IR. Notably, the
same figure shows that autophosphorylation of the 95-kDa
subunit
of IGF-IR (as well as the 200-kDa IGF-IR precursor) also increased in
V922E IGF-IR but not in WT or V922I IGF-IR, verifying the augmented in vitro kinase activity of V922E IGF-IR. Expression levels of
IGF-IRs were assumed to be similar in these experiments, as the levels
of tyrosine kinase activities maximally stimulated by IGF-I were
similar (Fig.1A). This was confirmed by parallel
experiments, in which COS-7 cells transfected with mutant IGF-IR cDNAs
were metabolically labeled. Proteins of 200, 135, and 95 kDa,
corresponding to the precursor,
, and
subunits of IGF-IR,
respectively, were precipitated by
IR3 from these transfected
COS-7 cells in similar amounts (Fig.1B).
Figure 1:
Transient
expression of Val IGF-IR mutants. A, semi-in
vivo kinase activity of IGF-IRs. WT, V922E, or V922I IGF-IR
transiently expressed in COS-7 cells were immunoprecipitated with
IR3 from lysates of cells that had been treated with or without
100 nM IGF-I for 5 min at 37 °C. Receptor tyrosine kinase
activity was measured as incorporation of radioactivity into either the
exogenous substrate poly(Glu, Tyr) 4:1 or the
subunit of IGF-IR.
Shown is an autoradiograph of SDS-polyacrylamide gel electrophoresis. pre, IGF-IR precursor; poly(E,Y), poly(Glu, Tyr) 4:1. B, metabolic
S labeling of COS-7 cells. COS-7
cells transfected with pcDNA1 (mock), WT, V922E, or V922I
IGF-IR cDNA were metabolically labeled, then treated with or without
100 nM IGF-I for 5 min at 37 °C, and their lysates were
precipitated with
IR3 and analyzed by SDS-polyacrylamide gel
electrophoresis. Shown is an autoradiograph. C, in vivo autophosphorylation of IGF-IR
subunit in COS-7 cells. COS-7
cells transfected with WT, V922E, or V922I IGF-IR cDNA were lysed, then
precipitated with
IR3. Precipitates were probed with anti-Tyr(P)
antibody RC20H. D, in vivo tyrosine phosphorylation of IRS-1
in SV40-BALB cells. SV40-BALB cells transfected with WT, V922E, or
V922I IGF-IR cDNA were lysed, then precipitated with 1D6 (anti-rat
IRS-1 antibody). Precipitates were probed with
RC20H.
We also
examined autophosphorylation of the subunit of V922E IGF-IR using
a different method, which represents in vivo tyrosine kinase
activity of this mutant. IGF-IRs transiently expressed in COS-7 cells
were precipitated by
IR3 and immunoblotted with anti-Tyr(P)
antibody RC20H. Constitutively augmented
-subunit
autophosphorylation of V922E IGF-IR, but not WT or V922I was observed (Fig.1C).
We next examined IRS-1 phosphorylation by the V922E mutant in intact cells. As 1D6 recognizes rodent IRS-1 but not monkey IRS-1, we used SV40-BALB cells instead of COS-7 for this experiment with transient transfection. Without IGF-I treatment, 1D6 precipitated IRS-1 with enhanced tyrosine phosphorylation of IRS-1 from SV40-BALB cells transfected with V922E IGF-IR cDNA (Fig.1D). This was not observed in the parallel transfection with WT or V922I cDNA.
In an effort to confirm these
results obtained from transient expression, we established CHO cell
lines overexpressing IGF-IR mutants. CHO-IGF-IR, CHO-V922E (Clones 1
and 2), or CHO-V922I cells specifically bound 3.7 to 4.1 times more I-IGF-I as compared to the parental CHO cells. The
expressed WT and mutant IGF-IRs had apparent K
values of 1.3 to 2.7
10
M for IGF-I (Fig.2A). The K
value of native IGF-IR in parental CHO cells was 1.0
10
M. Therefore, the stable CHO cell lines
did overexpress IGF-IRs with comparable affinities for IGF-I. We also
assessed the amount of IRS-1 in the stable CHO cell lines, as it has
been reported that a CHO cell line overexpressing a mutant IR retains
reduced amounts of IRS-1(24) . The lysates of CHO-IGF-IR,
CHO-V922E (Clones 1 and 2), or CHO-V922I were precipitated with 1D6 and
blotted with anti-IRS-1 antibody. The results clearly indicated that
the amounts of IRS-1 were similar among these cell lines (Fig.2B).
