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INTRODUCTION |
The insulin and IGF-11
receptors (IR and IGF-1R) belong to a family of tyrosine kinase
receptors that also includes the insulin-related receptor. They are
tetrameric receptors made up of two 
-subunits that bind the ligands
insulin, IGF-1 or IGF-2, and two 
-subunits that share high homology
in their kinase domains (reviewed in Ref. 1). These receptors are
homologous to a receptor found in the nematode
Caenorhabditis elegans and in
Drosophila, and they activate an evolutionarily conserved
metabolic and survival signaling pathway that includes insulin-related
substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3-K), the
serine/threonine kinase Akt, and the Forkhead family of
transcription factors (2-5).
There is considerable overlap in IR and IGF-1R function. The IR has a
primary role in regulating glucose metabolism and also promotes cell
survival and growth (1). The IGF-1R can regulate metabolism; it is
critical for growth during development; it promotes cell survival, and
it has an additional role in facilitating cellular transformation and
cancer progression (6). Cell survival and glucose metabolism are
tightly inter-linked, because glucose metabolism is essential for
Akt-mediated survival stimulated by IGF-1 and other growth factors (7,
8). In addition the potential of the IR and IGF-1R polypeptide chains
to associate and form hybrid receptors (9-11) gives them the capacity
to either compensate for or to inactivate one another. Lack of function
of either the IR or the IGF-1R can cause diabetes in mouse models
(12).
The IGF-1R has a well documented role in cancer development and
progression (6). Signals from the IGF-1R can enhance tumor cell
survival and growth and increase expression of genes that mediate
invasion and metastasis (6, 13, 14). The dependence of tumor cells on
IGF-1R function is supported by the observations that inhibition of
IGF-1R function by antibodies (15), triple helix formation (16), or
antisense strategies (17) can reverse the transformed phenotype and
lead to cell death.
Although there is a huge body of literature focusing on activation of
the PI3-K/Akt or other signaling pathway by IGF-1 and insulin, there is
a limited understanding of how the activity of the IR and IGF-1R is
regulated. Tyrosine phosphatases including LAR and PTP-1B
regulate IR kinase activity and glucose metabolism (18, 19). Recently,
we found that PTP-1B can also regulate IGF-1R kinase activity
and function in transformed cells (20). Another regulatory mechanism
for the IR and IGF-1R is proposed to operate through serine
phosphorylation of the receptors or IRS-1 (21). However, although
specific serines on IRS-1 are associated with inhibition of insulin
signaling (21), it is not known which serines in the IR or IGF-1R are
phosphorylated or how they could negatively regulate the activity of
these receptors. It is also not known if there are regulatory
mechanisms that act uniquely on the IGF-1R or the IR. Specific
regulatory mechanisms could be very important in distinguishing signals
necessary for the maintenance of normal cells from those necessary for
cancer progression.
A hint that the IGF-1R and IR have different signals and regulation
came from previous studies with mutants of the IGF-1R (22-25).
These indicated that domains of the IGF-IR C terminus with distinct
amino acid sequences from similarly located domains in the IR are
required for or have a regulatory effect on the anti-apoptotic and
transforming activity of the IGF-1R (22-25). The functions of the C
terminus in recruiting signaling molecules or regulating receptor
function have not yet been elucidated. To address this we undertook a
screen for proteins that could interact with the IGF-1R by using the
yeast two-hybrid system.
In this report we identify RACK1, a homologue of the -subunit of
heterotrimeric G proteins (26, 27), as an interacting protein for the
IGF-IR and IR. RACK1 associated with the IGF-1R and the IR in a
tyrosine kinase-independent manner but did not interact with an IGF-1R
that had serine 1248 mutated to alanine. Overexpression of RACK1 in R+
fibroblasts or in MCF-7 cells resulted in enhanced receptor kinase
activity, phosphorylation of IRS-2 and Shc, as well as enhanced
phosphorylation of Erks and JNK. However, IGF-1-induced phosphorylation
of Akt was greatly inhibited. Interestingly, although RACK1 enhanced
the growth rate of MCF-7 cells, it inhibited IGF-1-mediated protection
from etoposide killing. Altogether the data indicate that RACK1
interacts with the IGF-1R to negatively regulate activation of the
PI3-K pathway. Thus, RACK1 may have a broad role in regulating glucose
metabolism and cell survival.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant IGF-1 was purchased from PeproTech
(Rocky Hill, NJ). The anti-IGF-1R and anti-IR, anti-SHP-2, and
anti-c-Src polyclonal antibodies and the anti-c-Src monoclonal antibody
were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-IGF-1R
monoclonal antibody ND122 was from ImmunoGen (24). The
anti-phospho-Akt, anti-Akt polyclonal antibodies, and the
anti-phospho-p42/44 MAP kinase monoclonal antibody were from Cell
Signaling Technology (Beverly, MA). The anti-phosphotyrosine monoclonal
antibody, 4G10, the anti-Erk-2, anti-p85, and anti-IRS-2 monoclonal
antibodies, and the anti-phospho-Jun and anti-Shc polyclonal antibodies
were from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-Shc and anti-RACK1 monoclonal antibodies were from BD Transduction Laboratories (Heidelberg, Germany). The anti-HA antibody, 12CA5, was
from Roche Molecular Biochemicals, and the anti-actin monoclonal antibody was purchased from Sigma.
