Phosphorylation of the insulin receptor in cultured hepatoma cells and a solubilized system.
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Animals↗
Biomedical subjects
M F White
Publications and source records attributed to M F White.
Giant cell arteritis presenting as a breast lump.
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Aged↗
Phosphorylation and dephosphorylation of the insulin receptor: evidence against an intrinsic phosphatase activity.
We have studied the reversibility of insulin receptor phosphorylation to establish the relation between this autophosphorylation reaction and the initiation of insulin action and between dephosphorylation and the termination of insulin effects in cells. In cultured Fao hepatoma cells labeled with 32PO4(3-), insulin increased 5-fold the phosphorylation of the beta-subunit of the insulin receptor at serine, threonine, and tyrosine residues. Addition of anti-insulin antiserum to cells incubated with insulin caused dissociation of insulin from the receptor and concurrent dephosphorylation of the beta-subunit. 32PO4(3-) associated with the insulin-stimulated receptor could be decreased by the addition of sodium phosphate to the medium but with a slower time course. Insulin stimulated phosphorylation of insulin receptor purified partially on immobilized wheat germ agglutinin. This reaction utilized [gamma-32P] ATP and occurred exclusively on tyrosine residues. Addition of unlabeled ATP caused a decrease in the amount of PO4(3-) associated with the receptor. Insulin-stimulated phosphorylation was also observed if the receptors were further purified by immunoprecipitation with anti-insulin receptor antibody prior to the phosphorylation reaction; however, addition of unlabeled ATP to this system did not chase the labeled 32PO4(3-) from the beta-subunit. These data are consistent with the notion that phosphorylation and dephosphorylation of the insulin receptor parallel the onset and termination of insulin action. Phosphatase activity involved in the dephosphorylation of the insulin receptor appears to be a glycoprotein because it was retained after partial purification of the receptor on wheat germ agglutinin-agarose; however, this phosphatase activity is distinct from the insulin receptor because it was not retained after immunoprecipitation of the receptor with anti-insulin receptor antibodies.
Animals↗
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.
Adenosine Triphosphate↗
Phosphorylation of the solubilized insulin receptor by the gene product of the Rous sarcoma virus, pp60src.
Both the insulin receptor and the gene product of the Rous sarcoma virus, pp60src, are protein kinases which phosphorylate themselves and other proteins on tyrosine residues. Addition of the solubilized insulin receptor to purified pp60src increased the phosphorylation of the beta-subunit of the insulin receptor. Phosphorylation of the insulin receptor by pp60src occurred both in the absence and presence of insulin but did not alter the insulin dose response for autophosphorylation of the receptor. Increasing concentrations of pp60src increased the phosphorylation of the receptor and at high concentrations equaled the maximal effect produced by insulin. Our observations suggest a possible mechanism by which the metabolically regulated insulin receptor tyrosine kinase could be altered by other tyrosine kinases such as that associated with pp60src. Further studies will be required to determine if the insulin receptor is phosphorylated by pp60src in Rous sarcoma virus-infected cells.
Animals↗
Phorbol esters modulate insulin receptor phosphorylation and insulin action in cultured hepatoma cells.
The effect of the tumor-promoting agent phorbol 12-myristate 13-acetate (PMA) on insulin receptors and insulin action was studied in rat hepatoma cells in culture. PMA (0.1-1.0 micrograms/ml) did not affect insulin binding either acutely or chronically but inhibited insulin stimulation of glycogen synthase and tyrosine aminotransferase. PMA (1 microgram/ml) stimulated the phosphorylation of the beta subunit of insulin receptor purified from [32P]phosphate-labeled Fao cells by 1.3-fold in the absence of insulin. In contrast, insulin-stimulated phosphorylation in the presence of PMA was reduced. Phosphoamino acid analysis of the beta subunit after PMA stimulation revealed an increase of both phosphoserine and phosphothreonine residues, whereas insulin stimulated primarily phosphorylation of tyrosine and serine residues. Insulin stimulation of cells after PMA treatment revealed a decrease in phosphotyrosine when compared to cells stimulated by insulin alone. Tryptic peptide mapping of the beta subunit by a two-dimensional chromatographic/electrophoretic separation revealed nine phosphopeptides from the cells treated with PMA. Insulin stimulated phosphorylation at six new sites in the receptor, three of which appeared to be similar to those in PMA-treated cells. This report shows that phorbol esters stimulate insulin receptor phosphorylation, inhibit insulin-induced receptor phosphorylation and insulin action, and suggest a physiologic relation between insulin action and the calcium-activated and phospholipid-dependent protein kinase C.
