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J. Biol. Chem., Vol. 281, Issue 33, 23785-23791, August 18, 2006

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Role of the Activation Loop Tyrosines in Regulation of the Insulin-like Growth Factor I Receptor-tyrosine Kinase^*

From the Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794

Received for publication, June 1, 2006 , and in revised form, June 21, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The tyrosine kinase activity of insulin-like growth factor I receptor (IGF1R) is under tight control. Ligand binding to the extracellular portion of IGF1R stimulates autophosphorylation at three sites (Tyr¹¹³¹, Tyr¹¹³⁵, and Tyr¹¹³⁶) in the activation loop within the tyrosine kinase catalytic domain. Autophosphorylation at all three sites is required for maximum enzyme activity, and for IGF1-stimulated cellular activity of the receptor. Previous studies have not clarified the contributions of the individual tyrosines to enzymatic activation. Here, we produced single Tyr-to-Phe mutations at these positions, and compared activities of the purified kinase domains (unphosphorylated and phosphorylated) with wild-type IGF1R. Rates of autophosphorylation of the three mutants were more rapid than for wild-type IGF1R; this was most apparent for the Y1135F mutant. Substrate phosphorylation studies on the unphosphorylated forms of IGF1R confirmed that the value of V_max for Y1135F was elevated relative to wild-type IGF1R, consistent with a disruption of an autoinhibitory interaction. In contrast, activity measurements on the fully phosphorylated enzymes indicated that k_cat/K_m values were lowered relative to wild-type IGF1R; this effect was most dramatic for Y1136F. We confirmed these findings using limited proteolysis and tryptophan fluorescence experiments. The results demonstrate that Tyr¹¹³⁵ plays a particularly important role in stabilizing the autoinhibited conformation of the activation loop, while Tyr¹¹³⁶ plays the key role in stabilizing the open, activated conformation of IGF1R.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Insulin-like growth factor 1 receptor (IGF1R)² is a transmembrane tyrosine kinase that is essential for fetal and postnatal growth and development (1-5). It is expressed in most human tissues and cell types (the only known exceptions are hepatocytes and mature B cells). The receptor is activated by binding of the secreted growth factor ligand IGF1 (or the lower affinity ligand IGF2) to the extracellular domain of IGF1R. Ligand binding stimulates the intrinsic tyrosine kinase activity of the cytoplasmic domain. This in turn triggers a number of cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway (the main mechanism by which IGF1R protects cells from apoptosis), and the Erk/MAPK pathway leading to mitogenesis. Aberrant IGF1R signaling has been implicated in malignant transformation of cells, and the enzyme has emerged as a target for anticancer drug design (6-10).

IGF1R, insulin receptor (IR) and insulin-related receptor (IRR) belong to the same receptor-tyrosine kinase (RTK) family (11, 12). These receptors are composed of two extracellular subunits containing the ligand binding domain and two transmembrane subunits possessing tyrosine kinase activity. IGF1R and IR share 70% sequence identity overall, and 84% identity within the tyrosine kinase catalytic domains. The mechanism for receptor activation is thought to be similar for IR and IGF1R (2-4,13).

The structures of the IGF1R and IR catalytic domains display the typical two-lobed protein kinase fold (14-18). ATP binding and catalysis takes place in a deep cleft between the two lobes. These RTKs contain a segment known as the activation loop (A-loop), which contains the three principal autophosphorylation sites (Tyr¹¹³¹, Tyr¹¹³⁵, and Tyr¹¹³⁶ in IGF1R) (Fig. 1). Ligand binding to the extracellular subunit promotes a conformational change that leads to autophosphorylation of these sites. The first site of autophosphorylation is predominantly Tyr¹¹³⁵, followed by Tyr¹¹³¹ and then by Tyr¹¹³⁶ (16). The unphosphorylated, monophosphorylated, bisphosphorylated, and trisphosphorylated forms of IGF1R (IGF1R-0P, -1P, -2P, and -3P, respectively) have increasing levels of enzyme activity (16).