Yeast Two-hybrid Screen--
The yeast two-hybrid screen was
carried out using the reagents and protocols from the MATCHMAKER LexA
two-hybrid system (CLONTECH, Palo Alto, CA). To be
used as bait, the cDNA encoding the cytoplasmic domain of the wild
type IGF-1R (amino acids 930-1337 (28)) was fused to the LexA
sequence in the yeast expression vector pLexA under the control of the
ADH1 promoter. This plasmid was then transformed into
the yeast strain EGY48 (p80P-lacZ), which harbored the lacZ
reporter plasmid, and transformants were selected using Ura
, His
medium. These yeast cells were subsequently transformed with a cDNA
library derived from fetal brain expressed in the pB42AD vector and
simultaneously subjected to nutritional selection and selection for
growth of clones that had expressed activated LacZ (medium: Ura,
His, Trp, leu, gal+, and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)). Blue colonies that expressed potential interacting proteins were isolated and subjected to further selection for a true interaction using Shc as a positive control for an IGF-1R-interacting protein and
for interaction with a kinase-inactive IGF-1R -chain (K1003A). The
yeast plasmid DNA was recovered and transformed into the
Escherichia coli strain KC8 by electroporation. The cDNA
inserts were subjected to PCR analysis to exclude known
IGF-1R-interacting proteins including Grb-10 and p85. Unidentified
inserts were then sequenced and compared with the DNA data bases using
BLAST analysis.
RACK1 and IGF-1R Subcloning and Mutants--
Sequence analysis
of the two clones expressing RACK1 in the pB42AD vector revealed that
one of them encoded the full-length RACK1 protein. To obtain
full-length RACK1 in-frame with the HA epitope tag, oligonucleotide
primers complementary to the 5' and 3' ends of RACK1 were designed that
incorporated the restriction sites SalI and XbaI,
RACK1 forward 5'-TCGGTCGACCCATGACTGAGCAGATG and RACK1 reverse
5'-CATTCTAGACTAGCGTGCCAAT. PCR products were digested with
SalI and XbaI and analyzed by agarose gel
electrophoresis. Bands of the expected length were cut out and ligated
into the pKS vector, which had been digested with SalI and
Xba. After confirmation of the DNA sequence, RACK1 was
liberated from this vector using SalI/XbaI and
ligated into a pcDNA3 vector containing the HA-coding sequence that
had been digested with XhoI/XbaI.
The pcDNA3 vectors encoding IGF-1R full-length receptor, the
kinase-dead mutant (K1003R), and the tyrosine 1316 to phenylalanine mutant (Y1316F) were described previously (22). The -chain of the
IGF-1R (wild type, K1003R, and Y1316F) was subcloned into the
expression vector pIRES (CLONTECH) using
BamHI restriction sites on either end as described
previously (20). The double tyrosine 1162/1221 to phenylalanine mutant
(Y1162F/Y1221F), the serine 1248 to alanine mutant (S1248A), and
the serine 1252 to alanine (S1252A) mutant were all generated by
site-directed mutagenesis using "The TransformerTM" kit
from CLONTECH. The template used was a pKS vector
encoding a fragment of the IGF-1R from the unique HindIII
restriction site in the kinase domain to the stop codon (20). After
verification of the sequence of the mutants, the fragments were
subcloned into the pIRES expression vector already containing a wild
type IGF-1R -chain sequence by using the HindIII and
BamHI restriction sites. Full-length IGF-1R harboring each
of these mutations in the pcDNA3 vector was obtained by digestion
of pcDNA3 vector expressing wild type IGF-1R with
HindIII and BamHI and then replacing it with the
fragment containing each mutation. The sequence of all IGF-1R-encoding plasmids was verified by DNA sequencing. A prcCMV plasmid encoding the
full-length insulin receptor was kindly provided by Kenneth Siddle,
University of Cambridge, UK.
Cell Culture and Transfection--
The MCF-7 breast carcinoma
cell line, R cells (fibroblasts derived from the IGF-1R knock out
mouse), R+ cells (R cells that have been re-transfected with the
IGF-1R (29)), and COS cells were all maintained in Dulbecco's modified
Eagle's medium (Bio-Whittaker, Verviers, Belgium), supplemented with
10% (v/v) fetal calf serum, 10 mM L-Glu, and 5 mg/ml penicillin/streptomycin. COS cells or R+ cells were transiently
transfected with pcDNA3/HA-RACK1 or empty pcDNA3 vectors (4 µg of DNA) using LipofectAMINE Plus, (Invitrogen). After 24 h in
culture the transfected R+ cells were split into 6-well plates or 10-cm
plates and cultured for an additional 18 h, at which time cells
were starved for 3 h and stimulated with IGF-1, and protein
extracts were prepared for immunoprecipitation or Western blot analyses.
To generate stable transfectants of HA-RACK1, MCF-7 cells were
transfected as described for R+ cells, but 24 h after transfection cells were split into medium containing G418 (1 mg/ml) and maintained for 14 days, with regular replenishment of medium and drug. At this
time individual clones were selected, expanded, and screened for
expression of HA Rack by Western blotting. Clones of MCF-7 cells stably
overexpressing HA-RACK1 were maintained in Dulbecco's modified
Eagle's medium supplemented with 1 mg/ml G418. For analysis of
signaling responses cells were washed and starved from serum for 3 h.
Preparation of Cellular Protein Extracts and
Immunoprecipitation--
Cellular protein extracts were prepared by
washing cells with phosphate-buffered saline and then scraping into
lysis buffer consisting of Tris-HCl, pH 7.4, 150 mM NaCl,
1% Nonidet P-40 plus the tyrosine phosphatase inhibitor
Na3VO4 (1 mM) and the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), pepstatin
(1 µM), and aprotinin (1.5 µg/ml). After incubation at
4 °C for 20 min, nuclear and cellular debris were removed by
micro-centrifugation at 14,000 rpm for 15 min at 4 °C.
