From the Eppley Institute for Research in Cancer and
Allied Diseases, the ¶ Department of Biochemistry and
Molecular Biology, and the
Department of Pathology and
Microbiology, University of Nebraska Medical Center,
Omaha, Nebraska 68198-6805
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ABSTRACT |
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The intracellular mechanisms used by insulin and
insulin-like growth factors to block programmed cell death are unknown.
To identify receptor structures and signaling pathways essential for
anti-apoptotic effects on cells, we have created a chimeric receptor
(colony-stimulating factor-1 receptor/insulin receptor chimera
(CSF1R/IR)) connecting the extracellular, ligand-binding domain of the
colony-stimulating factor-1 (CSF-1) receptor to the transmembrane and
cytoplasmic domains of the insulin receptor. Upon activation with
CSF-1, the CSF1R/IR phosphorylates itself and intracellular
substrates in a manner characteristic of normal insulin
receptors. CSF-1 treatment protected cells expressing the
CSF1R/IR from staurosporine-induced apoptosis. A chimeric receptor
(CSF1R/IR960) with a deletion of 12 amino acids from its
juxtamembrane domain was constructed and expressed. CSF-1-treated cells
expressing the CSF1R/IR
960 are unable to phosphorylate IRS-1 and Shc
(Chaika, O. V., Chaika, N., Volle, D. J., Wilden, P. A.,
Pirrucello, S. J., and Lewis, R. E. (1997) J. Biol. Chem. 272, 11968-11974). CSF-1 stimulated glucose uptake,
mitogen-activated protein kinases, and IRS-1-associated
phosphatidylinositol 3' kinase in cells expressing the CSF1R/IR but not
in cells expressing the CSF1R/IR
960. Surprisingly, the
CSF1R/IR
960 was as effective as the CSF1R/IR in mediating CSF-1
protection of cells from staurosporine-induced apoptosis. These
observations indicate that the anti-apoptotic effects of the insulin
receptor cytoplasmic domain can be mediated by signaling pathways
distinct from those requiring IRS-1 and Shc.
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INTRODUCTION |
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Most proposed mechanisms of carcinogenic progression predict that any single cell with a selective growth advantage would, by virtue of clonal expansion, provide an enlarged target for the accumulation of additional carcinogenic mutations. The relative rarity of tumor formation in organisms with billions of proliferating cells has led to the hypothesis that most cells acquiring growth advantages are selectively deleted by apoptotic mechanisms (1). A corollary of this proposal is that suppression of the apoptotic pathway is a requirement for carcinogenesis.
Several growth factors and cytokines have been identified as suppressors of apoptosis in specific cell types (2-5). In particular, IGF I1 has a potent ability to inhibit apoptosis in a broad range of cells (2, 5-8). The ability of the IGF I receptor to block cell death is marked in oncogene-driven cells placed in a growth factor-deprived environment and is manifested in vivo even more dramatically than in vitro (9). Furthermore, human melanoma cells transfected with an antisense cDNA construct to the IGF I receptor were very weakly tumorigenic upon injection into nude mice compared with cells expressing wild type levels of IGF I receptors (10). Although it was initially postulated that such IGF I receptor-deficient cells simply failed to proliferate in vivo, further analysis revealed that the cells undergo apoptosis within 27 h of injection (10). In addition, IGF I and insulin have been demonstrated to suppress apoptosis in c-myc-induced Rat 1 fibroblasts, PC-12 cells, and IL-3-dependent hematopoietic cells (2, 3, 5). These observations lend strong support to the notion that IGF action mediated through the IGF I receptor is directly and intimately involved in suppression of apoptosis in many cell types. If inhibition of an IGF-regulated anti-apoptotic pathway is a requisite step in carcinogenesis, then knowledge of the essential components in that pathway may lead to strategies that can specifically invoke apoptosis of transformed cells.
The IGF I receptor and insulin receptor encode homologous cytoplasmic domains (11). In addition to a highly conserved tyrosine kinase domain, the insulin and IGF I receptor also retain homologous phosphorylation sites including sites that regulate the interaction of each receptor with the intracellular signaling proteins IRS-1 and Shc (12-15). The conservation of structure and signaling pathways shared by these related receptors explains their ability to generate similar biological responses in experimental systems (16-20). A primary goal in the study of insulin and IGF I receptor action is the identification of the intracellular pathways that mediate the biological actions of these hormones. One strategy to identify signaling pathways that regulate specific biological actions of insulin and IGF I receptors has been to create mutations within specific receptor sequences that disrupt one insulin or IGF I-regulated pathway while leaving others intact. Deletion mutagenesis of the insulin receptor cytoplasmic domain has generated insulin receptors with altered biological properties (21-24), suggesting that different regions of the insulin receptor cytoplasmic domain may modulate the distinct biological effects of insulin.
