Structural and biochemical characterization of the KRLB ... - Nature

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Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2 Jinhua Wu1, Yolanda D Tseng2, Chong-Feng Xu1, Thomas A Neubert1, Morris F White2 & Stevan R Hubbard1 Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast twohybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2.

The hormone insulin activates intracellular signaling pathways that regulate cell growth and metabolism. The pleiotropic effects of insulin are mediated by its cell-surface receptor, an a2b2 receptor tyrosine kinase. Upon insulin binding and receptor autophosphorylation (activation), several proteins are recruited to the two cytoplasmic (kinase-containing) domains of the receptor for downstream signal propagation. Among these are the IRS proteins, a family of four to six adaptor proteins that possess an N-terminal pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, followed by a stretch of more than 900 residues containing multiple sites of tyrosine, serine and threonine phosphorylation1. Gene-deletion studies in mice show that Irs1 and Irs2 are essential for normal organismal development and glucose homeostasis1. Although IRS1 and IRS2 have many common features at the protein level and overlapping tissue-expression patterns, the phenotypes for the Irs1 and Irs2 knockout mice are distinct. Irs1/ mice are 40% smaller than wild-type litter mates and show insulin resistance in peripheral tissues2,3. Irs2/ mice are only slightly smaller (10%) than wild-type mice but are insulin resistant and develop type II diabetes as a result of loss of pancreatic beta-cell function4. Many of the tyrosine phosphorylation sites in the C-terminal region of IRS1 and IRS2 reside in a YFXM motif (where F denotes a hydrophobic residue (often methionine) and X denotes any residue), which, when phosphorylated by the insulin receptor or insulin-like growth factor 1 (IGF1) receptor, serves as a recruitment site for the Src homology 2 (SH2) domains of phosphatidylinositol 3-kinase (PI3K)5. Activation of PI3K through engagement of tyrosine-phosphorylated IRS1 or IRS2 is required for glucose uptake into insulin-responsive cells6. Other tyrosine-phosphorylation sites in IRS1 and IRS2 recruit

the adaptor protein GRB2 and the protein tyrosine phosphatase SHP2 (ref. 1). In addition to tyrosine-phosphorylation sites, IRS1 and IRS2 contain numerous sites of serine and threonine phosphorylation that, in general, negatively regulate tyrosine phosphorylation, either in the course of normal negative feedback or in pathological insulin resistance7. The tandem PH-PTB domains in the N-terminal region of these adaptor proteins function to recruit IRS1 and IRS2 to the insulin receptor for phosphorylation. The PTB domain binds to phosphorylated Tyr972 (pTyr972) and adjacent residues (NPXY motif) in the juxtamembrane region of the insulin receptor8,9. The PH domain is also important for recruitment of IRS1 and IRS2 to the receptor10,11, although whether this occurs via phosphoinositide binding has not been firmly established. Previous yeast two-hybrid (Y2H) studies provided evidence for a second (in addition to the PTB domain) insulin receptor–interacting region in IRS2, which was named the kinase regulatory-loop binding (KRLB) region12,13 or receptor binding domain 2 (RBD2)14. This region of IRS2 interacts with the tyrosine kinase domain of the insulin receptor (IRK), and the interaction is dependent upon phosphorylation of the kinase activation loop12,14. The KRLB region was roughly defined from the Y2H studies to include residues 591–733 (refs. 12,14), starting approximately 300 residues C-terminal to the PTB domain (Fig. 1) and contains three tyrosine residues in a YFXM motif. Furthermore, this region has a high proportion of glycine, serine and proline residues, and secondary-structure predictions indicate that it is unstructured. Although the corresponding region in IRS1 contains considerable sequence similarity, including the three YFXM sites, it does not stably interact with IRK13,14. Mutagenesis studies in the KRLB region

1Structural Biology Program, Kimmel Center for Biology and Medicine of the Skirball Institute, and Department of Pharmacology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA. 2Howard Hughes Medical Institute, Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA. Correspondence should be addressed to S.R.H. ([email protected]).

Received 9 September 2007; accepted 10 January 2008; published online 17 February 2008; doi:10.1038/nsmb.1388

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Tyr727–IRK structure showed that, C-terminal to the substrate tyrosine, KRLBY628 and the Tyr727 peptide bind similarly (Fig. 2b). However, N-terminal to the substrate tyrosine, KRLBY628 makes far more interactions with the kinase domain (Fig. 2b,c). Fourteen residues of KRLBY628 are ordered in the structure, whereas only six residues of the Tyr727 peptide are ordered (comparable resolution: 1.65 A˚ (KRLBY628) versus 1.9 A˚ (Tyr727)). The total buried surface area of the KRLBY628–IRK complex is 2,121 A˚2, versus 1,033 A˚2 for the Tyr727–IRK complex and 1,256 A˚2 for the IGF1 receptor kinase (IGF1RK) complex with a YFXM peptide (IRS1 Tyr896)16. In the last complex, two more C-terminal residues of the peptide are ordered. Tyr628 (P residue) in the KRLBY628–IRK structure makes hydrogen bonds with the IRK catalytic residues Asp1132 and Arg1136, but is

IRS2Y628 IRS1Y727-ANP

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Figure 1 Schematic diagram of IRS2. Domain organization and location of tyrosine-phosphorylation sites are drawn to linear scale (mouse numbering, 1,321 residues). Tyrosine-phosphorylation sites that are recruitment sites for PI3K (YFXM motif) are labeled by *, the GRB2 site (Y911) is labeled by w and the two SHP2 sites (Y1242 and Y1303) are labeled by y. The KRLB region as determined by Y2H studies (residues 591–733) is shown with dashed lines, and the 15-residue region that was cocrystallized with IRK is shown in the gray box, with the sequence expanded below and Tyr628 underlined. The corresponding 15-residue sequence in IRS1 (mouse) is also shown.

