(Received for publication, June 13, 1994; and in revised form, January 4, 1995)
From the
Insulin receptor substrate-1 (IRS-1) is a major endogenous
substrate of the insulin receptor. To study the interaction of the
insulin receptor with IRS-1 in vitro, we expressed in Escherichia coli the amino acids 516-777 of human IRS-1
(hIRS-p30) covering five potential tyrosine phosphorylation sites
within YXXM motifs. Kinetic data for tyrosine phosphorylation
of hIRS-p30 by partially purified insulin receptor and insulin-like
growth factor I receptor and by baculovirus-expressed insulin receptor
kinase domain were determined. Native insulin receptor demonstrated the
highest affinity to hIRS-p30 (K =
6.8 ± 0.6 µM), followed by the insulin-like growth
factor I receptor (K
= 9.9
± 1.0 µM). We used the soluble recombinant insulin
receptor kinase domain, which phosphorylated hIRS-p30 with high
affinity (K
= 11.9 ± 0.8
µM), and affinity columns prepared by coupling hIRS-p30 to
NHS-activated Sepharose for binding assays. The insulin receptor kinase
domain phosphorylated the hIRS-p30 on the column, was bound by the
immobilized hIRS-p30, and was eluted with high salt buffer.
Autophosphorylated and EDTA-inactivated insulin receptor kinase domain
was bound only by immobilized hIRS-p30 protein that had been
prephosphorylated. Our results indicate that the recombinant hIRS-p30
protein is a high affinity substrate for insulin receptor and
insulin-like growth factor I receptor in vitro. Moreover, we
show that only tyrosine-phosphorylated hIRS-p30 is able to bind to the
insulin receptor.
Regulation of cellular metabolism and growth by insulin involves
binding of the hormone to its receptor leading to activation of the
intracellular receptor tyrosine kinase(1) . Autophosphorylation
of the receptor -subunit and phosphorylation of endogenous
substrates are the next steps occurring in the insulin signaling
system. Phosphoproteins of 160-185 kDa (pp185) were discovered as
major substrates of the insulin receptor(2, 3) . A
component of pp185, termed insulin receptor substrate-1 (IRS-1), (
)has been purified recently, and its cDNA sequence has been
determined(4, 5) . This protein possesses numerous
potential tyrosine phosphorylation sites including six YMXM
and three YXXM motifs, which are considered to associate with
Src homology-2 domains in the regulatory 85-kDa subunit of
phosphatidylinositol 3-kinase(6, 7) , in the adaptor
protein Grb-2 linking the guanine nucleotide exchange factor for
p21
(8, 9) , in the protein
tyrosine phosphatase 1D(10) , and in the adaptor protein
Nck(11) . Thus tyrosine-phosphorylated IRS-1 serves as a
multi-site docking protein for at least four Src homology-2 domain
proteins involved in signal transduction. IRS-1 is also a substrate of
insulin-like growth factor I (IGF-I) receptor, which is homologous to
the insulin receptor, linking IGF-I stimulation to activation of
phosphatidylinositol 3-kinase(12) . Signaling via a docking
protein distinguishes the insulin receptor and the IGF-I receptor from
other receptor tyrosine kinases, which interact directly with Src
homology-2 domain proteins. For review see (13, 14, 15, 16) .
Although
mutation of Tyr to Phe within the juxtamembrane domain of
the human insulin receptor does not affect the kinase activity of the
receptor toward a peptide substrate, the mutant was not biologically
active in Chinese hamster ovary cells. In particular, the mutation is
associated with impaired tyrosine phosphorylation of IRS-1 and impaired
activation of phosphatidylinositol 3-kinase and of mitogen-activated
protein kinases, suggesting that this region is involved in substrate
recognition(17, 18) . Complexes of insulin receptor
and IRS-1 are immunoprecipitable from insulin-stimulated cells but not
from unstimulated cells, suggesting that IRS-1 is partially bound by
the receptor in vivo(19) . We have started to look for
the domains within IRS-1 that are recognized by the insulin receptor
for binding of the protein.
In this paper, we show that a bacterial expressed 262-amino acid IRS-1 region covering a cluster of five tyrosine phosphorylation sites within YXXM motifs is an excellent substrate of the insulin receptor and of the IGF-I receptor in vitro. After tyrosine phosphorylation this IRS-1 domain is able to bind the insulin receptor. This study shows directly that the binding of the receptor is independent from an active receptor kinase.