Analysis of the ability of mutated insulin receptors to mediate signaling in cells is obscured by the activity of endogenous insulin receptors. We have avoided this dilemma by constructing a chimeric receptor (CSF1R/IR) consisting of the extracellular ligand binding domain of the human CSF-1 receptor (25) coupled to the transmembrane and cytoplasmic domains of the human insulin receptor (26-28). The CSF1R/IR chimera has CSF-1-dependent enzymatic and biological properties expected of the insulin receptor and IGF I receptor (20). CSF-1 inhibits apoptosis in CHO cells expressing the CSF1R/IR but has no protective effect on control cells. Inhibition of apoptosis by insulin in CHO cells is sensitive to the PI 3' kinase inhibitor wortmannin. However, deletions within the cytoplasmic domain of the CSF1R/IR that disrupt the ability of the insulin receptor cytoplasmic domain to activate MAP kinase and IRS-1-associated PI 3' kinase activity do not disrupt its anti-apoptotic activity. These observations indicate that the phosphorylation of IRS-1 and its subsequent association with and direct activation of PI 3' kinase are not requisite components of anti-apoptotic signaling by the insulin receptor kinase.
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EXPERIMENTAL PROCEDURES |
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Creation of Cell Lines Expressing the Wild Type and Mutant CSF1R/IR Chimera-- The construction of the chimera, transfection of the DNA, and isolation of clonal cell lines have been described previously (20).
Cell Culture and Growth Factor Stimulation-- CHO cells were cultured in F12 medium with 10% fetal bovine serum. Before stimulation with insulin or CSF-1 the medium was changed to serum-free medium. Insulin (100 nM) or CSF-1 (10 nM) were added to stimulate the cells.
Western Blot Analysis-- Proteins were transferred to polyvinylidene difluoride membranes, and Western blots were developed using anti-phosphotyrosine (PY-20, 1:1000) antibodies as specified by the manufacturer (Transduction Laboratories) or using CT-1 (1:5000) (29) antibodies. Blots were developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium using the appropriate secondary antibody conjugated to alkaline phosphatase.
DNA Ladder Assay--
1 × 106 cells were
plated in 100-mm dishes and incubated for 18 h at 37 °C in
serum-free medium with or without growth factors or 5 nM
staurosporine (30). The medium from each culture dish was removed and
pelleted in a bench top centrifuge for 10 min to capture any cells that
had detached from the tissue culture dish. 1 ml of Hirt lysis buffer
(50 mM Tris-HCl, pH 7.5, 0.6% SDS, and 10 mM
EDTA) with 0.5 mg/ml proteinase K was added to each tube. The
resuspended pellets were transferred back to the corresponding plate
from which they were taken. The plates were incubated at 37 °C for
1 h to lyse the cells. Following the incubation, 300 µl of 5 M NaCl was added dropwise to each dish, and lysates were
scraped into polypropylene tubes and incubated at 4 °C for 4-16 h.
The precipitated lysates were centrifuged for 30 min at 31,000 × g in a SS34 rotor (Sorvall). The supernatants were combined with an equal volume of isopropanol, mixed, and centrifuged for 45 min
at 31,000 × g in an SS34 rotor. Supernatants were
immediately decanted, and the pellets were allowed to dry briefly. The
pellets were resuspended in 300 µl of 10 mM Tris-HCl, pH
8, 1 mM EDTA containing 100 µg/ml RNase A. The suspension
was transferred to Eppendorf tubes and allowed to incubate at 25 °C
for 1 h. The DNA was extracted with an equal volume of
phenol/chloroform (1:1) and precipitated, and each sample was
resuspended in 100 µl of DNase-free water. The DNA was radiolabeled
with [-32P]dATP using a kit from Trevigen
(Gaithersburg, MD) and run on a 1.5% agarose gel. The gel was fixed in
10% acetic acid overnight, dried, and visualized using phosphor
storage technology (Molecular Dynamics).
Quantification of Pyknotic Nuclei--
Flame sterilized
coverslips were placed at the bottom of 35-mm tissue culture dishes and
secured with a drop of medium. 5 × 104 CHO cells
expressing CSF1R/IR or CSF1R/IR960 or transfected with the
expression vector pcDNA3 (Invitrogen) were plated onto the
coverslips. The day after plating the medium was replaced with fresh
serum-free medium, and the cells were treated for at least 9 h at
37 °C with or without 5 nM staurosporine, 1.7 µM insulin, or 10 nM CSF-1. The coverslips
were washed twice with PBS (2.7 mM KCl, 1.5 mM
KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4) and fixed with ice-cold
2.5% paraformaldehyde in PBS for 10 min at 4 °C. Following fixation
the cells were washed once with 50 mM NH4Cl in
PBS for 5 min at 25 °C, once with 70% ethanol for 5 min at
25 °C, and twice with PBS. The coverslips were then stained with 25 µl of 25 µg/ml Hoechst 33258 in PBS for 30 min. The coverslips were
washed twice with PBS and mounted on microscope slides with 2.5%
1,4-diazabicyclo-[2.2.2]octane in glycerol gelatin. At least 500 nuclei were counted for each treatment, and the percentage of pyknotic
nuclei present was calculated.