Animals↗
Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.
The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.
Binding, Competitive↗
Insulin binding and insulin-dependent phosphorylation of the insulin receptor solubilized from human erythrocytes.
Insulin receptors were extracted from human erythrocytes contained in 100 ml of blood with the nonionic detergent Triton X-100 with almost 100% yield. The solubilized receptor had binding characteristics similar to those of the intact cell. Using 125I-monoiodoinsulin as tracer and a computer-assisted statistical curve-fitting program, a cooperative model gave values of 1.7 X 10(9) M-1 for the Ke (affinity of the empty receptor) and of 1.6 X 10(8) M-1 for Kf (affinity of the filled receptor). Bovine desalanine-desasparagine insulin inhibited tracer binding with 3-6% the potency of porcine insulin. Serum (B-8) containing anti-insulin receptor antibodies inhibited binding by 70% at a dilution of 1:100. Receptor autophosphorylation reaction was studied by incubation of the Triton extract with [gamma-32P]ATP and Mn2+ in the presence or absence of insulin, and the receptor was identified by immunoprecipitation with anti-receptor antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Porcine insulin stimulated 4- to 5-fold the incorporation of 32P in a protein of Mr = 95,000, corresponding to the beta-subunit of the insulin receptor. Phosphoamino acid analysis revealed phosphorylation of the tyrosyl residues exclusively. The dose-response curve for insulin stimulation was sigmoidal; some effect of insulin was observed at 1 ng/ml but maximal effect was observed at 10 micrograms/ml. Bovine desalanine-desasparagine insulin, a noncooperative analogue of insulin, was able to fully stimulate the phosphate incorporation, but the dose-response curve was shifted to the right and steeper, consistent with the intrinsic affinity of this analogue for the insulin receptor. When insulin binding was performed under the same conditions as the phosphorylation, half-maximal stimulation of phosphate incorporation occurred with 6-29% of the fractional occupancy of the receptor. These data suggest that the insulin receptor of the human erythrocyte, as in other cells, is a tyrosine-specific protein kinase. Coupling between the receptor occupancy and kinase activation is complex. Furthermore, sufficient quantities of receptor can be easily obtained from a single individual to study the binding and kinase properties of the receptor opening the opportunity to a wide field of applications in human pathology.
Amino Acids↗
Characterization of the insulin receptor kinase purified from human placental membranes.
The insulin receptor purified from human placenta by sequential affinity chromatography on wheat germ agglutinin- and insulin-Sepharose to near homogeneity retained tyrosine-specific protein kinase activity. This purified insulin receptor kinase specifically catalyzed the incorporation of 32P from [gamma-32P]ATP into not only the beta-subunit of the insulin receptor but also histone H2B, a synthetic peptide which is sequentially similar to the site of tyrosine phosphorylation in pp60src (a gene product of the Rous sarcoma virus) and antibodies to pp60src present in the sera obtained from three rabbits bearing tumors induced by the Rous sarcoma virus. In each case, phosphorylation occurred exclusively on tyrosine residues. Insulin stimulated phosphorylation of these substrates 3- to 5-fold. Kinetic analysis using the synthetic peptide indicated that insulin acted by increasing the Vmax of peptide phosphorylation from about 3.1 to 9.5 nmol X mg-1 of protein X min-1, whereas the value of the Km for the peptide, about 1.5 mM, was not significantly changed. This kinase acted weakly on casein, alpha-S-casein, actin, and a tyrosine-containing peptide analogue of a serine-containing peptide used commonly as a substrate for the cyclic AMP-dependent protein kinases. These data show that the insulin receptor kinase displays specificity toward exogenous substrates similar to the substrate specificity observed for pp60src and the protein kinase activity associated with the receptor for epidermal growth factor. The data suggest that the catalytic sites of these three tyrosine kinases are similar and that insulin activates its receptor kinase by increasing the Vmax.
