(Received for publication, August 18, 1995)
From the
To compare the substrate specificities of the insulin and
insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, the
catalytic domains of the enzymes have been expressed in Escherichia
coli as fusion proteins. The purified proteins have kinase
activity, demonstrating that the catalytic domain of IGF-1 receptor,
like that of insulin receptor, is active independent of its
ligand-binding and transmembrane domains. The specificities of the two
enzymes for the divalent cations Mg and
Mn
are indistinguishable. A series of peptides has
been prepared that reproduces the major phosphorylation sites of
insulin receptor substrate-1, a common substrate for the two receptor
tyrosine kinases in vivo. Insulin and IGF-1 receptors show
distinct preferences for these peptides; whereas insulin receptor
prefers peptides based on Tyr-987 or Tyr-727 of insulin receptor
substrate-1, the IGF-1 receptor preferentially recognizes the Tyr-895
site. The latter site, when phosphorylated, is a binding site for the
SH2 domain-containing adapter protein Grb2. The ability of the two
receptor tyrosine kinases to be phosphorylated and activated by v-Src
has also been examined. The catalytic activity of IGF-1 receptor is
stimulated
3.4-fold by treatment with purified v-Src, while insulin
receptor shows very little effect of Src phosphorylation under these
conditions. This observation is relevant to recent findings of IGF-1
receptor activation in Src-transformed cells, and may represent one
method by which Src amplifies its mitogenic signal. Collectively the
data suggest that the catalytic domains of the two receptor kinases
possess inherently different substrate specificities and signaling
potentials.
The polypeptide hormones insulin and insulin-like growth factor
1 (IGF-1) ()are closely related growth factors that regulate
cell growth and metabolism. The two growth factors produce their
biological effects by binding to distinct receptors on the surface of
target cells. Although insulin and IGF-1 receptors (IR and IGF-1R,
respectively) have some functions in common, recent evidence suggests
that the receptors play significantly different biological roles (for
review, see Kahn(1985), Rechler and Nissey(1990), and Adamo et
al.(1992)). While insulin primarily stimulates physiological
processes such as glucose transport and biosynthesis of glycogen and
fat (Kahn, 1985), IGF-1 has been shown to be more potent in stimulating
cell growth by increasing DNA synthesis, and in promoting cell
differentiation (Lammers et al., 1989; Rechler and Nissey,
1990). In keeping with its mitogenic role, IGF-1R has been shown to be
important in the onset and maintenance of the transformed phenotype in vivo and in vitro (Kaleko et al., 1990;
Baserga, 1995).
The receptors for the two growth factors are highly
homologous. They share the same oligomeric structure: in each case the
receptor is made up of two extracellular subunits containing the
ligand-binding domain and two transmembrane
subunits possessing
tyrosine kinase activity (Yarden and Ullrich, 1988). The tyrosine
kinase catalytic domains of the insulin and IGF-1 receptors, located in
the cytoplasmic portions of the
subunits, possess
84%
sequence identity (Ullrich et al., 1986). The positions of N-linked glycosylation sites and cysteine residues in the
extracellular domains are also highly conserved. Activation of the
receptors is thought to occur in a similar manner. Binding of ligand to
the
subunits activates IR or IGF-1R, leading to
autophosphorylation of tyrosine residues in the
subunits (Yarden
and Ullrich, 1988). Signaling via the IR and IGF-1R has been
demonstrated to be dependent on their tyrosine kinase domains (Ebina et al., 1987; Chou et al., 1987; Kato et
al., 1993), which catalyze the phosphorylation of specific
substrates, including the 185-kDa insulin receptor substrate-1 (IRS-1)
protein (Sun et al., 1991). IRS-1 is a major substrate for
both IR and IGF-1R in vivo (Sun et al., 1991; Myers et al., 1993), and it serves as an intermediate docking
protein, providing binding sites for multiple downstream SH2
domain-containing proteins. For example, after IR and IGF-1R
activation, the tyrosine-phosphorylated form of IRS-1 binds to the
85-kDa regulatory subunit of the phosphatidylinositol 3-kinase, and
this interaction activates phosphatidylinositol 3-kinase (Backer et
al., 1992; Giorgetti et al., 1993; Myers et al.,
1993). In addition to phosphatidylinositol 3-kinase, IRS-1 can also
interact with the growth factor receptor bound-2 (Grb2) protein and the
SH2 containing tyrosine phosphatase Syp, in each case by SH2 domains in
the downstream proteins binding to phosphorylated tyrosine residues
within IRS-1 (Sun et al., 1993). Thus, insulin and IGF-1
appear to activate at least one common signaling pathway through IRS-1
phosphorylation and phosphatidylinositol 3-kinase activation. On the
other hand, because the binding of insulin and IGF-1 to their
respective receptors trigger distinct cellular responses, the signaling
pathways emanating from the receptors are presumably different from
each other, at least in part. At present the molecular basis for these
differences in signal transduction remain unclear, particularly in
light of the structural homology of the receptors. Effects specific for
IGF-1 may be mediated by additional substrates that have not yet been
identified, or by differential phosphorylation of sites on known
substrates such as IRS-1.
