Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.     

Stimulation of tyrosine-specific protein phosphorylation and phosphatidylinositol phosphorylation by orthovanadate in rat liver plasma membrane

Orthovanadate stimulated the incorporation of 32P from [gamma-32P]ATP by Triton X-100-solubilized rat liver plasma membrane into endogenous, trichloroacetic acid-precipitable materials as well as added (Glu4:Tyr1) copolymers. Extraction of incubation mixture with chloroform-methanol-HCl revealed that the increase in 32P incorporation by vanadate was predominantly into endogenous phospholipids. [32P]Phosphatidylinositol 4-phosphate (PtdIns-4-P) was identified by thin-layer chromatography as the major phosphorylated product of vanadate stimulation, which also resulted in elevated 32P, predominantly in P-Tyr in endogenous membrane proteins. Vanadate effects on protein tyrosine and phosphatidylinositol phosphorylation were concomitant and exhibited similar sensitivity. These effects of vanadate were enhanced by the presence of either dithiothreitol or NAD(P)H. Phosphatidylinositol phosphorylation could also be stimulated by a substrate of and inhibited by a synthetic inhibitory copolymer of tyrosine kinase. These results suggest that vanadate, an oxygen radical producer, stimulates a tyrosine kinase-PtdIns kinase coupled system much like those described for a number of growth factors and oncogene encoded products.     

Insulin stimulation of the insulin receptor kinase can occur in the complete absence of beta subunit autophosphorylation

The glutamic acid:tyrosine (Glu:Tyr) synthetic polymer was observed to inhibit the insulin receptor beta subunit autophosphorylation with an IC50 of 0.20 mg/ml in the absence and 0.15 mg/ml in the presence of insulin. Even though complete blockade of beta subunit autophosphorylation was observed at 4.0 mg/ml Glu:Tyr, insulin was still capable of stimulating the exogenous protein kinase activity of the insulin receptor toward Glu:Tyr. Histone H2B (1.3 mg/ml) was also observed to inhibit the beta subunit autophosphorylation by approximately 80% with an IC50 of 0.31 and 0.35 mg/ml in the absence and presence of insulin, respectively. Similar to the results with Glu:Tyr, insulin was found to stimulate histone H2B phosphorylation under these conditions. Comparisons between the time courses of beta subunit autophosphorylation with those of Glu:Tyr phosphorylation both in the presence and absence of insulin confirmed that insulin can stimulate the exogenous protein kinase activity of the insulin receptor in the complete absence of beta subunit autophosphorylation. Prephosphorylation of the insulin receptor (from 0 to 1.3 mol of phosphate/mol of insulin receptor) in the absence of insulin was found to have no significant effect on the exogenous protein kinase activity when assayed both in the presence and absence of insulin. Insulin was observed to stimulate the phosphorylation of Glu:Tyr approximately 3-fold independent of the extent of beta subunit autophosphorylation. In contrast, prephosphorylation of the insulin receptors in the presence of insulin was observed to enhance the exogenous protein kinase activity dependent on the extent of autophosphorylation, such that by 1.4 mol of phosphate incorporated per mol of insulin receptor, insulin was found to maximally stimulate the initial rate of Glu:Tyr phosphorylation (approximately 9-fold). These results demonstrate that the insulin-dependent autophosphorylation of the insulin receptor results in an amplification of the insulin stimulation of the exogenous protein kinase activity, whereas the insulin-independent autophosphorylation does not.     

Various proteins modulate the kinase activity of the insulin receptor

Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.     

Diacylglycerols modulate phosphorylation of the insulin receptor from human mononuclear cells

It has been found that 1,2- but not 1,3-diacylglycerols stimulated phosphorylation of the insulin receptor of cultured human monocyte-like (U-937) and lymphoblastoid (IM-9) cells both in the intact- and broken-cell systems. The stimulation of the receptor's beta-subunit phosphorylation was dose-dependent, with optimal effect at 100 micrograms/ml of diacylglycerol. The effects of insulin and 1,2-diacylglycerols on the phosphorylation of partially purified insulin receptors were additive. Phosphoamino acid analysis showed a major effect of diacylglycerols on phosphorylation of tyrosine residues. The diacylglycerols also stimulated tyrosine kinase activity of the partially purified U-937 and IM-9 insulin receptors 2.5-3.5-fold when measured by phosphorylation of an exogenous substrate, poly(Glu80Tyr20) in the absence of any added insulin, calcium or phospholipid. Since this diacylglycerol effect could not be reproduced under conditions optimal for protein kinase C activation and the purified protein kinase C did not stimulate phosphorylation of the beta-subunit of the insulin receptor in this system, it is unlikely that the diacylglycerol effect was mediated by protein kinase C. Since these exogenous 1,2-diacylglycerols at the same high concentration also inhibited 125I-insulin binding to the insulin receptor of the intact U-937 and IM-9 cells, diacylglycerols could modulate the function of the insulin receptor and insulin action in human mononuclear cells.     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

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.     

