FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2161573035> ?p ?o ?g. }

• W2161573035 endingPage "30214" @default.
• W2161573035 startingPage "30208" @default.
• W2161573035 abstract "We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 → Gln and Pro-1178 → Leu (Gln-1174 and Leu1178, respectively), both found in patients with inherited severe insulin resistance, markedly impaired receptor tyrosine autophosphorylation, with both mutant receptors being unable to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed in Chinese hamster ovary cells. However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be sufficient to mediate insulin's metabolic and mitogenic effects. In the present study, we have demonstrated that these mutations also impair the ability of the insulin receptor to activate the transcription factor Elk-1 and promote GLUT4 translocation to the plasma membrane. Although at low concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at higher insulin concentrations we confirmed that the cells expressing the mutant receptors showed significantly increased tyrosine phosphorylation of IRS-1 compared with parental nontransfected cells. In addition, at comparable insulin concentrations, the association of the p85α subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and the enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors. In contrast, no significant stimulation of the tyrosine phosphorylation of Shc, GTP loading of Ras, or mitogen-activated protein kinase phosphorylation was seen in cell lines expressing these mutant receptors. Thus, no activation of any measurable mitogenic or metabolic response was detectable, despite significant insulin-induced phosphorylation of IRS-1 and its association with PI3-kinase in cells stably expressing the mutant insulin receptors. These findings suggest that PI3-kinase activation alone may be insufficient to mediate a wide range of the metabolic and mitogenic effects of insulin. Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation. We have recently reported (1) that two naturally occurring mutants of the insulin receptor tyrosine kinase domain, Arg-1174 → Gln and Pro-1178 → Leu (Gln-1174 and Leu1178, respectively), both found in patients with inherited severe insulin resistance, markedly impaired receptor tyrosine autophosphorylation, with both mutant receptors being unable to mediate the stimulation of glycogen synthesis or mitogenesis by insulin when expressed in Chinese hamster ovary cells. However, these mutations did not fully prevent IRS-1 phosphorylation in response to insulin in these cells, suggesting that IRS-1 alone may not be sufficient to mediate insulin's metabolic and mitogenic effects. In the present study, we have demonstrated that these mutations also impair the ability of the insulin receptor to activate the transcription factor Elk-1 and promote GLUT4 translocation to the plasma membrane. Although at low concentrations of insulin, the mutant receptors were impaired in their ability to stimulate the tyrosine phosphorylation of IRS-1, at higher insulin concentrations we confirmed that the cells expressing the mutant receptors showed significantly increased tyrosine phosphorylation of IRS-1 compared with parental nontransfected cells. In addition, at comparable insulin concentrations, the association of the p85α subunit of phosphoinositide 3-kinase (PI3-kinase) with IRS-1 and the enzymatic activity of IRS-1-associated PI3-kinase were significantly enhanced in cells expressing the mutant receptors. In contrast, no significant stimulation of the tyrosine phosphorylation of Shc, GTP loading of Ras, or mitogen-activated protein kinase phosphorylation was seen in cell lines expressing these mutant receptors. Thus, no activation of any measurable mitogenic or metabolic response was detectable, despite significant insulin-induced phosphorylation of IRS-1 and its association with PI3-kinase in cells stably expressing the mutant insulin receptors. These findings suggest