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C R Kahn

Publications and source records attributed to C R Kahn.

At least 199 records · Page 11Linked to original sources
PubMed1990

Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats.

Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.

Animals↗
PubMed1990

A family of polypeptide substrates and inhibitors of insulin receptor kinase.

Previous studies have shown that reduced carbamoylmethylated lysozyme (RCAM-lysozyme, MW approximately 14.5K) is a substrate and inhibitor (Ki approximately 0.6 microM) of insulin receptor kinase (InsRK) autophosphorylation (Kohanski & Lane, 1986; Lane & Kohanski, 1986). In this study we have prepared a family of defined modified derivatives of RCAM-lysozyme and used them to probe the nature of the substrate and inhibitory sites of InsRK. All open-chain derivatives of lysozyme in which either the tryptophanyl, methionyl, cysteinyl, arginyl, or histidyl side chains were modified served as substrates and were potent inhibitors of InsRK autophosphorylation. This was true whether the substitutions were either hydrophilic or hydrophobic, although the hydrophilic derivatives had a higher inhibitory potency. Tryptic peptides derived from RCAM-lysozyme, however, were inactive as inhibitors, and a mixture of the three cyanogen bromide fragments (containing 12, 24, and 93 amino acids, respectively) was found to be less potent in inhibiting the receptor kinase. Derivatization of either tyrosyl or carboxyl side chains produced derivatives that were neither substrates nor capable of inhibiting receptor autophosphorylation. Derivatives with modified amino groups were substrates for InsRK but were not able to inhibit InsRK autophosphorylation. The present study suggests that (a) unphosphorylated InsRK has a large hydrophilic substrate binding domain and is effectively inhibited by long-chain polypeptides but not by short sequences, (b) some of the amino, carboxyl, and hydroxyphenyl side chains are essential to the inhibitory nature of these polypeptides, and (c) derivatives that fail to inhibit autophosphorylation can still be recognized and phosphorylated by active InsRK.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Augmented desensitization to epidermal growth factor (EGF) immediate actions: a novel mechanism for altered EGF growth response in mutant A431 cells.

Epidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphorylation of lipocortin 1, a major substrate for the EGF receptor kinase whose phosphorylation is calcium-dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF-induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF-induced increase in cytoplasmic calcium, lipocortin phosphorylation, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to this hormone.

Annexins↗
PubMed1990

Activation of liver and muscle insulin receptor tyrosine kinase activity during in vivo insulin administration in rats.

We have studied autophosphorylation and tyrosine kinase activity of the insulin receptor purified from liver and muscle of fasted rats before and after infusion of insulin (100 mU/h) during a 2.5 h glucose clamp. Recovery of insulin receptors and insulin binding to the solubilised receptors was unaffected by the glucose clamp. Autophosphorylation of the insulin receptor beta subunit was increased in liver receptors prepared from rats at the end of the glucose clamp compared to rats in the basal state both in the absence of insulin in vitro (109% increase, p less than 0.001) and after in vitro stimulation with 10(-7) mol/l insulin (clamped vs fasted; 96% increase, p less than 0.001). Insulin (10(-7) mol/l) stimulated autophosphorylation was also increased in muscle receptor preparations from clamped rats compared with rats in the basal state (58% increase, p less than 0.05). In both liver and muscle receptors, the clamp increased the amount of [32P]-phosphate incorporated into the beta subunit without changing the sensitivity of the insulin stimulation. HPLC analysis of the tryptic phosphopeptides derived from the beta subunit after insulin stimulated autophosphorylation of liver receptors revealed an increase of 32P in all phosphorylation sites without any change in the overall pattern. Tyrosine kinase activity of liver and muscle insulin receptors from clamped rats was also increased approximately twofold (p less than 0.05) when analysed using a synthetic substrate (poly Glu4 Tyr1). Our results support the notion that the insulin receptor exists in an active an inactive form, and that elevated plasma insulin concentrations increases the proportion of active receptors.

Animals↗
PubMed1990

T-cell vaccination in experimental myasthenia gravis: a double-edged sword.