For immunoprecipitation of endogenous or transfected proteins, protein
extracts from stimulated or unstimulated cells were initially
pre-cleared using bovine serum albumin-coated protein G-agarose beads
(15 µl of beads per 400 µg of total protein in 700 µl of lysis
buffer) by incubation at 4 °C for 1 h with gentle rocking. The
lysates were recovered from the beads by centrifugation at 3,000 rpm
for 3 min and transferred to fresh tubes for incubation with primary
antibody (3 µg of each antibody) overnight at 4 °C with gentle
rocking. Immune complexes were obtained by adding 20 µl of protein
G-agarose beads for 3 h at room temperature and were pelleted by
centrifugation at 3,000 rpm for 3 min at 4 °C. The beads were washed
(3 times) with ice-cold lysis buffer and then either used for in
vitro kinase assay or removed from the beads by boiling for 5 min
in 20 µl of 2× SDS-PAGE sample buffer for electrophoresis and
Western blot analysis.
Western Blot Analysis--
All protein samples for Western blot
analysis were resolved by SDS-PAGE on 4-20% gradient gels and then
transferred to nitrocellulose membranes, which were blocked for 1 h at room temperature in TBS containing 0.05% Tween 20 (TBS-T) and 5%
milk (w/v). All primary antibody incubations were overnight at 4 °C.
Secondary antibody incubations were carried out at room temperature.
Where indicated, membranes were stripped by incubation in 62.5 mM Tris-Cl, 1% SDS, and 0.7% -mercaptoethanol for 30 min at 50 °C followed by extensive washing in 0.2 and 0.05% TBS-T.
Secondary antibodies conjugated with horseradish peroxidase were used
for detection with enhanced chemiluminescence (SuperSignal from Pierce)
or ECL+ (Amersham Biosciences) following the manufacturers' instructions.
Assays for Proliferation and IGF-1-mediated Protection from Cell
Death--
MCF-7 Neo and MCF-7 cells overexpressing HA-RACK1 (clone
A1, B1, and C1) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (complete medium) at 3 × 104 cells per well in multiple wells of a 24-well plate. To
monitor cell growth at intervals, attached cells were removed from
triplicate wells to Eppendorf tubes using trypsin-EDTA and were
centrifuged at 3,000 rpm for 3 min. The cell pellets were then
resuspended in 100 µl of medium and counted using a hemocytometer and
trypan blue exclusion. Data are presented as the mean and S.D. of
counts from triplicate wells.
To assess cell survival in response to IGF-1 in the presence or absence
of the apoptosis-inducing drug etoposide, cells were seeded in complete
medium. After 18 h the cells were washed once in
phosphate-buffered saline and re-incubated in serum-free medium supplemented with either no additives, with added IGF-1 (100 ng/ml), with etoposide (8.5 µM), or with IGF-1 + etoposide. After
a further 36 h in culture, the cells were assessed for viability
and cell number in triplicate wells as described for proliferation assays.
In Vitro Kinase Assays--
MCF-7 cells were starved from serum
for 4 h and then stimulated with IGF-1 for 0, 15, or 30 min. Cell
lysates were prepared and immunoprecipitated with anti-IGF-1R
polyclonal antisera as described above. Protein G-agarose complexes
obtained from immunoprecipitations were washed in kinase buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 10 mM MnCl2) and then resuspended in 25 µl of a
kinase reaction mixture containing ATP (0.03 mM final
concentration), 2 µl of [32P]ATP (5 µCi/µl), and 2 µl of poly(Glu-Tyr) (Sigma). Following a 20-min incubation
period, samples of reaction mixture (5 µl) were removed to fresh
tubes containing 9 µl of H2O and 35 µl of 20 mM EDTA, pH 7.4. Triplicate samples were then transferred
to glass microfiber filters in 24-well plates and washed extensively with ice-cold trichloroacetic acid (10%) containing 10 mM
Na2HPO4. Following a final wash with 70%
ethanol, the filters were dried, and 32P was measured in a
scintillation counter (Beckman Instruments). The data are presented as
the mean and S.D. of counts/min for triplicate samples.
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RESULTS |
Identification of RACK1 as an IGF-1R-interacting Protein--
In
order to identify novel IGF-1R-interacting proteins, a yeast two-hybrid
screen was performed using the cytoplasmic portion of the IGF-1R
-chain fused to the LexA DNA-binding protein as bait. Preliminary
experiments demonstrated that this IGF-1R -chain was
autophosphorylated when expressed in yeast. It could also interact with
a series of proteins derived from B cell and HeLa cell cDNA
libraries including the p85 subunit or PI3-K, Csk, Grb-10, and Shc,
most of which were identified previously (30-32) as IGF-1R-interacting proteins by other investigators.
From a screen of a brain-derived cDNA library, the only known
protein that interacted with the IGF-1R was a cDNA encoding RACK1/GNB2L1. RACK1 interaction was observed with a kinase-active IGF-1R -chain in yeast, but unlike Shc, it was also observed with a
kinase-inactive (K1003R) mutant of the IGF-1R -chain (data not
shown) RACK1 was originally identified in brain (26) and is a known
adapter protein for the IFN receptor (33). It has the potential to
interact with several proteins through its 7 WD repeat motifs (34).
To determine whether RACK1 interacts with the IGF-1R in mammalian
cells, the cDNA encoding RACK1 was subcloned in-frame with the
epitope tag HA at the N terminus into the mammalian expression vector
pcDNA3. COS cells were then transiently co-transfected with
pcDNA3 plasmids encoding either HA-RACK1 or full-length IGF-1R. The
IGF-1R was immunoprecipitated from cells using a polyclonal antibody
that detects an epitope in its C terminus and was then analyzed for
associated HA-RACK1 by immunoblotting with anti-HA antibody. To
determine whether activated PKCs influenced the interaction of RACK1
with the IGF-1R (as it does with PKC and Src (36)), the transfected
cells were treated with the phorbol ester phorbol 12-myristate
13-acetate to activate PKC or were left untreated. The results shown in
Fig. 1A demonstrated that
HA-RACK1 was present in the IGF-1R immunoprecipitates at equivalent
amounts in the presence or absence of phorbol 12-myristate 13-acetate.
