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INTRODUCTION |
The IGF-IR,1 closely
related to the IR, is a receptor protein-tyrosine kinase consisting of
two heterodimers linked by disulfide bonds (1). Each heterodimer
contains an extracellular subunit, the
subunit, responsible for
ligand binding and a transmembrane subunit, the
subunit, which
contains the protein-tyrosine kinase activity in its intracellular
domain. When IGF-I binds to the IGF-IR, the receptor becomes
autophosphorylated on several tyrosine residues. Phosphorylation of the
receptor allows interactions with phosphotyrosine binding or Src
homology 2 (SH2) domain-containing proteins, thereby transducing
signals to downstream effectors. Insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) and Shc are major substrates of the IGF-IR that
subsequently bind other SH2 domain-containing proteins, including the
p85 subunit of phosphatidylinositol 3'-kinase, the adaptor proteins,
Grb2 and Nck, the protein-tyrosine-phosphatase, Shp 2, and the
C-terminal Src kinase, CSK (2-10). Binding of IGF-I to the IGF-IR
activates the Ras-mitogen-activated protein kinase signaling pathway
(11, 12).
Protein-tyrosine kinases of the Src family have been studied
extensively (for reviews see Refs. 13 and 14). Extracellular stimuli
activate Src kinases, stimulating cellular DNA synthesis (15), mitosis
(16, 17), proliferation, adhesion (18), and cytokine production (19).
Targeted disruption of the c-src proto-oncogene (20, 21)
results in osteopetrosis, suggesting that Src is required for
osteoclast differentiation function (22, 23). c-Src is involved in
transducing signals from several receptor protein-tyrosine kinases (13,
14), including the platelet-derived growth factor receptor, the
epidermal growth factor receptor, the fibroblast growth factor
receptor, and the nerve growth factor receptor. Src family kinases have
been implicated in insulin and IGF-I signaling pathways. For example,
it has been reported that tyrosine-phosphorylated IRS-1 binds to Fyn
(24), and the
subunit of the IGF-IR is phosphorylated by Src in
v-Src-transformed cells (25).
The c-Src protein has a domain organization consisting of an N-terminal
Src homology (SH) 4 domain containing a myristoylation and membrane
localization signal, followed by a unique domain, SH3 and SH2 domains,
the catalytic SH1 domain, and the C-terminal tail (see Ref. 13 for
review). c-Src kinase activity is regulated by phosphorylation of two
tyrosine residues, Tyr416, the autophosphorylation site,
and Tyr527 in the C-terminal tail, as well as by SH2 and
SH3 domain-mediated interactions. The three-dimensional structure of
c-Src helps explain the regulatory interactions (26). Phosphorylation
of Tyr527 creates a binding site for the Src SH2 domain,
locking the molecule in an inactive state. The SH3 domain contributes
to the stability of the inactive state by interacting with a
"linker" that joins the SH2 and catalytic domains. The compact
organization pushes the two lobes of the catalytic domain together,
disabling the active site.
CSK, the C-terminal Src kinase, is a ubiquitously expressed cytoplasmic
protein-tyrosine kinase. Its organization resembles that of Src,
including an SH3, SH2, and a catalytic domain (27, 28). However, CSK
lacks the catalytic domain autophosphorylation site, the C-terminal
regulatory tyrosine, and the N-terminal myristoylation signal of Src.
CSK phosphorylates Src Tyr527, down-regulating Src kinase
activity (27, 29). The regulation of CSK activity is not well
understood. Several studies (30-33) have suggested that relocalization
of CSK allows the molecule to phosphorylate Src C-terminal tyrosine.
Howell and Cooper (30) suggested a model in which CSK would interact
with Src substrates via its SH3 or SH2 domain in order to down-regulate
Src activity.
The observations that IGF-IR is a substrate of Src in v-Src-transformed
cells and that IGF-IR expression is required for Src-mediated transformation in mouse fibroblasts suggested that Src might
participate in IGF-IR signaling. In this study, we observed that CSK
bound to the IGF-IR in vitro and in vivo. By
using a yeast two-hybrid system to characterize the IGF-IR-CSK
interaction, we found that the CSK SH2 domain interacted with
Tyr943 and Tyr1316 of the IGF-IR. By contrast,
the CSK SH2 domain interacted only with Tyr1322 of the
insulin receptor (IR). We further observed that Src kinase activity was
rapidly decreased following IGF-I and insulin stimulation of human or
mouse fibroblasts. Therefore, CSK is a new interacting protein of the
IGF-IR and the IR. Our observations suggest a role for CSK and c-Src in
IGF-IR and IR signaling.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Vectors--
pCLXSN-hIGF-IR was constructed as
described previously.2
Hemagglutinin (HA)-tagged wild type CSK was generated by polymerase chain reaction (PCR) using convenient cloning sites for insertion into
the pEF-HA vector. HA-tagged CSK mutant S109C was made using the
QuickChange procedure (Stratagene) from wild type CSK subcloned into
pBluescript followed by subcloning into the pEF-HA vector. The
pGEX-KG-CSK from which these constructs were derived was a generous
gift from K. Neet (see Ref. 35). Both wild type and S109C mutant CSK
were verified by DNA sequencing.