The three-dimensional structures of IGF1R-0P, -2P, and -3P have been solved (16-18). In IGF1R-0P, the A-loop inhibits access to substrates (17). Phosphorylation of Tyr¹¹³¹, Tyr¹¹³⁵, and Tyr¹¹³⁶ triggers a large change in the A-loop conformation, which allows access of ATP and peptide substrate to the catalytic site. Thus, autophosphorylation of IGF1R is the crucial regulator of the catalytic activity. The details of how autophosphorylation controls enzymatic activity, however, are not completely understood. Steady state kinetic studies show that a significant decrease in the substrate K_m occurs in the transition from the 1P state (mainly Tyr¹¹³⁵) to the 2P state (Tyr¹¹³⁵ and Tyr¹¹³¹) (16). The conformation of the trisphosphorylated activation loop in IGF1R-3P is stabilized by numerous interactions between the activation loop and other segments of the kinase. The purpose of the present study was to define the roles of Tyr¹¹³¹, Tyr¹¹³⁵, and Tyr¹¹³⁶ in regulation of autophosphorylation.

View larger version (69K): [in this window] [in a new window]   FIGURE 1. Ribbon diagram of the IGF1RK-3P structure (16). The activation loop is shown in green. The side chains of the three phosphotyrosine residues (pY1131, pY1135, and pY1136) are shown in green, with phosphates in red. The side chains of Arg1137 and Lys1138 (the sites of trypsin cleavage) are shown in magenta. Trp1148 (the reporter for fluorescence experiments) is colored yellow and displayed in ball-and-stick format.

We previously reported a method to separately purify each of the phosphorylated forms of IGF1R (IGF1R-0P, -1P, -2P, and -3P) (16). Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor showed that each autophosphorylation increases enzyme turnover number and decreases K_m for ATP and peptide. In this study, we used similar methods to purify unphosphorylated and phosphorylated forms of IGF1R mutants containing single mutations at Tyr¹¹³¹, Tyr¹¹³⁵, and Tyr¹¹³⁶.We analyzed the conformation of the A-loop and the level of enzymatic activity in these forms of IGF1R. Our results indicate that each tyrosine is important for full activation of the IGF1R kinase domain. Furthermore, our results shed light on the individual roles of the three tyrosines. Autophosphorylation of Tyr¹¹³⁵ and Tyr¹¹³¹ appears to destabilize the autoinhibitory conformation of the activation loop, whereas Tyr¹¹³⁶ phosphorylation plays the key role in structural stabilization of the A-loop.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—10% Tris-HCl gels were from Bio-Rad. Mass spectrometry grade trypsin was obtained from Promega. ADP and potassium iodide were obtained from Sigma.

Mutagenesis and Purification of IGF1R Kinase Domain—IGF1R mutants were produced with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocols. The mutants were made in a pFastBac-1 baculovirus transfer vector (Invitrogen) encoding the kinase catalytic domain of IGF1R (IGF1RK). The mutations were verified by DNA sequencing on an ABI 373 automated DNA sequencer. Mutant forms of IGF1RK were expressed in Spodoptera frugiperda (Sf9) cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen) as described previously (16). Sf9 cells were cultured in Excel 410 (JRH Biosciences) with 5% heat-inactivated serum (Sigma) and 1% pencillin/streptomycin/amphotericin at 27 °C. The cells were harvested after 72 h of infection.

The purification of the mutants was accomplished using similar procedures as for wild-type IGF1RK (16). Unphosphorylated forms of the mutants were purified in three chromatographic steps on an FPLC system (GE Healthcare): 1) a Source Q-15 column, 2) a Superdex-75 gel filtration column, and 3) a Mono-Q HR10/10 column. To produce the completely phosphorylated forms of IGF1RK (i.e. IGF1RK-3P for wild-type enzyme, or IGF1RK-2P for the single site mutants), enzymes were incubated with 10 mM ATP and 30 mM MgCl₂ for 5 min at room temperature. The autophosphorylation reactions were terminated by addition of 100 mM EDTA. Native gel analysis confirmed that these reactions contained predominantly the completely phosphorylated forms. Sodium orthovanadate (200 µM) was added to prevent dephosphorylation. The reactions were then passed over Superdex 75 to remove nucleotides, and the phosphorylated forms were separated by Mono-Q chromatography (16). The pooled fractions were concentrated and stored at -80 °C in 20 mM Tris-HCl, pH 7.5. All final protein concentrations were determined by the Bradford method (Bio-Rad).