This indicates that HA-RACK1 associates with the IGF-1R in COS cells,
and this interaction is not altered by phorbol 12-myristate
13-acetate.
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Fig. 1.
RACK1 interacts with IGF-1R in COS cells and
in R+ cells. A, COS cells were transiently transfected
with plasmids encoding HA-RACK1. After 36 h cell lysates were
prepared, precleared, and subjected to immunoprecipitation with an
anti-IGF-1R polyclonal antiserum. The precipitated proteins were then
resolved by SDS-PAGE and transferred to nylon membranes for Western
blotting with an anti-HA antibody to detect HA-RACK1. The blots were
stripped and re-probed for IGF-1R content using an anti-IGF-1R
monoclonal antibody. B, R+ cells were starved for 3 h
and then stimulated with IGF-1 for the indicated times. Cell lysates
were prepared, and the IGF-1R was immunoprecipitated and then analyzed
for IGF-1R levels, phosphotyrosine content, or associated RACK1 by
Western blotting.
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We next investigated whether endogenous RACK1 interacts with the IGF-1R
and how this is affected by ligand stimulation of the IGF-1R. To do
this we used R+ cells (R fibroblasts derived from the IGF-1R knock
out mouse) that were re-transfected with the IGF-1R (29). Cells were
starved from serum and then stimulated with IGF-1 for the indicated
times, at which times the IGF-1R was immunoprecipitated and assessed
for both phosphotyrosine content and associated endogenous cellular
RACK1 by Western blotting. As can be seen in Fig. 1B, RACK1
was associated with the unphosphorylated form of the IGF-1R. Upon IGF-1
stimulation the IGF-1R -chain underwent autophosphorylation, as
detected by phosphotyrosine staining, and similar amounts of RACK1
protein were associated with the phosphorylated receptor. This
indicates that RACK1 is associated with the IGF-1R in either the
inactive or active state, and this interaction does not require
autophosphorylation of the IGF-1R.
RACK1 Interacts the Insulin Receptor with Kinase-inactive IGF-1R
but Not with a Serine Mutant of the IGF-1R--
Several proteins that
interact with the IGF-1R also interact with the IR, but there is at
least one IGF-1R-interacting protein that was identified in a yeast
two-hybrid system that does not interact with the IR (36). We were
therefore interested to determine whether RACK1 could interact with the
IR as well as with the IGF-1R. To test this, the endogenous IR or
IGF-1R proteins were immunoprecipitated from COS cells, and the
immunoprecipitates were investigated for associated endogenous RACK1 by
Western blotting. As can be seen in Fig.
2A, RACK1 interacted with both
the IR and IGF-1R in COS cells. This suggests that RACK1 interacts with
amino acid residues or receptor domains that are common to both the IR
and IGF-1R.
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Fig. 2.
RACK1 association with the insulin receptor
and IGF-1R mutants. A, COS cells were lysed, and the lysates
were split into two samples that were immunoprecipitated using either
anti-IR or anti-IGF-1R antibodies. The precipitated proteins were then
resolved on the same gel and transferred to a nylon membrane that was
sequentially probed with anti-IGF-1R, anti-IR, or anti-RACK1
antibodies. B, R cells were transiently transfected with
plasmids encoding the indicated IGF-1R -chains, either wild type
(wt) or the indicated point mutants. After 36 h the
cells were lysed, and the lysates were immunoprecipitated with
anti-IGF-1R polyclonal antisera or with antibody-coated protein
G-agarose beads as a control (Co). The precipitated proteins
were then analyzed by Western blotting to detect IGF-1R -chain or
associated cellular RACK1. C, R cells were transiently
transfected with plasmids encoding full-length IGF-1Rs (either wild
type or the indicated mutants) and with a plasmid encoding full-length
IR. After 24 h the cells were lysed and analyzed for association
of RACK1 with IGF-1R or IR as described for A and
B.
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To investigate further the residues in the IGF-1R necessary for
interaction with RACK1, we investigated a series of IGF-1R -chain
mutants transiently expressed in R fibroblasts. From each transfected
cell population, endogenous RACK1 association with the IGF-1R -chain
was analyzed by Western blotting with anti-RACK1 antibody. As can be
seen in Fig. 2B, RACK1 interacted with a wild type IGF-1R
-chain but also with a kinase-inactive (K1003R) mutant of the
-chain. This confirms the result obtained in the yeast two-hybrid
system and the observation that RACK1 interacts with un-stimulated
IGF-1R (Fig. 1B). RACK1 also interacted with IGF-1R
-chains containing mutated tyrosines, the Y1316F mutant, or the
double tyrosine mutant Y1162F/Y1222F. However, RACK1 did not
interact with an IGF-1R -chain mutated at a C-terminal serine,
S1248A, whereas it retained interaction with another -chain mutated
at serine, S1252A. Levels of expression of the mutants and wild type
IGF-1R -chain receptors were similar as shown by Western blotting
with anti-IGF-1R antibody.
To confirm that the pattern of interaction with the mutant -chain
receptors was physiologically relevant, the experiments were also
performed with full-length IGF-1R (wild type and mutants) transiently
transfected into R cells. Results are shown in Fig. 2C and
demonstrate that, as was observed with the -chain proteins, interaction of endogenous RACK1 was not observed with the S1248A mutant, whereas RACK1 interacted with the other mutants tested.
Altogether, these data demonstrate that RACK1 interacts with both the
IR and the IGF-1R. The interaction with the IGF-1R is not dependent on
an active tyrosine kinase nor does it require a number of tyrosines in
the IGF-1R. However, RACK1 interaction requires serine 1248 in the C
terminus of the IGF-1R. Interestingly, this serine is conserved in the
IR at amino acid position 1262, which suggests that it could mediate
RACK1 interaction with both receptors.