Antibodies--
Anti-IGF-IR, a rabbit polyclonal antibody,
directed against the C terminus of the
subunit, was described
previously.2 Antibody directed against the C terminus of
CSK was a gift from K. Neet (see Ref. 35); anti-phosphotyrosine
antibody (4G10) was from Upstate Biotechnology Inc. (New York); the
hybridoma for the monoclonal antibody to the hemagglutinin epitope tag
(anti-HA, monoclonal antibody 12CA5) was kindly provided by Dr. I. Wilson (The Scripps Research Institute); anti-IR
subunit antibody
was from Santa Cruz Biotechnology, Inc.; the hybridoma for the
monoclonal peptide antibody directed against amino acids 2-17 of
v-Src, which recognizes both c-Src and v-Src, was from Microbiological
Associates; and anti-Src hybridoma monoclonal antibody 327 was a
generous gift from Joan Brugge (see Ref. 36).
Immunoprecipitation--
Cells were incubated in Dulbecco's
modified Eagle's medium supplemented with 0.1% calf serum for 18 h before stimulation with LR3-IGF-I (JRH Biosciences) or insulin
(Sigma). Cells were lysed in RIPA buffer (37) modified by the addition
of 10 µg/ml aprotinin, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 1 mM sodium orthovanadate, 0.5 mM dithiothreitol at 4 °C. Lysates were precleared with formalin-fixed
Staphylococcus aureus (Pansorbin) (Calbiochem) by
centrifugation at 4 °C for 20 min at 14,000 rpm. For
co-immunoprecipitation studies, SDS and sodium deoxycholate were
omitted from the lysis buffer.
Similar amounts of lysate (about 1 ml), adjusted to contain equal
amounts of protein (0.5-1.0 mg), were incubated with 1 µg of
antibody for 2 h at 4 °C. For c-Src immunoprecipitation, rabbit anti-mouse secondary antibody was added for an additional 30 min. Protein A-agarose (Repligen) was added for 1 h at 4 °C and
washed 3 times with lysis buffer at 4 °C. Immune complexes were
boiled for 5 min in sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 20%
-mercaptoethanol) and separated on a
10% SDS acrylamide gel.
Immunoblotting--
After gel electrophoresis, proteins were
transferred to Immobilon polyvinylidene fluoride membrane (Millipore,
Bedford, MA) using a semi-dry transfer apparatus (Acrylictech) for 90 min at 200 mA. The membranes were blocked overnight at 4 °C in TBS-T (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05%
Tween 20) containing 5% bovine serum albumin. The membranes were
incubated with primary antibodies at room temperature for 2 h in
TBS-T plus 5% bovine serum albumin, washed in the same buffer without
bovine serum albumin, and incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h. Antibodies were
detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Conjugated horseradish peroxidase-protein A antibodies were used for
anti-IGF-IR, anti-IR, and anti-Csk blotting and horseradish peroxidase
anti-mouse antibodies for 4G10, 2-17, and 12CA5 blotting.
In Vitro Kinase Assay--
c-Src and Csk immunoprecipitates were
washed three times with lysis buffer and once with cold 20 mM PIPES, pH 7.0, 1 mM dithiothreitol. Twenty
µl of kinase buffer (20 mM PIPES, pH 7.0, 10 mM MnCl2, 1 mM dithiothreitol),
containing 20 µCi of [
-32P]ATP (specific activity 1 mCi/mM) and 2.5 µg of acid-denatured enolase (38) or 50 ng of baculovirus-expressed kinase-inactive Src K297R (39), was added
to the beads. After 15 min at 30 °C the reaction was stopped by
adding 2× sample buffer and analyzed by SDS-polyacrylamide gel
electrophoresis. The conditions used in this study are in the linear
range of the assay according to Cooper et al. (38). The
presence of equivalent amounts of enolase was verified by Coomassie
staining of the gel and equivalent amounts of K297R Src by
immunoblotting. Finally, the gel was dried and protein phosphorylation
revealed by autoradiography. Tryptic phosphopeptide mapping and
cyanogen bromide cleavage were done according to Boyle et
al. (40).