Animals↗
Simultaneous regulation of amino acid influx and efflux by system A in the hepatoma cell HTC. Ouabain simulates the starvation-induced derepression of system A amino acid transport.
In the cultured hepatoma cell HTC, amino acid starvation stimulated both influx and efflux of 2-(methylamino)isobutyric acid (MeAIB) across the plasma membrane with little effect on the ultimate cellular accumulation of this amino acid. In agreement, prior amino acid starvation had little effect on the cellular steady state levels reached for various natural amino acids during subsequent incubation in an amino acid-rich medium containing cycloheximide. Furthermore, efflux of [14C]MeAIB was markedly increased by amino acid starvation. These findings do not mean that adaptive regulation of neutral amino acid transport is pointless. If membrane transport rather than metabolism is the rate-limiting step for net amino acid production or consumption, or becomes so during times of elevated formation or catabolism of an amino acid, then proportionate changes of both the opposed fluxes should enhance its net generation or consumption. Amino acid starvation enhances MeAIB-dependent Na+ influx. Alteration of the external [Na+] changes the Km, not the Vmax, for MeAIB influx when the degree of System A derepression is stabilized with cycloheximide. In both starved and unstarved cells, Km/Vmax for MeAIB entry yields a linear function with the reciprocal of the external [Na+], supporting at least for influx a rapid equilibrium-ordered kinetic model in which Na+ binds to the carrier site before the amino acid. Elevated cellular [Na+] obtained by ouabain treatment increased MeAIB efflux in parallel. Trans-inhibition of MeAIB influx by accumulated MeAIB, and as a related phenomenon by cellular Na+, was as effective in unstarved as in starved cells, showing independence of this kinetic phenomenon from adaptive regulation. The decreased MeAIB accumulation resulting from decreased influx and increased efflux occurring at high internal [Na+] applies both to unstarved and starved cells. We conclude that cellular Na+ accumulations, produced by increasing levels of ouabain, reversibly reduce the ability of MeAIB to repress System A because its interior concentration is prevented from rising, although transport in both directions continues; accordingly, the repressive signal appears to come from the internal amino acid levels rather than from occupation of the carrier site for System A flux.
3-O-Methylglucose↗
The two-way flux of cationic amino acids across the plasma membrane of mammalian cells is largely explained by a single transport system.
We show in this report that the inward and outward transport of cationic amino acids through the plasma membrane of cultured fibroblasts and HTC cells is mediated mostly by a single saturable transport system, here designated y+. The kinetic behavior of System y+ can be accounted for by the alternating generation of at least two conformational states, one on each side of the plasma membrane. No predictions are made as to the mechanism of these carrier oscillations. The saturable flux of cationic amino acids in both directions is strongly increased by the presence of competing substrates on the side of the membrane opposite to the measured flux. The kinetic parameters describing these interactions vary as predicted by the classical mobile carrier hypothesis. These observations support the homogeneity of mediated cationic amino acid transport. Our results do not exclude other small flux contributions by a minor, apparently passive nonsaturable migration in each direction. The mediated arginine influx is half-maximally saturated at an external substrate concentration 1/10 to 1/20 as high as the apparent intracellular concentration that half-maximally saturates efflux. The maximum flux through System y+ attained by raising the substrate concentration to a saturating level is about equal in the two directions. The test amino acids accumulate 5- to 20-fold into HTC cells and human skin fibroblasts, findings which are consistent with the kinetic asymmetry of System y+.