The bacterial
expression vector pET-3d ((24) ; AGS Heidelberg) was linearized
with NcoI and BamHI. After generation of blunt ends
using Klenow polymerase (Boehringer Mannheim) and dephosphorylation by
calf intestinal alkaline phosphatase (Boehringer Mannheim), the plasmid
was gel-purified. A 786-base pair BglII-SmaI
subfragment of the 4.1-kilobase pair BamHI fragment of the
human IRS-1 gene locus (Fig. 1A) encoding the amino
acid sequence 516-777 of the human IRS-1 protein (5) was
gel-purified after filling in the BglII end by Klenow
polymerase. The IRS-1 fragment was ligated into the pET vector to
generate the expression plasmid hIRS-p30 and transformed into Escherichia coli strain DH5. The structure of constructs
with sense orientation of the inserted fragment relative to the T7 RNA
polymerase promoter of the vector was determined by restriction with XbaI and RsaI simultaneously and confirmed by
nucleotide sequence analysis.
Figure 1: Cloning strategy for the construction of hIRS-p30 expression plasmid and purification of the recombinant protein. A, the genetic map of 4.1- and 5.7-kilobase pair BamHI fragments of the human genomic IRS-1 gene locus containing the entire IRS-1 coding region (filledbox) is shown. The 786-base pair BglII-SmaI (bold) restriction fragment was used for the construction of hIRS-p30 expression plasmid (only the SmaI site used for the construction is shown). B, the BglII-SmaI restriction fragment was inserted by blunt-end ligation into the NcoI-BamHI linearized pET-3d vector. The nucleotide and deduced amino acid sequences around the insertion sites are depicted. Positions of restriction enzyme sites used for the construction and the Shine-Dalgarno (SD) sequence of the vector are shown. The restriction enzyme sites which were lost during the procedure are in italics. Amino acid numbering refers to the recombinant protein. The numbers given in brackets refer to the entire human IRS-1 protein(5) . C, SDS-PAGE analysis of hIRS-p30 bacterial expression and purification. Aliquots from the purification steps were analyzed by Coomassie Blue staining after 12% SDS-PAGE. Lane1, E. coli lysate before induction of pET-vector driven protein synthesis; lane2, E. coli lysate 3 h after induction of hIRS-p30 synthesis; lane3, crude fraction of soluble proteins; lane4, pooled peak fractions of HiTrap SP chromatography; lane5, pooled peak fractions of Mono-Q chromatography.
The insulin receptor kinase domain (IRKD) containing the human insulin receptor amino acid sequence 947-1343 (28) was expressed and purified from a baculovirus expression system(29) . For substrate phosphorylation assays and hIRS-p30 affinity chromatography assays, we used IRKD preparations, which were purified to 25-30% by DEAE-cellulose chromatography.
IRKD was autophosphorylated for 10 min at 22 °C in
kinase buffer B (30 mM Tris/HCl, 100 mM NaCl, 5
mM MgCl, 5 mM MnCl
, 1 mM DTT, 0.25 mM PMSF, 100 µM ATP, and 0.1
mCi/ml [
-
P]ATP, pH 7.4) supplemented with 1
µM poly(lysine). Aliquots were diluted to 1 ml of kinase
buffer B supplemented with 0.5 mM ATP to allow substrate
phosphorylation or to 1 ml of kinase buffer B supplemented with 20
mM EDTA (EDTA buffer) to inhibit any phosphorylation activity,
and applied to hIRS-p30 columns equilibrated with kinase buffer B
supplemented with ATP, or with EDTA buffer. IRKD was incubated for 10
min at 22 °C on the columns and than washed with 6 ml of washing
buffer (30 mM Tris/HCl, 100 mM NaCl, pH 7.4). The
first 2 ml of the flow-through were collected, and the protein was
trichloroacetic acid-precipitated. The columns were eluted either with
2 ml of elution buffer (30 mM Tris/HCl, 1 M NaCl, pH
7.4) or with a step gradient of 150-500 mM NaCl, each
step having a volume of 2 ml and an increase of 50 mM NaCl.