Yeast Two-hybrid System--
The yeast expression plasmids
pBTM116 and pLexA-lamin were provided by A. Vojtek (Seattle) (see Ref.
41) and J. Camonis (Paris, France), respectively. The yeast expression
plasmid encoding the Gal4 activation domain (AD) pACTII was from
CLONTECH (Palo Alto, CA). The cytoplasmic domains
of the IR and IGF-IR, subcloned in pBTM116 in frame with the DNA
binding domain of LexA, were described previously (9, 42). Plasmid
construction, cloning, and DNA sequencing were carried out according to
standard protocols (43). When necessary, PCR using thermostable
Pfu polymerase (Stratagene) was performed to generate
adequate cloning sites to allow the in-frame insertion into the
two-hybrid plasmids. The rat IRS-1 cDNA (a gift from J. Pierce,
National Cancer Institute, Bethesda) (residues 5-1235) was cloned in
frame with the Gal4 AD. The full-length human CSK cDNA was
PCR-amplified from pGST-CSK (35) and fused to the Gal4 AD-encoding
sequence in pACTII. The different subdomains of CSK (SH3/2, SH3, and
SH2 constructs) were PCR-amplified from pACTII CSK and fused to pACTII.
All point mutations of different proteins were generated by
site-directed mutagenesis using the TransformerTM Kit
(CLONTECH). Mutations were verified by DNA sequencing.
The two-hybrid system used in this study was described previously (41,
42). The yeast strain L40, which is MATa, trp1, leu2, his3,
LYS2::lexA-HIS3,
URA3::lexA-lacZ, was provided by A. Vojtek (Seattle) and J. Camonis (Paris, France). Growth conditions and
yeast maintenance were as described previously (44). Yeast L40 was
transformed simultaneously with the two indicated plasmids by the
lithium acetate method of Gietz et al. (45). The
transformants were grown on plates lacking tryptophan and leucine to
select for the pBTM116 and pACTII derivatives, respectively. After 3 days at 30 °C, 3 colonies of each transformation were tested for histidine prototrophy after plating on medium lacking tryptophan, leucine, and histidine and for
-galactosidase activity using a
liquid assay, performed essentially as described previously (46). In
brief, 1 ml of yeast cell extract was incubated with the substrate
o-nitrophenyl-
-D-galactopyranoside,
and the increase in A420 was measured after 10 or 30 min (47). Similar results were obtained by analyzing growth on
plates lacking histidine and
-galactosidase activity in a filter
color assay using X-gal as substrate. Results expressed in Tables
I-III represent a summary of the data obtained with these three methods.
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RESULTS |
CSK Associates with the IGF-IR in Vivo--
To examine the role of
c-Src or CSK in IGF-I and insulin signaling, we tested whether c-Src or
CSK associated with the IGF-IR in co-immunoprecipitation studies.
First, using several different conditions, we tried to
co-immunoprecipitate c-Src with the IGF-IR. We were unable to detect
c-Src in IGF-IR immunoprecipitates or the IGF-IR in c-Src
immunoprecipitates (data not shown).
Then, we tested whether CSK could be co-immunoprecipitated with the
IGF-IR. Human 293 cells were starved for 24 h in Dulbecco's modified Eagle's medium supplemented with 0.1% FCS and stimulated with IGF-I. In these experiments we used an analog of IGF-I, LR3-IGF-I, which is not bound and sequestered by IGF-binding proteins (48). We
confirmed that LR3-IGF-I activates the IGF-IR by immunoprecipitating the IGF-IR from stimulated cells and immunoblotting the receptor with
anti-phosphotyrosine antibody (see Fig.
1B). IGF-IR immunoprecipitates were tested for the presence of CSK by immunoblotting with anti-CSK antibodies. As shown in Fig. 1A (upper panel),
CSK was detected in association with the IGF-IR in stimulated cells but
not in unstimulated cells. The amount of CSK bound to the IGF-IR
compared with the total amount present in 293 fibroblasts, estimated
from immunoprecipitation of CSK from the remaining lysates after the IGF-IR immunoprecipitation, represented approximately 5% of total cellular CSK (Fig. 1A, lower panel).

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Fig. 1.
CSK associates with the IGF-IR. 293 cells were serum-starved for 24 h and stimulated with 1.3 × 10 7 M LR3-IGF-I. A, IGF-IR was
immunoprecipitated (IP) from non-transfected cells and CSK
detected by immunoblot (upper panel). After IGF-IR
immunoprecipitation, CSK was immunoprecipitated from the same lysates,
and CSK was detected by immunoblot. B, after various times
of stimulation, the IGF-IR was immunoprecipitated from 293 cells
cotransfected with vectors encoding the IGF-IR and CSK, and CSK
(upper panel), IGF-IR (middle), and
anti-phosphotyrosine (lower) were detected by
immunoblotting. C, immunoprecipitated HA-tagged CSK was
immunoblotted from cotransfected 293 cells with vectors encoding the
IGF-IR and CSK to detect the IGF-IR (upper panel) or the HA
tag (lower panel).