Kinase Assays—The kinetics of IGF1R autophosphorylation and substrate peptide phosphorylation were measured by a continuous spectrophotometric assay (16, 23). In this assay, the consumption of ATP is coupled to the oxidation of NADH, which is monitored as a reduction in NADH absorption at 340 nm. Reactions (50 µl) contained 100 mM Tris (pH 7.5), 10 mM MgCl₂, 1 mM phosphoenolpyruvate, 1.2 mg/ml NADH, 111 units/ml pyruvate kinase, 156 units/ml lactate dehydrogenase, and substrate peptide (KKEEEEYMMMMG). Autophosphorylation reactions contained 6 µM enzyme and 1 mM ATP. Peptide phosphorylation reactions contained 100 nM enzyme. For determinations of K_m (ATP), reactions contained a peptide concentration of 2 mM and varying concentrations of ATP (10-4000 µM). For determinations of K_m (peptide), reactions contained an ATP concentration of 1 mM and varying concentrations of peptide (10-5000 µM). Data were recorded every 6 s. The kinetic parameters were determined by fitting to the Michaelis-Menten equation.

Native-PAGE autophosphorylation assays were carried out using 6 µM enzyme, 10 mM ATP, and 30 mM MgCl₂ at room temperature (24). The reactions were stopped by addition of 100 mM EDTA at various time points. Autophosphorylation reactions were analyzed by 10% Tris-HCl native PAGE and visualized by Coomassie Blue staining.

Limited Proteolysis of IGF1RK—Limited proteolysis of wild-type and mutant forms of IGF1RK was carried out according to previous methods established for IRK (25). Proteins were diluted to 9 µM in 50 mM Tris-HCl, pH 7.0, 30 mM MgCl₂, and 2mM dithiothreitol, with or without 10 mM ADP. Trypsin was added at a ratio of trypsin to IGF1RK of 1:25-30 by mass. The reactions proceeded at room temperature for 15 min and were stopped by addition of 5x SDS-PAGE buffer. The digestion products were resolved by SDS-PAGE, and the fragments were visualized by Coomassie Blue staining.

Fluorescence Spectra and Iodide Quenching—Tryptophan fluorescence experiments were carried out in 100 mM Tris-HCl and 1 mM dithiothreitol using methods described for IRK (26). Experiments were carried out in volumes of 500 µl using 0.5 µM IGF1RK (wild-type or mutants). Potassium iodide was included at varying concentrations to quench emission of tryptophan 1148 of IGF1R. The ionic strength in the reactions was kept at 0.5 M by addition of KCl, and 0.1 mM sodium thiosulfite was included to inhibit the formation of I^-₃. The fluorescence emission spectra were obtained with a Cary Eclipse Fluorescence spectrophotometer. The excitation wavelength was 295 nm, and emission spectra were collected from 310-420 nm. The solvent accessibility of fluorescent tryptophan residues was characterized by the Stern-Volmer constant, K_SV, according to the relationship between the peak fluorescence intensity and the iodide quencher concentration [Q] in the solution, as defined by Equation 1,

(Eq.1)

where F₀ is the fluorescence intensity in the absence of quencher.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Autophosphorylation of Single Site Mutants—To analyze the importance of the individual A-loop tyrosines, we produced mutant forms of IGF1RK in which each tyrosine was changed to phenylalanine (Y1135F, Y1131F, and Y1136F). We expressed wild-type and mutant IGF1RKs using the Sf9/baculovirus system, and purified the unphosphorylated forms of the enzymes. In wild-type IGF1R, the first A-loop tyrosine to be phosphorylated is Tyr¹¹³⁵, followed by Tyr¹¹³¹, and then Tyr¹¹³⁶ (16). To determine whether elimination of single A-loop tyrosines blocked the autophosphorylation of the remaining two sites, we incubated the enzymes with Mg-ATP and followed the time course of autophosphorylation by native PAGE analysis (Fig. 2A). The various phosphoforms of IGF1R are readily resolved by native PAGE (Fig. 2A and Ref. 16). All three mutants were capable of autophosphorylation at the remaining two sites, indicating that no one site is obligatory for full phosphorylation (Fig. 2A).

To compare the rates of autophosphorylation for wild-type and mutant forms of IGF1RK, we used a continuous spectrophotometric assay. We incubated the enzymes with ATP in the absence of any exogenous substrate. As we observed previously (16), the enzyme progress curves were biphasic, because autophosphorylation activates IGF1RK. Autophosphorylation of all three single-site mutants was more rapid than for wild-type IGF1R (Fig. 2B), suggesting that to a certain extent all three tyrosines participate in autoinhibitory interactions. Activation was most apparent in the case of the Y1135F and Y1131F mutants; the final autophosphorylation rates for these mutants were 63.1 and 31.8 mOD/min., respectively, compared with 6.2 mOD/min for wild-type IGF1RK. The Y1136F mutant had a rate that was closer to wild-type IGF1R (12.2 mOD/min).