[3H]2-Deoxy-D-glucose Uptake in 3T3-L1
Cells--
3T3-L1 fibroblasts expressing the CSF1R/IR or
CSF1R/IR960 were differentiated into adipocytes with dexamethasone,
3-isobutyl-1-methylxanthine, and insulin as described previously (20).
Glucose uptake in 3T3-L1 adipocytes was measured using a modification
of the method described previously (31). 3T3-L1 adipocytes were used 14 days after induction of differentiation, at which time >90% of the cells expressed an adipocyte phenotype. Cell monolayers were washed twice with buffer A (130 mM NaCl, 2.7 mM KCl, 1 mM KH2PO4, 8.1 mM
Na2HPO4, 0.68 mM CaCl2,
0.49 mM MgCl2, pH 7.4) and then incubated for
2 h at 37 °C in 1 ml of serum-free Dulbecco's modified
Eagle's medium. To determine nonspecific glucose uptake the cells were treated for 10 min in serum-free Dulbecco's modified Eagle's medium containing 20 µM cytochalasin B and 200 µM
phloretin. Cell monolayers were washed once with 1 ml of KRP buffer
(130 mM NaCl, 5 mM KCl, 1.3 mM
CaCl2, 1.3 mM MgSO4, 10 mM Na2HPO4, pH 7.4) with or without CSF-1 or insulin as indicated and incubated for 20 min at 37 °C in 1 ml of the same buffer. The glucose uptake assay was initiated by
replacement of KRP buffer in each well with KRP buffer containing 0.1 mM 2-deoxy-D-[2,6-3H] glucose (1 µCi) for 10 min at 37 °C. Termination of glucose uptake was
performed by the rapid removal of assay buffer followed by four rapid
washes of each well with 1 ml of ice-cold buffer A containing 500 µM phloretin. Cells were removed from each well with 0.4 ml of 0.1% SDS and counted for radioactivity after the addition of 4 ml of Optifluor (Packard Instrument Co.).
MAP Kinase Assay--
CHO cells (1 × 105) were
seeded in 35-mm dishes. 16 h after plating, cells were starved in
serum-free F-12 medium for 18 h. After serum deprivation, the cell
monolayer was washed with PBS and stimulated with 10 nM
CSF-1 or 1 µM phorbol 12-myristate 13-acetate for 10 min.
Cells were snap frozen with liquid nitrogen and lysed in 1 ml of lysis
buffer (40 mM Tris-HCl, pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 0.5% Triton X-100, 10 mM
NaF, 10 mM sodium PPi, 10 µg/ml leupeptin, 5 µg/ml aprotinin), transferred to microcentrifuge tubes, and pelleted
at 14,000 × g for 10 min at 4 °C. Clarified lysates
were transferred into new microcentrifuge tubes containing 15 µl of protein G-agarose beads and 10 µl each of ERK-1 (C-16) and ERK-2 (C-14) antibodies (Santa Cruz Biotechnologies) and incubated for 3 h at 4 °C. Immunoprecipitates were washed three times with 1 ml of
lysis buffer (without sodium PPi and leupeptin) and three times
with ice-cold kinase buffer (40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 2 mM MnCl2,
25 µM ATP, 0.5 mM dithiothreitol). Myelin basic protein (0.5 mg/ml) and [-32P]ATP (0.7 µl, 10 µCi/µl, and 6000 µCi/nmol) were added to each tube. After a
30-min incubation at 30 °C, the reaction was stopped by adding 2×
sample buffer (250 mM Tris-HCl, pH 6.8, 5% SDS, 50% glycerol, 100 mM dithiothreitol) and boiling the samples
for 5 min. Samples were resolved by electrophoresis on a 12%
polyacrylamide gel. Each gel was stained with Coomassie Brilliant Blue
to detect myelin basic protein and dried. The relative amount of
32P incorporated into myelin basic protein was determined
using phosphor storage technology (Molecular Dynamics).
In Vitro PI 3' Kinase Assay--
CHO cells (5 × 105) were seeded in 6-well plates in duplicate. The day
after plating the cells were incubated in serum-free medium for 18 h. The cells were stimulated with 1 µM insulin or 10 nM CSF-1 for 2 min, snap frozen with liquid nitrogen, lysed with 450 µl/well of lysis buffer containing 40 mM
Tris-HCl, pH 7.4, 2 mM Na3VO4, 10 mM NaF, 10 mM sodium PPi, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride.