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To study further the association between CSK and the IGF-IR, 293 cells
were cotransfected with IGF-IR and CSK cDNAs, incubated for 24 h, starved for a further 24 h in Dulbecco's modified Eagle's medium supplemented with 0.1% FCS, and stimulated for various times
with IGF-I. As shown in Fig. 1B, CSK was detected in
association with the IGF-IR only in stimulated cells. The association
was rapid, occurring within 1 min of addition of IGF-I. The IGF-IR also
became phosphorylated on tyrosine within 1 min after stimulation with
IGF-I. Similar amounts of IGF-IR were present in immunoprecipitates at
all time points.
To characterize further the CSK-IGF-IR interaction, we tested whether
the IGF-IR could be observed in CSK immunoprecipitates. Since
co-immunoprecipitation of CSK with the IGF-IR was observed only after
IGF-I stimulation (Fig. 1A), suggesting that tyrosine phosphorylation of the IGF-IR was required for IGF-IR-CSK association, we also tested whether the interaction depended on a functional SH2
domain of CSK. Human 293 cells were cotransfected with the IGF-IR and a
vector encoding either wild type CSK or a CSK mutant with a defect in
the FLVRES motif of the SH2 domain, CSK S109C, which fails to bind
phosphotyrosine-containing proteins (35). Both CSK wild type and the
CSK S109C mutant contained a hemagglutinin epitope tag allowing them to
be separated from the endogenous CSK protein during
immunoprecipitation. The transfected cells were incubated, starved, and
stimulated with IGF-I as described above. HA-CSK immunoprecipitates
made with the 12CA5 anti-HA antibody were assayed for the presence of
the IGF-IR by immunoblotting. As shown in Fig. 1C, IGF-IR
was not observed in unstimulated immunoprecipitates. IGF-IR was present
in wild type CSK immunoprecipitates from IGF-I-stimulated cells but not
in CSK immunoprecipitates of the CSK S109C mutant, defective in SH2
domain binding to phosphotyrosine. We conclude that CSK associates
in vivo with the IGF-IR. Furthermore, the interaction
requires a functional SH2 domain of CSK and occurs only after
activation of the receptor by IGF-I.
CSK Associates through Its SH2 Domain with the IGF-IR and the IR in
Yeast--
To characterize further the association between the IGF-IR
and CSK and to compare the IGF-IR and the IR with regard to the sites
required for CSK binding, we used the yeast two-hybrid system of Fields
and Song (49). This system has been used to study the interaction of
the IGF-IR and the IR with their two major substrates, IRS1 and Shc, in
yeast (42). Tyrosine phosphorylation of these receptors in the yeast
two-hybrid system has been demonstrated previously (42). Constructs
encoding the intracytoplasmic domain of the IGF-IR or the IR fused to
the DNA binding domain of LexA, and full-length CSK fused to the
transcriptional activation domain of Gal4 (GAD) were used. Interaction
between the LexA-receptor hybrid and the CSK-GAD hybrid in the yeast
strain stimulates transcription of two reporter genes, HIS3
and lacZ, which are flanked upstream by LexA-binding sites.
The results of cotransfection with various constructs are presented
here as a summary of the growth on plates lacking histidine and
-galactosidase activity obtained from yeast liquid culture or filter
color assay.
We verified that the IGF-IR-LexA or the IR-LexA and the CSK-GAD hybrids
were expressed in yeast by immunoblotting of yeast lysates transfected
with these constructs (Ref. 42 and data not shown). We confirmed that
the constructs did not activate transcription of the reporter genes
alone or in combination with an unrelated protein, lamin, or the kinase
interaction domain, KID, of cyclic AMP response element-binding protein
(Table I). We also confirmed, as an
internal positive control, that the IGF-IR and the IR wild type and
mutants interacted with IRS-1 (Tables I-III). We used wild type
IGF-IR, or a kinase-inactive mutant of the IGF-IR (K1003A), and the
wild type IR, or a kinase-inactive mutant of the IR (K1018A), to test
the interaction with wild type CSK. Table I shows that
-galactosidase activity was stimulated, and growth on plates lacking
histidine was observed when wild type IGF-IR or IR was cotransfected
with wild type CSK, reflecting the interaction of CSK with the
receptors. In contrast, no interaction was observed when the
kinase-inactive mutants of the IGF-IR K1003A and the IR K1018A were
used. The results demonstrate that CSK interacts with the IGF-IR and
the IR in yeast and that the protein-tyrosine kinase activity of the
receptors is required for the interaction.