that PI3-kinase activation alone may be insufficient to mediate a wide range of the metabolic and mitogenic effects of insulin. Additionally, the data provide support for the notion that insulin activation of Ras is more closely linked with Shc, and not IRS-1, phosphorylation. Mutations of the insulin receptor gene have been identified in patients with a wide variety of genetic syndromes of severe insulin resistance (for reviews see Refs. 2Taylor S.I. Cama A. Accili D. Barbetti F. Quon M.J. de al Luz Sierra M. Suzuki Y. Koller E. Levy-Telodano R. Wertheimer E. Moncado V.Y. Kadowaki H. Kadowaki T. Endocr. Rev. 1992; 13: 566-595Crossref PubMed Scopus (278) Google Scholar and 3Krook A. O'Rahilly S. Sheppard M.C. Franklyn J.A. Ballieres Clinical Endocrinology and Metabolism. 10. Balliere Tindal, London1996: 97-122Google Scholar). Studies of the functional properties of these mutant insulin receptors have contributed to the understanding of molecular mechanisms involved in insulin signaling. The insulin receptor is a heterotetrameric receptor tyrosine kinase composed of two extracellular α subunits and two β subunits. Insulin binds to the α subunit of the receptor, an event which activates the tyrosine kinase function of the cytoplasmic domain of the β subunit. This results in autophosphorylation of the β subunit of the receptor itself as well as the phosphorylation of a number of cytoplasmic target proteins that include IRS-1, IRS-2, and Shc (for review see Ref. 4Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar). These tyrosine-phosphorylated molecules are involved in the coupling of the insulin receptor to downstream metabolic and mitogenic events. Thus, the phosphorylation of IRS-1 generates a number of recognition sites for interaction with SH2 domain-containing molecules, including the lipid kinase PI3-kinase, the phosphatase SHP-2, and the adaptor proteins Grb2 and Nck. The phosphorylation of Shc also facilitates its interaction with the SH2 domain of Grb2. Grb2, in tight association with the guanine nucleotide exchange factor Sos, is closely involved with insulin stimulation of Ras activation (5Medema R.H. de Vries-Smits A.M.M. van der Zon G.C.M. Maassen A.J. Bos J.L. Mol. Cell. Biol. 1993; 13: 155-162Crossref PubMed Scopus (159) Google Scholar). A large body of evidence exists to support the hypothesis that the activation of the tyrosine kinase activity of the insulin receptor is an essential first step for most, if not all, of the biological effects of insulin (6Murakami M.S. Rosen O.M. J. Biol. Chem. 1991; 266: 22653-22660Abstract Full Text PDF PubMed Google Scholar, 7Zhang B. Tavaré J.M. Ellis L. Roth R.A J. Biol. Chem. 1991; 266: 990-996Abstract Full Text PDF PubMed Google Scholar). Thus, mutant insulin receptors where the ATP binding site has been altered by site-directed mutagenesis or naturally occurring insulin receptor mutations abolishing receptor tyrosine kinase activity fail to mediate any biological effects of insulin when expressed in transfected cells (8Ebina Y. Araki E. Taira M. Shimada F. Mori M. Craig C.S. Siddle K. Roth R.A. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 704-708Crossref PubMed Scopus (267) Google Scholar, 9McClain D.A. Maegawa H. Lee J. Dull T.J. Ullrich A. Olefsky J.M. J. Biol. Chem. 1987; 262: 14663-14671Abstract Full Text PDF PubMed Google Scholar). The importance of IRS-1 tyrosine phosphorylation in the mediation of insulin's downstream effects has also been highlighted by several studies. Insulin receptors in which tyrosine 972 was replaced by phenylalanine were capable of insulin-stimulated autophosphorylation but were markedly impaired in their interaction and phosphorylation of IRS-1 (10White M. Livingston J. Backer J. Lauris V. Dull T.J. Ullrich A. Kahn C.R. Cell. 1988; 54: 641-649Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 11O'Neill T.J. Craparo A. Gustafson T.A. Mol. Cell. Biol. 1994; 14: 6433-6442Crossref PubMed Scopus (166) Google Scholar, 12Gustafson T.A. He W. Craparo A Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (327) Google Scholar). Cells expressing this mutant insulin receptor showed severely impaired insulin stimulation of glycogen synthesis, thymidine incorporation, and amino acid uptake, suggesting that receptor autophosphorylationper se might be less relevant to insulin signaling than the ability to phosphorylate downstream substrates (10White M. Livingston J. Backer J. Lauris V. Dull T.J. Ullrich A. Kahn C.R. Cell. 1988; 54: 641-649Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Further support for the importance of IRS-1 phosphorylation in mediating the insulin signal comes from studies of a truncated insulin receptor lacking the 82 C-terminal amino acids (Δ82) (13Yamamoto-Honda R. Kadowaki T. Momomura K. Tobe K. Tamori Y. Shibasaki Y. Mori Y. Kaburagi Y. Koshio O. Akanuma Y. Yazaki Y. Kasuga M. J. Biol. Chem. 1993; 268: 16859-16865Abstract Full Text PDF PubMed Google Scholar). This receptor displayed severely impaired autophosphorylation, whereas insulin-stimulated tyrosine phosphorylation of IRS-1 was unaffected. Despite this receptor-impaired ligand-induced autophosphorylation, Chinese hamster ovary (CHO) 1The abbreviations used are: CHO, Chinese hamster ovary; PI3-kinase, phosphoinositide 3-kinase; CMV, cytomegalovirus; GFP, green fluorescent protein; MAP, mitogen-activated protein; IR, insulin receptor. cells expressing this mutant insulin receptor were able to mediate insulin-stimulated thymidine incorporation into DNA as well as insulin-stimulated glucose uptake. Furthermore, in 32D cells, which normally do not contain either the insulin receptor or IRS-1 or -2, insulin-responsive mitogenesis cannot be conferred by transfecting either the insulin receptor or IRS-1 but only by coexpressing both molecules (14Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar). Although the targeted deletion of IRS-1 in mice is not lethal, −/− mice show significant impairment in insulin-stimulated glucose metabolism and reduced size, despite the normal phosphorylation of IRS-2 and Shc (15Araki E. Lipes M.A. Patti M.-E. Brüning J.C. Haag B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1095) Google Scholar,16Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (905) Google Scholar). These data provide further support for the central importance of IRS-1 phosphorylation in the mediation of insulin action. We have recently described two insulin receptor mutations, Gln-1174 and Leu-1178, found in patients with inherited forms of severe insulin resistance (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). When stably expressed in CHO cells, these receptors showed severely impaired autophosphorylation and in vitrotyrosine kinase activity toward artificial substrates but retained the ability to phosphorylate IRS-1. In contrast to findings with the Δ82 receptor (see above), these receptors were also severely impaired in their ability to mediate both the metabolic (as measured by glycogen synthesis) and mitogenic (as measured by thymidine incorporation) effects of insulin. We now present further characterization of the signaling properties of these insulin receptors. These new studies provide evidence that insulin-stimulated PI3-kinase activity, although undoubtedly important for insulin action, may not in itself be sufficient to mediate the full metabolic and mitogenic actions of insulin. They also provide supportive evidence that insulin activation of Ras may be more closely related to Shc rather than IRS-1 phosphorylation. Both mutations were identified in heterozygous form in adolescent females suffering from the Type A syndrome of insulin resistance. Insulin resistance was inherited in a dominant fashion in both families and co-segregated with the mutation. The identification and initial characterization of both mutations has been described previously (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 17Krook A. Kumar S. Lang I. Boulton A.J.M. Wass J.A.H. O'Rahilly S. Diabetes. 