Amino Acids, Diamino↗
Cationic amino acid transport into cultured animal cells. I. Influx into cultured human fibroblasts.
Cationic amino acid transport into human skin fibroblasts occurs by a saturable mediation which we designate System y+, which may prove identical with the earlier Ly. This plasma membrane agency is kinetically distinguishable from systems transporting neutral and acidic amino acids and serves to catalyze the flows of omega-guanidino amino acids and alpha, omega-diamino acids. Uptake of cationic substrates by System y+ is Na+-independent, pH-insensitive, stereoselective, and inhibitable by neutral amino acids in the presence of Na+. Uptake and exodus of System y+ substrates are strongly stimulated by cationic amino acids inside and outside the cell, respectively. Arginine and homoarginine accumulate in human fibroblasts reaching distribution ratios of more than 20 at external amino acid concentrations in the physiological range.
Amino Acids↗
Cationic amino acid transport into cultured animal cells. II. Transport system barely perceptible in ordinary hepatocytes, but active in hepatoma cell lines.
The transport of cationic amino acids across the plasma membrane of several hepatoma cell lines (HTC, McA-RH7777, McA-RH8994, characterized in detail in the first of these) occurs by a saturable mediation which we designate System y+. Identical experiments with cultured rat hepatocytes usually yield nonsaturating kinetic cures. Accordingly, System y+ contributes little, if at all, to the flux of cationic amino acids in these cells. Analogous to the findings with other tissues, the influx of cationic amino acids into hepatoma cells is Na+- and pH-independent, stereoselective, inhibitable by neutral amino acids in the presence of Na+, and stimulated by cationic amino acids inside of the cell. This final characteristic, called trans-stimulation, is a kinetic property associated with the cationic amino acid transport system in all other eukaryotic cell types studied and provides evidence supporting the operation of System y+. Influx of cationic amino acids into hepatocytes displays no significant trans-stimulation which strongly suggests the absence or alteration of System y+ in this cell. Transport of arginine into hepatocytes is the rate-limiting step for its hydrolysis by arginase. Therefore, the relatively low influx of this amino acid under physiologic conditions due to the attenuation of System y+ activity apparently provides a kinetic barrier separating the extrahepatic arginine pool from the active cytoplasmic enzymes of the hepatic urea cycle. Such a separation may be required for the nutrition and survival of extrahepatic tissues.
Amino Acids↗
The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells.
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Amino Acids↗
A flap suspension technique in blepharoplasty on lower lids.
Blepharoplasty on lower lids frequently results in a downward pull on the free border of the lid and inferomedially on the lateral canthus. Extending the incision in the lower lid upward and lateral to the outer canthus allows most of the excision to occur laterally. Closure then pulls superolaterally, which tends to prevent the commonest complications of blepharoplasty on lower lids.
Adult↗
Conservative face lift surgery.
Face lift surgery almost always is elective; and, because its purpose is to diminish aging problems, it most often is done in those who are aged. Local and general risks, therefore, should be minimized. Wide undermining dictates flaps dissected more deeply and wounds closed well short of the limits of stretch of skin. Less radical undermining described here produces much smaller total wounds, lessens hazards of hematoma, considerably shortens operative and anesthetic times, reduces risks to nerves and ducts, and allows snugger closures. Details of operative technique, including platysma plication, are demonstrated. Short- and long-term results are shown. They indicate improvement sufficient to justify expanded use of the suggested techniques, particularly in view of diminished morbidity and risks to the general condition of those in the older age group.
Aged↗
M-plasty techniques.
Conventional closure of surgical defects in skin may cause unsightly protrusions (dog ears). Fusiform excisions with angles of 30 degrees or less reduce protrusion but may require excision of larger volumes and lengths of healthy skin and subcutaneous tissue than may be desirable. M-plasties reduce these requirements while still allowing use of 30 degrees angles at maximum. Applications and technical details are described and illustrated.
Dermatologic Surgical Procedures↗
Use of double W-plasty in upper blepharoplasty.
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Eyelids↗