Overexpression of RACK1 Enhances Erk and JNK Activation by IGF-1
but Decreases Akt Activation--
Because RACK1 can interact with the
IGF-1R in the absence of kinase activation, this suggests that RACK1
acts to negatively regulate receptor activity rather than to mediate
signaling responses in response to ligand binding. Overexpression of
RACK1 has been shown previously to be inhibitory to the growth of 3T3
fibroblast cells, and this is associated with its ability to sequester
Src (35). Like these investigators we were unable to obtain clones of
3T3 fibroblasts that stably overexpressed HA-RACK1. However, we were
able to generate clones of MCF-7 cells that stably overexpressed HA-RACK1.
To determine whether RACK1 influences the signaling responses from the
IGF-1R, we analyzed this in R+ cells transiently overexpressing HA-RACK1 and MCF-7 cells stably overexpressing HA-RACK1. IGF-1-induced phosphorylation of Erks was assessed as a measure of MAP kinase activation; phosphorylation of Akt was assessed as a measure of PI3-K
activation; and c-Jun phosphorylation was assessed as a measure of JNK activation.
As can be seen in Fig. 3A,
IGF-1-induced phosphorylation of Akt, which was induced by 5 min in
vector-transfected R+ cells and peaked by 10 min, was greatly reduced
in HA-RACK1-transfected R+ cells. By contrast, phosphorylation of Erks
was enhanced at 5 min and was more sustained in the HA-RACK1-expressing
cells than in the vector controls. Under these conditions
phosphorylation of c-Jun was not detectable within 30 min of IGF-1
stimulation in the control cell but was clearly detectable by 30 min in
the HA-RACK1-expressing R+ cells (Fig. 3A). Levels of
HA-RACK1 overexpression were confirmed by staining with the anti-HA
antibody. These results indicate that the PI3-K pathway is inhibited by
overexpression of RACK1 in R+ cells, whereas the MAP kinase and JNK
pathways are enhanced.
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Fig. 3.
Analysis of IGF-1-mediated activation of
PI3-K, MAP kinase, and Jun kinase pathways. A, R+ cells were
transiently transfected with either empty pcDNA3 vector or pcDNA3
encoding HA-RACK1. After 36 h cells were starved for 3 h and
then stimulated with IGF-1 for the indicated times and lysed. The
lysates were then resolved by SDS-PAGE and analyzed by Western blotting
with anti-phospho-Akt (P-Akt), anti-phospho-Erk
(P-Erk), and anti-phospho-c-Jun (P-c-Jun)
antibodies. The blots were then stripped and reprobed with anti-Akt,
anti-HA, and anti-actin antibodies to demonstrate equal loading.
B, R+ cells transiently expressing either vector or HA-RACK1
were starved and stimulated with IGF-1 for the indicated times. Cell
lysates were prepared and immunoprecipitated (IP) with
anti-IRS-2 and anti-Shc antibodies. The immunoprecipitates were then
subjected to Western blotting with anti-phosphotyrosine antibody, and
the blots were re-probed with either anti-IRS-2 or anti-Shc antibody.
C, MCF-7 cells were transfected with either pcDNA3 empty
vector or HA-RACK1, and clones stably overexpressing HA-RACK1 were
isolated as described under "Experimental Procedures." Expression
of HA-RACK1 (lower arrow) and endogenous RACK1 (upper
arrow) was measured in these clones by Western blotting with
anti-RACK1 antibody. D, cells were starved from serum for
4 h and then stimulated with IGF-1 for the indicated times. To
measure phosphorylation of Akt and Erks cell lysates were prepared and
subjected to Western blotting with the indicated antibodies.
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To assess the effects of RACK1 on IGF-1R activity and substrate
phosphorylation, Shc and IRS-2 were immunoprecipitated from R+ and
assessed for phosphorylation in response to IGF-1 stimulation by
Western blotting with anti-phosphotyrosine antibody. This demonstrated that both IRS-2 and Shc phosphorylation were increased in the HA-RACK1-overexpressing cells compared with vector-expressing controls
(Fig. 3B). This suggests that IGF-1R activity is enhanced by
overexpression of HA-RACK1.
We next investigated the effects of RACK1 overexpression on signaling
responses in MCF-7 cells. Three clones of MCF-7 cells overexpressing
HA-RACK1 were isolated (clones A1, B1, and C1). Analysis of endogenous
RACK1 and HA-RACK1 expression levels is shown in Fig. 3C and
indicates that ~2-fold higher levels of HA-RACK1 are expressed in
these clones. IGF-1-mediated activation of the PI3-K and MAP kinase
pathways was investigated in all three clones with similar results, and
this is shown in Fig. 3D for clone A. As was seen with R+
cells, phosphorylation of Akt was decreased in response to IGF-1
stimulation, and phosphorylation of Erks was enhanced.
Overall the data indicate that RACK1 overexpression increases
phosphorylation of IRS-1 and Shc by IGF-1. At the same time it
attenuates activation of Akt but enhances activation of MAP kinase and
JNKs. This suggests that RACK1 has a selective role in modulation of
IGF-1R signaling and that it has a negative regulatory effect on the
Akt pathway.
Overexpression of RACK1 Enhances the Proliferation Rate of MCF-7
Cells but Decreases IGF-1-mediated Protection from Apoptosis--
The
data above indicate that overexpression of RACK1 abrogates
IGF-1-mediated activation of the PI3-K pathway but enhances the MAP
kinase pathway in both fibroblasts and MCF-7 cells. These pathways are
activated in survival and proliferative responses from the IGF-1R.
However, we and others (37) observed that RACK1 is inhibitory to the
growth of fibroblasts but apparently does not inhibit MCF-7 cells.
