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K T Yu

Publications and source records attributed to K T Yu.

35 records · Page 2Linked to original sources
PubMed1986

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit.

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Adsorption↗
PubMed1986

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.

Amino Acids↗
PubMed1985

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.

Amino Acids↗
PubMed1984

Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity.

The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.

Amino Acids↗
PubMed1984

The type I insulin-like growth factor receptor mediates the rapid effects of multiplication-stimulating activity on membrane transport systems in rat soleus muscle.

Multiplication-stimulating activity (MSA) stimulates the uptake of xylose and alpha-aminoisobutyric acid (AIB) by intact rat soleus muscle. It is approximately 50 times less potent than insulin. A native insulin receptor species with apparent Mr = 350,000 in soleus muscle is revealed by affinity cross-linking to 125I-insulin with disuccinimidyl suberate. 125I-labeled insulin-like growth factor (IGF) I with the cross-linker affinity labels two native receptor types with Mr = 360,000 (type I) and Mr = 220,000 (type II). In order to distinguish which of the three insulin and IGF receptor systems identified mediate the rapid insulin-like effects of MSA, the biological actions of the unlabeled ligands at various concentrations were correlated with their ability to inhibit the affinity labeling of these receptor species. The stimulatory action of native insulin is closely related to its ability to inhibit the labeling of the insulin receptor by 125I-insulin such that the uptake of xylose and AIB is maximally stimulated when 80% of insulin receptor 125I-affinity labeling is inhibited. In contrast, MSA only displaced 16% of the 125I-labeling of the insulin receptor when it maximally stimulates the uptake of xylose and AIB, indicating that the insulin receptor is not primarily involved in mediating these effects. The affinity of MSA to the type II IGF receptor is 10 times higher than that to the type I IGF receptor. There is close to a 1:1 relationship between the stimulatory effects of MSA on xylose and AIB uptake and its inhibitory action on the affinity labeling of the type I receptor by 125I-IGF I. In contrast, MSA almost abolishes the labeling of the type II IGF receptor at concentrations which have no detectable effects on the uptake of xylose and AIB by soleus muscle. Thus, a marked dissociation can be observed between the rapid insulin-like action of MSA and its inhibitory effects on the affinity labeling of the type II receptor by 125I-IGF I. We conclude that MSA acts through the type I IGF receptor in soleus muscle to stimulate hexose and amino acid transport. The type II IGF receptor appears to be incapable of modulating these effects in this tissue.

Aminoisobutyric Acids↗
PubMed1983

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle.

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process (F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167-173). The stimulatory effect of insulin (0.1 U/ml) on the uptake of D-[U-14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 mM). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 degrees C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.

2,4-Dinitrophenol↗
PubMed1982

Effect of ionophore A23187 on basal and insulin-stimulated sugar transport by rat soleus muscle.

The ionophore A23187 (10 micrograms/ml) did not affect the uptake of D-[U-14C]xylose by rat soleus muscle incubated under basal conditions. When muscles were incubated in a Ca2+/Mg2+-free (CMF) medium, A23187 promoted the efflux of intracellular Mg2+ and the efflux of 45Ca from preloaded muscles. Under these conditions, conditions, A23187 inhibited insulin-stimulated sugar transport without affecting 125I-insulin binding by the muscle. A23187 induced a slight fall in muscle ATP (16-18%); this does not appear to be responsible for the inhibitory effect of the ionophore on sugar transport. The inhibitory effect of A23187 was completely abolished when the CMF medium was supplemented with Mg2+ and partially reversed by Mn2+ or Zn2+; supplementation with Ca2+ did not reverse the inhibitory effect of the ionophore. These results suggest that insulin stimulates muscle sugar transport through a mechanism that involves intracellular Mg2+.

Adenosine Triphosphate↗
PubMed1981

Stimulation of sugar transport in rat soleus muscle by prolonged cooling at 0 degrees C.

The uptake of D-xylose by isolated rat soleus muscle (measured at 37 degrees C) was stimulated by prolonged cooling at 0 degrees C. The effect of cooling reached a maximum value after 3 h and was reversed on rewarming; reversal was temperature-dependent. Cooling stimulated xylose uptake sub-maximally compared with the effect of insulin (100 U/l). Xylose uptake in cooled muscle was further stimulated by insulin, but not by anoxia. The effect of cooling and its reversal were still demonstrable in the presence of ouabain (1 mmol/l), or when unidirectional efflux of calcium and magnesium from the muscle was induced by EDTA (5 mmol/l). The ionophore, A23187 (2.5 mg/l), depressed the effect of cooling in the presence of EDTA but not in the presence of EGTA. It is concluded that cooling disrupts and intracellular magnesium-p]ump and that muscle sugar transport is consequentially stimulated through an increase in cytoplasmic magnesium.