Substrate Phosphorylation Assays—We carried out steady-state kinetic measurements of phosphorylation of a synthetic peptide substrate by wild-type and single site mutants of IGF1RK. We first tested the unphosphorylated forms of the proteins. To ensure that the enzymes remained in their unphosphorylated states during the initial rate measurements, we analyzed samples from the reactions by native PAGE; there was no detectable conversion to 1P, 2P, or 3P forms in the peptide phosphorylation reactions (data not shown). The kinetic experiments showed that all three mutants had higher activity than wild-type IGF1RK (Table 1). This activation was driven primarily by changes in V_max. The values of V_max for Y1135F, Y1131F, and Y1136F were 6-fold, 3-fold, and 2-fold higher than wild-type, respectively. Changes in K_m for ATP were modest; the largest change was a 2-fold reduction for the Y1135F mutant. Values of K_m (peptide) were increased for all three mutants, with the largest change (3-fold) in the Y1136F mutant.

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Autophosphorylation of wild-type and mutant forms of IGF1RK. A, native PAGE analysis of autophosphorylation reactions. The assays were carried out using unphosphorylated enzymes. The reactions were stopped by addition of 100 mM EDTA at the indicated time points, and analyzed by 10% Tris-HCl native PAGE. IGF1RK was visualized by Coomassie Blue staining. B, continuous spectrophotometric assays of receptor autophosphorylation. Reactions contained 6 µM unphosphorylated enzyme and 1 mM ATP.

We carried out similar experiments on the fully phosphorylated forms of the proteins (IGF1RK-3P, Y1135F-2P, Y1131F-2P, and Y1136F-2P). Surprisingly, IGF1RK-3P was relatively resistant to trypsin cleavage in the A-loop, even in the presence of ADP (Fig. 3B). This result suggests that the triple phosphorylation of the A-loop stabilizes an "open" conformation that renders the Arg-Lys sequence inaccessible to trypsin. We were able to exploit this effect to compare the fully phosphorylated forms of the enzymes. In contrast to wild-type IGF1R, the phosphorylated mutants showed significant trypsin cleavage in the presence or absence of ADP (Fig. 3B). The highest extent of cleavage was seen for the Y1136F mutant, consistent with a role for Tyr¹¹³⁶ in stabilizing the open conformation of the A-loop.

As a complementary approach to study conformational changes in the IGF1RK A-loop, we measured tryptophan fluorescence. This method has been previously applied to IRK; the solvent accessibility of Trp¹¹⁷⁵ differs between IRK-0P and IRK-3P, as detected by solute quenching (26). The catalytic domains of both IR and IGF1R have five tryptophan residues. One is on the surface of the small lobe, and the other four are in the interior of the large lobe. Mutagenic analysis demonstrated that the fluorescence emission spectrum of IRK was dominated by Trp¹¹⁷⁵, a residue that is near the C terminus of the A-loop in the IRK active site (26). The solvent accessibility of IRK Trp¹¹⁷⁵ increases significantly upon autophosphorylation. For IRK, unphosphorylated enzyme gave a Stern-Volmer constant of 0.3 M^-1, whereas phospho-IRK gave a value of 1.5 M^-1. IR and IGF1R are highly conserved in this region, and the residue corresponding to IRK Trp¹¹⁷⁵ in IGF1R is Trp¹¹⁴⁸ (Fig. 1). We employed a similar fluorescence quenching strategy to monitor solvent accessibility in the active site of purified IGF1RK-0P and IGF1RK-3P. We carried out fluorescence experiments using an excitation wavelength of 295 nm, because tyrosine absorbance is negligible at this wavelength. We measured emission spectra over 310-420 nm, using increasing concentrations of potassium iodide as quencher. The fluorescence intensity over 310-420 nm was integrated and analyzed on Stern-Volmer plots (Fig. 4). As reported previously for IRK, the solvent accessibility of IGF1RK increases upon autophosphorylation: the Stern-Volmer constant for unphosphorylated IGF1RK was 0.29 M^-1, whereas the value for IGF1R-3P was 1.72 M^-1 (Fig. 4 and Table 3). Next, we performed similar studies on the three A-loop mutants of IGF1R, in both their unphosphorylated and fully phosphorylated (2P states). All unphosphorylated mutants showed higher values of K_SV than wild-type IGF1RK-0P, with the most significant increase for Y1135F (Table 3). These results are consistent with our other experiments, and suggest that Tyr¹¹³⁵ plays a particularly important role in stabilizing the autoinhibited conformation. Tyr¹¹³¹ and (to a lesser extent) Tyr¹¹³⁶ are also involved in autoinhibition. The Stern-Volmer constants for the doubly phosphorylated forms of the A-loop mutants were less than the value of K_SV for fully phosphorylated wild type (Table 3). The most significant decrease was observed for Y1136F-2P, consistent with a role for Tyr¹¹³⁶ in stabilizing the "open" conformation.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Limited proteolysis of wild type and mutant forms of IGF1RK. A, purified unphosphorylated enzymes were incubated with or without 10 mM ADP. Trypsin (1:30, w/w) was added, and the cleavage reactions were stopped by addition of SDS-PAGE sample buffer after 15 min. The digestion products were resolved by SDS-PAGE and visualized by Coomassie Blue staining. Molecular weight markers were run in the first and last lanes of the gel. The arrowheads indicate the positions of the IGF1RK cleavage products. B, similar experiments were carried out on the purified fully phosphorylated enzymes. In this case, 1:25 (w/w) trypsin was used.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Iodide quenching of IGF1RK-0P and IGF1RK-3P fluorescence. Stern-Volmer plot of fluorescence quenching of IGF1RK-0P (circles) and IGF1RK-3P (squares) by potassium iodide. The excitation wavelength was 295 nm and emission spectra were collected from 310-420 nm. Experiments were carried out at constant ionic strength using 0.5 µM IGF1RK.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our autophosphorylation assays (Fig. 2) showed that removal of individual A-loop tyrosines did not block the autophosphorylation of the remaining sites. Furthermore, for each mutant, rates of autophosphorylation at the remaining two sites exceeded the rate for wild-type IGF1RK (Fig. 2B). This effect was most pronounced for the Y1135F mutant. The Y1131F mutant also showed a significantly higher autophosphorylation rate than wild-type, while the Y1136F mutant was only slightly increased. The data suggest that the three A-loop tyrosines are involved in IGF1R autoinhibition in the following order of importance: Tyr¹¹³⁵>Tyr¹¹³¹>Tyr¹¹³⁶. This possibility was borne out by steady-state kinetic measurements using the unphosphorylated forms of the enzymes (Table 1). For the mutants (particularly for Y1135F), values of V_max for phosphorylation of a synthetic peptide substrate were elevated as compared with wild-type IGF1R kinase.