Some experimental systems for comparing
the activities of IR and IGF-1R have been described. Using highly
purified preparations of IGF-1R and IR from human placenta, Sahal et al.(1988) demonstrated intrinsic differences in substrate
specificity between the two kinases toward polymeric substrates.
Although (Glu,Tyr
)
and
(Glu
,Ala
,Tyr
)
served as substrates for both tyrosine kinases, IGF-1R
phosphorylated the former polymer approximately four times more
efficiently than the latter polymer. In contrast, insulin receptor
phosphorylated the two polymeric substrates nearly equally. In
addition, insulin receptor was at least 10-fold more sensitive to
inhibition by polymers such as (Tyr,Ala,Glu)
and
(Tyr-Ala-Glu)
than the IGF-1 receptor. The
signaling potentials of insulin and IGF-1 receptors have also been
compared in studies using chimeric receptors. A receptor consisting of
the ligand-binding domain of IR and the cytoplasmic domain of IGF-1R is
10 times more active in stimulating DNA synthesis than the IR itself
(Lammers et al., 1989), suggesting that dissection of the
substrate specificities of IR and IGF-1R is possible. We hypothesize
that a difference in substrate specificity between the two catalytic
domains allows IGF-1R to transmit signals which are distinct from those
mediated by IR. The experiments described in this paper attempt to
identify ``downstream'' events which are specific for IGF-1
through an examination of substrate recognition by the IGF-1 receptor.
The polymerase chain reaction primers had 25 nucleotides of complementarity with the template and encoded unique restriction sites (BamHI at the 5` end and EcoRI at the 3` end). Amplified fragments consisted of the following regions: for the IGF-1R kinase domain, amino acids 956 to 1246; for the IR kinase domain, amino acids 971 to 1262. Polymerase chain reaction products were digested with BamHI and EcoRI, purified from agarose gels, and subcloned into the EcoRI-BamHI sites of the expression vector pGEX 2T. The DNA sequences of the inserted regions were determined using the Sequenase kit (U. S. Biochemical Corp.).
Expression of the GST fusion proteins was
carried out in bacterial strain NB42 as described previously (Smith and
Johnson, 1988; Garcia et al., 1993), with the following
modifications. Overnight cultures (50 ml) of E. coli strain
NB42 harboring each plasmid were inoculated into 1 liter of LB broth
containing ampicillin (50 µg/ml). The cultures were incubated for 2
h at 37 °C, at which time isopropyl
-D-thiogalactopyranoside was added to a final
concentration of 0.2 mM. The cultures were grown for an
additional 5 h at 30 °C. E. coli cells expressing the GST
fusion proteins were lysed in a French pressure cell. After
centrifugation of the cell lysates (13,000
g, 30 min),
cell pellets were resuspended in 1.5% N-lauroylsarcosine
(Sigma), 25 mM triethanolamine, 1 mM EDTA, pH 8.0.
This mixture was rocked at 4 °C for 30 min, then recentrifuged at 4
°C (10,000
g, 10 min). This supernatant was
combined with that from the initial centrifugation and applied to
glutathione-agarose (Molecular Probes, Inc.). After washing with 50
mM HEPES (pH 7.4), 100 mM EDTA, purification of the
IR and IGF-1R constructs was carried out by elution with excess reduced
glutathione (Smith and Johnson, 1988). In both cases, purification of
the fusion proteins on glutathione-agarose yielded proteins which were
>95% pure, as judged by SDS-polyacrylamide gel electrophoresis with
Coomassie Blue staining. Proteins were stored in 40% glycerol at
-20 °C. After purification, tyrosine kinase activity was
demonstrated toward synthetic peptides using the phosphocellulose paper
assay (Casnellie, 1991).
Although the GST-IR and GST-IGF-1R fusion proteins
were isolated in a soluble form, a substantial portion (75%) of the
protein was lost as a pellet in the clarifying centrifugation step
after the bacterial cells were sonicated. This loss might be due to the
insolubility of the expressed protein or to coaggregation with
bacterial membranes. Because of the low yield of soluble fusion protein
(
100 µg/liter of cells), we were unable to carry out the
thrombin cleavage reaction (Smith and Johnson, 1988) to produce the
catalytic domain in sufficient quantities for kinetic analyses.