1994; 43: 357-368Crossref PubMed Google Scholar, 18Moller D.E. Cohen O. Yamaguchi Y. Assiz R. Grigorescu F. Eberle A. Morrow L.A. Moses A.C. Flier J.S. Diabetes. 1994; 43: 247-255Crossref PubMed Google Scholar). Construction of CHO cell lines expressing Gln-1174 or Leu-1178 insulin receptors has been described previously (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). CHO cells were grown in F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C under an atmosphere of 95% air and 5% CO2. Anti-IRS-1 and anti-Shc antibodies (polyclonal) were raised against GST fusion proteins containing IRS-1 amino acids 511–860 and Shc amino acids 366–473, respectively. A monoclonal anti-p85α antibody (U2) was a gift from Dr. Mike Waterfield (University College London, UK). Phospho-specific p44/42 MAP kinase antibody was purchased from New England Biolabs, and anti-Ras antibody Y13-259 was purchased from Oncogene Science. Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (clone 4G10). CHO.K1 cells at ∼80% confluence in 12-mm dishes were transfected for 2 h with four plasmids: (i) pCMV.hIR, the wild-type IR or mutants thereof, under the control of the nonregulated CMV promoter, (ii) pGL3.G5E4D[Δ]38, the firefly luciferase reporter plasmid, which possesses five GAL4 binding sites upstream of the firefly luciferase gene in the plasmid pGL3 (Promega Corp.), (iii) pRL.CMV, Renilla luciferase under the control of the CMV promoter, and (iv) pSG424.Elk1 (83–428), fusion of the GAL4 DNA binding domain and Elk-1 activation domain. Transfections were performed using Tfx-50 (Promega Corp.) transfection reagent (2.2 μg of Tfx-50/μg of DNA in 5% serum-free Ham's F-12). The dishes were further incubated in Ham's F-12 containing 5% fetal calf serum for 4 h, then washed with phosphate-buffered saline and serum-starved for 2 h. The cells were stimulated with 100 nm insulin for 16 h. The cells were then extracted using Stop and GloR lysis buffer and assayed sequentially for the firefly and Renilla luciferases according to the manufacturer's instructions (Promega Corp.). In brief, 10 μl of crude cell lysate was incubated with 50 μl of luciferin reagent (LARII). After 15 s at room temperature, the luminescence was recorded for 30 s in a Berthold Lumat LB9501 luminometer. 50 μl of Stop and GloR reagent was added, and the specific luminescence from the Renilla luciferase was recorded for an additional 30 s. CHO.K1 cells on 22-mm-diameter glass coverslips were co-microinjected as described previously (19Dobson S.P. Livingstone C. Gould G.W. Tavaré J.M. FEBS Lett. 1996; 393: 179-184Crossref PubMed Scopus (45) Google Scholar) with two plasmids; pCMV.hIR (or mutants thereof) at 50 μg/ml and pcDNAneoI possessing a chimera between green fluorescent protein (GFP) fused to the N terminus of GLUT4 at 200 μg/ml. Cells were incubated in growth medium for 16–24 h before serum starvation for 2 h. The cells were subjected to fluorescence analysis in Hepes-buffered Krebs (10 mm Hepes, pH 7.4, 2 mm NaHCO3, 140 mm NaCl, 3.6 mm KCl, 0.6 mmNa2HPO4, 0.5 mm MgSO4, 1 mm CaCl2, and 5.5 mm glucose) using a Zeiss Axiovert 100TV microscope with a 40 × oil immersion objective. GFP excitation/emission was achieved with a High Q fluorescein isothiocyanate filter set (Chroma Technology Corp., Brattleboro, VT). Treatment with insulin (100 nm) was for 60 min. Immunoprecipitation and Western blotting was carried out essentially as described previously (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), except blots were probed with125I-labeled secondary antibodies and proteins were visualized by autoradiography and quantified by phosphoimaging using a Fujix BAS 2000 phosphoimager. PI3-kinase activity was assayed using a protocol adapted from Jackson et al. (20Jackson T.R. Stephens L.R. Hawkins P.T. J. Biol. Chem. 1992; 267: 16627-16636Abstract Full Text PDF PubMed Google Scholar). Cells were grown until confluency in 3.5-cm (6 well) plates, incubated overnight in serum-free Ham's F-12 media, and then treated with insulin (100 nm) for 