Following centrifugation for 10 min at 14,000 × g,
clarified lysates were incubated for 1.5-2 h at 4 °C with 3 µl of
antibodies to IRS-1. Protein G-agarose beads (12.5 µl) were added to
each immune complex and precipitated for 1.5-2 h at 4 °C. The
immunoprecipitates were washed twice with 1% Triton X-100 and PBS,
twice with 100 mM Tris-HCl, pH 7.5, and 0.5 M
LiCl, and twice with 50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.1 mM
Na3VO4. The washed beads were resuspended in 30 µl of a buffer containing (30 mM MgCl2, 30 nM HEPES, 0.6 mM EGTA) and left on ice. At this
time the sonication mixture containing 30 mM HEPES, 0.6 mM EGTA, and 0.6 mg/ml phosphoinositol (Avanti) was
prepared and placed on ice. The mixture was sonicated at 4 °C for
5-7 s with a probe sonicator (Heat Systems) set at position 9, and 15 µl of the sonicated mixture was added to the immunoprecipitates.
[-32P]ATP (0.8 µl, 10 µCi/µl, 6000 µCi/nmol)
was quickly added, and the mixtures were mixed rigorously at 25 °C
for 10 min. The reaction was stopped with 12 µl of 6 M
HCl and 120 µl of CHCl3/MeOH (1:1). 20 µl of the
organic phase were spotted on a silica gel 60 (Merck) TLC plate that
had been previously coated with 1.3% potassium oxalate in
H2O/MeOH (3:2). Radiolabeled phospholipids were resolved in
a solvent system consisting of
CHCl3/methanol/acetone/acetic acid/H2O
(35:13:15:12:8). The relative amount of PI 3' phosphate generated was
determined using phosphor storage technology (Molecular Dynamics).
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RESULTS |
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CSF-1-dependent Phosphorylation of the CSF1R/IR and
CSF1R/IR960--
Treatment of cells expressing the CSF1R/IR with
CSF-1 for 10 min resulted in the phosphorylation of the 170-kDa
chimeric receptor (Fig. 1). CSF-1
stimulates phosphorylation of the CSF1R/IR
960 to a lesser extent
than it stimulates phosphorylation of the intact CSF1R/IR (Fig. 1). The
tyrosine-phosphorylated CSF1R/IR and CSF1R/IR
960 were
immunoprecipitated with the monoclonal antibody CT-1, which is specific
for the carboxyl terminus of the insulin receptor. The amounts of
CSF1R/IR and CSF1R/IR
960 in each cell line were equal as determined
by a Western blot using the CT-1 antibody (Fig. 1), thus showing that
the difference in tyrosine phosphorylation between the CSF1R/IR and the
CSF1R/IR
960 is due to the 12-amino acid deletion of the
juxtamembrane domain that is present in the CSF1R/IR
960 and not to
clonal variation.
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Inhibition of DNA Fragmentation by CSF-1 in Cells Expressing the CSF1R/IR-- CHO cells expressing the CSF1R/IR were examined for their ability to resist apoptosis upon treatment with CSF-1. Cells were incubated with CSF-1 (0.1-10 nM) for 1 h and then induced to undergo apoptosis with the addition of 5 nM staurosporine. Cells treated with staurosporine for 9 h showed the presence of DNA fragmentation (Fig. 2). CSF-1 decreased DNA fragmentation in a dose-dependent manner.
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The Effects of a Juxtamembrane Deletion on Anti-apoptotic Signaling
by the CSF1R/IR--
The juxtamembrane region contains an important
phosphorylation site at Tyr960 (32) that facilitates
tyrosine phosphorylation of IRS-1 and Shc by the insulin receptor
kinase (12-14). Deletion of 12 amino acids within the juxtamembrane
domain (960) of the insulin receptor or the CSF1R/IR blocks the
ability of the insulin receptor tyrosine kinase to phosphorylate IRS-1
or Shc (20, 33). To determine whether or not the anti-apoptotic
signaling of the CSF1R/IR required juxtamembrane phosphorylation sites,
we looked at the ability of the CSF1R/IR
960 to protect cells from
staurosporine-induced apoptosis. Cells transfected with the expression
vector alone, expressing the CSF1R/IR, or expressing the CSF1R/IR
960
were treated with 5 nM staurosporine and with or without 10 nM CSF-1 or 1.7 µM insulin for 18 h at
37 °C. The percentage of pyknotic nuclei present was determined as a
measure of the level of apoptosis. Cells transfected with the
expression vector alone contained detectable pyknotic nuclei after a
9-h treatment with staurosporine alone (Fig.
3). Physiological concentrations of IGF I
inhibit apoptosis in different cultured cell lines (2). Previous
studies have demonstrated that CHO cells express relatively low numbers
(1,000-10,000 receptors/cell) of insulin receptors and are relatively
insensitive to physiological concentrations of insulin. High
concentrations of insulin were used to ensure activation of endogenous
IGF I receptors. Insulin blocked the formation of pyknotic nuclei upon staurosporine treatment, whereas 10 nM CSF-1 had no effect
(Fig. 3). Cells expressing the CSF1R/IR were also susceptible to
staurosporine-induced apoptosis in the absence of insulin or CSF-1.
