©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Human Insulin Receptor Substrate-1 Protein
TYROSINE PHOSPHORYLATION AND IN VITRO BINDING OF INSULIN RECEPTOR KINASE (*)

(Received for publication, June 13, 1994; and in revised form, January 4, 1995)

Gerhard Siemeister (1)(§) Hadi Al-Hasani Helmut W. Klein Silvia Kellner (1) Rüdiger Streicher (1) Wilhelm Krone (1) Dirk Müller-Wieland (1)(¶)

From the From Klinik II und Poliklinik für Innere Medizin, University of Cologne, D-50924 Cologne, Diabetes-Forschungsinstitut, University of Düsseldorf, D-40225 Düsseldorf, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 beta-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), (^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.


EXPERIMENTAL PROCEDURES

Construction of a hIRS-p30 Expression Plasmid

Generation of a rat IRS-1 cDNA fragment containing nucleotides 586-1149 (4) from RNA isolated from rat liver (20) was performed by reverse transcription using murine leukemia virus reverse transcriptase (Pharmacia Biotech Inc.) and subsequent amplification by polymerase chain reaction using (21) Taq polymerase (Boehringer Mannheim) and oligonucleotide primers synthesized on a Pharmacia Gene Assembler (5`-primer, AGCATGGCGAGCCCTCCGGATACCGAT; 3`-primer, GAGCTTCACAAAGCTGATGGTCTTGCTG). Recombinant DNA techniques and screening of a human genomic library (Stratagene 946203) with P-labeled DNA probes were performed using standard procedures(22) . Identity of the rat IRS-1 fragment and of the human IRS-1 gene locus were verified by restriction endonuclease analyses and nucleotide sequencing(23) .

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 DH5alpha. 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.



Bacterial Expression and Purification of hIRS-p30 Protein

For bacterial expression, the plasmid construct was transformed into E. coli strain BL21(DE3) carrying an inducible T7 RNA polymerase gene ((24) ; AGS Heidelberg). 2 ml of an overnight culture of transformed BL21(DE3) bacteria grown at 37 °C in 10 ml of LB medium containing 100 µg/ml ampicillin were used to inoculate 200 ml of LB medium containing 100 µg/ml ampicillin. The culture was grown in shaking flasks at 37 °C to an A of 0.8. Isopropyl-beta-D-thiogalactoside was added to a final concentration of 0.4 mM, and the culture was grown for another 3 h. Bacterial cells were harvested by centrifugation for 10 min at 5,000 rpm (Sorvall SS-34 rotor) and 4 °C. The pellet was resuspended in 20 ml of TBS buffer (20 mM Tris/HCl, 150 mM NaCl, 1 mM PMSF, 100 KIU/ml aprotinin, pH 7.5) and centrifuged again for 10 min at 5,000 rpm and 4 °C. The pellet was frozen at -80 °C, thawed at 37 °C, resuspended in 20 ml of buffer A (50 mM Tris/HCl, 10 mM 2-mercaptoethanol, 2 mM EDTA, 5% (v/v) glycerol, 1 mM PMSF, 100 KIU/ml aprotinin, 0.2 mg/ml lysozyme, 10 µg/ml DNase I, pH 8.0), and incubated for 30 min at 22 °C. The suspension was sheared by five high speed treatments of 20 s in an Ultra-Turrax dispersing apparatus and incubated for 10 min at 22 °C. The mixture was cooled on ice and sonicated six times for 15 s with the microtip of a MSE sonifier. After the addition of sodium deoxycholate and Nonidet P-40 to a final concentration of 0.05% and 1% (w/v), respectively, the mixture was incubated for 10 min at 4 °C and than centrifuged at 10,000 rpm (Sorvall SS-34 rotor) and 4 °C for 45 min. The supernatant containing the recombinant hIRS-p30 protein was dialyzed three times against 100 ml of buffer SP-A (50 mM MES, 1 mM DTT, 0.25 mM PMSF, 1 KIU/ml aprotinin, pH 5.5) for 30 min at 4 °C. The solution was clarified by centrifugation for 10 min at 4 °C and 14,000 rpm in an Eppendorf centrifuge. Fractions of 2 ml of the solution were applied using a fast performance liquid chromatography apparatus (Pharmacia) at a flow rate of 0.5 ml/min to a HiTrap SP Sepharose cation exchange column (Pharmacia) equilibrated with buffer SP-A. After washing the column with 8 ml of buffer SP-A and with 10 ml of buffer SP-A supplemented with 300 mM NaCl (SP-A/300 mM NaCl), the recombinant protein was eluted in 0.5-ml fractions by a linear step gradient of 1 ml to buffer SP-A/400 mM NaCl followed by 4 ml of buffer SP-A/400 mM NaCl. The three peak fractions of each chromatography were collected and dialyzed three times at 22 °C against 100 ml of a buffer Q-A (50 mM Tris/HCl, 1 mM DTT, 0.25 mM PMSF, 1 KIU/ml aprotinin, pH 9.0) supplemented with DTT to a final concentration of 5 mM. The solution was applied at a flow rate of 1 ml/min to a Mono-Q HR5/5 anion exchange column (Pharmacia) equilibrated with buffer Q-A/20 mM NaCl. The column was washed with 10 ml of buffer Q-A/20 mM NaCl followed by a linear gradient of 5 ml to 150 mM NaCl. The hIRS-p30 protein was eluted at a flow rate of 0.5 ml/min in 0.25 ml fractions at 150-170 mM NaCl during a linear gradient of 8 ml to 200 mM NaCl. The nine peak fractions containing the purified hIRS-p30 protein were pooled, dialyzed three times at 4 °C against 100 ml of buffer B (50 mM HEPES/NaOH, 150 mM NaCl, 1 mM DTT, 0.25 mM PMSF, 1 KIU/ml aprotinin) for substrate phosphorylation assays or dialyzed against coupling buffer (see below) for the preparation of hIRS-p30 affinity columns, and stored at -20 °C. Protein concentrations were determined by the method of Bradford(25) .