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Table I
Interaction between CSK and the IGF-IR or the IR
Yeast two-hybrid interaction between various domains of CSK and the
wild type or kinase-deficient IGF-IR and the IR. The results
represent a summary of the data obtained by analyzing growth on plates
lacking histidine and -galactosidase activity in a filter color
assay using X-gal as substrate or in a yeast liquid culture using
o-nitrophenyl- -D-galactopyranoside as
substrate.
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To test the ability of the SH2 and SH3 domains of CSK to bind to the
receptors, we fused the SH2 and SH3 domains, alone or in combination,
to the GAD, and we tested the ability of the hybrid proteins to
interact with wild type or kinase-inactive mutants of the IGF-IR and IR
when coexpressed in yeast. Table I shows that
-galactosidase
activity was stimulated, and growth on plates lacking histidine was
observed when the CSK SH2 or CSK combined SH3 and SH2 (CSK SH3/2)
domains were cotransfected with the wild type IGF-IR and IR receptors,
but not when the kinase-inactive receptors were used. The CSK SH3
domain did not stimulate
-galactosidase activity or growth on
plates lacking histidine when cotransfected with either wild type or
kinase-inactive receptors. These results demonstrate that CSK
associates with tyrosine-phosphorylated IGF-IR or IR through its SH2
domain in yeast.
The SH2 Domain of CSK Associates with Tyr943 and
Tyr1316 of the IGF-IR and Tyr943 of the
IR--
The recognition specificities of the SH2 domain of CSK (50)
suggested that amino acids Thr, Ala, or Ser in position +1 after the
phosphorylated tyrosine and Val, Ile, Met, or Arg in position +3 could
constitute a high affinity binding motif for the SH2 domain of CSK.
Examination of the amino acid sequences of the IGF-IR and the IR
revealed two such motifs in the IGF-IR, Tyr943-Ala-Ser-Val
and Tyr1316-Ala-His-Met, and one in the IR,
Tyr1322-Thr-His-Met. To test the importance of these
tyrosine residues for CSK binding to the receptors, we mutated each
tyrosine to phenylalanine, incorporated the mutated sequences into
vectors containing the LexA DNA binding domain, and tested them for
interaction with CSK in the yeast two-hybrid system. The IGF-IR
mutants, Y943F, Y1316F, and the double mutant, Y943/1316F, interacted
with IRS1 in the two-hybrid system demonstrating that the mutant
receptors had normal tyrosine kinase activity and could interact with
their substrate in this system (Table
II). The mutants were cotransfected with
full-length CSK, the CSK SH2 domain, or the combined CSK SH3 and SH2
domains (Table II). The single mutants of the IGF-IR, Y943F and Y1316F,
stimulated
-galactosidase activity and growth on plates lacking
histidine in the transfected cells. However, when the double mutant,
IGF-IR Y943/1316F, was used,
-galactosidase activity and growth on
plates lacking histidine was not observed. These results confirmed that
the SH2 domain of CSK interacts with phosphorylated Tyr943
and Tyr1316 of the IGF-IR.
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Table II
Phosphotyrosines 943 and 1316 of the IGF-IR binds the CSK SH2
domain
Yeast two-hybrid interaction between CSK and various mutants of the
IGF-IR. The results represent a summary of the data obtained by
analyzing growth on plates lacking histidine and -galactosidase
activity in a filter color assay using X-Gal as substrate or in a yeast
liquid culture using
o-nitrophenyl- -D-galactopyranoside as
substrate. NA, not applicable.
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We performed similar experiments using mutants of the IR, Y960F, Y1316F
and Y1322F, cotransfected with full-length CSK, the CSK SH2 domain, or
the combined CSK SH3 and SH2 domains (Table III). The Y960F (binding site for IRS1)
and Y1316F mutants stimulated
-galactosidase activity and growth on
plates lacking histidine when cotransfected with wild type CSK. The
Y1316F mutant also stimulated
-galactosidase activity and growth on
plates lacking histidine when cotransfected with the SH2 or the SH3/SH2
domains of CSK. However, both a deletion mutant of the IR lacking the 7 amino acids containing the two tyrosines, 1316 and 1322 (42), and the
Y1322F mutant failed to stimulate activity or growth with any of the
CSK constructs. We conclude that phosphorylated Tyr1322 of
the IR is required for interaction with the CSK SH2 domain in
yeast.
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Table III
Phosphotyrosine 1322 of the IR binds the CSK SH2 domain
Yeast two-hybrid interaction between CSK and various mutants of the
IR. The results represent a summary of the the data obtained by
analyzing growth on plates lacking histidine and -galactosidase
activity in a filter color assay using X-Gal as substrate or in a yeast
liquid culture using
o-nitrophenyl- -D-galactopyranoside as
substrate.