Figure 2:
Stable expression of V922 IGF-IR mutants
in CHO cells. A, radioactive IGF-I binding to CHO cell lines.
The binding of I-IGF-I to parent CHO (
), CHO-IGF-IR
(
), CHO-V922E (Clones 1 (
) and 2 (
)), or CHO-V922I
(
) cells was measured in the presence of the indicated
concentrations of IGF-I (
I-labeled plus unlabeled).
Results are means ± S.D. of three independent experiments. B, amount of IRS-1 in the stable CHO cell lines. Lysates of
CHO-IGF-IR, CHO-V922E (Clones 1 and 2 (C1 and C2)), or CHO-V922I were
precipitated with 1D6 and blotted with a polyclonal anti-IRS-1
antibody. C and D, in vivo tyrosine kinase activities
of Val
mutants in CHO cell lines. CHO cell lines were
treated with or without 10 nM IGF-I for 5 min at 37 °C. WT
or mutant IGF-IRs and IRS-1 were immunoprecipitated with
IR3 (C) and 1D6 (D), respectively, from lysates of cells
and then immunoblotted with anti-Tyr(P)
antibody.
From stable CHO cell lines, we
immunoprecipitated both IGF-IRs and IRS-1 and detected them with
anti-Tyr(P) antibody immunoblotting. In the absence of IGF-I, augmented
autophosphorylation of the IGF-IR subunit was observed in
CHO-V922E (Clones 1 and 2) but not in CHO-IGF-IR or CHO-V922I (Fig.2C). Besides, tyrosine phosphorylation of IRS-1
was stimulated in the absence of IGF-I in CHO-V922E (Clones 1 and 2)
but not in CHO-IGF-IR or CHO-V922I (Fig.2D). The
levels of tyrosine phosphorylation of both IGF-IR
subunit and
IRS-1 maximally activated by 10 nM IGF-I were similar among
these cell lines.
As the established target of IRS-1 activated by IGF-IR is PI 3-kinase, we measured IRS-1-associated PI 3-kinase activity in CHO-V922E cells. By precipitating IRS-1 from lysates of CHO-V922E cells, the PI 3-kinase activity was found to be significantly elevated in these cells (Fig.3A). We quantitated the PI 3-kinase-stimulating effect of V922E IGF-IR by comparing it with those effects of various concentrations of IGF-I in CHO-IGF-IR. The effect of V922E IGF-IR on PI 3-kinase was virtually equivalent to that of 1 nM IGF-I in CHO-IGF-IR (Fig.3A). This was also the case when the effect was assessed by tyrosine phosphorylation of IRS-1 (Fig.3B). Thus, V922E mutation turns on the IGF-IR signal as strongly as does 1 nM IGF-I stimulation of WT IGF-IR.
Figure 3: IRS-1-associated PI 3-kinase activity in CHO cell lines. CHO cell lines were treated without (lanes 1, 5, and 6) or with 0.1 nM (lane 2), 1 nM (lane 3), and 10 nM (lanes 4 and 7) IGF-I for 5 min at 37 °C. IRS-1 was immunoprecipitated with 1D6 from cell lysates. The precipitates were measured with the associated PI 3-kinase activity (A) and immunoblotted with anti-Tyr(P) antibody (B). PIP marker indicates the position of migration of PI 4-phosphate standard on TLC.