Insulin and IGF-I Receptor Kinase

Human hepatoma cells HepG2 (26) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). For receptor preparation HepG2 cells from 10 15-cm dishes (80% confluent) were incubated for 24 h in medium supplemented with 0.5% serum. WGA-purified insulin receptor and IGF-I receptor were prepared essentially as described in (27) .

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.

Substrate Phosphorylation Assay

For hIRS-p30 phosphorylation by WGA-purified receptors, WGA-column eluted protein was preincubated for 90 min at 4 °C with 10 nM insulin or IGF-I in a solution containing 50 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM MgCl(2), 5 mM MnCl(2), 1 mM DTT, 0.25 mM PMSF, 0.1% Triton X-100, and 1 KIU/ml aprotinin. Autophosphorylation was initiated by the addition of ATP and [-P]ATP to a concentration of 100 µM and 0.25 mCi/ml, respectively, and continued for 10 min at 22 °C. Aliquots of hIRS-p30 protein in concentrations ranging from 0.5 to 60 µM in kinase buffer A (50 mM HEPES/NaOH, 150 mM NaCl, 5 mM MgCl(2), 5 mM MnCl(2), 1 mM DTT, 0.25 mM PMSF, 100 µM ATP, 0.25 mCi/ml [-P]ATP, and 1 KIU/ml aprotinin, pH 7.4) were dispersed into reaction tubes. Substrate phosphorylations were initiated by the addition of an equal volume of the autophosphorylated receptor (equivalent to 2.5 µg of WGA-eluted protein) to the aliquots of hIRS-p30 protein (to achieve substrate concentrations of 0.25 to 30 µM), allowed to proceed for 150 s at 22 °C, and terminated by the addition of 5 times sample buffer (1 times sample buffer = 25 mM Tris/HCl, 50 mM DTT, 5% glycerol, 1% SDS, 0.05% bromphenol blue, pH 6.8) and boiling for 5 min. Phosphorylated proteins were separated on 12% SDS-polyacrylamide gels (30) . Their positions were determined by autoradiography of the Coomassie-stained and dried gels, and phosphate incorporation was determined by Cerenkov counting of excised fragments. IRKD was autophosphorylated for 10 min at 22 °C in kinase buffer A. Aliquots of autophosphorylated IRKD were used to perform substrate phosphorylation of hIRS-p30 for 150 s at 22 °C. Substrate concentrations ranged from 0.25 to 50 µM. The reactions were terminated by the addition of sample buffer and subsequent boiling for 5 min. The analysis of phosphate incorporation was as described above. The K(m) values were determined using the Enzfitter program (Elsevier Biosoft); conversion of protein concentration into molar units was based on the calculated molecular mass of hIRS-p30 of 30.3 kDa.

Preparation of hIRS-p30 Columns and Affinity Chromatography

HiTrap NHS-activated Sepharose columns of 1-ml bed volume (Pharmacia) were used to immobilize 150 µg (5 nmol) of purified recombinant hIRS-p30 protein. The coupling procedure was performed as described by the supplier, except that a coupling buffer of lower pH was used (0.2 M NaHCO(3), 0.5 M NaCl, pH 6). Coupling efficiencies of approximately 70% were achieved.