Immunization with antigen-specific T cells has been used successfully in the treatment of several T cell-mediated experimental autoimmune diseases, including experimental allergic encephalomyelitis, thyroiditis, and adjuvant arthritis. The aim of this study was to determine whether T-cell vaccination could be used to down-regulate specifically the antibody response to AChR in experimental autoimmune myasthenia gravis (EAMG), an antibody-mediated disorder. We produced T cells specific for the acetylcholine receptor (AChR) by immunizing Lewis rats with torpedo AChR, harvesting the regional lymph node cells, and restimulating them in vitro with AChR. This cell population was expanded with IL2. The cells were then activated with concanavalin A (Con-A) and exposed to high hydrostatic pressure to augment their immunogenicity. We found that rats vaccinated with these cells did not manifest decreased antibody titers to AChR, when challenged. In fact, the antibody response to AChR was consistently potentiated by the vaccine treatment. This result could not be attributed to antigen carryover by the vaccinating cells or to induction of anti-idiotypic antibodies. Despite these results showing overall enhancement of the AChR antibody response, we found evidence of AChR-specific suppressor cells in the spleens of the vaccinated animals. Our observations indicate that T-cell vaccination can elicit both a positive immune response and a suppressive response in the same animal. If the T-cell vaccination strategy is to be useful for the treatment of MG, methods for amplifying the suppressive effect will need to be developed.

Animals↗
PubMed1990

The insulin receptor with phenylalanine replacing tyrosine-1146 provides evidence for separate signals regulating cellular metabolism and growth.

We have studied the function of a mutant insulin receptor (IR) molecule in which Tyr-1146, one of the first autophosphorylation sites in the beta subunit, was replaced with phenylalanine (IRF1146). Autophosphorylation of the partially purified IRF1146 was reduced 60-70% when compared to the wild-type IR but was still stimulated by insulin. The phosphotransferase activity of the dephospho form of both the IR and IRF1146 toward exogenous substrates was stimulated 3- to 4-fold by insulin. However, the wild-type IR was activated 12-fold by autophosphorylation, whereas the IRF1146 was activated only 2-fold. When the IRF1146 was expressed in Chinese hamster ovary (CHO) cells, insulin binding was normal, whereas autophosphorylation was reduced 80% when compared to cells expressing the wild-type IR. Endogenous substrates of the insulin receptor kinase were not detected during insulin stimulation of CHO cells expressing the IRF1146. Moreover, the IRF1146 did not internalize insulin rapidly or stimulate DNA synthesis in the presence of insulin. In contrast, both the IR and IRF1146 stimulated glycogen synthase equally in CHO cells. These data suggest that activation of the IR tyrosine kinase can be resolved into two components: the first is dependent on insulin binding and the second is dependent on the subsequent insulin-stimulated autophosphorylation cascade. Thus, at least two signal transduction pathways diverging from the IR are implicated in the mechanism of insulin action.

Animals↗
PubMed1990

Insulin receptor carbohydrate units contain poly-N-acetyllactosamine chains.

The insulin receptor was immunoprecipitated from cultured human lymphocytes (IM-9) and rat hepatocytes (Fao) after biosynthetic labeling with [3H]glucosamine or [3H]mannose, and the nature of the carbohydrate units was investigated. Digestion of the receptor from IM-9 lymphocytes with E. freundii endo-beta-galactosidase increased the migration of the insulin receptor alpha- and beta-subunits on sodium dodecyl sulfate-polyacrylamide gels and sharpened the electrophoretic bands; the alpha-subunit was converted from an apparent mol wt (Mr) of 123,000 to a Mr of 118,000, and the beta-subunit from a Mr of 92,000 to 89,000. The susceptibility of the insulin receptor to this enzyme indicates that its carbohydrate units contain poly-N-acetyllactosamine sequences. Affinity chromatography of receptor glycopeptides on Concanavalin-A-Sepharose revealed that the poly-N-acetyllactosamine units were attached to multiantennary glycopeptides that accounted for over 75% of the [3H]glucosamine incorporated into the IM-9 lymphocyte insulin receptor; the remaining radioactivity was present in polymannose units (primarily Man8GlcNAc2) and biantennary complex saccharides. Several differences in the carbohydrate chains of the insulin receptor from the Fao and IM-9 cells indicated that glycosylation was cell specific despite the occurrence of poly-N-acetyllactosamine chains in both cell types. The IM-9 lymphocyte receptor glycopeptides were larger (Mr, 3,200-9,500) and more susceptible to endo-beta-galactosidase than those from the Fao receptor (Mr, 3,000-5,000). Moreover, the released saccharides from the Fao receptor were found by exoglycosidase digestions and chromatographic comparison to standards to contain terminal sialic acid in both alpha 2----3 and alpha 2----6 linkage to galactose, whereas the IM-9 carbohydrate units contained only alpha 2----3-linked sialic acid.