5 min. Incubations were terminated by aspirating the medium and rinsing briefly with ice-cold phosphate-buffered saline before the addition of 500 μl of ice-cold freshly prepared PI3-kinase lysis buffer A (20 mm Tris, pH 8.0, 137 mm NaCl, 2.7 mm KCl, 1 mmMgCl2, 500 μm NaVO3, 1% Nonidet P-40, 10% (w/v) glycerol, 10 μg/ml leupeptin, and 200 μm phenylmethylsulfonyl fluoride). The lysates were cleared by centrifugation and incubated with anti-IRS-1 antibody (1 in 100 dilution) and 50 μl of protein A-Sepharose (50 mg/ml pre-equilibrated in lysis buffer A) by tumbling end over end at 4 °C for 2 h. The immunoprecipitates were then washed 3 times with lysis buffer A, 2 times in buffer B (500 mm LiCl, 100 mm Tris-HCl, pH 8.0, at 4 °C), once in buffer C (150 mm NaCl, 10 mm Tris-HCl, 1 mm EDTA, pH 7.6, at 4 °C), and once in buffer D (20 mmHepes, 1 mm dithiothreitol, 5 mmMgCl2, pH 7.6, at 4 °C). The beads were then resuspended in 40 μl of buffer E (10 mm β-glycerophosphate, 5 mm Na4P2O7, 30 mm NaCl, 1 mm dithiothreitol, pH 7.2, at 4 °C). 20 μl of phosphatidylinositol/cholate solution (3 mg/ml in 1% (w/v) sodium cholate) was added to each tube, and the reaction was started by the addition of 5 μCi of [γ-32P]ATP in 40 μl of reaction mix (3 μm Na2ATP, 7.5 mm MgCl2) and incubated at 37 °C for 15 min. Reactions were terminated by the addition of 450 μl of CHCl3:CH3OH (1:2 v/v). The product was then extracted by the addition of 150 μl of CHCl3 and 150 μl of 0.1 m HCl and then again by the addition of 300 μl of CHCl3 and 300 μl of 0.1 m HCl. Extracted lipid was dried down under vacuum before redissolving in 25 μl of CHCl3, CH3OH, 0.1 m HCl (200:100:1). Reaction products were separated by thin layer chromatography (run in a pre-equilibrated tank containing methanol:chloroform:ammonia:water, 300:210:45:75) and quantified using a Fujix Bas 2000 phosphoimager. Analysis of the GDP/GTP ratio on Ras was determined as described previously (21Ouwens D.M. van der Zon G.C.M. Pronk G.J. Bos J.L. Möller W. Cheatham B. Kahn C.R. Maassen J.A. J. Biol. Chem. 1994; 269: 33116-33122Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were grown until confluency in 4-cm dishes, serum-starved overnight, then labeled with [32P]orthophosphate before being stimulated with insulin (100 nm, 5 min). Cells were then lysed in a buffer containing 1% Triton X-114. Lysates were cleared by centrifugation, and the Triton X-114-soluble material was collected by a short temperature shift. The detergent phase was then used to immunoprecipitate Ras. Guanine nucleotides were eluted from the immune complex and resolved by thin layer chromatography. The plates were visualized by autoradiography and quantitated using a Fujix Bas 2000 phosphoimager. We have previously reported that the mutant insulin receptors Gln-1174 or Leu-1178 are unable to autophosphorylate and that CHO cells stably expressing these receptors are severely defective in insulin-stimulated glycogen synthesis and thymidine incorporation (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Unexpectedly, significant insulin-stimulated IRS-1 phosphorylation was seen in both cell lines expressing the mutant insulin receptors using anti-phosphotyrosine blotting of total cell lysates and anti-IRS-1 immunoprecipitates (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). These observations suggested to us that IRS-1 phosphorylation per se may be insufficient to mediate downstream insulin signaling events. This possibility was further explored by more detailed characterization of the signaling properties of the CHO cell lines stably expressing similar numbers of wild-type, Gln-1174, or Leu-1178 insulin receptors termed wild-type, Gln-1174, or Leu-1178 cell lines, respectively. To more precisely define the dose response characteristics of IRS-1 tyrosine phosphorylation, all cell lines were treated with various concentrations of insulin for 2 min, and IRS-1 immunoprecipitates were analyzed by anti-phosphotyrosine blotting (Fig.1). At 100 nm insulin, the