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Biomedical subjects

Y Zick

Publications and source records attributed to Y Zick.

At least 73 records · Page 4Linked to original sources
PubMed1986

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase.

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.

Animals↗
PubMed1986

Multisite phosphorylation of the alpha subunit of transducin by the insulin receptor kinase and protein kinase C.

The GDP-bound alpha subunit of transducin, but not the guanosine 5'-[gamma-thio]triphosphate-bound one, undergoes phosphorylation on tyrosine residues by the insulin receptor kinase and on serine residues by protein kinase C. Holotransducin is poorly phosphorylated by the insulin receptor kinase and is not phosphorylated by protein kinase C. Neither holotransducin nor any of its subunits were phosphorylated by the cAMP-dependent protein kinase. That a given subunit of transducin undergoes multisite phosphorylation depending on the type of nucleotide bound to it or the nature of the kinase suggests that hormone-dependent phosphorylation could provide a versatile mode for regulation of guanine nucleotide-binding protein (G protein) function. In particular, the findings that certain G proteins serve as substrates for both the insulin receptor kinase and protein kinase C implicate G proteins in playing a key role in mediating the action of insulin and ligands that act to activate protein kinase C.

Animals↗
PubMed1985

Characterization of insulin-like growth factor I-stimulated tyrosine kinase activity associated with the beta-subunit of type I insulin-like growth factor receptors of rat liver cells.

We previously reported that insulin-like growth factor I (IGF-I) stimulates the phosphorylation of a Mr 98,000 protein thought to be the beta-subunit of the type I IGF receptor of BRL-3A2 rat liver cells, as well as phosphorylation of the exogenous tyrosine-containing substrate poly(Glu,Tyr), 4:1. The present study provides additional evidence that the type I IGF receptor possesses intrinsic tyrosine kinase activity and characterizes the properties of this receptor kinase. IGF-I stimulates receptor phosphorylation and phosphorylation of poly(Glu,Tyr), 4:1, by lectin-purified receptor preparations with the same concentration dependence; half-maximal stimulation was observed with approximately 3 nM IGF-I and approximately 3-fold higher concentrations of insulin. Although IGF-I-dependent receptor phosphorylation was observed within 2 min and was maximal after 10 min, phosphorylation of exogenous substrate did not begin to increase until 8 min after addition of [gamma-32P] ATP and poly(Glu,Tyr), 4:1. When IGF-I-receptor complexes were preincubated with unlabeled ATP for 10 min before addition of substrate, however, IGF-I-dependent 32P incorporation was observed within 2 min after addition of poly(Glu,Tyr), 4:1, and increased linearly for 20 min. We propose that this activation of type I IGF receptor tyrosine kinase activity results from autophosphorylation of the receptor kinase. Kinase activation is an intramolecular reaction, is specific for ATP, occurs within 3 min after addition of unlabeled ATP, and requires the presence of IGF-I before or concomitant with activation by ATP. IGF-I acts rapidly, stimulating substrate phosphorylation within 3 min. The properties of the type I-IGF receptor kinase closely resemble those of the insulin receptor kinase, suggesting that the homologies between the two receptors extend to their kinase domains.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Insulin-stimulated receptor phosphorylation appears normal in cultured Epstein-Barr virus-transformed lymphocyte cell lines derived from patients with extreme insulin resistance.

Insulin-stimulated phosphorylation of the insulin receptor was studied in cultured B-lymphocytes transformed by Epstein-Barr virus. In studies with cell lines derived from six normal subjects, insulin (10(-7) M) caused an average increase of approximately 200% in 32P incorporation into the 95K subunit of the insulin receptor. Phosphorylation was rapid (detectable within 1-2 min) and reached a maximum level by 15 min. Dose-response curves for receptor occupancy and phosphorylation were nearly superimposable, indicating few or absent spare receptors for this response to insulin. These data suggest that insulin receptor phosphorylation is an early response to insulin in cultured lymphocytes transformed with Epstein-Barr virus. We studied insulin receptor phosphorylation in cell lines derived from nine patients with clinical syndromes associated with extreme insulin resistance, all of whom had normal [125I] insulin binding. While the magnitude of insulin's stimulation varied widely among the individual cell lines, no significant differences were found between cell lines from normal subjects and those from patients with extreme insulin resistance.

Cell Line↗
PubMed1984

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor.

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.

Animals↗
PubMed1984

Defect in phosphorylation of insulin receptors in cells from an insulin-resistant patient with normal insulin binding.