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c-Src Kinase Activity Decreases after IGF-I and Insulin
Stimulation--
To determine whether c-Src kinase activity was
modified following IGF-I or insulin stimulation, human 293 or mouse NIH
3T3 fibroblasts (Fig. 2) were treated
with both hormones. c-Src was immunoprecipitated and its activity
assayed in an in vitro kinase assay using enolase as
substrate. Stimulation with either LR3-IGF-I or insulin decreased the
kinase activity of Src by about 40 or 30%, respectively, in human 293 and 50 or 20%, respectively, in NIH 3T3 fibroblasts, as measured by
the amount of 32P incorporated into enolase or into c-Src
itself (Fig. 2). For each reaction, the amount of immunoprecipitated
c-Src was assayed by immunoblotting, and the amount of enolase was
determined by Coomassie staining. The amounts of both Src and enolase
were similar in all samples.

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Fig. 2.
Src kinase activity decreases following
stimulation with IGF-I and insulin. 293 or NIH 3T3 cells were
serum-starved for 24 h and stimulated with IGF-I and insulin. Src
was immunoprecipitated (IP), and its activity was assayed in
an in vitro kinase assay using enolase as a substrate. In
the upper panel, 293 cells were stimulated for 2 min with
1.3 × 10 7 M LR3 IGF1 or 1.7 × 10 7 M insulin. The lower panel is
an immunoblot for Src protein.
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To characterize further the down-regulation of c-Src by IGF-I, human
293 cells were incubated in medium containing 0.1% FCS for 24 h
and then stimulated for various times with LR3-IGF-I at a concentration
of 1.3 × 10
7 M. c-Src enzymatic
activity was tested by in vitro kinase assay as described
above. A maximum 35% decrease in c-Src kinase activity, measured by
32P incorporation into enolase, was observed after 2 min of
IGF-I stimulation and a return to basal activity occurred after about 20 min (Fig. 3A). c-Src
autophosphorylation, as measured by 32P incorporation into
c-Src, showed a maximum decrease of 25% and followed a similar time
course (Fig. 3B).

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Fig. 3.
The IGF-I-induced decrease in the kinase
activity of Src is transient and dose-dependent. 293 cells were serum-starved for 24 h and stimulated with IGF-I.
Results represent mean ±S.E. of five experiments. A, 293 cells were stimulated for various periods with 1.3 × 10 7 M LR3-IGF-I and Src activity was assayed
by -32P incorporation into enolase. B, in the
same experiment as in A, we also determined the
-32P incorporation of Src. C, 293 cells were
stimulated with various amounts of LR3-IGF-I for 2 min. Src activity
was assayed as above. D, in the same experiment
as in C, Src -32P incorporation was assayed.
E, 293 cells were stimulated with various amounts of insulin
for 2 min. Src activity was assayed as above. F,
in the same experiment as in E, Src -32P
incorporation was assayed.
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Human 293 cells were stimulated with increasing concentrations of
LR3-IGF-I ranging from 1.3 × 10
10 to 6.5 × 10
7 M. Fig. 3C shows that the
kinase activity of c-Src, measured by 32P incorporation
into enolase, was decreased to a plateau of about 65% of the untreated
cell level by concentrations of LR3-IGF-I of 1.3 × 10
7 M or higher. c-Src autophosphorylation,
measured by 32P incorporation into c-Src, was decreased to
a plateau of about 75% of the untreated cell level (Fig.
3D). Human 293 cells were also stimulated with insulin under
the same conditions used in Fig. 3, C and D. The
kinase activity of c-Src, measured by 32P incorporation
into enolase, was decreased to about 65% of the untreated cell level
after stimulation with concentrations of insulin of 8.6 × 10
7 M, without reaching a plateau as for
IGF-I stimulation (Fig. 3E). c-Src autophosphorylation,
measured by 32P incorporation into c-Src, was decreased to
a plateau of about 80% of the untreated cell level (Fig.
3F).
From these results, we conclude that stimulation of human 293 or mouse
3T3 fibroblasts by IGF-I or insulin causes a rapid and transient
decrease in the kinase activity of c-Src which is dependent on the
concentration of the hormone.
c-Src Down-regulation after IGF-I Stimulation Requires CSK Binding
to the IGF-IR but Increased CSK Activity Is Not Detected after
Stimulation--
To determine whether c-Src down-regulation induced by
IGF-I was dependent on the interaction between CSK and the IGF-IR, we tested whether disruption of this interaction would affect c-Src down-regulation. Wild type CSK, SH2 mutant S109C of CSK, and the vector
alone as a control were transfected in 293 cells. After 24 h
starvation in 0.1% FCS, cells were stimulated with 1.3 × 10
7 M IGF-I for 2 min, and c-Src was
immunoprecipitated and its activity tested in an in vitro
kinase assay using enolase as substrate. As shown in Fig.