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KRLB IRS2 620 -AYNPYPEDYGDIEIG- 634 IRS1 574 -PTHSYPEEGLEMHHL- 588

RESULTS Crystal structure of KRLBY628 bound to IRK We cocrystallized a 15-residue peptide representing IRS2 residues 620– 634 (AYNPYPEDY628GDIEIG), which will be referred to as KRLBY628, with IRK phosphorylated on the activation loop (pTyr1158, pTyr1162 and pTyr1163). The monoclinic crystals contained one KRLBY628–IRK complex in the asymmetric unit. We determined the crystal structure (refined at 1.65-A˚ resolution) by molecular replacement, using as the search model a crystal structure of phosphorylated IRK in complex with a YFXM peptide substrate (IRS1 Tyr727) and AMP-PNP15. In the crystal structure of the complex, this region of IRS2 is bound across the front face of the kinase domain with Tyr628 positioned in the active site (Fig. 2a). A superposition of this structure with the

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identified two non-YFXM tyrosine residues, Tyr624 and Tyr628 (mouse numbering), that are crucial for binding to IRK13. In the present study, we characterized the precise mode of binding between the KRLB region of IRS2 and IRK through cocrystallization of IRK (human) with a 15-residue KRLB peptide (mouse) that encompasses Tyr624 and Tyr628. Biochemical experiments in vitro and in cells demonstrated that the KRLB region serves as a negative regulatory element to control the extent of tyrosine phosphorylation on IRS2. This regulatory element is likely to be a key feature that distinguishes IRS2 function from that of IRS1.

Y5 38 Y5 * 9 Y6 4 * Y628 Y 649 * 7 Y7 1 * Y 734 * 58 Y8 * 14 *

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Figure 2 Crystal structure of the KRLBY628–IRK complex. (a) IRK is shown as a molecular surface, with the N lobe colored dark gray and the C lobe colored light gray. The activation loop (residues 1150–1171) is colored green and the catalytic loop (residues 1130–1137) is colored orange. IRS2 KRLBY628 is shown in stick representation. The final 2Fo – Fc electron density (1.65-A˚ resolution, 1s contour) is shown in blue mesh. The N and C termini of the peptide are labeled. (b) Comparison of IRK binding modes for KRLBY628 and IRS1 Tyr727 (YFXM motif). Superimposed (C-lobe residues 1080–1283) on the KRLBY628–IRK structure are the Tyr727 peptide substrate (pink) and AMP-PNP (ANP) from the ternary IRK structure (PDB code 1IR3)15. Sulfur atoms are green and phosphorus atoms are black. The IRK molecular surface is semitransparent. (c) Comparison of KRLBY628 binding and conformation of the unphosphorylated IRK activation loop. Superimposed (catalytic loop residues 1130–1137) on the KRLBY628–IRK structure is the unphosphorylated activation loop (dark green) from the structure of autoinhibited IRK (PDB code 1IRK)17. Select hydrogen-bonding interactions between KRLBY628 and IRK residues are shown as dashed lines. (d) Comparison of KRLBY628, KRLBY628+ATP and KRLBpY628 bound to IRK. The IRK molecular surface (semitransparent) is from the KRLBY628– IRK structure. KRLBY628 from the structure with ATP is colored cyan, and KRLBpY628 is colored rose. Images were prepared using PyMOL (http://pymol.org).


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IRK Blot: anti-pTyr IRK KRLB

Coomassie KRLB602–637 R L –K B, + R L IR +K B, K R +IG L +K B, F1 R +IR RK LB K +K , + I R L GF +K B(Y 1R K R F +I LB ), G ( + F1 YF IR K R ), K

–K


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Figure 3 In vitro binding of the KRLB region to IRK and IGF1RK. (a) His-tagged KRLB protein, wild-type protein (WT) or various mutants, including a deletion mutant (residues 602–637), were used in pull-down experiments with phosphorylated IRK. The pull-downs (above) were resolved by SDS-PAGE, and immunoblotting was carried out with an antiphosphotyrosine antibody (PY99). IRK and KRLB protein levels were demonstrated by PY99 immunoblots (middle) and Coomassie Blue staining (bottom), respectively. (b) His-tagged KRLB protein, wild-type protein or Y628F mutant (KRLB(YF)), were used to pull down phosphorylated IRK or phosphorylated IGF1RK. The pull-downs were resolved by SDS-PAGE, and immunoblotting was carried out with PY99 (above). The middle blot shows levels of phosphorylated IRK and IGF1RK in the samples. KRLB protein levels are shown in the bottom gel stained with Coomassie Blue.

IRK, IGF1RK Blot: anti-pTyr IRK, IGF1RK KRLB, KRLB(YF)

Coomassie

displaced 1.5 A˚ (Ca position) relative to Tyr727 of the YFXM peptide (Fig. 2b,c). This displacement is made possible sterically by glycine (Gly629) at the P+1 position in the peptide, an atypical P+1 residue for an insulin receptor substrate. The branched side chain of Ile631 (P+3) compensates for the lack of a hydrophobic side chain at the P+1 position by straddling the P+1 and P+3 binding pockets in the peptide binding groove of the kinase (Fig. 2b). As is the case for YFXMpeptide binding, backbone hydrogen bonds (five) are made between peptide residues C-terminal to Tyr628 and the IRK activation loop. A superposition of the KRLBY628–IRK structure with the structure of unphosphorylated, autoinhibited IRK17 revealed a marked similarity between the mode of binding of KRLBY628 to IRK and stabilization of the IRK activation loop in its inactive state (Fig. 2c). Tyr624 (P–4 residue), Asp627 (P–1) and Tyr628 (P) of KRLBY628 mimic the interactions with the kinase domain of Tyr1158, Asp1161 and Tyr1162, respectively, in the unphosphorylated activation loop. In the active site, Tyr628 (KRLBY628) and Tyr1162 (activation loop) are superimposable, that is, there is no displacement. Evidently, the displacement of Tyr628 relative to Tyr727 of the YFXM peptide, facilitated by Gly629 (P+1), is required for residues N-terminal to Tyr628 (namely, Tyr624 and Asp627) to make their respective interactions with the kinase domain. The observed interactions of Tyr624 and Tyr628 with IRK explain the previous mutagenesis results for these two tyrosines13. The side chain of Tyr621 (P–7) is situated in the ATP binding pocket where the adenine base would bind, hydrogen-bonded (as is adenine) to the backbone nitrogen of Met1079 in the linker between the N lobe and C lobe (Fig. 2c,d). This observation raises the possibility that Tyr621 may compete with ATP for binding to the kinase. To partially address this, we soaked magnesium-ATP into crystals of KRLBY628–IRK and determined the structure of the ternary complex at 2.1-A˚ resolution. Indeed, in this structure with ATP present, Tyr621 is displaced from the ATP binding pocket and is now disordered along with the adjacent residues (Ala620 and Asn622; Fig. 2d). ATP is bound in the cleft between the two kinase lobes, with a single Mg2+ ion coordinated by the a- and b-phosphate groups of ATP, as well as by kinase residues Asp1150 (activation loop) and Asn1137 (catalytic loop). Binding of the KRLB region to IRK and IGF1RK On the basis of the crystal structure, numerous residues proximal to Tyr628 in the KRLB region contribute to the interaction with IRK. To