Mononuclear blood cells were obtained from a patient with type A insulin resistance. The cells showed a normal ability to bind iodine 125-labeled insulin. Analysis of solubilized insulin receptors from the patient's cells revealed a defect in insulin-stimulated tyrosine kinase activity, which is closely associated with the receptor itself. The enzyme failed to phosphorylate the insulin receptor and showed a markedly reduced ability to phosphorylate exogenously added substrates. It appears that receptors from this insulin-resistant patient have a defect distal to the insulin-binding site (the alpha subunit of the receptor). The defect could be located in the beta subunit, which has an adenosine triphosphate-binding site, or in another receptor component that transfers a signal of insulin binding into kinase activity. This dissociation between the normal binding and the defective protein kinase component of the insulin receptor represents the first biochemical defect of the receptor distal to ligand binding.

Caseins↗
PubMed1984

Insulin-like growth factor-I (IGF-I) stimulates tyrosine kinase activity in purified receptors from a rat liver cell line.

Solubilized, lectin-purified receptor preparations from BRL 3A2 rat liver cells are rich in Type I and Type II IGF receptors, but possess few insulin receptors. High concentrations of IGF-I or insulin stimulate phosphorylation of a Mr congruent to 98K membrane protein in these preparations. Phosphorylation of a synthetic polymer of tyrosine and glutamic acid was stimulated by IGF-I greater than IGF-II congruent to insulin. These relative potencies, together with the results of immunodepletion experiments using an autoantibody to the insulin receptor, suggest that the effects of each of these hormones is mediated by the Type I IGF receptor. Our results are consistent with the Type I IGF receptor having intrinsic tyrosine kinase activity capable of phosphorylating the receptor itself and other substrates.

Animals↗
PubMed1984

Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter.

Cells, Cultured↗
PubMed1983

The insulin-stimulated receptor kinase is a tyrosine-specific casein kinase.

Insulin stimulates a kinase that phosphorylates tyrosines in the insulin receptor; this kinase is tightly associated with the insulin receptor itself. We now show that the insulin-stimulated casein kinase, present in solubilized, lectin-purified receptor preparations from rat liver, is indistinguishable from the insulin receptor kinase. As with phosphorylation of the insulin receptor, insulin selectively enhanced by 2-3-fold the phosphorylation of tyrosines in casein. The insulin-stimulated activities of both kinases were inactivated at 37 degrees C with the same t0.5 of 5 min and were identically affected by alkylating agents. Both receptor and casein kinase activities were specifically coprecipitated by anti-receptor antibodies or by insulin and anti-insulin antibodies. When the latter type of immune complexes were incubated with an excess of insulin, both kinase activities were quantitatively recovered. We therefore conclude that insulin-stimulated receptor and casein phosphorylations are probably catalyzed by a single enzyme which is tightly associated with the receptor itself. Now, by replacing casein for receptor as substrate, it is possible to measure the enzymatic activity of this receptor-related kinase itself, i.e. independent of the receptor as substrate. Detection of this activity is improved in the presence of certain alkylating agents. Use of artificial substrates (in combination with alkylating agents) is particularly important to dissect the functional components of the receptor complex, to study mechanisms of enzyme regulation and especially in situations where the available receptor for study is limited, e.g. fresh or cultured cells from patients.

Amino Acids↗
PubMed1983

Insulin stimulates phosphorylation of serine residues in soluble insulin receptors.

Using lectin affinity-purified receptor preparations from human hepatoma cells, insulin (10(-7)M) specifically stimulated phosphorylation of the 95,000 dalton (beta) subunit of its own receptor. Phospho-amino acid analysis of the receptor subunit revealed that insulin increased at least 2.5-fold the content of phosphoserine and of phosphotyrosine. In intact cells, the major effect of insulin is to increase the phosphoserine content of its receptor. These findings are the first demonstration of an insulin-stimulated serine kinase in a cell-free system.

Carcinoma, Hepatocellular↗
PubMed1983

Insulin-stimulated phosphorylation of the insulin receptor precursor.

The alpha and beta subunits of the insulin receptor, Mr = 135K and 95K, appear to be synthesized via a single polypeptide precursor of Mr = 190K. We have investigated whether insulin stimulates the phosphorylation of this proreceptor, as is the case with mature receptor. Rat liver endoplasmic reticulum membranes were solubilized in Triton X-100 and chromatographed sequentially on wheat-germ agglutinin-agarose and lentil lectin-agarose columns. Phosphorylation of the lentil eluate with [gamma 32P]ATP revealed an insulin-stimulated phosphoprotein of Mr = 192K, which was recognized by antireceptor antibody, compatible with the receptor precursor. This suggests that further processing of the Mr = 190K insulin receptor precursor is not necessary for insulin binding, kinase activation, and receptor phosphorylation.

Animals↗
PubMed1983

Protein kinase activity of the insulin receptor in human circulating and cultured mononuclear cells.