Gln-1174 and Leu-1178 cell lines show significant enhancement of insulin-stimulated tyrosine phosphorylation of IRS-1 compared with parental nontransfected CHO cells, although this was somewhat reduced compared with the wild-type cell line. The levels of IRS-1 phosphorylation seen in the Gln-1174 and Leu-1178 cell lines stimulated with 100 nm insulin and the wild-type cell line stimulated with 1 nm insulin were comparable. Thus, while we confirmed the ability of the mutant receptors to promote insulin-stimulated tyrosine phosphorylation of IRS-1, these mutant receptors did so less well than wild-type receptors. In parallel immunoblotting studies, insulin ability to promote the association of the p85α subunit of PI3-kinase with IRS-1 was examined. Consistent with the studies of IRS-1 phosphorylation, association of p85α with IRS-1 in response to insulin was significantly enhanced in the Gln-1174 and Leu-1178 cell lines compared with parental CHO cells (Fig. 1). Again, similar levels of IRS-1-associated p85α were seen when the Gln-1174 and Leu-1178 cell lines were treated with 100 nm insulin versus 1 nm insulin treatment of the wild-type cell line. To examine whether the insulin-stimulated association of p85α seen in cells expressing the mutant receptors could result in the functional activation of PI3-kinase, IRS-1-associated PI3-kinase activity was measured in all cell lines. Parental CHO cells and Gln-1174 and Leu-1178 cells were incubated with 100 nm insulin for 5 min, whereas wild-type cells were incubated with either 1 or 100 nm insulin for 5 min. PI3-kinase activity in IRS-1 immunoprecipitates from wild-type cells (stimulated with either 1 or 100 nm insulin) or Gln-1174 and Leu-1178 cells showed a stimulation that was significantly greater (p < 0.03) than that seen in parental CHO cells (Fig.2). Also, PI3-kinase activity was comparable in wild-type cells stimulated with 1 nm insulin and Gln-1174 and Leu-1178 cells stimulated with 100 nminsulin. Thus the Gln-1174 and Leu-1178 mutant receptors, despite showing undetectable autophosphorylation, are capable of phosphorylating IRS-1 such that it can interact with and activate PI3-kinase. Of note, using the identical cell lines, we have previously reported that exposure of the wild-type cell line to 1 nm insulin results in a marked enhancement of glycogen synthesis (∼60% maximal), whereas no significant enhancement of glycogen synthesis was seen in the Gln-1174 and Leu-1178 cell lines when studied at concentrations of 100 nm insulin or higher (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). There is therefore a clear discordance between the abilities of these mutant receptors to activate IRS-1-associated PI3-kinase activity and to stimulate glycogen synthesis. To provide additional confirmation that metabolic signaling through the mutant receptors was markedly impaired, we monitored the translocation of GLUT4 to the plasma membrane in CHO cells by taking advantage of the recently described fluorescent chimera between GLUT4 and GFP (19Dobson S.P. Livingstone C. Gould G.W. Tavaré J.M. FEBS Lett. 1996; 393: 179-184Crossref PubMed Scopus (45) Google Scholar). In CHO.T cells that stably overexpress wild-type insulin receptors, we have previously demonstrated that this chimera translocates to the cell surface in response to insulin in approximately 60% of cells (19Dobson S.P. Livingstone C. Gould G.W. Tavaré J.M. FEBS Lett. 1996; 393: 179-184Crossref PubMed Scopus (45) Google Scholar). We confirmed this observation in CHO.K1 cells using co-microinjection to transiently overexpress wild-type insulin receptors and GFP-GLUT4 (Fig.3). Cells injected with GFP-GLUT4 and control vector mediated no significant increase in GFP-GLUT4 translocation in response to insulin. In addition, neither the Gln-1174 or Leu-1178 mutant insulin receptors mediated insulin-stimulated GFP-GLUT4 translocation. These results provide additional confirmation that metabolic signaling is severely impaired in the Gln-1174- and