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probe their respective contributions, we introduced point mutations in the full-length KRLB region (residues 591–733), as defined by the Y2H studies12,14, and measured the ability of the KRLB mutants to bind to IRK through in vitro pull-down experiments. A substantial loss of IRK binding occurred with mutation to alanine (or phenylalanine) of many KRLB residues: Tyr621 (P–7), Tyr624 (P–4), Asp627 (P–1), Tyr628 (P), Gly629 (P+1), Ile631 (P+3) and Ile633 (P+5) (Fig. 3). The pull-down result for G629A confirmed that the 1.5-A˚ shift of Tyr628 relative to Tyr727 of the YFXM peptide (Fig. 2b), which Gly629 affords, is a crucial feature of the KRLB binding mode. Loss of IRK binding for Y621A supports the hypothesis that this tyrosine could influence the phosphorylation kinetics of Tyr628 by competing with ATP for binding to the kinase domain (Fig. 2d). We also tested a truncated version of the KRLB region, residues 602–637, in a pull-down experiment alongside the full-length KRLB region. This shorter form lacks the three YFXM phosphorylation sites, but includes the 15 residues observed in the cocrystal structure. The pull-down experiment showed that the short form of the KRLB region bound approximately as well to IRK as the longer form (Fig. 3a), indicating that the extent of the IRK-interacting region is probably limited to the residues in the vicinity of Tyr628, as viewed in the crystal structure. The sequence identity between IRK and IGF1RK is 82%. We therefore examined the ability of the KRLB region to interact with IGF1RK. The notable result was that the KRLB region bound substantially better to IRK than to IGF1RK (Fig. 3b), [ATP] (mM):

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KRLB KRLBY621A Blot: anti-pTyr IRK for KRLB IRK for KRLBY621A KRLB Coomassie KRLBY621A

Figure 4 Role of Tyr621 in the high Km(ATP) for Tyr628 phosphorylation. Wild-type KRLB protein or a Y621A mutant was phosphorylated by IRK at the indicated ATP concentration. The reaction components were resolved by SDS-PAGE, and immunoblotting was carried out using the antiphosphotyrosine antibody PY99. IRK levels are shown in the middle two anti-pTyr blots, and KRLB protein levels are shown below in the two Coomassie Blue–stained gels. Testing of a Y628F mutant under these assay conditions indicates that only Tyr628 (not any of the three YFXM sites present) is appreciably phosphorylated for wild-type protein and Y621A (data not shown).

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Figure 5 Inhibition of IRK substrate phosphorylation by KRLBY628 and KRLBpY628. (a) Effect of peptides KRLBY628 (Y628) and KRLBpY628 (pY628) on IRK phosphorylation of IRS1 Tyr983 (Y983) using the continuous spectrophotometric assay. The negative slope of the linear portion of the curve (fitting range 20–120 s) is proportional to the rate of ADP generation, with contributions from IRK substrate phosphorylation and IRK ATPase activity (reaction 2, no peptide). For clarity, the raw data are shown for a representative subset of the reactions, all of which are given in b. (b) Relative rates of ADP production obtained from the data in a are plotted, normalized to the basal IRK ATPase rate (reaction 2). Reaction 1 contains 2 mM ATP and 20 mM MgCl2, but no IRK or peptide substrate (not shown in a). Reaction 2 includes, in addition, 200 nM phosphorylated IRK. Reactions 3–10 include, in addition, the indicated concentrations of peptide. Error bars represent s.d. for the reactions performed in triplicate. The histogram bars are shaded according to the reaction contents: for example, light gray, IRK +Y983 (100 mM or 200 mM); black, IRK + Y983 (100 mM) + pY628 (50 mM, 100 mM or 200 mM).

despite the fact that all of the IRK residues that interact directly with the KRLB peptide are conserved in IGF1RK. We used isothermal titration calorimetry to measure the binding affinity of the KRLB peptide for phosphorylated IRK and determined a dissociation constant (Kd) of 1.3 mM (Supplementary Fig. 1 online), which is considerably lower (higher affinity) than the Kd determined by viscometric analysis for a YFXM peptide substrate (IRS1 Tyr939): 0.2 mM (Km ¼ 0.05 mM)18. As another comparison of the KRLB binding strength, the measured Kd for binding of the IRS1 PTB domain to a phosphorylated peptide representing the insulin receptor juxtamembrane region (pTyr972) was 87 mM19 (which is relatively high for a PTB domain–phosphopeptide interaction).

for a typical YFXM site18. Using the same MS method, we measured a Km(ATP) of 80 mM for IRS1 Tyr983 (YFXM) (data not shown). To determine whether Tyr621 contributes to the high Km(ATP), as suggested by the crystal structure (Fig. 2d), we compared IRK phosphorylation of a KRLB Y621A mutant with that of the wild type at various ATP concentrations. Phosphorylation of the wild-type KRLB region plateaued at an ATP concentration of B1.6 mM, consistent with our quantitative determination of the Km(ATP) for the 15-residue KRLB peptide, whereas phosphorylation of the Y621A mutant plateaued near 0.4 mM ATP (Fig. 4). Thus, the high Km(ATP) for Tyr628 phosphorylation is due, at least in part, to Tyr621.

Phosphorylation of the KRLB region by IRK Previous biochemical studies on an 11-residue KRLB peptide (residues 623–633) containing Tyr624 and Tyr628 suggested that this peptide was not a substrate of the insulin receptor13. In light of the KRLBY628– IRK crystal structure, in which Tyr628 is bound in the active site, we revisited this issue by carrying out an in vitro kinase reaction with glutathione S-transferase–tagged IRK (GST-IRK) as the enzyme and the KRLB region (residues 591–733) as the substrate. MALDI-Q-TOF and LC-ESI-MS/MS Q-TOF were used to map the sites of tyrosine phosphorylation. The data (not shown) revealed that Tyr628 was indeed phosphorylated, as well as the YFXM sites Tyr594, Tyr649 and Tyr671. There was no evidence for phosphorylation of Tyr621 or Tyr624. The above in vitro phosphorylation experiment was carried out at a high ATP concentration (5 mM), whereas in the previous study13 the 11-residue peptide was not phosphorylated at 30 mM ATP. We measured the Km(ATP) for IRK phosphorylation of Tyr628 in the 15-residue KRLB peptide (KRLBY628) using MALDI-TOF (Supplementary Fig. 2 online) and determined a Km(ATP) of 1.7 mM, which is substantially higher than the 40 mM Km(ATP)