Consequently, we used the intact GST fusion proteins to test for
phosphorylation of the IRS-1 peptide substrates. Tyrosine kinase
activity was tested using the phosphocellulose paper assay. These
results ( Table 2and Table 3) indicated that insulin and
IGF-1 receptor catalytic domains, expressed in E. coli as
described above, possess functional tyrosine kinase activity. The
specific activities of the fusion proteins were similar to those
reported for other recombinant tyrosine kinase catalytic domains (e.g. Herrera et al., 1988; Morgan et al.,
1991; Garcia et al., 1993), and the specificities appear to be
unaltered, as judged from comparison of the GST-IR protein to a
partially purified preparation of IR holoreceptor toward synthetic
peptides (see below). These observations show that the catalytic domain
of IGF-1R, like that of insulin receptor (Herrera et al.,
1988; Cobb et al., 1989) is active in the absence of its
ligand-binding and transmembrane domains.
Figure 1:
Divalent cation
specificity of insulin and IGF-1 receptor kinases. Enzyme reactions
were carried out in 50 mM Tris-HCl (pH 7.4), and contained 1
mg/ml bovine serum albumin, 200 µM
[-
P]ATP (100-500 cpm/pmol), 1.0
mM Peptide Y727, and the indicated concentrations of
MgCl
and MnCl
. Results are given as mean
± S.D.
As shown in Table 2, all of the IRS-1 peptides served as good substrates for
the catalytic domain of the IGF-1R kinase. Experiments were performed
at saturating concentrations of ATP (200 µM) and
Mg (10 mM) to arrive at values of K
and V
for the peptides.
The IGF-1R catalytic domain exhibited distinct preferences for the
IRS-1 peptides; these preferences tended to be dominated by K
rather than by V
. K
values ranged from a low of 26 µM (Y895) to a high of 249 µM (Y628), whereas V
values fell between 0.5 and 1.6 nmol/min/mg (Table 2). These peptides are among the best reported in
vitro substrates for the IGF-1 receptor tyrosine kinase to date.
In terms of k
/K
, the most
meaningful parameter for substrate specificity comparison (Fersht,
1985), the best substrate was Y895 (Table 2). When
phosphorylated, the site surrounding Y895 in IRS-1 has been shown to be
a binding site for Grb2 (Sun et al., 1993), a small adapter
molecule that contains one SH2 domain and two SH3 domains. The poorest
substrate of this series was Peptide Y987, with k
/K
reduced
14-fold
from Peptide Y895.
With the exception of Y895 and Y1172, all of the
peptides listed in Table 1have been tested as substrates for the
insulin receptor tyrosine kinase (Shoelson et al., 1992).
These results showed that the preferred sequence for phosphorylation
was Y987, although for IR the peptides were all excellent substrates
and only displayed a 2-fold range of k/K
(Shoelson et
al., 1992). Peptides Y895, Y987, and Y1172 were therefore compared
as substrates for the catalytic domain of the IR (Table 3).
Peptide Y987 had the lowest value of K
(30
µM) and the highest value of k
/K
(2.6
10
M
min
) of
these three peptides. These results contrasted with those for IGF-1R (Table 2), in which Y895 was preferred over Y987 and Y1172. The
results obtained for IGF-1R and IR catalytic domains, along with those
obtained previously for IR, are compared directly in terms of relative k
/K
in Fig. 2. It
can be seen from these data that the IGF-1R has a stricter substrate
specificity for the IRS-1 phosphorylation sites, with Y895 being the
best and Y628, Y987, and Y1172 being more poorly recognized. In
contrast, the insulin receptor kinase preferentially phosphorylates
peptides corresponding to IRS-1 sites Y727 and Y987. These differences
in YMXM substrate specificity may also represent a difference
in signaling potential between IR and IGF-1R.
Figure 2:
Phosphorylation of IRS-1 peptides by IGF-1
and insulin receptors. In each case k/K
values are
normalized to 1.0 for the best substrate (for IGF-1R (&cjs2113;),
Peptide Y895; for IR (&cjs2110;), Peptide Y987). For IGF-1R, k
/K
values are
taken from Table 2. For insulin receptor, k
/K
values for
Peptides Y895, Y987, and Y1172 are from Table 3. Values of k
/K
for the
remaining peptides with insulin receptor are taken from Shoelson et
al.(1992).