When insulin or CSF-1 were present, the number of pyknotic nuclei
detected was greatly reduced (Fig. 3). Cells expressing the
CSF1R/IR
960 were also protected from staurosporine-induced death by
insulin or CSF-1.
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The Effects of a Juxtamembrane Deletion on Stimulation of Glucose
Uptake by the CSF1R/IR--
The CSF1R/IR and the CSF1R/IR960 were
compared for their ability to stimulate glucose uptake in 3T3-L1
adipocytes. Saturating concentrations of insulin (100 nM)
stimulated [3H]2-deoxyglucose uptake 8-9-fold in
adipocytes expressing either chimera (Fig.
4). 10 nM CSF-1 is sufficient
to maximally activate the CSF1R/IR kinase (20). Adipocytes expressing
the CSF1R/IR were also able to increase glucose uptake approximately
7.7-fold in response to 10 nM CSF-1 (Fig. 4). In contrast,
the juxtamembrane deletion severely inhibited the ability of CSF-1 to
stimulate glucose uptake in cells expressing the CSF1R/IR
960 (Fig.
4).
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Regulation of MAP Kinase Activation by the Juxtamembrane Region of
the Insulin Receptor Cytoplasmic Domain--
IRS-1 and Shc are
important mediators of insulin-induced Ras activation and the
subsequent activation of MAP kinases (34-36). We tested the ability of
the 12-amino acid deletion in the CSF1R/IR to affect CSF-1-induced
activation of MAP kinases in CHO cells. CHO cells transfected with the
expression vector alone activated MAP kinases approximately 3.5-fold in
response to phorbol esters but were unresponsive to CSF-1 (Fig.
5). In cells expressing the CSF1R/IR,
CSF-1 was able to activate MAP kinases at least as well as phorbol
esters (Fig. 5). The 12-amino acid juxtamembrane deletion (960)
blocked the ability of CSF-1 to stimulate the MAP kinases but had no
effect on the action of phorbol esters (Fig. 5).
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The Effect of PI 3' Kinase Inhibition on the Anti-apoptotic Action of the Insulin Receptor-- PI 3' kinase has been shown to play a role in inhibiting apoptosis in some cells (3, 37). The contribution of PI 3' kinase activity to anti-apoptotic signaling of the insulin receptor kinase in CHO cells was tested with the PI 3' kinase inhibitor wortmannin. CHO cells expressing the human insulin receptor were treated with 5 nM staurosporine with or without 1 µM wortmannin and 100 nM insulin for 18 h. Wortmannin alone had no effect on the extent of apoptosis in staurosporine-treated cells (Fig. 6). Insulin significantly reduced the number of pyknotic nuclei detectable upon staurosporine treatment (Fig. 6). However, the addition of wortmannin significantly reversed the anti-apoptotic effect of insulin (Fig. 6).
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The Effect of Carboxyl-terminal Truncation on the
Anti-apoptotic Action of the CSF1R/IR--
PI 3' kinase can be
activated through association with phosphorylated IRS-1 and to a lesser
extent with the phosphorylated carboxyl terminus of the insulin
receptor (38-40). We examined anti-apoptotic signaling in cells
expressing the CSF1R/IRCT, which contains a 43-amino acid truncation
of the carboxyl terminus of the chimera or cells expressing the
CSF1R/IR
960
CT, which combines the carboxyl-terminal truncation
with the juxtamembrane deletion. Cells expressing the mutated forms of
the chimera were treated for 18 h at 37 °C with 5 nM staurosporine and with or without 100 nM
insulin or 10 nM CSF-1. Cells expressing the CSF1R/IR
CT showed resistance to apoptosis in the presence of insulin or CSF-1, reducing the level of pyknotic nuclei approximately 3- and 4-fold when
treated with insulin or CSF-1, respectively (Fig.
7). Cells expressing the
CSF1R/IR
960
CT also showed resistance to staurosporine-induced apoptosis in the presence of insulin or CSF-1, reducing pyknotic nuclei
formation approximately 8- and 4-fold, respectively (Fig. 7). However,
the anti-apoptotic action of the CSF1R/IR
960
CT was reversed by
the addition of wortmannin (Fig. 7). These data demonstrate that
although activation of PI 3' kinase may be essential to the
anti-apoptotic action of the insulin receptor kinase, the loss of PI 3'
kinase binding sites from the carboxyl terminus does not appear to
impair the anti-apoptotic activity of the insulin receptor cytoplasmic
domain.