Inhibition of IRK-substrate phosphorylation by KRLBY628 We initially attempted to measure the steady-state kinetic values of the 15-residue KRLB peptide by using a continuous spectrophotometric assay, in which the phosphorylation rate is derived from the rate of ADP production20. Addition of a typical YFXM peptide substrate to IRK in the presence of magnesium-ATP increases the rate of ADP production (as a consequence of substrate phosphorylation) above a basal rate of IRK ATPase activity (Fig. 5, reactions 3 and 4 versus reaction 2). When using KRLBY628 as a substrate (at 2 mM ATP), we were surprised to observe that the rate of ADP production decreased substantially, well below the basal ATPase rate (Fig. 5, reactions 5 and 6 versus reaction 2). This suggested that the peptide was binding to IRK and either not being phosphorylated—that is, acting as a pseudo substrate inhibitor—or being phosphorylated but with low turnover (with suppression of the basal ATPase rate in either case). MALDI-TOF carried out on the postreaction sample showed that the KRLB peptide was phosphorylated on Tyr628, consistent with the latter explanation. Because the KRLB peptide binds with relatively high affinity (compared to a YFXM peptide) and, as a substrate, is turned over poorly,

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ARTICLES Figure 6 Tyrosine phosphorylation of IRS2 in CHO-IR cells. (a) Lysates from insulin-stimulated (or unstimulated) CHO-IR cells transfected with WT Y624F, Y628F [Insulin] wild-type IRS2 (WT) or the double mutant Y624F, Y628F were (nm): 0 1 3 10 30 100 0 1 3 10 30 100 immunoprecipitated with an anti-IRS2 antibody, and the immunoprecipiBlot: IRS2 tates were resolved by SDS-PAGE. Immunoblotting was performed using anti-pTyr an anti-phosphotyrosine antibody (4G10; anti-pTyr, above) or an anti-IRS2 antibody (below) to show approximately equal levels of IRS2 in the Blot: immunoprecipitates. (b) Lysates from insulin-stimulated (or unstimulated) anti-IRS2 CHO-IR cells transfected with either wild-type IRS2 or Y628F were IP: anti-IRS2 immunoprecipitated with an anti-IRS2 antibody, and the immunoprecipitates were resolved by SDS-PAGE. Immunoblotting was carried out using WT Y628F WT Y628F Y621A phosphospecific antibodies to pTyr612 of human IRS1 (pTyr649 of mouse Insulin: – + + Insulin: – + + + IRS2; PI3K site), to pTyr896 of human IRS1 (pTyr911 of mouse IRS2; Blot: IRS2 Blot: IRS2 anti-pTyr649 GRB2 site) or an anti-IRS2 antibody to show approximately equal levels of anti-Tyr IRS2 in the immunoprecipitates. (c) Lysates of insulin-stimulated (or Blot: IRS2 Blot: unstimulated) CHO-IR cells transfected with either wild-type IRS2 or KRLB anti-pTyr911 anti-IRS2 mutants Y628F or Y621A were immunoprecipitated with an anti-IRS2 Blot: IP: anti-IRS2 antibody, and the immunoprecipitates were resolved by SDS-PAGE. anti-IRS2 Immunoblotting was carried out using an anti-phosphotyrosine antibody IP: anti-IRS2 (PY99) or an anti-IRS2 antibody to show approximately equal levels of IRS2 in the immunoprecipitates. The first three lanes in the anti-IRS2 blot (below) are identical to those in the anti-IRS2 blot in b, as the samples are derived from the same experiment. The unstimulated phosphorylation levels of Y628F and Y621A are not appreciably different from that of wild-type IRS (data not shown).

a


b

we tested the ability of KRLBY628 and the Tyr628-phosphorylated form of the peptide (KRLBpY628) to inhibit IRK phosphorylation of substrates. Addition of KRLBY628 to a reaction containing IRK and an IRS1 Tyr983 peptide (YFXM) lowered the rate of ADP production in a dose-dependent manner (Fig. 5b, reactions 7–9 versus reaction 3), demonstrating that the KRLB peptide can inhibit phosphorylation of a YFXM peptide. The phosphorylated KRLB peptide also inhibited IRS1 Tyr983 phosphorylation (Fig. 5b, reactions 10–12 versus reaction 3), but less effectively, as would be expected (the affinity of the product is less than that of the reactant). Using a different assay, KRLBY628 inhibited activation-loop phosphorylation of a kinase-dead IRK by GST-IRK, as did KRLBpY628, whereas the IRS1 Tyr983 peptide had no inhibitory effect (Supplementary Fig. 3 online). When the tyrosine kinase domain from Src was substituted for IRK, the KRLB peptide acted as a normal tyrosine substrate (that is, the rate of ADP production increased; data not shown), demonstrating that the slow turnover of the KRLB substrate is specific for IRK. Because the phosphorylated KRLB peptide was capable of inhibiting substrate phosphorylation, we cocrystallized KRLBpY628 with IRK and obtained a structure of the complex at 1.95-A˚ resolution. KRLBpY628 binds to IRK in a manner similar to KRLBY628 (Fig. 2d), including the positioning of Tyr621 in the ATP binding cleft. The pTyr628 phosphate group is within hydrogen-bonding distance of Asp1132 and Asn1137 in the catalytic loop. The fact that KRLBpY628 could be cocrystallized with IRK is further indication that the affinity of the phosphorylated peptide for IRK is appreciable; previous efforts to cocrystallize a phosphorylated YFXM peptide with IRK had proved unsuccessful. In-cell phosphorylation of IRS2 by the insulin receptor To understand the functional role of the KRLB region for IRS2 signaling, we transfected full-length IRS2, either the wild type or a KRLB (double) mutant, Y624F-Y628F, into Chinese hamster ovary cells stably transfected with the insulin receptor (CHO-IR). The transfected cells were stimulated with various doses of insulin, and the phosphorylation level of IRS2 was monitored by immunoblotting of anti-IRS2 immunoprecipitates. Loss of KRLB binding to the insulin receptor (via mutation of Tyr624 and Tyr628) substantially increased insulin-stimulated tyrosine phosphorylation of IRS2 (Fig. 6a).