Thus, it is shown that V922E IGF-IR has autoactive intrinsic tyrosine kinase, and this causes constitutive activation of the IRS-1/PI 3-kinase cascade. Next, we investigated biological outputs of this mutant by examining metabolic and mitogenic stimulation. In CHO-IGF-IR cells, exposure to IGF-I (20 min) resulted in a dose-dependent enhancement of the uptake of radiolabeled 2-deoxyglucose (Fig.4A). CHO-V922E cells (Clone 1) exhibited significantly elevated basal uptake of glucose, as strongly as did 0.1 nM IGF-I stimulation in CHO-IGF-IR cells. As for mitogenic stimulation, thymidine uptake assay and cell proliferation assay with XTT were performed. Very interestingly, in clear contrast to glucose uptake, CHO-V922E cells (Clone 1) showed no basal elevation in thymidine uptake and XTT values and exhibited IGF-I-dependent stimulation similar to that observed in CHO-IGF-IR with both assays (Fig.4, B and C). Similarly, Clone 2 of CHO-V922E cells also showed basal elevation of glucose uptake but not of cell proliferation assays (data not shown).
Figure 4:
Biological outputs of V922E IGF-IR. A, 2-deoxyglucose uptake in CHO cell lines. CHO-IGF-IR ()
and CHO-V922E (
) were stimulated with the increasing
concentrations of IGF-I for 20 min at 37 °C and incubated for
another 20 min in the presence of
2-deoxy-D-[1,2-
H]glucose. The
incorporated radiolabeled glucose was determined. Incorporated
radiolabeled glucose in the presence of 100 nM wortmannin
(CHO-IGF-IR, 5868 ± 107 cpm; CHO-V922E, 3843 ± 39 cpm)
was subtracted from each value, and results were expressed as
percentage of the uptake at 10
M IGF-I
(unsubtracted values are: CHO-IGF-IR, 11,536 ± 327; CHO-V922E,
9,507 ± 27 cpm) in each CHO cell line. Results are means
± S.E. of three independent experiments. The panel shows a
representative result from three separate experiments. In all
experiments presented in this figure, Clone 1 was used as CHO-V922E. *, p < 0.01, unpaired t-test; asterisks indicate significant differences of the incorporated radiolabeled
glucose between CHO-V922E and CHO-IGF-IR. B, thymidine uptake
in CHO cell lines. CHO-IGF-IR (WT,
) and CHO-V922E (V922E,
)
were stimulated with IGF-I or 10% FCS for 16 h at 37 °C and
incubated for another 2 h in the presence of
[
H]thymidine. Then the incorporated radiolabeled
thymidine was determined. Results are means ± S.E. of three
independent experiments, expressed as percentage of 10% FCS stimulation
(CHO-IGF-IR, 39,121 ± 1,010 cpm; CHO-V922E, 24,072 ± 534
cpm) of each cell line. The panel shows a representative result from
seven separate experiments. C, XTT assay of proliferation in
CHO cell lines. CHO-IGF-IR (WT) and CHO-V922E (V922E) were cultured
with IGF-I for 36 h at 37 °C and incubated for another 30 min in
the presence of XTT labeling mixture. The spectrophotometrical
absorbance (A
nm/A
nm)
was measured using a microtiter plate reader. Results are means
± S.E. of three independent experiments, expressed as percentage
of 10% FCS stimulation (S indicates 10% FCS stimulation). The
panel shows a representative result from four separate
experiments.
Finally, we made an effort to address the question of why this autoactive IGF-IR mutant fails to stimulate cellular mitogenesis. Since the Ras-MAP kinase pathway has been implicated in tyrosine kinase-induced mitogenesis(22, 25, 26, 27, 28) , we focused on this pathway and examined both Ras activation and MAP kinase activation in CHO-V922E cells. The former assay clearly showed that no basal elevation was observed in the Ras-GTP amount, while IGF-I treatment stimulated the Ras activity in CHO-V922E cells to an extent similar to that observed in CHO-IGF-IR (Fig.5A). Activities of MAP kinases were measured by immune-complex kinase assay of ERK1 (Fig.5B) and in-gel kinase assay of ERKs 1 and 2 (Fig.5C). Again, CHO-V922E cells showed no basal elevation in the MAP kinase activities, and IGF-I-dependent stimulation of MAP kinases was similar between CHO-V922E and CHO-IGF-IR cells. These data clearly indicate that the V922E mutant fails to activate the Ras-MAP kinase cascade in CHO cells in the absence of IGF-I. The activation failure in this pathway provides a reasonable explanation for the failure of constitutive mitogenic stimulation by the V922E mutant.
