Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.     

Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity

The ability of insulin to activate the insulin receptor protein kinase is shown to be completely dependent on prior beta subunit tyrosine autophosphorylation. Autophosphorylation in the presence of insulin is a highly concerted reaction; tryptic digestion of insulin receptor beta subunits derived from preparations whose kinase activation ranges from under 5% to 100% of maximal yields the same array of [32P]Tyr(P)-containing peptides over the entire range. Of special note is the significant contribution of multiply phosphorylated forms of tryptic peptides corresponding to proreceptor residues 1144-1152 (from the "tyrosine kinase" domain) and 1314-1329 (near the carboxyl terminus) to overall beta subunit phosphorylation at kinase activations of 5% and under. Thus, partially activated/autophosphorylated receptor preparations consist of mixtures of unactivated unphosphorylated receptors and activated fully (or nearly fully) phosphorylated receptors. The latter can be selectively removed by adsorption to antiphosphotyrosine antibodies. This abrupt multiple phosphorylation of individual receptor molecules explains why, in the presence of insulin, overall beta subunit tyrosine phosphorylation tracks closely with kinase, up to approximately 90% activation. Insulin stimulates phosphorylation into all domains (involving at least 6 of the 13 tyrosines on the intracellular portion of the beta subunit) but does not cause the appearance of "new" 32P-labeled species. Rather, insulin directs 32P incorporation preferentially into those domains most productive of kinase activation. Phosphorylation of the tyrosine residues at 1146, 1150, and 1151 correlates most closely with kinase activation. These residues show the largest 32P incorporation during rapid kinase activation; moreover, in comparisons of receptors with similar overall autophosphorylation but very different activations (or similar activations but different extents of autophosphorylation), achieved by omitting insulin or varying [ATP], the phosphorylation of peptide 1144-1152 tracks closely with kinase activation, and phosphorylation of sites and Mr 4000-5000 tryptic peptide (presumably Tyr 953 and/or 960) tract nearly as well. By contrast the extent of phosphorylation of the carboxy-terminal peptide is frequently dissociated from the extent of kinase activation. Phosphorylation of this latter domain probably underlies a beta subunit function other than tyrosine kinase activity.     

Kinetic properties and sites of autophosphorylation of the partially purified insulin receptor from hepatoma cells

Autophosphorylation of the insulin receptor was studied using a glycoprotein fraction solubilized and purified partially from the rat hepatoma cell line, Fao. Incubation of this receptor preparation with [gamma-32P] ATP, Mn2+, and insulin yielded a single insulin-stimulated phosphoprotein of Mr = 95,000 which corresponds to the beta-subunit of the insulin receptor. At 22 degrees C, incorporation of 32P was half-maximal at 30 s and about 90% complete after 2 min. At steady state, about 200 pmol of 32P were incorporated per mg of protein; this value corresponded to about 2 molecules of phosphate per insulin binding site estimated from Scatchard plots. Insulin increased the Vmax for autophosphorylation of the insulin receptor kinase nearly 20-fold with no effect on the Km for ATP. Mn2+ stimulated autophosphorylation by decreasing the Km of the kinase for ATP, whereas Mg2+ had no effect. Dilution of the insulin receptor over a 10-fold concentration range did not decrease the rate of autophosphorylation suggesting that it may occur by an intramolecular mechanism. When the phosphorylated beta-subunit of the insulin receptor was digested with trypsin, at least 5 phosphopeptides could be separated by high performance liquid chromatography on a mu Bondapak C18 reverse-phase column. Insulin stimulated the phosphorylation of all sites. These phosphate acceptor sites varied in their rate and degree of phosphorylation. Phosphopeptides pp4 and pp5 were phosphorylated very rapidly and reached steady state within 20 s, whereas phosphorylation of pp1 and pp2 required several minutes to reach steady state.     

Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.     

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.     

An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta.

The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.     

Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [gamma-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the beta-subunit digested with trypsin revealed one minor and two major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the beta-subunit of the insulin-stimulated receptor labeled in vivo. In contrast, the elution position of one of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Our results indicate that: tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessibility of the phosphorylation sites are different in vivo and in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.     

Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of insulin receptor tyrosine residues autophosphorylated in vitro

To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.     

Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tyrosine phosphorylation of insulin receptor beta subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells

The phosphorylation characteristics of insulin receptor from control and insulin-treated rat H-35 hepatoma cells 32P-labeled to equilibrium have been documented. The 32P-labeled insulin receptor is isolated by immunoprecipitation with patient-derived insulin receptor antibodies in the presence of phosphatase and protease inhibitors to preserve the native phosphorylation and structural characteristics of the receptor. The unstimulated insulin receptor contains predominantly [32P] phosphoserine and trace amounts of [32P]phosphothreonine in its beta subunit. In response to insulin, the insulin receptor beta subunit exhibits marked tyrosine phosphorylation and a 2-fold increase in total [32P]phosphoserine contents. High pressure liquid chromatography of the tryptic hydrolysates of the 32P-labeled receptor beta subunit from quiescent cells results in the resolution of up to 9 fractions containing [32P]phosphoserine. The insulin-stimulated tyrosine phosphorylation is concentrated in two of these receptor phosphopeptide fractions, whereas the increase in [32P]phosphoserine content is scattered in low abundance over all receptor tryptic fractions. Insulin receptors affinity-purified by lectin- and insulin-agarose chromatographies from insulin-treated, 32P-labeled cells exhibit a 22-fold increase in the Vmax of receptor tyrosine kinase activity toward histone when compared to controls. The elevated kinase activity of the insulin receptor derived from insulin-treated cells is not due to the presence of hormone bound to the receptor because the receptor kinase activity is assayed while immobilized on insulin-agarose. Furthermore, the insulin-activated receptor kinase activity is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase in vitro. The correlation between the insulin-stimulated site specific tyrosine phosphorylation on receptor beta subunit and the elevation of receptor tyrosine kinase activity strongly suggests that the insulin receptor kinase is activated by hormone-stimulated autophosphorylation on tyrosine residues in intact cells, as previously demonstrated for the purified receptor.     

An endogenous substrate for the insulin receptor-associated tyrosine kinase

Insulin binding to its receptor stimulates a tyrosine-specific protein kinase. This enzyme phosphorylates the insulin receptor, as well as a variety of exogenous substrates in vitro. In the present studies, we have identified an endogenous substrate for the insulin receptor-associated kinase. We studied insulin-stimulated protein phosphorylation in partially purified insulin receptor preparations from the livers of dexamethasone-treated rats. In this cell-free system, insulin stimulated the phosphorylation of its own receptor as well as of a phosphoprotein of apparent Mr = 120,000 (termed pp120). pp120 was not immunoprecipitated by three anti-receptor antisera, nor was the receptor immunoprecipitated by antisera raised against pp120, suggesting that pp120 is not antigenically related or tightly bound to the insulin receptor. Dose-response curves for receptor and pp120 phosphorylation stimulated by pork insulin were essentially identical, and showed the appropriate specificity (insulin much greater than proinsulin) for a receptor-mediated event. Phosphoamino acid analysis revealed that insulin stimulated the incorporation of 32P predominantly into tyrosine residues of pp120. Casein, an artificial substrate for the insulin receptor kinase, competed with pp120 for insulin-stimulated phosphorylation. Phosphorylation of pp120 was rapid (half-maximal effect within 2 min at 24 degrees C) and, like receptor phosphorylation, was supported with Mn2+ or Mg2+ as divalent cation and ATP as the phosphate donor. While receptor autophosphorylation and artificial substrate phosphorylation were not altered by prior treatment of the rats with dexamethasone, insulin-stimulated pp120 phosphorylation was enhanced in preparations derived from dexamethasone-treated rats, suggesting an alteration of pp120, not the receptor, as a result of dexamethasone-treatment. Further studies of this newly identified endogenous substrate may help clarify the physiologic role of the insulin receptor-associated kinase.     

Identification of autophosphorylation sites of HER2/neu.

HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2.     

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.     

Phosphorylation of synthetic insulin receptor peptides by the insulin receptor kinase and evidence that the preferred sequence containing Tyr-1150 is phosphorylated in vivo

Three peptides were synthesized corresponding to potential autophosphorylation sites of the beta subunit of the human insulin receptor. These were peptide 1150 corresponding to amino acids 1142-1153 of the pro-receptor, peptide 960 corresponding to amino acids 952-961 of the proreceptor, and peptide 1316 corresponding to amino acids 1313-1329 of the proreceptor. Peptide 1150 served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the Src peptide (corresponding to the sequence surrounding the autophosphorylation site at Tyr-416). Microsequencing of the phosphorylated peptide 1150 indicated that Tyr-1150 rather than Tyr-1146 or Tyr-1151 was phosphorylated in the in vitro reaction. The insulin receptor was then isolated from 32P-labeled IM-9 cells that had been exposed to insulin. Tryptic digestion of the beta subunit revealed one peptide whose phosphorylation was dependent upon insulin and occurred exclusively on Tyr. This peptide was selectively immunoprecipitated by an antipeptide antibody directed to the Tyr-1150-containing sequence. We conclude that Tyr-1150 is preferentially phosphorylated by the purified receptor kinase and that one of the autophosphorylation reactions elicited by insulin in intact cells occurs in a sequence that contains this residue.     

Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.     

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.