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c

Although the overall tyrosine-phosphorylation level of wild-type IRS2 is lower than that of the KRLB mutant, it is conceivable that, through trans-phosphorylation by the other kinase domain in the receptor dimer, particular tyrosine sites in IRS2 might be phosphorylated to a greater extent as a consequence of KRLB binding in the kinase active site. To gain a sense of whether such facilitation might occur, we made use of two IRS1 phosphotyrosine-specific antibodies that cross-react with the corresponding sites in IRS2. One of the antibodies recognizes a PI3K binding site in IRS1 (pTyr612) and IRS2 (pY649; 21 residues C-terminal to Tyr628), and the other recognizes a GRB2 binding site in IRS1 (pTyr896) and IRS2 (pY911; 283 residues C-terminal to Tyr628). A third phosphotyrosine-specific antibody, to a SHP2 binding site in IRS1 (pTyr1179), was also tested, but it was not found to cross-react with IRS2 (pTyr1242) (data not shown). The insulin-stimulated phosphorylation levels of both pTyr649 and pTyr911 were lower in wild-type IRS2 than in the KRLB single mutant Y628F (Fig. 6b). To determine whether Tyr621 has a role in modulating IRS2 phosphorylation in cells as it does in vitro, by setting a high Km(ATP) of B1.7 mM for Tyr628 phosphorylation (Fig. 4), we compared insulin-stimulated phosphorylation of IRS2 Y621A with that of the wild type and Y628F. Mutation of Tyr621 led to an increase in IRS2 phosphorylation to a level similar to that of Y628F (Fig. 6c). These data suggest that, at cellular ATP levels, competition of Tyr621 with ATP for binding to IRK (Fig. 2b) is important for suppression of IRS2 phosphorylation by the KRLB region. DISCUSSION Mechanism and specificity of KRLB binding to IRK The crystal structure of KRLB residues 620–634 in complex with phosphorylated (activated) IRK revealed that this region of IRS2 binds in the active site of the kinase domain with Tyr628 poised for phosphorylation (Fig. 2a). In binding to IRK, the Tyr628 region mimics binding of a YFXM peptide substrate C-terminal to Tyr628 (Fig. 2b) and the unphosphorylated IRK activation loop N-terminal to Tyr628 (Fig. 2c). The last observation explains why KRLB binding is contingent upon prior activation-loop autophosphorylation and the concomitant structural rearrangement15. Notably, a naturally occurring mutation in the activation loop of the insulin receptor (R1152Q),

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ARTICLES identified in several patients with type II diabetes, leads to non–insulindependent binding of the KRLB region to the mutant receptor21. This arginine is not well ordered in the structure of unphosphorylated IRK17, yet we presume that the R1152Q mutation partially destabilizes the autoinhibited conformation of the activation loop, facilitating binding of the KRLB region in the kinase active site. Although previous Y2H studies12,14 had roughly defined the extent of the KRLB region as residues 591–733, our crystallographic and in vitro pull-down studies (Figs. 2 and 3) indicate that this second insulin receptor–interacting region in IRS2 is limited to approximately 15 residues encompassing Tyr628 (residues 620–634). In this region (Fig. 1), IRS1 lacks not only the equivalent of Tyr628 (Gly582) but also the equivalents of Asp627 (P–1), Gly629 (P+1) and Ile633 (P+5), all of which are important for high-affinity binding to IRK (Fig. 3a). This explains why simply substituting Gly582 with tyrosine in IRS1 was not sufficient to generate KRLB functionality in IRS1 (ref. 13). A database pattern search indicated that IRS2 is likely to be the only protein with a KRLB-like sequence for binding to the insulin receptor. Whether there are sequences with similar functionality for other protein tyrosine kinases is difficult to predict, owing to the limited structural information on peptide substrate–kinase interactions, especially for residues N-terminal to the substrate tyrosine. Despite the high sequence conservation (82% identity) in the kinase domains of the insulin and IGF1 receptors, the KRLB region showed a strong binding preference for IRK (Fig. 3b), which is consistent with previous observations22. The molecular determinants that underlie this discrimination are not clear, because the IRK residues involved in binding of the KRLB peptide are conserved in IGF1RK. Conceivably, this differential binding could derive from two amino acid differences in the hinge region of the kinases16, as the N terminus of the KRLB peptide in the crystal structure is proximal to the hinge region. Alternatively, there may be subtle differences in the conformation of the active state in the two kinases (for example, the peptide binding groove) that account for the difference in affinity. Functional role of the KRLB region in IRS2 IRS1 and IRS2 possess tandem PH-PTB domains in their N-terminal regions that function to recruit these proteins to the activated insulin and IGF receptors for efficient phosphorylation of multiple tyrosine residues in the extensive C-terminal region1. Upon discovery of a second insulin receptor–interacting region (KRLB) in IRS2 (refs. 12,14), it was reasonable to assume that this region would function to enhance receptor coupling and, potentially, phosphorylation of IRS2 over IRS1, which lacks this binding modality. The binding of KRLB residue Tyr628 in the IRK active site was unexpected, because this tyrosine was not known or predicted to be an insulin-receptor phosphorylation site. Thus, this second coupling mechanism for IRS2 involves the kinase active site rather than a distal site, as is commonly used for substrate recruitment to a receptor tyrosine kinase (for example, PTB-domain binding to the juxtamembrane region). The original Y2H studies12–14 provided evidence that the interaction between the 15-residue KRLB sequence and IRK is not a typical substrate-kinase interaction. Common tyrosine substrates of the insulin receptor (YFXM) and of other tyrosine kinases interact transiently in the active site—they are phosphorylated and released—and regions containing such sites do not score positive in a Y2H experiment (for example, the corresponding region of IRS1 (refs. 13,23)). Our in vitro biochemical experiments demonstrated that the Km(ATP) for Tyr628 phosphorylation is substantially higher than for a YFXM substrate (B1.7 mM versus B40 mM; Supplementary