We also investigated how these three autophosphorylation sites contribute to the activity of the fully phosphorylated forms of the enzyme toward an exogenous substrate. If destabilization of the autoinhibited conformation is sufficient for full enzyme activity, we would expect the fully phosphorylated forms of the mutants to reach a level of activity that is comparable to wild-type IGF1RK. Our kinetic studies on the purified phosphorylated forms of the mutants (Table 2) showed that this is not the case. The k_cat/K_m values for Y1135F, Y1131F, and Y136F were decreased by 9.3-fold, 2.2-fold, and 18.6-fold, respectively. For Y1136F, the K_m for peptide substrate was raised by over 20-fold. These results indicate that the phosphorylated A-loop tyrosines are required for full enzyme activity, in the order of importance Tyr¹¹³⁶>Tyr¹¹³⁵>Tyr¹¹³¹.

We confirmed the enzyme activity results using two probes of the A-loop conformation: limited trypsin proteolyis and fluorescence quenching. Unphosphorylated IGF1RK behaved similarly to insulin receptor in these studies (the Stern-Volmer constant for unphosphorylated IR was 0.3 M^-1, compared with 0.29 M^-1 for IGF1RK). In the proteolysis experiments on unphosphorylated forms of IGF1RK, we found that only the Y1135F mutant showed significant cleavage in the absence of ADP (Fig. 3A). Similarly, fluorescence experiments showed the largest degree of iodide quenching for Y1135F (Table 3). Y1131F and Y1136F also gave greater fluorescence quenching than wild-type, with the same rank order as was observed for the enzyme activity assays. The data suggest that removal of these tyrosine residues (particularly Tyr¹¹³⁵) results in a more flexible conformation of the A-loop.