Figure 5:
Failure of activation of the Ras-MAP
kinase cascade by V922E IGF-IR in the absence of IGF-I. A, Ras
activation in CHO cell lines. CHO-IGF-IR (WT) and CHO-V922E (V922E)
were P-labeled for 6 h at 37 °C and stimulated with
100 nM IGF-I for 3 min at 37 °C. Ras protein was recovered
by immunoprecipitation with anti-Ras monoclonal antibody Y13-259, and
bound nucleotides were separated by TLC. Shown is a representative
result from three independent experiments. Left, an
autoradiogram of the TLC plate. The positions of GDP and GTP are
indicated. Right, the right panel shows the
quantification of the data in the left panel. The relative
molar ratio of GTP and GDP was corrected for the number of
phosphates/mol of guanosine, and the percentage of the Ras-GTP complex
relative to the total amount of Ras was calculated. B, MAP
kinase activation in CHO cell lines with immune-complex kinase assay. C, MAP kinase activation in CHO cell lines with in-gel kinase
assay. CHO-IGF-IR (WT) and CHO-V922E (V922E) were stimulated with IGF-I
for 10 min at 37 °C. MAP kinase activity was measured by
immune-complex kinase assay on anti-ERK1 immunoprecipitates (B) and in-gel kinase assay on cell lysates (C). The
data in B and C are from independent experiments. B, an autoradiogram of MBP phosphorylated by
immunoprecipitated ERK1 is shown. The incorporated
P
values (cpm) into MBP were (from left to right):
CHO-IGF-IR, 271, 292, 644, 795; CHO-V922E, 269, 470, 515, 1053. The
panel shows a representative result from two separate experiments. C, in-gel kinase assay was subjected to quantitative analysis
using a phosphoimage analyzer (Fuji BAS2000), and the radioactivity of
ERK1 (44 kDa) and ERK2 (42 kDa) bands was determined. The panel shows a
representative result from three separate
experiments.
We have herein shown that the transmembrane V922E mutation
turns on the tyrosine kinase activity of IGF-IR. As the substitution of
the same Val to Ile does not turn on the kinase activity,
V922E should specifically act on the receptor conformation and switch
on the intrinsic tyrosine kinase. The results also show that the V922E
mutant causes phosphorylation of IRS-1 and stimulation of the
IRS-1-associated PI 3-kinase activity. Thus, V922E IGF-IR can turn on
the consecutive downstream pathway of IGF-IR in intact cells. Mediation
by IR of the action of V922E IGF-IR is less likely, because BALB/c 3T3
cells, in which this mutant can transmit the signal to IRS-1, lack
detectable amounts of IR(29) . Four examples of autoactive
receptor tyrosine kinases by point mutations have so far been reported:
oncogenic Neu with V664E(12) , IR with V938D(14) ,
c-Kit with V560G or D814V(15) , and CSF-1 receptor/c-Fms with
L301S or Y969F(13) . Although these mutants were shown to carry
augmented tyrosine kinase activity, it has been less defined whether
their downstream pathways are biochemically activated in intact cells.
For the first time, this study thus shows that mutationally activated
receptor tyrosine kinase turns on the downstream consecutive signaling
cascade, like ligand-dependent activation.
V922E submaximally activated IGF-IR as compared to the maximal stimulation by IGF-I. The effects of V922E IGF-IR on IRS-1 and PI 3-kinase were equivalent to those of 1 nM IGF-I. As 1 nM is a concentration at which IGF-I can exert its physiological functions, this mutant should generate physiologically significant outputs of cells. Submaximal activation by V922E IGF-IR may reflect that a partial population of this mutant is activated, with each mutant being completely turned on. Alternatively, each molecule of V922E mutant may be in partially activated conformation, with all the population of this mutant being in such a half-activated state.