Eluted proteins were trichloroacetic acid-precipitated, washed with 96%
ethanol, dissolved in 1
sample buffer, and analyzed by SDS-PAGE
followed by autoradiography.
For competition binding experiments
IRKD (2 µM) was autophosphorylated in kinase buffer B at
22 °C for 10 min either with 4 mCi/ml
[-
P]ATP or without radioactivity. Kinase
reactions were stopped by the addition of EDTA buffer to achieve
concentrations of 0.1 pM [
P]IRKD
(tracer) and 0.2 µM P-IRKD. Aliquots of 0.1 pmol of
[
P]IRKD were supplemented with P-IRKD to achieve
final concentrations of autophosphorylated IRKD in the range of 0.1
µM to 0.1 pM. IRKD was applied in EDTA buffer to
prephosphorylated hIRS-p30 on columns, and the binding was analyzed as
described in the preceding paragraph.
A BglII-SmaI restriction fragment was inserted into the
pET3d vector to overexpress the amino acids 516-777 of the human
IRS-1 protein (hIRS-p30) in E. coli using the T7 RNA
polymerase-driven pET expression system (24) (Fig. 1B). Purification of the recombinant
protein from the soluble fraction of the bacterial extract was achieved
by sequential chromatography on SP-Sepharose and on Mono-Q with an
overall yield of 20-25 mg/liter bacterial culture. The purity of
hIRS-p30 exceeded 90% based upon evaluation of Coomassie-stained gels (Fig. 1C). Thus hIRS-p30 covers a domain of 262 amino
acids of the IRS-1 protein containing a cluster of five of the overall
nine potential tyrosine phosphorylation sites within YMXM or
YXXM consensus motifs. Recently, one of these sites within
hIRS-p30, Tyr, corresponding to Tyr
of the
human sequence, had been identified as a recognition site for the SH2
domain of p85 subunit of the phosphatidylinositiol
3`-kinase(32) . In addition the hIRS-p30 expression plasmid
encodes besides the start codon carboxyl-terminal 20 amino acids from
vector sequence without a termination codon (Fig. 1B).
Based on the structure of the hIRS-p30 expression plasmid, a molecular
mass of 30.3 kDa was calculated, although the apparent size was 38 kDa
as determined by SDS-PAGE. This abnormal behavior on SDS-PAGE is also
reflected by the native IRS-1 protein with an apparent molecular mass
of 165-185 kDa versus 132 kDa, which was calculated by
evaluation of the cDNA sequence, and it has been postulated to be a
result of serine/threonine phosphorylation(5) .
For
substrate phosphorylation of hIRS-p30 by WGA-purified insulin receptors
or IGF-I receptors or by baculovirusexpressed IRKD, saturable
Michaelis-Menten type kinetics were observed which yielded linear
Lineweaver-Burk blots (Fig. 2). Phosphorylation reactions
remained linear for at least 5 min (data not shown), validating the
conditions used for determination of K values. The
protein concentrations required for half-maximal saturation (K
) were in the range of 6-12
µM. WGA-purified insulin receptor demonstrated the highest
affinity to hIRS-p30, followed by the IGF-I receptor. Kinetic constants
had been determined by the use of peptides with YXXM and
YMXM motifs of IRS-1(35) , and K
values of 24 µM to 300 µM were
reported. Tyrosine phosphorylation revealed hIRS-p30 as a high affinity
substrate of the insulin receptor with a K
value
that is at least 4 times lower than the values determined using IRS-1
peptides, although hIRS-p30 covers five of the peptides tested
including the one with the lowest K
. These data
might be a result of complex protein-protein interactions of IRS-1 and
insulin receptor, which are more closely resembled by hIRS-p30 protein
than by peptides. Interestingly, the affinity of IGF-I receptors for
hIRS-p30 phosphorylation is only about 40% lower as compared to insulin
receptors, supporting that IGF-I receptors use signaling via IRS-1 with
high efficiency as was shown for stimulation of phosphatidylinositiol
3`-kinase by insulin and IGF-I(12) . Recombinant IRKD still
phosphorylated hIRS-p30 with high affinity, although the K
value was 2-fold higher as compared to native
receptor. Thus the recombinant hIRS-p30 and the soluble recombinant
intracellular kinase domain of the insulin receptor, which in addition
to the kinase covers the juxtamembrane domain and the C-terminal
region, proved to be a good model system to study the interaction of
IRS-1 and insulin receptor in vitro.
