Kinetic evidence for activating and non-activating components of autophosphorylation of the insulin receptor protein kinase

Reduced and carboxamidomethylated-lysozyme (RCAM-lysozyme) is an excellent substrate (Km = 13 microM) and a potent inhibitor of receptor autophosphorylation (Ki = 0.6 microM). By using these properties of RCAM-lysozyme autophosphorylation was resolved into two kinetically and functionally distinct components involving formation of phosphotyrosine on the receptor's beta-subunits: 1. Insulin-stimulated autophosphorylation is independent of autophosphorylation at other sites; activation of insulin receptor-catalyzed substrate phosphorylation is dependent upon this component of autophosphorylation, which is inhibited by RCAM-lysozyme. 2. Autophosphorylation at saturating RCAM-lysozyme concentration is insensitive to insulin and has little effect on substrate phosphorylation. Thus, only insulin-dependent receptor autophosphorylation is responsible for activation of kinase-catalyzed substrate phosphorylation.     

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.     

Control of insulin receptor autophosphorylation by polypeptide substrates: inhibition and stimulation by interaction with the catalytic subunit.

Autophosphorylation of purified insulin receptor, in the absence of insulin, was stimulated by selected polypeptide substrates. In the presence of 1 microM insulin these peptides inhibited autophosphorylation. Stimulation was observed with reduced [S-(carboxamidomethyl)cysteinyl]lysozyme (RCAM-lysozyme) and three peptides generated by CNBr cleavage, V8 proteinase digestion and/or chemical modification. We also generated two peptide substrates from RCAM-lysozyme which did not stimulate receptor autophosphorylation and were very weak inhibitors. As a control peptide, the simple substrate angiotensin inhibited receptor autophosphorylation in the absence or presence of insulin. However, stimulatory peptide, but not insulin, significantly shifted the concentration dependence for inhibition by angiotensin. The stimulatory peptides also increased autophosphorylation of the cloned cytoplasmic domain of the kinase [R-BIRK; Villalba, M., Wente, S. R., Russell, D. S., Ahn, J., Reichelderfer, C. F., & Rosen, O. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7848]. Therefore, stimulation occurs by interaction with the cytoplasmic process of the beta-subunit and not through interaction with the insulin binding alpha-subunit of the native receptor. Autophosphorylation was analyzed by mapping 32P-labeled tryptic phosphopeptides from the beta-subunit and from R-BIRK. Nearly identical phosphopeptide maps were found, comparing first, basal R-BIRK and basal native receptor, second, peptide- and insulin-stimulated native receptor, and third, peptide-stimulated R-BIRK and insulin-stimulated native receptor. Therefore, R-BIRK functions as a basal-state enzyme and can be stimulated in an insulin-like manner. On the basis of these observations, stimulation by insulin and by peptides yields similar functional results, but by apparently different mechanisms.     

Specificity of the tyrosine-phenylalanine transport system in Bacillus subtilis

l-Tyrosine and l-phenylalanine enter cells of Bacillus subtilis via a system of active transport that exhibits complex kinetic behavior. The specificity of the transport system was characterized both at low concentrations of transport substrate (where affinity for l-tyrosine or l-phenylalanine is high but capacity is low) and at high concentrations (where affinity is low but capacity is high). Specificity was not found to differ significantly as a function of either l-tyrosine or l-phenylalanine concentration. Kinetic analysis showed that the relationship between the uptake of l-phenylalanine and l-tyrosine is strictly competitive. Neither l-tyrosine nor l-phenylalanine uptake was competitively inhibited by other naturally occurring l-amino acids, indicating the importance of the phenyl side chain to uptake specificity. Hence, it is concluded that l-tyrosine and l-phenylalanine are transported by a common system that is specific for these two amino acids. The abilities of analogue derivatives of l-tyrosine and l-phenylalanine to inhibit the uptake of l-[(14)C]tyrosine and l-[(14)C]phenylalanine competitively were determined throughout a wide range of substrate and inhibitor concentrations. In this manner, the contributions of the side chain, the alpha-amino group and the carboxyl group to uptake specificity were established. It is concluded that the positively charged alpha-amino group contributes more significantly to uptake specificity than does the negatively charged carboxyl group. The recognition of a phenyl ring is an essential feature of specificity; other amino acids with aromatic side chains, such as the indole and imidazole rings of l-tryptophan and l-histidine, do not compete with l-tyrosine and l-phenylalanine for uptake. The presence of the p-hydroxy substitutent in the side chain (as in l-tyrosine) enhances the uptake of the aryl amino acid analogues investigated.     