Animals↗
PubMed1978

Permissive effect of ATP on insulin-stimulated sugar transport by rat soleus muscle.

The stimulatory effect of insulin (0.1 U/ml) on D-xylose uptake was progressively lost when rat soleus muscles were preincubated at 37 degrees C under anaerobic conditions for longer than 30 min; after 90 min these muscles were completely insensitive to insulin. This effect was associated with the loss of muscle ATP. When the breakdown of ATP was retarded either by lowering the preincubation temperature or by preincubation with 5 mM glucose, the effect of insulin in anaerobic muscle was correspondingly prolonged. Under certain conditions, externally added ATP promoted an effect of insulin in otherwise insulin-unresponsive muscles. This effect was small in magnitude and was complicated by the degradation of the added ATP in the incubation medium and by the fact that ATP also tended to inhibit insulin-stimulated xylose uptake. These results indicate that there is a relationship between insulin-stimulated sugar transport and muscle ATP levels. This supports the proposal that there may be some ATP-dependent reaction(s) involved in the mechanism whereby insulin promotes the process of muscle sugar transport.

Adenosine Triphosphate↗
PubMed1978

Effect of prolonged anaerobiosis on 125I-insulin binding to rat soleus muscle: permissive effect of ATP.

The specific binding of 125I-insulin by rat soleus muscle was depressed when muscle ATP was depleted, either by prolonged anoxia or more rapidly with 2,4-dinitrophenol. Insulin binding was not eliminated in ATP-depleted muscle, but was reduced by 70--80%. Insulin binding by aerobic muscle could be resolved into two components; a high-affinity, low-capacity site (KD = 7.8 nM) and a low-affinity, high-capacity site (KD = 390 nM). The stimulatory effect of insulin on xylose uptake could be correlated with binding to the high-affinity site. These results indicate that there is some ATP-dependent process involved in the regulation of insulin binding by soleus muscle. It is suggested that this could be a phosphorylation-dephosphorylation system, acting either on the receptor itself or on some closely related membrane protein.

Adenosine Triphosphate↗
PubMedDate not supplied

Liver biopsies in patients with lysosomal storage disease: experience with effective sedation.

We prospectively applied a protocol used to sedate children who required a liver biopsy. Sixty liver biopsies were performed on thirty pediatric patients to assess the effects of treatment. Sixteen patients had Type 1 Gaucher's disease of which seven had a platelet count between 50-100,000/mm3. All seven had bleeding time performed and when indicated, intravenous DDAVP (1-deamino-8-D-arginine vasopressin) was used to improve hemostasis. Fourteen patients had Niemann-Pick disease type C of which eight were significantly demented and uncooperative. Before liver biopsy, all patients were sedated with the following regimen: oral chlorpromazine (1 mg/kg) followed one hour later by intravenous meperidine (1 mg/kg) and pentobarbital (maximum dosage 6 mg/kg) administered by slow intravenous injection. Liver biopsies were obtained safely on all patients. Only 1 patient (2%) developed a potentially serious complication: an obstructed airway which was readily corrected by simple repositioning. Transient less serious complications occurred in another 7 patients (12%). There was no long term sequalae of the biopsy procedures. Our study indicates that with appropriate patient selection, this sedation protocol may be useful in pediatric patients requiring a liver biopsy.

Adolescent↗
PubMedDate not supplied

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)

Adenosine Triphosphate↗
PubMedDate not supplied

Cellular signaling by the insulin receptor.

Cellular signaling by insulin is initiated by specific membrane receptors that have been characterized as large multimeric disulfide-linked protein complexes with a minimal subunit structure of (beta-S-S-alpha)-S-S-(alpha-S-S-beta), where the alpha- and beta-subunits are about 125,000 and 90,000 daltons, respectively. The disulfides in this structure are of two classes based on their differential sensitivity to reductants (Massague, J., and Czech, M. P., J. Biol. Chem. 1982; 257:6729-35). An important recent discovery is that the insulin receptor, either in crude detergent extracts or after purification by affinity chromatography, is associated with insulin-activatable tyrosine phosphokinase activity and is itself autophosphorylated (Kasuga, M., et al., Proc. Natl. Acad. Sci. USA 1983; 80:2137-41). We demonstrate here that insulin receptor kinase activity is readily monitored while the receptor is absorbed onto insulin-agarose, using [gamma-32]ATP and histone as substrate. Phosphorylation of histone and the receptor beta-subunit on tyrosine residues is dependent on time, temperature, and Mn2+ in this system. The immobilized insulin receptor kinase is activated by prior phosphorylation with ATP, indicating that the autophosphorylation plays an important role in regulating receptor kinase activity. That the insulin receptor tyrosine kinase activity may be involved in initiating the mechanism of insulin action is currently an attractive hypothesis. A second working model of insulin action proposes that one or more soluble factors are released into the cell in response to insulin as suggested by studies using muscle and fat cell extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