Figure 2:
Insulin receptor-, IGF-I receptor-, and
IRKD-catalyzed phosphorylation of hIRS-p30. Substrate (hIRS-p30)
phosphorylations were catalyzed by WGA-purified insulin receptor (A), WGA-purified IGF-I receptor (B), and
baculovirus-expressed insulin receptor kinase domain (C). Vversus substrate concentration and
double-reciprocal plots are shown. Mean values ± S.D. of four
independent experiments were depicted in the plots. Methods used for
phosphorylation reactions and determination of Kvalues are described under
``Experimental
Procedures.''
Figure 3: Affinity chromatography of IRKD on immobilized hIRS-p30. Aliquots of purified hIRS-p30 protein were coupled to NHS-activated Sepharose columns. IRKD was autophosphorylated and incubated on the columns with non-phosphorylated hIRS-p30 in kinase buffer (lanes1 and 2), with prephosphorylated hIRS-p30 in EDTA buffer (lanes3 and 4), and with non-phosphorylated hIRS-p30 in EDTA buffer (lanes 5 and 6) in the presence of 0.1 M NaCl. IRKD protein in flow-through fractions (lanes1, 3, and 5) and in fractions of high salt (1 M NaCl) elution (lanes2, 4, and 6) was analyzed by 12% SDS-PAGE and autoradiography. For control autophosphorylated IRKD (lane7) and hIRS-p30 phosphorylated by IRKD (lane8) were coelectrophoresed.
Figure 4:
Specific binding of P-labeled
IRKD to immobilized hIRS-p30. IRKD was
P-labeled by
autophosphorylation. Binding of [
P]IRKD to
prephosphorylated hIRS-p30 on the column was studied in EDTA buffer in
the presence of the indicated concentrations of autophosphorylated
IRKD, which had not been labeled. Mean values ± S.D. of three
independent experiments are depicted.
The results of the IRKD affinity chromatography on hIRS-p30 columns demonstrate the ability of hIRS-p30 to bind to insulin receptor. The kinase activity of the receptor is not necessary for substrate binding, suggesting that kinase activity and binding of IRS-1 are independent functions within the receptor. Furthermore, the retardation assays revealed tyrosine phosphorylation of hIRS-p30 as an essential prerequisite for high affinity binding to the receptor. This is in agreement with the observation that in Chinese hamster ovary cells overexpressing insulin receptor and IRS-1 both proteins coimmunoprecipitate after insulin treatment, but none does from untreated cells(19) . Tyrosine-phosphorylated IRS-1 is only partially associated with insulin receptors after insulin stimulation of the cells (19) indicating a rapid exchange of IRS-1. This is consistent with our observation of moderate salt concentrations necessary to elute IRKD from the hIRS-p30 columns in the retardation experiments.
Addendum-After submission of this manuscript, O'Neill et al.(36) reported on the characterization of the interaction between the insulin receptor and IRS-1 using the yeast two-hybrid system. They found a 356-amino acid region encompassed by amino acids 160-516 of IRS-1 to be sufficient for interaction with the active insulin receptor. In contrast, IRS-1 constructs containing amino acids 516-865 or 516-1242 failed to interact with the insulin receptor in the yeast system. Using a COS-cell expressing system, they showed that the insulin receptor was unable to phosphorylate an IRS-1 protein from which the amino acids 45-516 had been deleted.
Our results revealed hIRS-p30, which contains the IRS-1 amino acids 516 to 777, as an excellent substrate of the receptor in vitro. In contrast to non-phosphorylated hIRS-p30, which does not bind to the receptor, phosphorylated hIRS-p30 is able to bind to the receptor with high affinity. Taking the findings of O'Neill et al. and our results together, we suppose at least two different domains within IRS-1 that are able to interact with the insulin receptor. One domain, contained within amino acids 45-516, promotes the binding of non-phosphorylated IRS-1 to the receptor resulting in IRS-1 phosphorylation. A second domain, located between amino acids 517 and 777, gets phosphorylated and binds to the receptor, presumably to enable a stable interaction of the two proteins.