Figure 3: Activation of IGF-1R and insulin receptors by v-Src. GST fusions of receptor tyrosine kinases were incubated in the presence or absence of purified v-Src as indicated. After reisolating the receptor kinases, their activities were measured toward Peptide Y727 using the phosphocellulose paper assay. In some experiments Yersinia tyrosine phosphatase (PTPase) was added together with v-Src. The amount of Src kinase activity which nonspecifically associates with the glutathione-agarose beads is shown in the right-hand panel of the figure. Assays were carried out in triplicate and are given as mean ± S.D.
To confirm that activation of IGF-1R in the presence of Src depended on the tyrosine phosphorylation of the receptor, the reactions were also carried out in the presence of the Yersinia tyrosine phosphatase. After exposure to Src and the Yersinia phosphatase, IGF-1R and IR were isolated by adsorption onto glutathione-agarose and tested for tyrosine kinase activity toward the synthetic peptide. As shown in Fig. 3, treatment with the tyrosine-specific phosphatase reversed the activation by Src, indicating that the increased IGF-1R activity was due to increased tyrosine phosphorylation of the receptor. Thus, in an experimental system consisting of only the catalytic domains of the receptor tyrosine kinases, the ability of v-Src to activate IGF-1R was reproduced. The degree of activation seen here correlates well with the ligand-independent increase in kinase activity measured when IGF-1R was immunoprecipitated from Src-expressing cells (Peterson et al., 1994).
The insulin and IGF-1 receptors exhibit a large degree of
similarity, both with respect to their enzymatic properties and to
their structural organization. On the other hand, the biological
responses elicited by the two receptors are different, suggesting that
specific signaling pathways must exist. The IGF-1 receptor appears to
be intrinsically more effective at stimulating DNA synthesis than the
insulin receptor (Kahn, 1985; Rechler and Nissey, 1990; Adamo et
al., 1992). Of particular interest are recent results which show
that IGF-1 receptor is required for the establishment and maintenance
of the transformed phenotype in vivo and in vitro.
For example, when overexpressed in NIH3T3 cells, the IGF-1 receptor
causes uncontrolled cell growth and neoplastic transformation (Kaleko et al., 1990). Identification of specific components of the
signaling pathways would provide a major step forward in the
understanding of IGF-I and insulin action. The experiments described
here approach this problem by focusing on the in vitro tyrosine kinase activity of the isolated catalytic domains. The
substrate specificities of the bacterial fusion proteins used in this
study appear to be similar to those reported for intact receptors; for
example, K values for phosphorylation of several
of the YMXM-containing substrates are nearly identical to
those reported for a partially purified preparation of insulin receptor
(Shoelson et al., 1992) ( Table 3and data not shown).
One signaling element that is common to both insulin and IGF-1 receptor pathways is the 185-kDa protein IRS-1. This protein becomes tyrosine phosphorylated in response to insulin or IGF-1 treatment, and subsequently associates with the phosphatidylinositol 3`-kinase and other SH2-containing proteins (Sun et al., 1993). Because of the large number of potential sites for tyrosine phosphorylation in IRS-1 (Sun et al., 1991), the possibility exists that different sites (or combinations of sites) are recognized and phosphorylated by the insulin and IGF-1 receptors, giving rise to different docking sites for downstream signaling molecules containing SH2 domains. Additionally or alternatively, individual SH2 domain-containing proteins might be expressed preferentially with one or the other receptor tyrosine kinase.
To test whether the sites of
IRS-1 are recognized differentially by the two enzymes, we have tested
a series of synthetic peptides which reproduce the major tyrosine
phosphorylation sites on IRS-1. Interestingly, we observe differences
in substrate specificity between the two enzymes in these studies ( Table 2and Table 3; summarized in Fig. 2). Whereas
the insulin receptor displays a rather broad specificity, with Peptides
Y727 and Y987 being the best substrates, IGF-1 receptor has a more
restricted specificity and prefers Peptide Y895. While we cannot
identify the precise amino acid determinants for phosphorylation by the
two enzymes, it is interesting to note that substitutions at either
methionine residue in YMXM motifs invariably leads to
decreased catalytic efficiency for the insulin receptor, suggesting
that the Met and Met
residues play
important roles in enzyme-substrate recognition (Shoelson et
al., 1992). The IGF-1 receptor kinase appears to be less dependent
on C-terminal methionine residues; Peptide Y895, which contains the
sequence Tyr-Val-Asn-Ile, is the preferred site. These experiments
demonstrate for the first time distinctions between the substrate
specificities of the insulin and IGF-1 receptors using amino acid
sequences that are physiologically relevant. The results are consistent
with earlier studies on random copolymers of amino acids, which also
showed different preferences for the two receptor kinases (Sahal et
al., 1988).