Therefore, we asked whether overexpression of RACK1 had an effect on
the proliferation rates or IGF-1-mediated protection from apoptosis in
MCF-7 cells.
To compare the proliferation rates of MCF-7 cells
overexpressing HA-RACK1, triplicate cultures of each of the three
clones A1, B1, and C1 were assessed for accumulated cell numbers in
medium supplemented with fetal bovine serum compared with
vector-expressing cells, Neo. This demonstrated that in each MCF-7 cell
clone overexpressing HA-RACK1 the rate of cellular proliferation was
increased, and the doubling time was approximately twice as high as in
Neo cells (Fig. 4A). This
suggests that overexpression of RACK1 provides a proliferative
advantage to these tumor cells, which is the opposite effect to that
observed in fibroblasts. The enhanced growth correlates with enhanced
IGF-1-mediated activation of MAP kinases (Fig. 2) and also suggests
that decreased Akt activation does not affect the growth of MCF-7
cells.
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Fig. 4.
Proliferation is enhanced, but protection
from etoposide killing is decreased in MCF-7 cells overexpressing
HA-RACK1. Three clones of MCF-7 cells overexpressing HA-RACK1 (A1,
B1, and C1) and one overexpressing vector (Neo) were seeded
at a density of 3 × 104 per ml in multiple wells of
24-well plates. At the indicated time points cells from triplicate
wells were removed using trypsin/EDTA and centrifuged. Cell numbers and
viability were determined by trypan blue exclusion, and data are
presented as mean and S.D. of live cell numbers in triplicate wells.
B, to measure IGF-1-mediated protection from etoposide
killing, MCF-7/Neo or MCF-7/HA-RACK1 cells (clone A1) were
seeded in multiple wells of a 24-well plate at 3 × 104 per ml. After 18 h culture cells were washed and
resuspended in serum-free medium either with no additions
(control), or with IGF-1 added, or with etoposide (8.5 µM) added, or with IGF-1 + etoposide added. After a
further 36 h in culture, the cells were harvested and assessed for
viability and number using trypan blue exclusion. Data are presented as
mean and S.D. of live cell number from triplicate wells.
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We next investigated IGF-1-mediated protection from apoptosis in
MCF-7 cells overexpressing RACK1. Cells were cultured in serum-free
medium and were then treated with etoposide in the presence or absence
of IGF-1. As can be seen in Fig. 4B, in serum-free medium
IGF-1 stimulated an increase in cell number, which was ~30% greater
in the HA-RACK1 A1 clone than in the Neo cells. This is in agreement
with the observations in Fig. 4A that the proliferation rate
of the HA-RACK1-overexpressing cells is increased in serum-supplemented medium. When cells were treated with etoposide a similar decrease in
cell number occurred in both Neo and A1 cells, which indicates a
similar level of cell killing. However, IGF-1 rescued the Neo cells and
also increased the cell number in these cultures by over 100%. By
contrast, in the A1 cells IGF-1 only afforded a very slight increase in
cell number. This indicates that MCF-7 cells overexpressing HA-RACK1
have diminished IGF-1-mediated protection from etoposide killing. Since
this decrease can be correlated with the decrease in IGF-1-induced
phosphorylation of Akt observed in these cells, it suggests that the
blunted Akt activation is responsible for the lack of protection from
etoposide killing.
Altogether the data demonstrate that overexpression of RACK1 enhances
the proliferative rate of MCF-7 cells in the presence of serum or IGF-1
but inhibits IGF-1-mediated protection from induction of cell death.
IGF-1R Kinase Activity Is Enhanced in MCF-7 Cells That Overexpress
RACK1--
The status of IGF-1-mediated activation of the PI3-K and
MAP kinase pathways is differentially affected in
HA-RACK1-overexpressing cells, and this may be responsible for the
inhibition of fibroblast cell growth as well as the enhanced
proliferation combined with decreased protection from apoptosis in
MCF-7 cells. However, we were also interested to determine whether the
kinase activity of the IGF-1R is altered in MCF-7 cells overexpressing
RACK1. To do this, in vitro kinase assays were performed
with IGF-1R immunoprecipitated from MCF-7 cells by measuring
incorporation [32P]ATP into the peptide substrate
poly(Glu-Tyr). Results shown in Fig. 5
demonstrate that the basal tyrosine kinase activity of the IGF-1R
toward poly(Glu-Tyr) was slightly higher in the MCF-7/HA-RACK1 cells
compared with control cells in the unstimulated state. IGF-1
stimulation caused an ~25% greater increase in kinase activity
toward poly(Glu-Tyr) in these cells compared with vector controls. This
indicates that overexpression of RACK1 in MCF-7 cells enhances IGF-1R
kinase activity. This could contribute to the enhanced IRS-2 and Shc
phosphorylation observed in R+ cells, to the enhanced MAP kinase and
JNK activation, and to the enhanced proliferative rates observed with
these cells (Fig. 4). However, it is remarkable that IGF-1-induced Akt
phosphorylation is diminished so much even in the presence of this
enhanced IGF-1R activity.
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Fig. 5.
In vitro kinase activity of IGF-1R
is enhanced in RACK1-overexpressing cells. MCF-7 cells either
expressing empty vector (Neo) or HA-RACK1 (clone
A1) were starved from serum for 3 h and then stimulated with
IGF-1 for the indicated times. Cells were lysed, and the lysates were
immunoprecipitated using an anti-IGF-1R polyclonal antisera and protein
G-agarose beads. The beads were then washed in kinase buffer (see
"Experimental Procedures") and resuspended in a kinase reaction mix
that contained kinase buffer, [32P]ATP, and an exogenous
peptide substrate poly(Glu-Tyr). After a 20-min incubation the reaction
mix was removed and precipitated on fiberglass filters using
trichloroacetic acid, and after extensive washing the filters were
dried and counted by liquid scintillation counting. Inset
shows the levels of IGF-1R in each sample determined by Western
blotting with an aliquot of the immunoprecipitated proteins.