Effect of phenylarsine oxide on insulin-dependent protein phosphorylation and glucose transport in 3T3-L1 adipocytes

We have reported previously that phenylarsine oxide (PAO) blocks insulin-stimulated glucose transport in 3T3-L1 adipocytes (Frost, S. C., and Lane, M. D. (1985) J. Biol. Chem. 260, 2646-2652). As shown in the present study, the locus of inhibition is post-receptor. Insulin stimulated the extent of receptor autophosphorylation in solution and in the intact cell by approximately 4-fold. PAO had no effect on this activity. Using reduced and carboxamidomethylated lysozyme as a substrate for the tyrosine-specific receptor, insulin stimulated the rate of receptor kinase-catalyzed substrate phosphorylation by 2-fold; PAO had no effect on this stimulation. However, the insulin-stimulated, serine-specific phosphorylation of two endogenous phosphoproteins (pp24 and pp240) in the intact cell was blocked by 25 microM PAO. These complementary in situ and in vitro studies demonstrate that the inhibition by PAO must be distal to the insulin receptor's protein tyrosine kinase activity.     

Convenient approach to polypeptide copolymers derived from native proteins

A convenient approach for the synthesis of narrowly dispersed polypeptide copolymers of defined compositions is presented. The controlled denaturation of the proteins serum albumin and lysozyme followed by an in situ stabilization with polyethylene(oxide) chains yields polypeptide side chain copolymers of precisely defined backbone lengths as well as the presence of secondary structure elements. Supramolecular architectures are formed in solution because of the presence of hydrophobic and hydrophilic amino acids along the polypeptide main chain. Polypeptide copolymers reported herein reveal excellent solubility and stability in aqueous media and no significant cytotoxicity at relevant concentrations, and they can be degraded via proteolysis, which is very attractive for biomedical applications. This "semi-synthetic chemistry" approach is based on a novel and convenient concept for producing synthetic polypeptides from native protein resources, which complements traditional polypeptide synthesis and expression approaches and offers great opportunities for the preparation of diverse polypeptides with unique architectures.     

Function of the alkyl side chains of Deltalac-acetogenins in the inhibitory effect on mitochondrial complex I (NADH-ubiquinone oxidoreductase)

We synthesized a series of Deltalac-acetogenins in which the two alkyl side chains were systematically modified, and examined their inhibitory effect on bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). The results revealed that the physicochemical properties of the side chains, such as the balance of hydrophobicity and the width (or bulkiness) of the chains, are important structural factors for a potent inhibitory effect of amphiphilic Deltalac-acetogenins. This is probably because such properties decide the precise location of the hydrophilic bis-THF ring moiety in the enzyme embedded in the inner mitochondrial membrane.     

Chemical mutations of the catalytic carboxyl groups in lysozyme to the corresponding amides

In a two-step process, esterification and ammonolysis, Glu-35 and Asp-52 in lysozyme were amidated to glutamine and asparagine residues. Since the side chains of glutamine and asparagine are almost equal in size to those of glutamic acid and aspartic acid, these conversions would provide appropriate derivatives to elucidate the catalytic participations of these residues. The enzymatic activities of the resulting [Gln35]lysozyme and [Asn52]lysozyme were found to be less than 4% of that of native lysozyme in a pH range of 3.4-8.0. As these derivatives were inactive, we could determine the dissociation constants (Ks values) for the binding of beta-1,4-linked n-mer, a hexasaccharide of N-acetyl-D-glucosamine, to [Gln35]lysozyme and [Asn52] lysozyme. The values of Ks at pH 5.5 and 40 degrees C were 1.6 X 10(-5) M for [Gln35]lysozyme and 2.7 X 10(-5) M for [Asn52]lysozyme. These values are similar to that for native lysozyme. The results are direct proof for the involvements of Glu35 and Asp52 in the catalytic action of lysozyme. A method for ammonolysis of ester groups in proteins in liquid ammonia is described and will be useful for amidation of carboxyl groups of proteins.     

Affinity-directed cross-linking of membrane-bound acetylcholine receptor polypeptides with photolabile alpha-bungarotoxin derivatives

Photolabile derivatives of [125I]-alpha-bungarotoxin that retain specific binding to Torpedo californica acetylcholine receptor have been utilized as structural probes of the receptor complex of polypeptide components in its membrane-associated form. The derivatized toxins contained aryl azide side chains poised to form covalent cross-links to both associated and adjacent polypeptides following toxin-receptor complex formation. The results demonstrate that, depending on the possible radius of extension of the photoactivated group from the parent toxin, either (1) both the polypeptide to which the toxin derivative binds and an adjacent polypeptide can be derivatized upon photolysis or (2) only the adjacent polypeptide is labeled. The results lend strong support to the notion that the nicotinic receptor from T. california is composed of a complex of different polypeptides.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.     

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.     

Solid-state conformation of some basic sequential polypeptides

We have studied the structure of solid films obtained by x-ray diffraction, from several basic polypeptides with a defined sequence. The alterating polypeptides poly(Ala-Lys), poly(Leu-Lys), poly(Val-Lys), and poly(Arg-Leu) all show a cross-β-structure in which layers of hydrophilic side chains alternate with layers containing hydrophobic side chains. The other polypeptides studied are not in the β-conformation and appear to be in the α-helical conformation. The helices obtained from poly(Lys-Ala-Ala) and poly(Lys-Ala-Ala-Lys) appear to be packed in an unusual fashion, which favors interaction between alanine side chains. Such behavior is not found in poly(Lys-Leu-Ala).     

Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc-tyrosine aminomalonate, effectively block the IRK-catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti-lipolytic effect of the hormone. Only tBocTyr-aminomalonate was found to inhibit all the effects of insulin measured: insulin-stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin-dependent lipogenesis and the insulin-dependent anti-lipolytic effect. This inhibitor is the first blocker which is reported to block insulin-dependent anti-lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin-independent lipolysis, on [U14C]fructose assimilation or on (-)isoproterenol-stimulated lipolysis. These studies suggest that insulin-dependent lipogenesis and anti-lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.     

Mechanistic insights into protein precipitation by alcohol

Ethanol is used to precipitate proteins during various processes, including purification and crystallization. To elucidate the mechanism of protein precipitation by alcohol, we have investigated the solubility and structural changes of protein over a wide range of alcohol concentrations. Conformation of hen egg-white lysozyme was changed from native to α-helical rich structure in the presence of ethanol at concentrations above 60%. The solubility of lysozyme was reduced with increasing ethanol concentration, although gel formation at ethanol concentrations between 60% and 75% prevented accurate solubility measurements. SH-modified lysozyme showed largely unfolded structure in water and α-helical structure in the presence of ethanol. More importantly, solubility of the chemically modified lysozyme molecules decreased with increasing ethanol concentration. There is no indication of increased solubility upon unfolding of the lysozyme molecules by ethanol, indicating that any favorable interaction of ethanol with the hydrophobic side chains, if indeed occuring, is offset by the unfavorable interaction of ethanol with the hydrophilic side chains and peptide bonds.     

Autophosphorylation of the insulin receptor in vitro. Designation of phosphorylation sites and correlation with receptor kinase activation

Chemical degradation and antipeptide antibodies were used to study alterations in the structure and function of the human placental insulin receptor following autophosphorylation in vitro. Antibodies elicited to residues 1143-1162 (P2) of the human insulin proreceptor immunoprecipitated the native, phosphorylated receptor but not the unphosphorylated receptor. Since this antibody recognizes both forms of the receptor on immunoblots, it was concluded that the accessibility of the P2 domain to the antibody is increased by in vitro autophosphorylation. Chemical cleavage at either tryptophan or methionine residues followed by immunoprecipitation with antipeptide antibodies was used to map the in vitro autophosphorylation sites of the beta subunit of the insulin receptor. Two phosphorylated fragments were resolved. One, recognized by antibody elicited to amino acid residues 1328-1343 (P5), is derived from the carboxyl terminus of the beta subunit and includes tyrosine 1316. The other, recognized by antibody to P2, is located in a domain that includes tyrosine 1150. The rate of phosphorylation of this latter site correlates with the rate of activation of the insulin receptor kinase during in vitro autophosphorylation. The results support the following conclusions: autophosphorylation alters the conformation of the beta subunit of the insulin receptor; autophosphorylation in vitro leads to phosphorylation of tyrosine residues near the carboxyl terminus of the protein and in the P2 domain that includes tyrosine 1150; activation of the insulin receptor kinase correlates with autophosphorylation of the domain containing tyrosine 1150.     

Synthesis of some analogs of estradiol

As part of a search for estradiol derivatives designed for conjugation to carboxyl or amine functions of anti-cancer agents or suitable derivatives thereof, estradiol analogs with side chains at the C-16 or -17 position were prepared for biological assay. These analogs include several which have a substituted nitrogenous function at C-17. The avidity of some of these analogs for binding to estrogen receptor was found to be of a low order.     

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.     

Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.     

Substrate specificity of the amino acyl adenylate activation sites of gramicidin S-synthetase (GSS).

An investigation was made of the intermolecular forces which determine substrate recognition and binding as well as of the topography and localized environment of the different binding sites of the substrate amino acids of gramicidin S-synthetase (GSS) using substrate derivatives as molecular probes. It is demonstrated that among the aminoacyl adenylate binding sites of the heavy component of GSS the activation site of L-ornithine is distinguished by a relatively high substrate variability. The active centres of GSS are less restrictive for the activation of substrate analogues modified at the carboxyl group than for derivatives substituted at the alpha-amino group.     

Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog.

The crystal structure of the phosphorylated, activated form of the insulin receptor tyrosine kinase in complex with a peptide substrate and an ATP analog has been determined at 1.9 A resolution. The activation loop (A-loop) of the kinase undergoes a major conformational change upon autophosphorylation of Tyr1158, Tyr1162 and Tyr1163 within the loop, resulting in unrestricted access of ATP and protein substrates to the kinase active site. Phosphorylated Tyr1163 (pTyr1163) is the key phosphotyrosine in stabilizing the conformation of the tris-phosphorylated A-loop, whereas pTyr1158 is completely solvent-exposed, suggesting an availability for interaction with downstream signaling proteins. The YMXM-containing peptide substrate binds as a short anti-parallel beta-strand to the C-terminal end of the A-loop, with the methionine side chains occupying two hydrophobic pockets on the C-terminal lobe of the kinase. The structure thus reveals the molecular basis for insulin receptor activation via autophosphorylation, and provides insights into tyrosine kinase substrate specificity and the mechanism of phosphotransfer.