4A (upper panel),
c-Src activity of cells transfected with the vector alone decreased to
82% after stimulation in comparison to unstimulated cells. As
expected, overexpression of wild type CSK further decreased c-Src
activity to 64% after IGF-I stimulation in comparison to unstimulated
cells. However, in 293 cells transfected with the S109C SH2 mutant of
CSK, c-Src activity after stimulation was decreased to 80%, a level
similar to that observed in cells transfected with the vector. Protein
levels of the wild type and SH2 CSK mutant S109C were similar and did
not change after stimulation, as shown by CSK immunoblot (Fig.
4A, lower panel).

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Fig. 4.
CSK interaction with the IGF-IR is required
for c-Src down-regulation following stimulation with IGF-I but its
activity remains unchanged. 293 cells were serum-starved for
24 h and stimulated for 2 min with 1.3 × 10 7
M LR3-IGF-I. A, cells were transfected with
cDNA from HA-tagged either wild type CSK or SH2 domain mutant,
S109C, CSK. c-Src activity was tested in in vitro kinase
assays using enolase as substrate (upper panel). Expression
of the various CSK constructs was detected after anti-HA
immunoprecipitates and anti-HA immunoblot (lower panel).
B, c-Src and CSK immunoprecipitates from three different
experiments were tested for c-Src activity and CSK activity,
respectively, in in vitro kinase assays using enolase or
baculovirus-expressed kinase-inactive c-Src, K295R, as substrates. *,
p < 0.05; **, p = 0.1 in t
test analysis, n = 3.
|
|
Because the kinase activity of c-Src can be decreased by
phosphorylation of Tyr527 by CSK, we also tested whether
CSK activity was changed following stimulation of human 293 cells by
IGF-I. CSK activity was measured by an in vitro kinase assay
of immunoprecipitated CSK using a baculovirus-expressed kinase-inactive
mouse c-Src mutant, K297R, as substrate. c-Src activity was measured as
described above, using the same 293 cell lysates. The results of three
separate experiments, summarized in Fig. 4B, showed
significant decreases of the kinase activity of c-Src, measured by
-32P incorporation into enolase and c-Src. In all of the
samples showing a decrease in c-Src activity, a small but reproducible increase in CSK activity was observed following stimulation with IGF-I,
but this difference was not statistically significant. However, this
experiment, testing the activity of total cellular CSK after IGF-I
stimulation, cannot rule out that CSK activity changes significantly in
the fraction of CSK bound to the IGF-IR, which represents approximately
5% of total cellular CSK (Fig. 1A).
We attempted to examine further the molecular mechanisms involved in
the down-regulation of the kinase activity of c-Src following IGF-I
treatment. In order to determine whether the decrease in c-Src activity
could be attributed to phosphorylation of c-Src Tyr527, we
tried to detect differences in c-Src phosphorylation in vivo following stimulation of 293 cells with IGF-I. Human 293 cells, radiolabeled overnight with [32P]orthophosphate, were
stimulated with IGF-I. Immunoprecipitated c-Src was analyzed by tryptic
phosphopeptide mapping or cyanogen bromide cleavage. No significant
changes were observed in the peptide maps of Src isolated from these
cells after stimulation with IGF-I compared with unstimulated cells
(data not shown).
From these data, we conclude that the association between the activated
IGF-IR and CSK is likely to be required for c-Src activity
down-regulation by IGF-I.
 |
DISCUSSION |
The results presented here show that CSK associates with the
IGF-IR and the IR. For both receptors, the association requires a
functional protein-tyrosine kinase activity and occurs through the SH2
domain of CSK, which binds phosphorylated Tyr943 and
Tyr1316 of the IGF-IR and Tyr1322 of the IR in
a yeast two-hybrid assay. The ligand-stimulated binding of CSK to the
IGF-IR and the IR suggests that CSK participates in IGF-I and insulin
signaling. We observed a concomitant decrease in the kinase activity of
c-Src which was rapid and transient following stimulation of human 293 cells with IGF-I or insulin. Also, CSK interaction with the activated
IGF-IR is likely to be required for c-Src down-regulation, although CSK
activity does not change after hormone stimulation.
It is likely that the interaction of CSK with the receptors observed in
the yeast two-hybrid system is a direct one, although it is possible
that a yeast protein might mediate the interaction. Assuming that the
interaction is direct, CSK is a new binding protein of the IGF-IR and
the IR.