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Association of Src, p85, and SHP-2 with RACK1--
One way in
which RACK1 could enhance IGF-1R kinase activity and differentially
modulate IGF-1R-activated signaling pathways is through recruitment of
Src, which has been shown previously to phosphorylate the IGF-1R on key
sites that stimulate its activation (38) or through sequestration of
proteins necessary to activate the different signaling pathways.
Sequestration of Src by RACK1 has been suggested previously (37) to
account for the inhibition of fibroblast growth. Therefore we
investigated Src recruitment to endogenous RACK1 in response to IGF-1
stimulation in R+ cells and in MCF-7 cells. Similar results were
obtained for both cell lines and are shown in Fig.
6A. Src was found to be
associated with RACK1 in unstimulated cells, but in response to IGF-1
stimulation the Src protein was slowly released. By 15 min there was a
significant decline in the amount of Src associated with RACK1, and by
30 min there was no Src associated with RACK1. This indicates that the
RACK1-Src complex is responsive to and is altered by IGF-1 stimulation.
However, the kinetics of Src dissociation is much slower than those for
Akt activation or Erk activation in response to IGF-1. This suggests
that Src activity does not account for the increase in MAP kinase
activation, but it may contribute to JNK activation after 30 min of
IGF-1 stimulation. However, because the kinetics of Src release is
similar in both R+ and MCF-7 cells, we conclude that Src is not
responsible for the differential effects of RACK1 on cell growth in
fibroblasts and MCF-7 cells.
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Fig. 6.
Dissociation of RACK1 and Src in response to
IGF-1 stimulation and association of p85 and SHP-2 with RACK-1.
A, R+ cells (top panel) or MCF-7 cells
(bottom panel) were starved for 4 h and stimulated with
IGF-1 for the indicated times. Cells were lysed and immunoprecipitated
(IP) with anti-Src polyclonal antisera. The precipitated
proteins were then analyzed by Western blotting for RACK1 or Src using
anti-RACK1 and anti-Src monoclonal antibodies. In the middle
panel the expression of the IGF-1R and phosphorylation status of
the IGF-1R -chain are shown by Western blot analysis of the lysates
of R+ cells. B, R+ cells were transiently transfected with
HA-RACK-1, cultured for 48 h, starved for 4 h, and stimulated
with IGF-1 for the indicated times. Cell lysates were prepared and
immunoprecipitated with anti-HA or control antibody (Co).
The immunoprecipitates were then subjected to Western blotting with
either anti-p85 or anti-SHP-2 antibody, and blots were re-probed with
anti-HA antibody. A sample of cell lysate is included to show total
cellular levels of these proteins.
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We next investigated whether the decreased activation of Akt observed
with overexpression of RACK1 was correlated with sequestration of
proteins that promote activation of this pathway, the p85 subunit of
PI-3K and SHP-2, which has been shown previously to associate with p85
and to be essential for IGF-1-mediated activation of Akt (39). To do
this, HA-RACK1 was immunoprecipitated from transiently transfected R+
cells and then analyzed for associated p85 and SHP-2 by Western
blotting. The results shown in Fig. 6B demonstrate that p85
and SHP-2 are both associated with RACK1 in the absence of IGF-1
stimulation and remain associated in response to 5 mi of IGF-1
stimulation. At this time Akt activation is diminished (Fig. 3). RACK1
does not interact with Shc in these cells (not shown). Overall the data
indicate that the decreased Akt activation observed in the presence of
RACK1 overexpression can be correlated with the sequestration of Src,
p85, and SHP-2 by RACK1.
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DISCUSSION |
We have identified RACK1 as an IGF-1R and IR-interacting protein
whose overexpression has a negative effect on activation of the PI-3K
pathway but has a positive effect on activation of the MAP kinase and
JNK pathways. This was correlated with increased proliferation rates
but decreased IGF-1-mediated protection from etoposide killing in MCF-7
cells. RACK1 interaction with the IGF-1R occurred independently of
tyrosine kinase activity, but it required serine 1248, which is also
present in a conserved amino acid stretch in the IR. This suggests that
RACK1 is a regulator of IGF-1R and IR function.
RACKs are a family of proteins that share homology with the
B-subunits of heterotrimeric G proteins and are also members of an ancient group of regulatory proteins, which are made up of a series
of Trp-Asp (WD repeats). RACKs generally are composed of 5-7 WD
repeats (seven in the case of RACK1), which confers on them the
potential to act as a scaffold or adapter proteins (40-49). Recent
evidence suggests that RACKs act as a -propeller structure where
each WD repeat forms a different blade on the -propeller (27).
Interestingly the WD repeats in RACK1 are conserved from chlamydomonas
to humans and can be used by viruses to interact with cellular proteins
(27, 44).
RACKS were originally identified as molecules that bind only to
activated forms of PKC, facilitating their translocation and anchoring
to membranes or cytoskeletal structures in proximity to its substrates
(26, 46, 47). However, RACK1 has also been found to interact with Src
family members (37), phospholipase C
, PTPµ (47),
cAMP-specific phosphodiesterase-4 (43), the B-subunit of
integrins, and certain pleckstrin homology domains in vitro,
including dynamin and -spectrin (27). Only a subset of these
interactions depends on PKC stimulation, suggesting that RACK1 can
facilitate signaling complexes in response to distinct cellular
stimuli. RACK1 also associates with the type 1 interferon receptor (48)
and the common -chain of the
interleukin-5/interleukin-3/GM-CSF receptor (43), and it is
thought to promote signaling from these receptors by its ability to
also associate with signal transducers and activators of transcription.