Animals↗
PubMed1989

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells.

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.

Amino Acid Sequence↗
PubMed1989

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies.

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.

Amino Acid Sequence↗
PubMed1989

Nonphosphorylatable substrate analogs selectively block autophosphorylation and activation of the insulin receptor, epidermal growth factor receptor, and pp60v-src kinases.

The receptors for insulin and epidermal growth factor undergo tyrosine autophosphorylation in response to ligand stimulation, while pp60v-src is an unregulated tyrosine kinase. In this report we show that each of the kinases phosphorylates an exogenous peptide that corresponds to the insulin proreceptor sequence 1142-1153. When the kinases were pre-phosphorylated, saturable Michaelis-Menten kinetics were observed. However, when the kinases had not been pre-phosphorylated biphasic kinetics were observed; at progressively higher substrate concentrations (greater than Km) less substrate phosphorylation was seen. Furthermore, when the kinases had not been pre-phosphorylated kinase autophosphorylation was inhibited at high substrate concentrations. On this basis we postulated that the substrate inhibition of substrate phosphorylation resulted directly from substrate inhibition of kinase autophosphorylation. To test this we designed additional peptides to function specifically as inhibitors of the kinases. Each of the 3 tyrosine residues within the substrate sequence were replaced either by 4-methoxyphenylalanine or phenylalanine, residues structurally similar to tyrosine but unable to accept phosphoryl transfer. Both analogs inhibited insulin and epidermal growth factor receptor autophosphorylation, whereas only the Phe-substituted analog inhibited pp60v-src phosphorylation. These data suggest that autophosphorylation of tyrosine residues near the kinase active site is a generalized mechanism for tyrosine kinase activation and that activation can be selectively blocked by substrates and nonphosphorylatable analogs.

Adenosine Triphosphate↗
PubMed1989

Analysis of mRNA heterogeneity by ribonuclease H mapping: application to the insulin receptor.

The major species of human insulin receptor mRNA (5.9, 7.5, 8.5 and 10.2 kb) and those in rat tissues (7.4 and 9.6 kb) are each much larger than the 4.2 kb required to encode the insulin receptor precursor. To evaluate the structural basis for this mRNA size heterogeneity, we performed a ribonuclease H mapping technique. A small insulin receptor cDNA insert was annealed to human and rat poly(A) RNA, followed by site-specific enzymatic cleavage with ribonuclease H. Subsequent Northern blot analysis with cDNA probes specific to the 5' end of the cDNA revealed a single fragment from each of the human and rat insulin receptor mRNA species. The size of this fragment indicated that each mRNA contains approximately 0.4 kb of 5' untranslated mRNA. In contrast, a 3' region probe demonstrated multiple mRNA fragments after cleavage. The sizes of these fragments indicated that the human insulin receptor mRNA species contain from 1.5 to 5.4 kb, and the rat insulin receptor mRNAs either 2.8 or 5.3 kb, of 3' untranslated RNA. Thus, the presence of varied, but extensive, 3' untranslated sequences in insulin receptor mRNA transcripts accounts for their size heterogeneity and may affect mRNA stability and/or translation efficiency.

Animals↗
PubMed1989

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells.

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.

Algorithms↗
PubMed1989

Cascade of autophosphorylation in the beta-subunit of the insulin receptor.

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.

Animals↗
PubMed1989

Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased secretion of proinsulin relative to insulin and rapid release of newly synthesized proinsulin. Moreover, the mutant cell lines did not store the prohormone nor did they release it upon stimulation with secretagogues. These data indicate that a significant fraction of the mutant prohormone is released via the constitutive secretory pathway rather than the regulated pathway, thereby bypassing granule-related processing and regulated release.

Animals↗
PubMed1989

Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells.

The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or [32P]phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of 125I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.

2,4-Dinitrophenol↗
PubMed1989

Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats.

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6 +/- 0.7; diabetic: 2.6 +/- 0.4% per milligram protein, P less than 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and alpha-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease in insulin-stimulated autophosphorylation (P less than 0.05). 125I-insulin cross-linking revealed an 140-kD alpha subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at greater than 200 kD were also detected. The molecular weight of the insulin receptor beta subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Animals↗