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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phenylarsine oxide (PhAsO), a dithiol reagent that blocks insulin stimulation of glucose transport in 3T3 L1 cells, also altered insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cultured cells. PhAsO (2 microM) similarly inhibited the insulin-induced glycogen and lipid syntheses without modifying the basal level of these processes, cell viability or the ATP content. Prior incubation of the cells with PhAsO did not prevent insulin binding to the cells, or activation of the receptor tyrosine kinase, while it minimally (16%) altered receptor internalization. These results indicate that cellular dithiols located at a post-receptor step are involved in the transduction of the insulin signal to intracellular glucose metabolism.
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PMID:Evidence for the involvement of vicinal sulfhydryl groups in the insulin stimulation of intracellular glucose metabolism in Zajdela hepatoma cells. 218 43

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.
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PMID:Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling. 255 Apr 26

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.
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PMID:Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors. 255 27

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)
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PMID:Regulation of insulin receptor kinase by multisite phosphorylation. 300 Apr 58

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.
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PMID:Characterization of phosphatidylinositol kinase activity associated with the insulin receptor. 300 26

We have compared the effect of phorbol 12-myristate 13-acetate (PMA) with that of insulin on three targets of insulin action in H4IIEC3 (H4) rat hepatoma cells. These parameters are the phosphorylation state and tyrosine kinase activity of the insulin receptor, the activation state of glycogen synthase, and the accumulation of p33 mRNA. Under conditions where insulin treatment of H4 cells clearly activated receptor serine and tyrosine phosphorylation on the insulin receptor beta-subunit in situ, activated receptor tyrosine kinase activity in vitro, and activated glycogen synthase and p33 mRNA accumulation in situ, PMA alone did not influence the insulin receptor phosphorylation state or tyrosine kinase activity and did not affect glycogen synthase activity, but markedly increased p33 mRNA accumulation. When PMA was added in the presence of insulin, particularly if PMA was preincubated, the receptor phosphorylation state and the tyrosine kinase activity again were not affected, but insulin-activated glycogen synthase was significantly diminished or abolished. In contrast, increased p33 mRNA accumulation by PMA was additive with that of insulin. Thus, under conditions where no effect was observed on the insulin receptor phosphorylation state or the tyrosine kinase activity, PMA acted in an insulin-antagonistic manner on glycogen synthase and in an insulin-like manner on p33 mRNA accumulation, indicating that these actions of PMA are unrelated to early events in the pathway of the insulin action. Effects on glycogen synthase are most readily explained by an effect of protein kinase C-activated phosphorylation of glycogen synthase.
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PMID:Contrasting interactions between phorbol ester and insulin on the regulation of glycogen synthase activity and p33 mRNA accumulation in rat hepatoma cells. 312 51

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.
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PMID:Tyrosine phosphorylation of insulin receptor beta subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells. 395 14

Activation of the insulin receptor tyrosine kinase is thought to initiate a chain of phosphorylation changes which regulate various metabolic and growth processes. Insulin has been shown to regulate some kinases by affecting their phosphorylation states; however, direct connections between activation of the receptor tyrosine kinase and subsequent altered phosphorylations of these kinases have yet to be established. To define the constituents of these regulated phosphorylation cascade systems, the time course of insulin-induced phosphorylation changes has been examined. 32P-Labeled H4IIE hepatoma cells were treated with insulin for 5-300 s, and then cell extracts were subjected to two-dimensional gel electrophoresis. Computer analysis of autoradiograms was employed to quantify insulin-induced phosphorylation changes. Phosphotyrosine was assessed by KOH digestion, by anti-phosphotyrosine immunoprecipitation, and by phenylarsine oxide treatment. Insulin induced a hierarchy of phosphorylation changes over the 300-s time course, with most increases being detectable within 10 s. Of these, only four did not undergo enhanced tyrosine phosphorylation. Eight proteins were found to undergo transient phosphorylation changes. Phenylarsine oxide elicited the appearance of novel low molecular weight phosphoproteins. These results support the concept of insulin-directed phosphorylation cascades, but indicate the magnitude of potential targets of the insulin receptor has been underestimated.
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PMID:Insulin regulation of protein phosphorylation in H4 hepatoma cells. Examination of rapid effects using two-dimensional electrophoresis. 768 56

Based on homology to the tyrosine kinase domain of the chick erythroblastosis virus oncogene v-sea, we cloned a cDNA encoding a novel receptor tyrosine kinase from a human hepatoma HepG2 cDNA library. The encoded protein, which we termed 'Sky', contains an intracellular tyrosine kinase domain and a unique extracellular domain with two immunoglobulin (Ig)-like domains and two fibronectin type III (FN III) domains. The overall structure of Sky is homologous to the reported sequence of the oncogenic Axl/Ufo receptor tyrosine kinase. Phylogenetic analysis in the tyrosine kinase domain shows that Sky, Axl/Ufo, Ark, and v-Ryk form a sub-family distinct from other tyrosine kinases. Northern blot analysis revealed that sky mRNA is expressed predominantly in brain and faintly in tissues of other organs. As the combination of Ig-like and FN III domains is often observed in neural cell adhesion molecules and receptor protein tyrosine phosphatases, Sky may be involved in cell adhesion processes, particularly in the central nervous system.
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PMID:Cloning of the cDNA for a novel receptor tyrosine kinase, Sky, predominantly expressed in brain. 810 12

By low-stringency screening of a human hepatoma HepG2 cell cDNA library, using the genomic fragment of chick c-sea receptor tyrosine kinase as a probe, we isolated overlapping cDNAs encoding a novel protein kinase, which we termed LIM-kinase (LIMK).* The predicted open reading frame encodes a 647-amino-acid polypeptide containing a putative protein kinase structure in the C-terminal half. In addition, LIMK has two repeats of cysteine-rich LIM/double zinc finger motif at the most N-terminus. To our knowledge, this is the first protein kinase seen to contain the LIM motif(s) in the molecule. Although the protein kinase domain of LIMK has highly conserved sequence elements of protein kinases, phylogenetic analysis revealed that LIMK cannot be classified into any subfamily of known protein kinases. Northern blot analysis revealed that the single species of LIMK mRNA of 3.3 kb was expressed in various human epithelial and hematopoietic cell lines. In rat tissues, LIMK mRNA was expressed in the brain, at the highest level. LIM is suggested to be involved in protein-protein interactions by binding to another LIM motif. As the LIM domain is frequently present in the homeodomain-containing transcriptional regulators and oncogenic nuclear proteins, LIMK may be involved in developmental or oncogenic processes through interactions with these LIM-containing proteins.
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PMID:Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif. 818 54


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