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Activation of PI 3' Kinase by Intact and Mutated Chimeric
Receptors--
PI 3' kinase is activated by association with proteins
phosphorylated on tyrosine by the insulin receptor kinase (38). We examined whether or not cells expressing the CSF1R/IR, the
CSF1R/IR960, or the CSF1R/IR
960
CT could activate PI 3' kinase
associated with tyrosine phosphorylated proteins in general and IRS-1
in particular. Serum-starved cells expressing the CSF1R/IR,
CSF1R/IR
960, or CSF1R/IR
960
CT were treated with 10 nM CSF-1 and 1 µM wortmannin for 10 min. The
cells were lysed and immunoprecipitated with antibodies to
phosphotyrosine. Analysis of PI 3' kinase activity in the
immunoprecipitates revealed that the CSF1R/IR strongly stimulated PI 3'
kinase activity in cells treated with CSF-1 (Fig.
8A). Simultaneous treatment with wortmannin blocked the activation of PI 3' kinase by the CSF1R/IR.
Deletion of the juxtamembrane domain also severely impaired the ability
of the CSF1R/IR
960 to activate PI 3' kinase associated with tyrosine
phosphorylated proteins (Fig. 8A). Wortmannin treatment removed the small CSF-1 stimulated increase in PI 3' kinase activity bound to tyrosine phosphorylated proteins in cells expressing the
CSF1R/IR
960. The additional deletion of the carboxyl-terminal tail
in the CSF1R/IR
960
CT completely abolished the ability of CSF-1 to
activate PI 3' kinase bound to tyrosine phosphorylated proteins (Fig.
8A). This observation suggests that a small portion of PI 3'
kinase activity associated with tyrosine phosphorylated proteins was
associated with tyrosine phosphorylation sites within the carboxyl
terminus of the chimeric receptor.
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DISCUSSION |
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We have used a chimeric receptor consisting of the extracellular domain of the CSF-1 receptor and the transmembrane and cytoplasmic domains of the insulin receptor to study anti-apoptotic signaling mechanisms of the insulin receptor cytoplasmic domain. The anti-apoptotic properties of the insulin receptor cytoplasmic domain were examined by deletion of structures within the receptor that couple it to known intracellular mediators of insulin action. Our data demonstrate that a deletion within the receptor juxtamembrane domain that inhibits the phosphorylation of IRS-1 and Shc by the insulin receptor (33) and the CSF1R/IR (20) also suppresses glucose transport (Fig. 4), MAP kinase activation (Fig. 5), and the activation of PI 3' kinase associated with IRS-1 (Fig. 8). However, the same juxtamembrane deletion does not suppress anti-apoptotic signaling by the CSF1R/IR (Fig. 3). The observation that wortmannin treatment reverses the anti-apoptotic actions of insulin on staurosporine-treated CHO cells supports previous data indicating that PI 3' kinase activity is an important component of the survival pathway (3, 37). However, experiments with mutated chimeric receptors (Figs. 7 and 8) indicate that neither the activation of PI 3' kinase associated with the insulin receptor kinase nor the activation of PI 3' kinase associated with other tyrosine phosphorylated proteins is a critical mediator of insulin-generated anti-apoptotic signals. These observations suggest that the activation of PI 3' kinase by the insulin receptor cytoplasmic domain to inhibit apoptosis can occur by other as yet unidentified mechanisms.
CSF-1 activates the insulin receptor kinase within the CSF1R/IR chimera in a time- and dose-dependent manner characteristic of the intact insulin receptor (20). Furthermore, CSF-1-stimulated phosphorylation of the CSF1R/IR in intact cells mimics the pattern of phosphorylation of the intact insulin receptor (20). The ability of the CSF1R/IR to inhibit apoptosis (Fig. 3) or stimulate glucose transport (Fig. 4) is probably not due to the formation of hybrid receptors formed between the CSF1R/IR and endogenous insulin or IGF I receptors. The levels of IGF I receptors in CHO cells are too low to readily detect hybrid formation in the CHO cells studied. However, previous experiments (20) demonstrated that there was no cross-phosphorylation between the insulin receptor and the CSF1R/IR when both receptors were expressed to high levels in CHO cells or in 3T3-L1 cells. These data argue against hybrid formation between the CSF1R/IR and endogenous IGF I receptors in CHO cells.
The 12-amino acid deletion within the juxtamembrane domain of the CSF1R/IR blocks the ability of the chimeric receptor to stimulate glucose uptake (Fig. 4) but does not impair the ability of CSF1R/IR to inhibit apoptosis (Fig. 3) or stimulate adipogenesis (20). These data suggest that the juxtamembrane region including Tyr960 plays a critical role in the ability of the insulin receptor cytoplasmic domain to stimulate glucose uptake.