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Fig. 2), that the Tyr628 region binds with relatively high affinity to IRK (Kd ¼ 1.3 mM; Supplementary Fig. 1) and that the phosphorylated peptide retains appreciable binding affinity (Fig. 5b and Supplementary Fig. 3). These characteristics lead to poor substrate turnover, which probably explains the positive Y2H result for the KRLB region. What are the functional consequences of KRLB binding in the active site of the insulin receptor? Our data in CHO-IR cells indicate that the KRLB region acts to suppress IRS2 tyrosine phosphorylation. When Tyr624 and Tyr628 were mutated to phenylalanine, binding to the kinase active site was markedly diminished (Fig. 3a), yet the tyrosine-phosphorylation level of IRS2 increased substantially (Fig. 6a). The mechanism by which the KRLB region inhibits IRS2 tyrosine phosphorylation by the insulin receptor evidently involves occupation and blockage of the kinase active site, both before and after phosphorylation. Of note, there are approximately 330 residues (predicted to be unstructured) in IRS2 between the C-terminal end of the PTB domain and Tyr628. This tether would seem to be of sufficient length to allow the PTB domain and the KRLB region to bind to the insulin receptor either in cis, to the same cytoplasmic domain, or in trans, engaging both cytoplasmic domains. Because the KRLB-mediated suppression of IRS2 phosphorylation was greater than two-fold (Fig. 6a), docking of Tyr628 in the active site of one kinase domain might restrict the ability of other tyrosines in IRS2 to reach the active site of the other kinase domain (although there are many intervening residues between Tyr628 and the more C-terminal phosphorylation sites). The KRLB region could also inhibit phosphorylation of other insulin receptor substrates, such as the adaptor protein APS, which could conceivably be recruited to the receptor simultaneously with IRS2; APS binds to the phosphorylated activation loop rather than to the juxtamembrane region24. When the KRLB region was overexpressed in a skeletal muscle cell line stably expressing the insulin receptor, recruitment of endogenous IRS2 to the insulin receptor was diminished, as was IRS2 tyrosine phosphorylation25. The overexpressed KRLB region competes against endogenous IRS2 for binding to the insulin receptor. Notably, recruitment of endogenous IRS1 to the insulin receptor was increased in the KRLB-overexpressing cells25. This is evidently due to reduced competition from the PTB domain of endogenous IRS2 for binding to the juxtamembrane site in the receptor. The fact that IRS1 phosphorylation was also increased indicates that enhanced recruitment of IRS1 to the receptor outweighs KRLB-mediated inhibition of the kinase domain. A proteomics study carried out in 3T3-L1 adipocytes showed that Tyr628 of IRS2 (endogenous) undergoes a 6.4-fold increase in its phosphorylation level after 5 min of insulin stimulation26. Six other IRS2 tyrosine-phosphorylation sites were detected in this study, with insulin-stimulated phosphorylation levels increasing from 1.2- to 4.9-fold. By comparison, the four tyrosine-phosphorylation sites detected in IRS1 showed higher insulin-stimulated increases, ranging from 5.6- to 29-fold. Although these data are not comprehensive— information for many sites in IRS1 and IRS2 are lacking—they are nevertheless consistent with the hypothesis that the KRLB region acts to suppress tyrosine phosphorylation of IRS2. A possible physiological role for KRLB-mediated suppression of IRS2 tyrosine phosphorylation by the insulin receptor might be to establish a differential signaling threshold for IRS2. There are nine YFXM sites in IRS2 to which PI3K could potentially be recruited, yet there is only one GRB2 site (similarly for IRS1) (Fig. 1). It is plausible that the reduced level of tyrosine phosphorylation in IRS2 may still be sufficient for activation of PI3K for metabolic signaling but

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ARTICLES insufficient for activation of Ras (through GRB2) for mitogenic signaling. This mechanism would enable IRS2 to couple selectively to metabolic signaling pathways upon insulin stimulation, yet activate mitogenic signaling pathways upon IGF1 stimulation. IRS1 would be capable of coupling to both metabolic and mitogenic pathways downstream of insulin and IGF1. The knockout studies of Irs1 (growth and metabolic phenotypes) and Irs2 (metabolic and IGF1-mediated beta-cell growth phenotypes) are generally consistent with this scenario2,3. Finally, the millimolarlevel Km(ATP) for Tyr628 phosphorylation and the tighter binding of Tyr628 versus pTyr628 in the kinase active site could conceivably serve as a regulatory mechanism, whereby the degree of insulin-stimulated tyrosine phosphorylation of IRS2 is dependent on cellular ATP levels. Further studies of Irs1 and Irs2 knockout and knock-in mice, and cell lines derived thereof, will be necessary to establish definitively the physiological role of the IRS2 KRLB region. METHODS

Table 1 X-ray data collection and refinement statistics KRLBY628-IRK Data collection Space group Cell dimensions a, b, c (A˚)

KRLBY628-IRK-ATP

KRLBpY628-IRK

P 21

P 21

P 21

a, b, g (1)

46.76, 84.93, 50.71 90.0, 113.2, 90.0

46.75, 84.43, 50.60 90.0, 112.9, 90.0

46.74, 84.72, 51.35 90.0, 113.1, 90.0

Resolution (A˚)

50–1.65 (1.71–1.65)

50–2.10 (2.18–2.10)

50–1.95 (2.02–1.95)