It may be significant that the SH2 domain of CSK interacts with two
tyrosine residues in the IGF-IR, Tyr943 and
Tyr1316, but only one in the IR, Tyr1322. It is
generally accepted that the IR plays a metabolic role in glucose
homeostasis and that the IGF-IR is responsible for cell growth and
proliferation. Therefore, it is possible that the interaction of CSK
with Tyr943 of the IGF-IR might be responsible for
IGF-IR-specific events such as cell proliferation. It is intriguing
that CSK also binds to phosphorylated IRS-1 following insulin
stimulation and is involved in insulin-stimulated dephosphorylation of
focal adhesion proteins, possibly through inactivation of Src family
kinases (8). This observation is in agreement with observations
suggesting that tyrosine phosphorylation of the focal adhesion kinase
is decreased after insulin treatment (8), although focal adhesion
kinase phosphorylation level was increased after IGF-I (34). These differences suggest that focal adhesion kinase tyrosine phosphorylation may not be a direct effect of insulin or IGF-I and may require further investigation.
c-Src catalytic activity is regulated by phosphorylation of two
tyrosine residues (13, 28). Phosphorylation of Tyr416, the
autophosphorylation site, is required for full activation of the kinase
(13, 14). Phosphorylation of Tyr527 by CSK decreases the
kinase activity of Src kinase by allowing intramolecular binding of
Tyr527 to the Src SH2 domain, leading to the inactive
"closed" conformation of c-Src. Howell and Cooper (30) examined the
features of CSK required for suppression of c-Src activity. They found
that mutations in the SH2, SH3, and catalytic domains of CSK rendered
CSK incapable of suppressing c-Src activity. They suggested that
colocalization of CSK with c-Src, rather than a change in CSK activity,
was responsible for regulation of the kinase activity of c-Src by CSK
and that colocalization might be facilitated by binding of CSK to
substrates of c-Src.
In a similar way, binding of CSK to ligand-stimulated IGF-IR and IR
could lead to localization of CSK to the membrane and to subsequent
effects on the tyrosine-protein kinase activity of c-Src. Indeed, when
we used an SH2 domain mutant of CSK, S109C, that does not bind the
activated IGF-IR, we observed that c-Src activity was similar to the
control (Fig. 4A). Our results suggest that the interaction
between CSK and the activated IGF-IR is required for the IGF-I induced
c-Src down-regulation. We also observed that this interaction does not
result in a significant change in the kinase activity of total cellular
CSK, although an increase in activity of the fraction of CSK bound to
the IGF-IR cannot be excluded. Therefore, we suggest that
colocalization of CSK and c-Src following IGF-I treatment is
responsible for the transient decrease in the kinase activity of c-Src
in fibroblasts. The similar timing of the binding and the decrease in
c-Src activity is consistent with this notion. However, since the
interaction between c-Src and CSK is not well understood, more complex
mechanisms for c-Src down-regulation following IGF-I stimulation are possible.
We were unable to observe a significant change in the phosphorylation
pattern of c-Src following stimulation of cells with IGF-I, which might
have implicated CSK further in the down-regulation of c-Src activity.
The lack of an observable change was not unexpected, since c-Src is
already phosphorylated on Tyr527 in unstimulated cells, and
observing a small change in phosphorylation of a specific peptide
following stimulation is difficult. Furthermore, the results from Fig.
1A, showing that only 5% of CSK binds to the IGF-IR,
suggest that it would be very difficult to detect a change in
phosphorylation of a particular peptide, using non-quantitative methods
such as phosphopeptide mapping or cyanogen bromide cleavage. Also, the
absence of an observable increase in kinase activity in CSK may reflect
the fact that only a fraction of the cellular CSK is binding to the
IGF-IR. Indeed, it is possible, according to the observations of Howell
et al. (30), that binding of CSK SH2 domain to the IGF-IR
changes its catalytic activity. In this case, the change in activity
should involve only a fraction of cellular CSK, thereby resulting in
phosphorylation of c-Src Tyr527 in only a fraction of c-Src
molecules. Therefore, observation of a significant increase in CSK
activity or observation of a major decrease in the kinase activity of
c-Src would be unlikely.
In conclusion, we demonstrated that CSK is a new binding protein of the
IGF-IR and the IR, associating with activated IGF-IR and IR through
binding of the SH2 domain of CSK to phosphorylated tyrosines in the
activated receptors. To explore further the role of CSK and Src in
signaling from the IGF-IR and the IR, it will be important to identify
downstream targets that are directly affected by IGF-I or
insulin-induced CSK binding to the IGF-IR or the IR and down-regulation
of c-Src.