The observation that RACK1 interacts with kinase-inactive and
unphosphorylated IGF-1R combined with the finding that serine 1248 is
necessary for the interaction suggests that RACK1 associates with the
inactive receptor and remains there when the receptor kinase becomes
activated. We cannot rule out the possibility that other serines or
domains in the receptor are also involved in RACK1 interaction, but
mutation of the single amino acid (serine 1248) is sufficient to
disrupt RACK1 association. This suggests that serine 1248 in the IGF-1R
or its cognate serine 1262 in the IR could act as regulatory sites on
these receptors. It is not known if these serines are phosphorylated
in vivo or how they contribute to IGF-IR or IR function.
Overexpression of RACK1 led to enhanced IGF-1R kinase activity and
IGF-1-induced phosphorylation of IRS-2 and Shc. This raised the
following interesting question: why does overexpression of RACK1 lead
to increased MAP kinase and JNK activation but decreased Akt
activation? Because the interaction of RACK1 with the IGF-1R apparently
does not change in response to kinase activation, the effects on
signaling modulation are likely to be mediated through proteins that
are already associated with RACK1 in unstimulated cells. Our
observation that two proteins that promote IGF-1-mediated activation of
AKT, the p85 subunit of PI-3K and the phosphatase SHP-2, are
constitutively associated with RACK1 suggests that sequestration of
these proteins may be the cause of the observed Akt inhibition.
However, it is possible that other RACK1-interacting proteins are also
involved in regulating Akt activity. For example the regulatory
phosphatase PTPµ interacts with RACK1 and was found to be
active in regulating focal adhesions via PKC
(47, 49). It will be
necessary to do a complete analysis of RACK1-associated proteins in
response to IGF-1 or insulin stimulation of cells to get a
comprehensive picture of how RACK1 regulates signaling from these receptors.
Sequestration of Src has been proposed previously (37) as the growth
inhibitory mechanism for RACK1 in NIH-3T3 cells. We also observed RACK1
interaction with c-Src in R+ cells and MCF-7 cells, but the complex
dissociated completely after 15 min of IGF-1 stimulation. Thus, Src is
associated with RACK1 during the 5-10-min post-IGF-1 stimulation that
Akt and Erks become phosphorylated. Dissociation of Src could
contribute to enhanced IGF-1-mediated JNK activation observed at 30 min
because we have found previously (50) that JNK is activated by IGF-1 in
a PI3-K and MAP kinase-independent manner. In all of our experiments
the differential effects of RACK1 on activation of the Akt and the MAP
kinase pathway were correlated with different consequences for the
growth and survival of fibroblasts and MCF-7 cells. It has been shown
previously (51) that -subunits of G proteins can activate MAP
kinases in a PKC-independent manner in response to IGF-1R or IR
stimulation but not in response to platelet-derived growth factor,
fibroblast growth factor, or epidermal growth factor. In addition
sequestration of G-subunits could block IGF-1R mitogenic activity
but had no effect on insulin or IGF-1R metabolic activity (52). Our
observation that RACK1 overexpression enhances IGF-1-mediated
activation of MAP kinase suggests that RACK1 may act like or assist
-subunits to enhance directly IGF-1-mediated activation of MAP
kinase and JNKs and also to promote cellular growth in MCF-7 cells. It
is also noteworthy that although enhanced MAP kinase signaling can
confer a growth advantage on MCF-7 cells, inhibition of Akt activation
by overexpression of RACK1 is sufficient to block IGF-1-mediated
protection from the cytotoxic insult of etoposide. This suggests that
reduced Akt activation by itself is not sufficient to halt the growth of these tumor cells because the MAP kinase pathway is hyperactive, but
in combination with an apoptotic signal from etoposide reduced Akt
activation can inhibit cell survival and growth.
RACK1 interacts with both the IGF-1R and IR, so it is likely that it
can regulate signaling from both receptors. Although the IR and IGF-1R
have many overlapping functions, they can have significant differences
in signaling output (53-56), some of which has been attributed to
tissue distribution, differences in the structure or amino acid
sequence of the receptors, or to the usage of different kinds of G
proteins (22, 29, 53, 57). It has also been demonstrated that
activation of the class 2 PI3-Ks via IRS-1 may be a unique signal from
the IR to regulate glucose metabolism (57). Thus, activation of PI3-K
and Akt may be more important for metabolism and cell survival than for
cellular proliferation or growth of tumor cells. If RACK1 selectively
regulates the Akt pathway, then it may have a particular role in
regulating glucose metabolism and cell survival. Because its
interaction with the IGF-1R is dependent on serine 1248, which is also
conserved in the IR at position 1262, the effects of RACK1 on the PI3-K
pathway may be dependent on the activity of cellular serine kinases
that phosphorylate this serine in the IR or IGF-1R.
Serine phosphorylation of the IR, IGF-1R, and IRS-1 has been proposed
to be a negative regulatory mechanism (21, 58) and cause insulin
resistance. Tumor necrosis factor can stimulate phosphorylation of
serine 307 in IRS-1 (59) and insulin resistance. Although a series of
kinases including PKC
, IKKB, PKC, and JNK have been implicated
(1, 21), it is not known which kinase directly phosphorylates this
serine. Because RACK1 is a receptor for the C kinases, it is also
possible that its associated kinases are involved in tumor necrosis
factor -mediated insulin resistance. It will be of interest to
investigate whether a serine kinase is necessary to maintain RACK1
interaction with the IGF-1R or IR and whether RACK1 has a role in
insulin resistance.
In summary we have identified RACK1 as an IGF-1R and IR-interacting
protein that has the potential to enhance activation of the MAP kinase
or JNK pathways but that inhibits Akt activation. Thus RACK1 may be an
important regulator of cell survival and metabolism.
,
TOP
ABSTRACT
INTRODUCTION