Internalization and activation of the rat liver insulin receptor kinase in vivo

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.     

Inhibition of the activation and catalytic activity of insulin receptor kinase by zinc and other divalent metal ions

The autophosphorylation reaction responsible for conversion of insulin receptor (from human placenta) to an active tyrosyl-protein kinase was shown to be inhibited by Zn2+ and other divalent metal ions. The order of inhibitory potency was found to be Cu2+ greater than Zn2+, Cd2+ greater than Co2+, Ni2+. Autophosphorylation of insulin receptor was almost completely blocked by 10 microM Zn2+. Zn2+, however, did not appear to affect the binding of insulin to its receptor. Histidine, a chelator of Zn2+, protected against the inhibitory effects of Zn2+. The failure of histidine to regenerate the competence of the Zn2+-inhibited receptor to undergo autophosphorylation suggested that the inhibition by Zn2+ was irreversible. In addition to inhibiting autophosphorylation, Zn2+ inhibited the tyrosyl-protein kinase activity of highly purified phosphorylated receptor. Zn2+ was also observed to inhibit phosphotyrosyl-protein phosphatase activity present in preparations of partially purified insulin receptor. These inhibitory effects of Zn2+ should be considered in the design of protocols for the isolation and handling of insulin receptor and possibly other tyrosine kinases. Additionally, the possible physiological significance of the inhibition of insulin receptor kinase by Zn2+ is discussed in light of the fact that Zn2+ is accumulated in and secreted from pancreatic islet cells together with insulin.     

Insulin receptor autophosphorylation and kinase activity in streptozotocin diabetic rats. Effect of a short fast

Insulin receptor associated kinase activity and its relationships with the insulin resistance of streptozotocin-induced diabetes were investigated in rats, using solubilized, partially purified insulin receptors from liver membranes. Insulin receptor kinase activity was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. Diabetes was associated with a 45% reduction in kinase activity, in the same number of insulin receptors, with no change in insulin binding affinity. To investigate the independent roles of hyperglycemia and hypoinsulinemia on the observed impairment of receptor kinase activity, diabetic rats were fasted for 24 h in order to normalize blood glucose levels only. After this short fast, no change in kinase activity, from the values measured in fed diabetic animals, was observed. Our findings suggest that streptozotocin diabetes is associated with a reduction of insulin receptor kinase activity, which a short fast is not able to reverse.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity.

To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.     

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)     

The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.

We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.     

猪脾脏酪氨酸蛋白激酶的研究

 采用DEAE-Sepharose CL-6B离子交换层析和Tyrosine-agarose亲和层析,首次从猪脾脏30000Xg颗粒中部分纯化了酪氨酸蛋白激酶,其比活性约为12000pmol.min~(-1).mg~(-1)。磷酸氨基酸分析表明,该酶能催化合成多肽poly(Glu.Ala.Tyr)_n(6:3:1)中的酪氨酸(Tyr)磷酸化,对该多肤废物和ATP的Km值分别约为2mg/mL和15μmol/L。Mn~(2+)对部分纯化的酪氨酸蛋白激酶的最大激活活性高于Mg~(2+),其AC_(50)分别约为1.4mmol/L和8mmol/L;发现红花素能强烈地抑制该酶活性,其IC_(50)约为125μmol/L。     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase.

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.     

Phorbol ester-mediated protein kinase C interaction with wild-type and COOH-terminal truncated insulin receptors.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin were compared in wild-type human insulin receptors (HIRc cells) and human insulin receptors lacking 43 COOH-terminal amino acid residues (HIR delta CT cells). TPA increased total phosphorylation of the wild-type insulin receptor and inhibited insulin-stimulated autophosphorylation by 32 +/- 10% in HIRc cells. TPA inhibited insulin-stimulated autophosphorylation by 46 +/- 14% in HIR delta CT cells and also caused a 65% decrease in basal phosphorylation. Insulin-stimulated tyrosine kinase activity for poly(Glu4/Tyr1) was inhibited by TPA in HIRc and HIR delta CT cells by 50 and 40%, respectively. TPA decreased insulin-stimulated glucose incorporation into glycogen by 50% in HIRc cells and to near basal levels in HIR delta CT cells; this inhibitory effect of TPA was reversed in both cell lines by staurosporine. In conclusion, 1) TPA-induced inhibition of insulin receptor tyrosine autophosphorylation was linked to concomitant inhibition of the biological effects of insulin in cells expressing either wild-type or COOH-terminal truncated insulin receptors; and 2) the inhibitory effects of TPA were not dependent upon phosphorylation of COOH-terminal residues and furthermore appeared to be independent of phosphorylation of any insulin receptor serine/threonine residues. These findings suggest a novel protein kinase C mechanism that results in altered insulin receptor function without increasing phosphorylation of the receptor.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor

On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.