Rsym (%) I/sI

4.6 (34.7)a 22.9 (2.8)a

6.4 (32.5)a 13.7 (2.7)a

7.1 (33.2)a 12.0 (2.4)a

Completeness (%) Redundancy

99.6 (99.5)a 3.1

99.3 (100.0)a 3.2

98.6 (99.9)a 2.7

Resolution (A˚) No. reflections

50–1.65 43,554

50–2.10 21,034

50–1.95 26,660

Rcryst/Rfree (%) No. atoms

20.2/22.0

19.4/23.9

20.0/23.8

IRK KRLB

2,335 117

2,312 97

2,357 120

Mg-ATP Water

– 267

32 215

– 213

B-factors (A˚2) IRK

24.3 23.2

29.1 28.0

27.5 26.8

KRLB Mg-ATP

26.7 –

31.7 38.4

30.9 –

Refinement

Protein expression and purification. Human IRK Water 32.3 34.0 32.1 (residues 978–1283) was expressed in baculovirusinfected Sf 9 insect cells, purified and autophos- R.m.s. deviations Bond lengths (A˚) 0.009 0.010 0.009 phorylated in vitro as described previously15. A Bond angles (1) 1.28 1.32 1.17 GST-tagged (N-terminal) version of IRK was generated by subcloning residues 956–1283 of the aValues in parentheses are for highest-resolution shell. One crystal was used for each data set. human insulin receptor into the baculovirus transfer vector pAcG2T (BD Biosciences). The fusion protein was expressed in baculovirus-infected Sf 9 cells and purified using a In vitro pull-down experiments. 150 mg of purified wild-type or mutant KRLB GSTrap column (GE Healthcare) and a Superdex-75 (GE Healthcare) sizeprotein (residues 591–733, His-tagged) were mixed with 15 mg of trisexclusion column. phosphorylated IRK or tris-phosphorylated IGF1RK in 300 ml binding buffer A 6His-tagged (C-terminal) and GST-tagged (N-terminal) KRLB con(150 mM NaCl and 50 mM Tris-HCl (pH 7.5)). 20 ml were taken from each struct was generated by subcloning cDNA encoding mouse IRS2 residues 591– pull-down mixture for SDS-PAGE to show approximately equal amounts of 733 into the vector pET41 (Novagen). A shorter construct encoding residues IRK or IGF1RK, and immunoblotting was carried out with anti-phosphotyr602–637 was also generated. Site-directed mutagenesis was carried out using osine antibody PY99 (Santa Cruz). 100 ml of a His-Select bead slurry (Sigmathe QuikChange system (Stratagene). All constructs were transformed into Aldrich) were equilibrated with the binding buffer, and the pull-down mixtures E. coli strain Rosetta(DE3) (Novagen). Bacteria were grown in LB supplemenwere incubated with the beads for 1 h at 4 1C. The beads were then loaded ted with 50 mg l1 kanamycin and 35 mg l1 chloramphenicol at 37 1C to an into a spin filter column (Pierce), spun in a microcentrifuge at 100g for OD600 of 0.7. Expression was induced with 1 mM IPTG at 30 1C for 5 h. See 1 min to remove any unbound protein, and washed three times with binding Supplementary Methods online for purification details. buffer plus 20 mM imidazole in the spin filter column. The washing buffer Crystallization. KRLBY628 (AYNPYPEDYGDIEIG) and KRLBpY628 (AYNPYwas removed by spinning at 100g. Bound proteins were eluted with elution PEDpYGDIEIG) were synthesized (GeneMed Synthesis) and solubilized in buffer (150 mM NaCl, 500 mM imidazole and 50 mM Tris-HCl 200 mM Tris-HCl (pH 7.5). Tris-phosphorylated IRK (pTyr1158/1162/1163) (pH 7.5)), boiled with Laemmli sample buffer and resolved by SDS-PAGE (14%). Gels were either stained with Coomassie Brilliant Blue to monitor the and KRLBY628 or KRLBpY628 were mixed in a 1:3 molar ratio. Crystals of KRLBY628–IRK and KRLBpY628–IRK were obtained at 4 1C by vapor diffusion in amount of KRLB protein, or transferred to a nitrocellulose membrane (Millihanging drops containing 1 ml of protein solution (7 mg ml1 IRK, 1 mg ml1 pore) for immunoblotting with PY99 to detect the amount of IRK or IGF1RK KRLBY628 or KRLBpY628, in 20 mM Tris-HCl (pH 7.5), 180 mM NaCl and in the pull-down. 1 mM DTT) and 1 ml of reservoir buffer (28% (w/v) PEG 8000 and 0.1 M HEPES (pH 7.5)). Microseeding was used to improve crystal quality. Crystals of In vitro phosphorylation of the KRLB region. The GST tag of wild-type or KRLBY628–IRK–ATP were obtained by soaking KRLBY628–IRK crystals Y621A KRLB (residues 591–733) was removed by tobacco etch virus (TEV) overnight in a cryosolvent (28% (w/v) PEG 8000, 0.1 M HEPES (pH 7.5) protease. 1 mM of purified KRLB or Y621A was incubated with 30 nM of and 20% (w/v) ethylene glycol) containing 10 mM ATP and 20 mM MgCl2. tris-phosphorylated IRK in the reaction buffer (50 mM MgCl2, 20 mM Crystals of KRLBY628–IRK or KRLB pY628–IRK were transferred into the same Tris-HCl (pH 7.5), 100 mM NaCl and 50 mM Na3VO4) containing various cryosolvent briefly and flash-frozen in liquid nitrogen. The crystals for the three concentrations of ATP at 25 1C for 2 min. Reactions were stopped by adding complexes all belong to monoclinic space group P21 with one complex per Laemmli sample buffer and boiling. The samples were separated by SDS-PAGE asymmetric unit and a 53% solvent content. Data collection and refinement (14%) and transferred to a nitrocellulose membrane. The phosphorylation statistics are summarized in Table 1. See Supplementary Methods online for levels of KRLB and Y621 were detected by immunoblotting with antidata collection, structure determination and model building. phosphotyrosine antibody PY99.


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In vitro inhibition of IRK phosphorylation. Continuous spectrophotometric assays were carried out as previously described27, using a SpectraMax 190 microplate reader (Molecular Devices). Along with the coupling reagents27, reactions contained 200 nM tris-phosphorylated IRK, 100 mM Tris-HCl (pH 7.5), 2 mM ATP, 20 mM MgCl2 and various concentrations of IRS1 Tyr983 peptide (KKSRGDYMTMQIG), KRLBY628 and KRLBpY628. Rates of ATP consumption were calculated between 20 s and 120 s. Cell transfection, immunoprecipitation and immunoblotting. Mouse Irs2 cDNA was cloned into the pcDNA3 vector. Point mutations in Irs2 were generated by using QuikChange II XL (Stratagene) and confirmed by DNA sequencing. CHO-IR cells were cultured in a-MEM containing 10% FBS plus gentamicin. The cells were seeded in 10-cm tissue culture dishes and transfected at 60% confluence. 60 ml of Lipofectamine 2000 (Invitrogen) and 24 mg of Irs2 DNA or empty vector were used for transfection at 37 1C for 6 h. After 24 h, the cells were serum-starved for 3 h in Ham’s F-12 medium (Invitrogen). Cells were stimulated with insulin at the desired dosage for 10 min. Each dish was washed twice with ice-cold PBS. Cells were lysed by adding 1 ml of ice-cold lysis buffer (50 mM Tris-HCl (pH 8.0), 135 mM NaCl, 1% (w/v) Triton X-100, 1 mM EDTA, 1 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM NaF and protease inhibitors cocktail (Roche)) and incubating on ice for 30 min. Cell lysates were clarified by centrifugation. For IRS2 immunoprecipitation, the clarified lysates were incubated overnight with anti-IRS2 antibody (M-19, Santa Cruz) at 4 1C. Protein G–agarose (Roche) was used to precipitate the antibody and bound proteins by incubating for 2 h at 4 1C. Protein G–agarose was washed extensively with lysis buffer and solubilized in SDS-PAGE sample buffer. The bound proteins or cell lysates were resolved by SDS-PAGE (8.8%) gels, transferred to nitrocellulose membrane, and detected by immunoblotting with anti-IRS2 antibody, anti-phosphotyrosine antibody (PY99), anti-IRS1,2 pTyr612 (Santa Cruz), anti-IRS1 pTyr896 (Millipore) and anti-IRS1 pTyr1179 (Santa Cruz). For the dose-dependent insulin-stimulation experiments (Fig. 6a), FuGENE 6 transfection reagent (Roche) was used for transfection, and IRS2 immunoprecipitation was carried out by overnight incubation with anti-IRS2 antibody, followed by 1 h incubation with Protein G–Sepharose (Amersham) and washing three times with lysis buffer. Accession codes. Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes as follows: KRLBY628–IRK, 3BU3; KRLBY628–IRK+ATP, 3BU5; KRLBpY628–IRK, 3BU6. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGMENTS This work was supported by the US National Institutes of Health (NIH) grant DK052916 (to S.R.H.) and a Howard Hughes Medical Institute (HHMI) Research Training Fellowship for Medical Students (to Y.D.T.). M.F.W. is an investigator at the HHMI. We thank W. Li and W.T. Miller (Stony Brook University, New York, USA) for purified IGF1RK, and W.T. Miller for manuscript comments. Beamline X4A at the National Synchrotron Light Source, Brookhaven National Laboratory, a US Department of Energy facility, is supported by the New York Structural Biology Consortium. The New York University Protein Analysis Facility is supported by NIH Shared Instrumentation Grant S10 RR017990, National Institute of Neurological Disorders and Stroke grant P30 NS050276 and US National Cancer Institute core grant P30 CA016087 (to T.A.N.). AUTHOR CONTRIBUTIONS J.W. designed and performed the crystallographic studies, the in vitro biochemical experiments and a portion of the in-cell phosphorylation experiments, and contributed to manuscript preparation. Y.D.T. designed and performed a portion of the in-cell phosphorylation experiments and contributed to manuscript preparation. C.-F.X. acquired the MS data and contributed to manuscript preparation. T.A.N. supervised the MS experiments and contributed to manuscript preparation. M.F.W. designed and supervised the in-cell biochemical experiments and contributed to manuscript preparation. S.R.H. supervised the project and was the principal manuscript author.