The 960 mutation also disrupts the ability of the CSF1R/IR to
interact with and phosphorylate IRS-1 (20), which is required for the
subsequent activation of PI 3' kinase (38). Experiments in IRS-1
/
mice have suggested that IRS-1 contributes to the activation of
insulin-stimulated glucose transport (42, 43). Data from other
laboratories also suggest that PI 3' kinase activation is an important
component of insulin-stimulated glucose transport (44, 45). However,
recent experiments have suggested that mere activation of PI 3' kinase
is not sufficient to induce glucose uptake because the activation of PI
3' kinase associated with IRS-1 by IL-4 in transfected L6 cells is not
sufficient to mimic the effects of insulin (46). Similarly, agents that
inhibit the function of IRS-1 have been shown to block some biologic
effects of insulin without impairing its ability to stimulate glucose uptake (47, 48). Our data suggest that the ability of the insulin
receptor kinase to stimulate glucose uptake requires the insulin
receptor juxtamembrane domain. However, disruption of that domain may
inhibit its interaction with other substrates in addition to IRS-1,
each of which is capable of contributing to insulin-stimulated glucose
transport. Related signaling molecules IRS-2 (49), IRS-3 (50), and
IRS-4 (51) are all potential candidates. These data further suggest
that the ability of PI 3' kinase to mediate a specific biological
effect is context-dependent. Thus, activation of PI 3'
kinase bound to IRS proteins may be relevant to glucose uptake only in
the context of IRS protein interaction with and phosphorylation by the
insulin receptor kinase. Conversely, PI 3' kinase bound to IRS-1 may
not be an essential transducer of the anti-apoptotic effects of the
lipid kinase if it is not proximal to the relevant mediator of the
survival pathway.
IRS-1 and Shc couple the insulin receptor kinase to mechanisms that
activate Ras and its downstream effectors (34-36). Consistent with the
inability of the CSF1R/IR960 to phosphorylate IRS-1 and Shc (20),
this mutated chimera is unable to significantly activate MAP kinases
(Fig. 5). Although it is possible that imperceptible amounts of IRS-1
and/or Shc phosphorylation is catalyzed by the CSF1R/IR
960, these
data suggest that the phosphorylation of IRS-1 and Shc by the chimera
is not essential for transduction of survival signals from the insulin
receptor kinase.
Previous reports conflict regarding a role for MAP kinases in anti-apoptotic signaling within different cell types. Our observations are supported by previous experiments in UV-treated Rat 1 fibroblasts (37) and serum-starved primary sympathetic neurons (52) in which the MAP ERK kinase inhibitor PD098059 (53) failed to block IGF I-mediated inhibition of UV-induced apoptosis. Similarly, survival in undifferentiated PC12 cells was found to be unaffected by inhibition of the MAP kinase pathway by dominant-negative RasN17 (3). Furthermore, the ability of the oncogene v-abl to suppress apoptosis in an IL-3-dependent mast cell line was concomitant with the phosphorylation of Shc but independent of activation of MAP kinases (54). These results contrast to observations in which IGF I-mediated survival of differentiated PC12 cells was blocked by PD098059 (55). The ability of an activated MAP ERK kinase 1 transgene to suppress apoptosis in differentiated PC12 cells also supports a role for MAP kinase in anti-apoptotic signaling in these cells. Taken together, these data suggest that the viability of some cell types may be dependent on an intact MAP kinase pathway. However, our data suggest that activation of the MAP kinase pathway by growth factor receptors may not be required to generate anti-apoptotic signals.
Inhibition of apoptosis by PI 3' kinase is believed to be mediated, at
least in part, through the activation of the kinase Akt by PI 3'
phospholipids (37, 56, 57). We also examined the role of PI 3' kinase
in the insulin-stimulated, anti-apoptotic signaling pathway by
treatment of CHO cells with insulin, staurosporine, and the PI 3'
kinase inhibitor wortmannin. Wortmannin was able to significantly
reverse insulin-mediated inhibition of apoptosis (Fig. 6). However, the
poor stability of wortmannin in aqueous solution and the long time
course of the pyknotic nuclei assay (9-18 h) required use of a high
concentration of the fungal metabolite. The high (1 µM)
concentration of wortmannin used in these experiments may not be
specific for PI 3' kinase. Thus, the ability of wortmannin to reverse
the anti-apoptotic effect of insulin (Fig. 6) and CSF-1 in cells
expressing the CSF1R/IRCT (Fig. 7) could also be due to its action
on other enzymes. Nevertheless, our data are consistent with previous
reports that PI 3' kinase activity is a necessary component of
anti-apoptotic signaling (3, 37).
PI 3' kinase is primarily activated by the insulin receptor through its
association with tyrosine phosphorylated IRS-1 (38). PI 3' kinase
activity can also be activated through direct association of the lipid
kinase with the carboxyl terminus of the insulin receptor cytoplasmic
domain (39, 40). We tested the anti-apoptotic activity of the
CSF1R/IRCT, which contains a truncation of 43 amino acids from the
carboxyl terminus. The carboxyl-terminal truncation removes the
tyrosine phosphorylation sites to which PI 3' kinase can bind directly
(40). The carboxyl-terminal deletion alone had no deleterious effect on
the anti-apoptotic signaling properties of the CSF1R/IR
CT (Fig. 7).