Limited proteolysis experiments on purified IGF1RK-3P showed that the A-loop was inaccessible to trypsin cleavage under these conditions, even in the presence of ADP (Fig. 3A). Phosphorylation appears to anchor the A-loop in such a way as to inhibit trypsin cleavage. In the structure of IGF1RK-3P, the phosphate group of Tyr¹¹³⁵ is salt bridged to Arg¹¹³⁷ within the trypsin cleavage site and the phosphate group of pY1136 is salt-bridged to Lys¹¹³⁸ (16). This might explain, at least in part, why trypsin can access the Arg-Lys site only when the A-loop has a flexible conformation. The phosphorylated forms of the mutants all showed enhanced cleavage relative to wild-type IGF1R, suggesting that the open form of the A-loop was destabilized in the mutants. The effect was most pronounced for Y1136F. Iodide quenching experiments showed the expected increase in quenching for IGF1RK-3P as compared with IGF1RK-0P, due to a change in solvent accessibility in the open conformation (26). The phosphorylated forms of the Y1135F, Y1131F, and Y1136F mutants showed progressively larger decreases in iodide quenching relative to IGF1RK-3P (Table 3). The results suggest that the A-loop in the single-site mutants adopts a conformation that is intermediate between IGF1RK-0P and IGF1RK-3P. The results are consistent with enzyme activity assays and point to a key role for Tyr¹¹³⁶ in stabilizing the activated conformation.

Our results on the roles of the A-loop tyrosines are consistent with the available structural data. In the unphosphorylated forms of IGF1RK (17) or IRK (15), the A-loop adopts an autoinhibitory conformation with Tyr¹¹³⁵ (IRK residue 1162) bound in the active site. Tyr¹¹³⁵ of IGF1R interacts with Asp¹¹⁰⁵, Arg¹¹⁰⁹, and Pro¹¹⁴⁵ (17). The side chain of Tyr¹¹³¹ is hydrogen-bonded to Ser¹⁰⁵⁹. Phosphorylation of Tyr¹¹³⁵ and Tyr¹¹³¹ would be expected to incrementally destabilize the autoinhibited conformation of the A-loop. In the structure of IGF1R-3P (Fig. 1), the phosphotyrosine side chains interact with numerous residues in the A-loop and in other regions of the enzyme (16). The phosphate group of Tyr¹¹³⁵ interacts with Arg¹¹³⁷, whereas the phosphate group of Tyr¹¹³⁶ is salt-bridged to Lys¹¹³⁸ and Arg¹¹²⁸. The latter interaction is conserved in the structure of the tris-phosphorylated form of insulin receptor. In the crystal structure of IGF1R-2P, in which residues Tyr¹¹³¹ and Tyr¹¹³⁵ are phosphorylated but Tyr¹¹³⁶ is not, the central part of the activation loop is disordered (18). Tyr¹¹³⁶ appears to trigger the final lobe closure in which ATP and substrate are brought close together, facilitating catalysis. Our data indicate that Tyr¹¹³⁶ autophosphorylation is necessary to stabilize the activated state of IGF1R.

Our results are relevant for the development of conformationally selective inhibitors of IGF1R. Most of the clinically tested inhibitors of tyrosine kinases are compounds that target the ATP-binding site. There were initial concerns that the conserved architecture of kinase ATP binding sites would preclude the development of specific inhibitors. A large body of evidence now demonstrates that different ATP-binding sites have distinct features (shape of the pockets, composition of amino acids) that can serve as unique drug targets. In particular, it has been argued that the inactive conformations of protein kinases may differ more dramatically than the fully active conformations (27). Gleevec is an example of a tyrosine kinase inhibitor that targets the unphosphorylated, inactive conformation (28). Thus, it is possible to design inhibitors that not only discriminate among related tyrosine kinases, but also between conformational states of a given tyrosine kinase. Among other advantages of targeting the unphosphorylated form of IGF1R, there are greater differences in the ATP-binding pocket between IGF1R-0P and IR-0P than between their triply phosphorylated counterparts (17). Furthermore, the active site of IGF1R-2P is not as tightly closed as that of IGF1R-3P, suggesting that it could accommodate inhibitors that are too large to fit in the relatively small, closed IGF1R-3P active site (18). The variety of conformational states of IGF1R present potential targets for inhibitor design. For example, the cyclolignan PPP was recently shown to block the phosphorylation of Tyr¹¹³⁶, but not the other two A-loop tyrosines (29). A full understanding of the enzymatic and conformational changes that occur with each A-loop phosphorylation is an important prelude to the development of conformation-specific inhibitors.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant CA28146 (to W. T. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: todd.miller{at}stonybrook.edu.

² The abbreviations used are: IGF1R, insulin-like growth factor I receptor; RTK, receptor-tyrosine kinase; FPLC, fast protein liquid chromatography; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; pY, phosphotyrosine.

	ACKNOWLEDGMENTS

 
We thank Aftabul Haque and Hongling Zhao for performing site-directed mutagenesis, and Dr. Stevan Hubbard for critical review of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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