These distinctions in specificity toward IRS-1
peptides in vitro may parallel a difference in signaling by
IGF-1 and insulin receptors through IRS-1. Recently it has been shown
that a 262-amino acid portion of IRS-1 (residues 516-777), which
contains five potential YMXM or YXXM phosphorylation
sites, is recognized similarly by insulin and IGF-1 receptors (K = 6.8 and 9.9 µM,
respectively) (Siemeister et al., 1995). However, Tyr-895,
which lies outside of this region of IRS-1, may be a determinant for
downstream signaling that is favored by IGF-1 receptor. After
phosphorylation, the Tyr-895 site in IRS-1 constitutes a binding site
for the Grb2 adapter protein (Sun et al., 1993). Genetic and
biochemical evidence suggests that Grb2 is an upstream regulator of the
GTP exchange protein mSOS, which stimulates the formation of an active
p21
-GTP complex (Egan et al., 1993; Gale et
al., 1993; Olivier et al., 1993; Rozakis-Adcock et
al., 1993). Thus, promotion of stable binding between IRS-1 and
Grb2 may play a role in the mitogenic signaling of IGF-1. On the other
hand, alternate pathways to IRS-1 may exist for insulin or IGF-1
signaling, as suggested by experiments with transgenic IRS-1 knockout
mice (Araki et al., 1994; Tamemoto et al., 1994). In
the case of insulin receptor, it has recently been shown that Grb2
associates rapidly and transiently with IRS-1 after insulin treatment,
but that Shc plays a more important role than IRS-1 in the binding of
Grb2 and formation of p21
-GTP (Sasaoka et al.,
1994).
We also show that the insulin and IGF-1 receptors differ with
regard to their potential for activation by v-Src (Fig. 3). It
has been shown recently that the IGF-1 receptor becomes tyrosine
phosphorylated in cells expressing v-Src, and that the in vivo increase in phosphorylation parallels an increased in vitro tyrosine kinase activity of the IGF-1 receptor (Peterson et
al., 1994). In these studies it was not shown whether IGF-1
receptor acted as a direct substrate for v-Src in vitro, or
where on the IGF-1 receptor the site(s) for Src phosphorylation
occurred. Under the in vitro conditions reported here, the
kinase activity of the IGF-1 receptor catalytic domain was stimulated
approximately 3.4-fold by v-Src, whereas the activity of insulin
receptor showed only a modest increase after treatment with v-Src under
identical conditions. Activation was due to increased tyrosine
phosphorylation, as demonstrated by reversal of the effect by a
tyrosine-specific phosphatase. In these experiments Src may catalyze
the direct phosphorylation of IGF-1R catalytic domain, or act
indirectly, by promoting receptor autophosphorylation. We favor the
former explanation since it has been shown that v-Src can cause
tyrosine phosphorylation of an inactive mutant of the IGF-1 receptor in vivo (Peterson et al., 1994). Our in vitro experiments indicate that the IGF-1 receptor has the potential to
act as a direct substrate for v-Src, and that at least one target for
Src phosphorylation may be present in the catalytic domain of IGF-1R
itself. The level of activation observed (3.4-fold) is similar to
the level of increased IGF-1 receptor kinase activity seen in vivo (
4-fold). Activation of IGF-1 receptor (or other receptor
tyrosine kinases) could be a regulatory mechanism by which v-Src
amplifies its signaling potential. In the case of IGF-1 receptor, this
may be particularly important because of the role of the receptor in
stimulating mitogenesis and because of its oncogenic potential (Kaleko et al., 1990). Overexpression of IGF-1 receptor in NIH3T3
cells leads to ligand-dependent morphological transformation and colony
growth in soft agar, and cells overexpressing IGF-1R cause the
formation of tumors when introduced into nude mice (Kaleko et
al., 1990).
In conclusion, we have demonstrated distinctions
between the tyrosine kinase catalytic domains of the IGF-1 receptor and
insulin receptor. Studies with chimeric receptors indicate that the
biological specificity of these two polypeptide hormones can be
attributed to the cytoplasmic portions of their subunits. For
example, a chimeric receptor composed of the extracellular domain of
the insulin receptor and the cytoplasmic portion of the IGF-1 receptor
behaves similarly to the wild-type IGF-1 receptor (Lammers et
al., 1989). This implies that a detailed knowledge of receptor
kinase substrate specificity may shed light on the different downstream
signaling pathways triggered by insulin and IGF-1.