IRKD was autophosphorylated for 10 min at 22 °C in kinase buffer B (30 mM Tris/HCl, 100 mM NaCl, 5 mM MgCl(2), 5 mM MnCl(2), 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 times 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.


RESULTS AND DISCUSSION

Bacterial Expression of hIRS-p30 Protein

The human IRS-1 gene locus was isolated from a genomic gene bank by low stringency hybridization of a rat IRS-1 cDNA probe. From initially eight positive plaques out of 400,000 plaque-forming units tested, six were purified and analyzed by restriction endonuclease mapping (Fig. 1A), subcloning, and partial sequence analysis (data not shown). No restriction endonuclease polymorphisms were detected within the coding region of the IRS-1 gene as compared to the published nucleotide sequence(5, 31) .

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) .

Phosphorylation Kinetics of hIRS-p30

Although in vivo IRS-1 is phosphorylated on tyrosine, serine, and threonine during insulin stimulation (19) and the insulin receptor seems to possess an intrinsic serine kinase activity(33, 34) , purified insulin receptor phosphorylated hIRS-p30 exclusively on tyrosine as determined by phosphoamino acid analysis (data not shown). This result is in agreement with the observation of others who used IRS-1 protein in in vitro kinase assays(19, 32) .

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(m) values. The protein concentrations required for half-maximal saturation (K(m)) 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(m) 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(m) 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(m). 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(m) 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.''



Binding of Insulin Receptor Kinase to Immobilized hIRS-p30

Affinity columns with purified hIRS-p30 as ligand were prepared by coupling the protein to NHS-activated Sepharose. Autophosphorylated IRKD was used to study the conditions of stable interaction between the proteins. Incorporation of P into immobilized hIRS-p30 demonstrated that active IRKD phosphorylated hIRS-p30 on the column (data not shown). Chromatography of active IRKD under conditions of substrate phosphorylation revealed binding of IRKD by hIRS-p30 on the column (Fig. 3, lanes 1 and 2). Using autophosphorylated IRKD under conditions of strongly inhibited kinase activity (20 mM EDTA), retardation of IRKD on the column was observed with prephosphorylated hIRS-p30 (Fig. 3, lanes3 and 4). In contrast, EDTA-inactivated IRKD was not bound by non-phosphorylated hIRS-p30 (Fig. 3, lanes5 and 6). All column retardation assays were performed in the presence of 0.1 M NaCl to prevent unspecific binding. Using step gradients of 0.1 M to 0.5 M NaCl, IRKD eluted between 0.20 and 0.25 M NaCl (data not shown). To investigate the affinity of IRKD binding to prephosphorylated hIRS-p30, binding of P-autophosphorylated IRKD was assayed in the presence of increased concentrations of autophosphorylated IRKD which was not P-labeled. A concentration of 0.2-0.3 nM unlabeled IRKD blocked half of the binding of the labeled IRKD (Fig. 4).


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.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB232 and SFB351 and the Fritz Thyssen Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institute of Molecular Medicine, Tumor Biology Center, 79011 Freiburg i. Br., Germany.

To whom correspondence should be addressed: Klinik II und Poliklinik für Innere Medizin, University of Cologne, Joseph-Stelzmann-Str. 9, D-50924 Cologne, Germany. Tel.: 49-221-478-5477; Fax.: 49-221-478-6458.

(^1)
The abbreviations used are: IRS-1, insulin receptor substrate-1; DTT, dithiothreitol; IGF-I, insulin-like growth factor I; IRKD, insulin receptor kinase domain; KIU, kallikrein inactivating unit(s); PMSF, phenylmethanesulfonyl fluoride; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin.


ACKNOWLEDGEMENTS

We thank M. Schiller for excellent technical assistance.