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1. White, M.F. IRS proteins and the common path to diabetes. Am. J. Physiol. Endocrinol. Metab. 283, E413–E422 (2002). 2. Araki, E. et al. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372, 186–190 (1994). 3. Tamemoto, H. et al. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372, 182–186 (1994). 4. Withers, D.J. et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391, 900–904 (1998). 5. Myers, M.G., Jr et al. IRS-1 activates phosphatidylinositol 3¢-kinase by associating with src homology 2 domains of p85. Proc. Natl. Acad. Sci. USA 89, 10350–10354 (1992). 6. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O. & Ui, M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J. Biol. Chem. 269, 3568–3573 (1994). 7. Pirola, L., Johnston, A.M. & Van Obberghen, E. Modulation of insulin action. Diabetologia 47, 170–184 (2004). 8. Craparo, A., O’Neill, T.J. & Gustafson, T.A. Non-SH2 domains within insulin receptor substrate-1 and SHC mediate their phosphotyrosine-dependent interaction with the NPEY motif of the insulin-like growth factor I receptor. J. Biol. Chem. 270, 15639–15643 (1995). 9. Eck, M.J., Dhe-Paganon, S., Trub, T., Nolte, R.T. & Shoelson, S.E. Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor. Cell 85, 695–705 (1996). 10. Yenush, L. et al. The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1. J. Biol. Chem. 271, 24300–24306 (1996). 11. Burks, D.J. et al. Heterologous pleckstrin homology domains do not couple IRS-1 to the insulin receptor. J. Biol. Chem. 272, 27716–27721 (1997). 12. Sawka-Verhelle, D., Tartare-Deckert, S., White, M.F. & Van Obberghen, E. Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591–786. J. Biol. Chem. 271, 5980–5983 (1996). 13. Sawka-Verhelle, D. et al. Tyr624 and Tyr628 in insulin receptor substrate-2 mediate its association with the insulin receptor. J. Biol. Chem. 272, 16414–16420 (1997). 14. He, W. et al. Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors. J. Biol. Chem. 271, 11641–11645 (1996). 15. Hubbard, S.R. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572–5581 (1997). 16. Favelyukis, S., Till, J.H., Hubbard, S.R. & Miller, W.T. Structure and autoregulation of the insulin-like growth factor 1 receptor kinase. Nat. Struct. Biol. 8, 1058–1063 (2001). 17. Hubbard, S.R., Wei, L., Ellis, L. & Hendrickson, W.A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746–754 (1994). 18. Ablooglu, A.J. & Kohanski, R.A. Activation of the insulin receptor’s kinase domain changes the rate-determining step of substrate phosphorylation. Biochemistry 40, 504–513 (2001). 19. Farooq, A., Plotnikova, O., Zeng, L. & Zhou, M.M. Phosphotyrosine binding domains of Shc and insulin receptor substrate 1 recognize the NPXpY motif in a thermodynamically distinct manner. J. Biol. Chem. 274, 6114–6121 (1999). 20. Barker, S.C. et al. Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry 34, 14843–14851 (1995). 21. Miele, C. et al. Differential role of insulin receptor substrate (IRS)-1 and IRS-2 in L6 skeletal muscle cells expressing the Arg1152 - Gln insulin receptor. J. Biol. Chem. 274, 3094–3102 (1999). 22. Van Obberghen, E. et al. Surfing the insulin signaling web. Eur. J. Clin. Invest. 31, 966–977 (2001). 23. O’Neill, T.J., Craparo, A. & Gustafson, T.A. Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system. Mol. Cell. Biol. 14, 6433–6442 (1994). 24. Hu, J., Liu, J., Ghirlando, R., Saltiel, A.R. & Hubbard, S.R. Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor. Mol. Cell 12, 1379–1389 (2003). 25. Oriente, F. et al. Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C æ activation by insulin in L6hIR cells. J. Biol. Chem. 276, 37109–37119 (2001). 26. Schmelzle, K., Kane, S., Gridley, S., Lienhard, G.E. & White, F.M. Temporal dynamics of tyrosine phosphorylation in insulin signaling. Diabetes 55, 2171–2179 (2006). 27. Li, S., Covino, N.D., Stein, E.G., Till, J.H. & Hubbard, S.R. Structural and biochemical evidence for an autoinhibitory role for tyrosine 984 in the juxtamembrane region of the insulin receptor. J. Biol. Chem. 278, 26007–26014 (2003).

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