In combination with the juxtamembrane deletion, the deletion also
inhibited apoptosis (Fig. 7) but completely blocked the activation of
PI 3' kinase associated with tyrosine phosphorylated proteins (Fig. 8).
These data indicate that the activation of PI 3' kinase activity bound directly to the insulin receptor cytoplasmic domain or bound to other tyrosine phosphorylated intracellular proteins is not essential for the inhibition of apoptosis.
The 960 mutation disrupted the ability of the chimeric receptor to
activate PI 3' kinase associated with IRS-1 (Fig. 8) but did not affect
anti-apoptotic signaling by the insulin receptor cytoplasmic domain
(Fig. 3). These data suggest that PI 3' kinase activity associated with
IRS-1, in contrast to its reported contribution to insulin-stimulated
glucose uptake, is also not essential for the inhibition of apoptosis
by the insulin receptor kinase. Furthermore, these data imply that as
yet undetermined intracellular mechanisms may activate PI 3' kinase to
transmit survival signals from the insulin receptor cytoplasmic domain.
Potential mediators of the survival signal may include other members of
the IRS family (49-51). However, the presence of phosphotyrosine
binding domains within each of these related proteins predicts that the
ability of all IRS proteins to mediate signaling by the insulin
receptor kinase may be dependent upon interaction with the
juxtamembrane region of the insulin receptor cytoplasmic domain. PI 3'
kinase may also be activated independent of its association with
tyrosine phosphorylated proteins. Interaction of the p110 catalytic
subunit of PI 3' kinase with Ras activates its lipid kinase (58, 59)
and can inhibit apoptosis (60). The inability of the CSF1R/IR to
phosphorylate IRS-1 or Shc (20), stimulate glucose uptake, or activate
MAP kinases (Fig. 5) implies that PI 3' kinase is not activated through Ras by this mutated chimera. However, the possibility exists that PI 3'
kinase might also be activated through an unidentified member of the
Ras superfamily or another unknown protein.
Our observations indicate that the juxtamembrane domain and 43 amino acids of the carboxyl-terminal tail are dispensable for anti-apoptotic signaling by the insulin receptor kinase. However, mutation of Tyr1251 or mutation of His1293 and Lys1294 within the cytoplasmic domain of the IGF I receptor abolished the anti-apoptotic action of IGF I (61), suggesting that structures within the cytoplasmic tail of this receptor might be required for inhibition of apoptosis. These amino acids within the IGF I cytoplasmic domain are not conserved within the homologous insulin receptor carboxyl-terminal tail, but they flank a region conserved between the IGF I receptor and insulin receptor in which one-third of the amino acids are acidic. Deletion of this acidic region with the remainder of the carboxyl-terminal tail did not prevent the anti-apoptotic functions of IGF I (61). Thus, structures conserved between the insulin receptor and IGF I receptor kinase that regulate anti-apoptotic signaling have yet to be identified. Inhibition of apoptosis by the IGF I receptor is dependent on an active tyrosine kinase (61). Taken together, these observations and the data presented here suggest that a novel cytoplasmic substrate may transduce the anti-apoptotic signals of the insulin and IGF I receptor kinase.
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ACKNOWLEDGEMENTS |
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We express appreciation to J. Whittaker for providing the human insulin receptor cDNA; P. Wilden for CHO cells expressing the human insulin receptor; C. Sherr for providing the cDNA of the human CSF-1 receptor; and M. Roussel, L. Sweet, and K. Siddle for providing antibodies. Special thanks to D. Volle and N. Chaika for the production of CHO cell lines expressing the intact and mutated CSF1R/IR and for glucose uptake assays. We thank the Genetics Institute (Cambridge, MA) for the invaluable gift of recombinant human CSF-1.
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FOOTNOTES |
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* This work was supported by Grants BE-260 from the American Cancer Society, DK52809 from the National Institutes of Health, and NE-92-GB-2 from the Nebraska affiliate of the American Heart Association (to R. E. L.) and Grant P30 CA36727 from the National Cancer Institute to the Eppley Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Training Grant CA09476 from the National Cancer Institute.
** To whom correspondence should be addressed: Eppley Cancer Inst., University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. Tel.: 402-559-8290; Fax: 402-559-4651; E-mail: rlewis{at}unmec.edu.
1 The abbreviations used are: IGF I, insulin like growth factor I; CSF-1, colony-stimulating factor-1; CSF1R/IR, colony-stimulating factor-1 receptor/insulin receptor; IRS-1, insulin receptor substrate 1; CHO, Chinese hamster ovary; KRP, Krebs-Ringer phosphate; MAP, mitogen-activated protein; PBS, phosphate buffered saline; PI, phosphatidylinositol; IL, interleukin.
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REFERENCES |
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