IGF-IR has pleiotropic outputs of cells, both mitogenic and metabolic. Recent studies prove that this receptor serves a wider range of biological roles than previously thought(30, 31, 32, 33, 34) . Also, IGF-IR has multiple sets of different downstream signaling pathways. However, little has been known about the role that each pathway plays for the cellular outputs of IGF-IR. This study clearly showed that V922E IGF-IR stimulated glucose uptake in the absence of IGF-I in CHO cells. It was also shown that V922E IGF-IR constitutively activated the IRS-1/PI 3-kinase pathway in the same cells. These data strongly suggest a close relationship between the PI 3-kinase stimulation and cellular response of glucose uptake. Accumulated evidence (35, 36, 37) has so far indicated that PI 3-kinase mediates glucose uptake stimulated by insulin. Our results are thus consistent with this notion. Further analysis of the dose-response relationship revealed that glucose uptake stimulation by V922E IGF-IR was 0.1 nM IGF-I-equivalent in expressing CHO cells. Given that PI 3-kinase activation by this mutant was 1 nM IGF-I-equivalent in the same cells, this suggests that other IGF-I-dependent machinery may potentiate PI 3-kinase-mediated glucose uptake and allow its optimal activation by IGF-I stimulation.
This study also indicated that V922E IGF-IR failed to promote mitogenesis in the absence of IGF-I. Does this signify that mitogenesis is not a physiological output of IGF-IR? We believe that mitogenesis is certainly the native output of this receptor and assumed that the V922E mutant could not turn on the native IGF-IR-responsive set of intracellular signals necessary for cell growth in the absence of IGF-I. This idea was verified by the results obtained from multiple independent assays all showing that V922E mutant could not activate the Ras-MAP kinase pathway, which is thought to be required for mitogenesis by receptor tyrosine kinases(22, 25, 26, 27, 28) . These data thus demonstrate that a transmembrane point mutation differentially turns on the downstream signaling pathways of IGF-IR. The mechanism for such differential activation of signals seems to be related to the partial tyrosine phosphorylation of IRS-1 by V922E mutant (see above). A simple interpretation of these data is that V922E-induced conformational change of IGF-IR allows its intrinsic tyrosine kinase to phosphorylate the PI 3-kinase-binding tyrosine on IRS-1 but not some of other sites on IRS-1. Alternatively, it might be related to differential phosphorylation of other IGF-IR kinase substrates such as Shc(38) . We must thus await precise analysis of V922E-dependent tyrosine phosphorylation sites of IRS-1 and comprehensive investigation of all possible IGF-IR downstream pathways turned on by this mutation.
The differential activation of the two
downstream pathways by V922E IGF-IR also suggests that the
physiological level of activation of PI 3-kinase does not lead to the
activation of the Ras-MAP kinase cascade, signifying that PI 3-kinase
may not be the native upstream machinery of the Ras system. This is
consistent with multiple reports using the same CHO cells showing that
a dominant negative mutant of p85, the adaptor subunit of PI 3-kinase,
inhibits insulin-induced PI 3-kinase activation but not Ras activation (36) or a dominant negative mutant of tyrosine-specific
phosphatase Syp and that of Ras guanylnucleotide exchange factor SOS
both inhibit insulin-induced Ras activation but not PI 3-kinase
activation(39, 40) . In further support of this
notion, 50 nM wortmannin completely inhibits insulin
stimulation of PI 3-kinase in CHO cells, whereas the same concentration
of wortmannin has no effect on insulin-induced Ras activation. ()On the other hand, Hu et al.(41) have
recently shown that expression of the constitutively active mutant of
p110, the catalytic subunit of PI 3-kinase, activates Ras-MAP kinase
cascade in Xenopus oocytes. The discrepancy between their and
our results may be attributed to the difference of cell systems
studied. Alternatively, the V922E mutant activated endogenous PI 3-kinase submaximally in our experiments, whereas
constitutively active PI 3-kinase has been exogenously overexpressed in
their experiments. Both stimulation intensity and spatial localization
of PI 3-kinases might be potentially different between these studies.
It is therefore quite interesting to compare precisely the action of
V922E IGF-IR with that of the activated p110 mutant using the same
experimental systems.