REFERENCES

  1. Kasuga, M., Karlsson, F. A., and Kahn, C. R. (1982) Science 215, 185-187 [Medline] [Order article via Infotrieve]
  2. White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186 [Medline] [Order article via Infotrieve]
  3. White, M. F., Stegmann, E. W., Dull, T. J., Ullrich, A., and Kahn, C. R. (1987) J. Biol. Chem. 262, 9769-9777 [Abstract/Free Full Text]
  4. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F. (1991) Nature 352, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  5. Araki, E., Sun, X. J., Haag, B. L., Chuang, L. M., Zhang, Y., Yang- Feng, T. L., White, M. F., and Kahn, C. R. (1993) Diabetes 42, 1041-1054 [Abstract]
  6. Myers, M. G., Jr., Backer, J. M., Sun, X. J., Shoelson, S., Hu, P., Schlessinger, J., Yoakim, M., Schaffhausen, B., and White, M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10350-10354 [Abstract]
  7. Yonezawa, K., Ueda, H., Hara, K., Nishida, K., Ando, A., Chavanieu, A., Matsuba, H., Shii, K., Yokono, K., Fukui, Y., Calas, B., Grigorescu, F., Dhand, R., Gout, I., Otsu, M., Waterfield, M. D., and Kasuga, M. (1992) J. Biol. Chem. 267, 25958-25965 [Abstract/Free Full Text]
  8. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) Cell 70, 431-442 [Medline] [Order article via Infotrieve]
  9. Skolnik, E. Y., Lee, C. H., Batzer, A., Vicentini, L. M., Zhou, M., Daly, R., Myers, M. J., Jr., Backer, J. M., Ullrich, A., White, M. F., and Schlessinger, J. (1993) EMBO J. 12, 1929-1936 [Abstract]
  10. Kuhné, M. R., Pawson, T., Lienhard, G. E., and Feng, G. S. (1993) J. Biol. Chem. 268, 11479-11481 [Abstract/Free Full Text]
  11. Lee, C. H., Li, W., Nishimura, R., Zhou, M., Batzer, A. G., Myers, M. G., Jr., White, M. F., Schlessinger, J., and Skolnik, E. Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11713-11717 [Abstract]
  12. Myers, M. G., Jr., Sun, X. J., Cheatham, B., Jachna, B. R., Glasheen, E. M., Backer, J. M., and White, M. F. (1993) Endocrinology 132, 1421-1430 [Abstract]
  13. Keller, S. R., and Lienhard, G. E. (1994) Trends Cell Biol. 4, 115-119 [CrossRef]
  14. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem. 269, 1-4 [Free Full Text]
  15. Myers, M. G., Jr., and White, M. F. (1993) Diabetes 42, 643-650 [Abstract]
  16. Kahn, C. R., and Folli, F. (1993) Horm. Res. 39(Suppl. 3), 93-101 _
  17. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649 [Medline] [Order article via Infotrieve]
  18. Kaburagi, Y., Momomura, K., Yamamoto-Honda, R., Tobe, K., Tamori, Y., Sakura, H., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1993) J. Biol. Chem. 268, 16610-16622 [Abstract/Free Full Text]
  19. Sun, X. J., Miralpeix, M., Myers, M. G., Jr., Glasheen, E. M., Backer, J. M., Kahn, C. R., and White, M. F. (1992) J. Biol. Chem. 267, 22662-22672 [Abstract/Free Full Text]
  20. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  21. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491 [Medline] [Order article via Infotrieve]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  24. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorf, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  25. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  26. Knowles, B. B., Howe, C. C., and Aden, D. P. (1980) Science 209, 497-499 [Medline] [Order article via Infotrieve]
  27. Kasuga, M., White, M. F., and Kahn, C. R. (1985) Methods Enzymol. 109, 609-621 [Medline] [Order article via Infotrieve]
  28. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761 [Medline] [Order article via Infotrieve]
  29. Al-Hasani, H., Paßlack, W., and Klein, H. W. (1994) FEBS Lett. 349, 17-22 [CrossRef][Medline] [Order article via Infotrieve]
  30. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  31. Nishiyama, M., and Wands, J. R. (1992) Biochem. Biophys. Res. Commun. 183, 280-285 [Medline] [Order article via Infotrieve]
  32. Sun, X. J., Crimmins, D. L., Myers, M. G., Jr., Miralpeix, M., and White, M. F. (1993) Mol. Cell. Biol. 13, 7418-7428 [Abstract]
  33. Baltensperger, K., Lewis, R. E., Woon, C. W., Vissavajjhala, P., Ross, A. H., and Czech, M. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7885-7889 [Abstract]
  34. Heidenreich, K., Paduschek, M., Mölders, M., and Klein, H. W. (1994) Biol. Chem. Hoppe-Seyler 375, 99-104 [Medline] [Order article via Infotrieve]
  35. Shoelson, S. E., Chatterjee, S., Chaudhuri, M., and White, M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2027-2031 [Abstract]
  36. O'Neill, T. J., Craparo, A., and Gustafson, T. A. (1994) Mol. Cell. Biol. 14, 6433-6442 [Abstract]

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