In lectin-purified receptor preparations from human monocyte-like cell (U-937), insulin (10(-7)M) stimulated phosphorylation of the 95,000 dalton subunit of its own receptor. In addition, insulin stimulated phosphorylation of exogenously added substrates like casein, (T,G)-A--L, and histones. Phosphorylation of the synthetic peptide (T,G)-A--L indicates the presence of at least one insulin-dependent tyrosine kinase in these cell extracts. Insulin receptor preparations from freshly isolated human mononuclear blood cells were also shown to possess insulin-dependent casein and (T,G)-A--L kinase activity. Phosphorylations in these systems are specific for insulin and dependent on insulin concentration. A simple and rapid method is described that is relevant for clinical investigations of early postbinding events.

Cells, Cultured↗
PubMed1983

Viable mouse thymocytes as a model system for studying the onset of hormone-induced cellular refractoriness.

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (beta-agonists, prostaglandin E1 and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, 'locked' desensitized state.

Adenylyl Cyclases↗
PubMed1983

Insulin stimulated phosphorylation of its own receptor. Activation of a tyrosine-specific protein kinase that is tightly associated with the receptor.

In solubilized, (wheat germ) lectin-purified preparations of rat liver membranes, insulin stimulated the incorporation of 32P from [gamma-32P]ATP into tyrosine residues of insulin receptor, casein, and histones. Despite the presence of both protein kinase and phosphatase activities in these preparations, no decrease in the 32P content of receptors (preincubated with or without insulin (0.5-100 nM)) was detected whether 32P incorporation was terminated by excess ATP, ATP + Mn2+, EDTA, or phosphatase inhibitors. Similarly, there was no decrease in the 32P content of phosphoreceptors incubated for up to 60 min with fresh receptor preparations in the presence or absence of insulin. Dephosphorylation of the insulin receptor to 20% of original 32P content only occurred when alkaline phosphatase was added to the preparations. It is concluded that endogenous receptor phosphatase(s) are either missing or inactive in these preparations, and consequently, insulin stimulates phosphorylation of its own receptor by activating a protein kinase. The kinase activity is tightly associated with the receptor itself; insulin also stimulated the phosphorylation of both receptor subunits in purified insulin-receptor complexes that had been immunoprecipitated by anti-insulin antibodies. However, the phosphorylating machinery is much more sensitive to heat inactivation than the binding function (90% less 32P incorporation versus 15% less binding during 60-min incubation at 37 degrees C), suggesting that the kinase is not associated exclusively with the insulin-binding domain.

Animals↗
PubMed1983

Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system.

Insulin stimulates phosphorylation of both alpha- and beta- subunits of its own receptor in a cell-free system. A solubilized lectin-purified preparation of insulin receptors from rat liver membranes was preincubated with or without insulin at 4 degrees C and labeled for 10 min with Mn[gamma- 32P]ATP; the receptor subunits were isolated by specific immunoprecipitation with anti-receptor antibodies, followed by gel electrophoresis in sodium dodecyl sulfate. In gels run under reduced conditions, two bands (Mr = 135,000 and 95,000) were selectively labeled. These correspond exactly to the position of the alpha- and beta-subunits of the insulin receptor. Labeling of the Mr = 95,000 band was approximately 5-fold that of the Mr = 135,000 band. No labeled bands were detected when identical samples were immunoprecipitated in control serum. Phosphorylation of the receptor subunits required the presence of the divalent cation Mn2+ or Co2+; other cations such as Mg2+, Cr3+, Ca2+, and Zn2+ were ineffective. [gamma- 32P]ATP served as the 32P donor, whereas [gamma- 32P]GTP was ineffective. Phosphorylation of both subunits was stimulated 4-6-fold after a 60-min exposure to 10(-7) M pork insulin. Insulin-stimulated phosphorylation was half-maximal after 5 min of incubation with 10(-7) M insulin or after 18 h with 3 X 10(-10) M hormone. The enhanced phosphorylation was specific for insulin and its analogs; guinea pig insulin was about 2% as potent as pork insulin, whereas epidermal growth factor, adrenocorticotropic hormone, and glucagon, as well as cAMP, were ineffective. The rapidity and specificity of this reaction, as well as the presence of all necessary components in the plasma membrane, suggest that insulin-mediated receptor phosphorylation is one of the earliest biochemical steps following insulin binding.

Adenosine Triphosphate↗
PubMed1982

Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine.

Rat hepatoma cells were labeled with [32P]orthophosphate and the insulin receptor subunits were identified by immunoprecipitation and sodium dodecyl sulfate-acrylamide gel electrophoresis. In the basal state, only the Mr = 95,000 (beta) subunit of the insulin receptor was phosphorylated. The covalent labeling with 32P of this subunit was stimulated about 3-fold by insulin (10(-6) M). This stimulation was due to an increase in the content of phosphoserine, the appearance of phosphotyrosine, and a possible increase in phosphothreonine as well. These results suggest phosphorylation of the insulin receptor at multiple sites is an early event in insulin action.

Amino Acids↗