Leu-1178-expressing cells. However, as we did not specifically compare wild-type and mutant receptors at insulin concentrations producing comparable degrees of PI3-kinase activation, we cannot use these data to draw any direct conclusions regarding the role of PI3-kinase in the mediation of this particular process. With respect to the mitogenic effects of insulin, we have previously demonstrated that the Gln-1174 and Leu-1178 mutant receptors are unable to mediate insulin stimulation of thymidine incorporation into DNA or to activate the enzymatic activity of MAP kinase (1Krook A. Moller D.E. Dib K. O'Rahilly S. J. Biol. Chem. 1996; 271: 7134-7140Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To examine events relating to insulin's mitogenic actions in more detail under conditions where cells expressing mutant and wild-type receptors demonstrated comparable levels of IRS-1 phosphorylation and PI3-kinase activation, the characteristics of MAP kinase phosphorylation were examined in all cell lines. Whereas cells overexpressing wild-type insulin receptors showed a 3-fold stimulation of MAP kinase tyrosine phosphorylation in response to 1 nm insulin and an approximate 5-fold stimulation following 100 nm insulin treatment, no such phosphorylation was seen in the Gln-1174 and Leu-1178 cell lines (Fig. 4). To provide independent confirmation of the inability of these mutant receptors to activate downstream effects related to mitogenesis, we examined the ability of these mutant receptors to activate the ternary complex factor Elk-1. This factor forms a complex with serum response factor at the serum response element within the c-fos promoter and is responsible for the observed insulin effect on c-fosinduction in CHO cells. 2M. R. Griffiths, E. J. Black, M. Dickens, P. E. Shaw, D. A. F. Gillespie, and J. M. Tavaré, submitted for publication. The activation of this transcription factor is closely associated with the proliferative response of cells to growth stimuli (23Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). A GAL4-Elk-1 fusion protein was expressed in CHO.K1 cells by transient transfection. The activity of this chimeric transcription factor was monitored with a co-transfected luciferase reporter plasmid, where the luciferase gene is placed under the control of a promoter possessing five GAL4 binding sites. A third plasmid, pRL.CMV, possessing the Renilla retinformis luciferase was included in these transfections to monitor cell transfection efficiency. Finally, a fourth plasmid was included to overexpress wild-type, Gln-1174, or Leu-1178 mutant insulin receptors. As shown in Fig. 5, parental CHO.K1 cells exhibited little or no stimulation of Elk-1 in response to insulin, as determined by luciferase activity. When the wild-type insulin receptor was overexpressed, insulin induced an approximate 2-fold increase in luciferase activity after incubation with 1 nm insulin and an approximate 5-fold increase in luciferase activity after incubation with 100 nm insulin. In contrast, neither mutant insulin receptor was capable of activating Elk-1 (Fig.5).Figure 5CHO cells expressing Gln-1174 and Leu-1178 mutant insulin receptors fail to mediate insulin stimulation of a GAL4-Elk1 transcription factor. CHO.K1 cells were transiently transfected with pRL.CMV, pSG424.Elk1(83–428), and pGL3.G5E4D[Δ]38. Also included was a fourth plasmid, either empty vector (vector), vector encoding wild-type insulin receptor cDNA (wt), or vector encoding either Gln-1174 or Leu-1178 mutant insulin receptors, as indicated. Transfected cells were then treated in the absence (open bars) or presence of 100 nminsulin (shaded bars) or 1 nm insulin (hatched bar). After 16 h, the cells were harvested, and lysates were assayed for firefly and Renilla luciferases as described under “Experimental Procedures.” Results shown are the ratio of the firefly luciferase/Renilla luciferase activities (i.e. corrected for transfection efficiency) and are standardized to the maximal response of insulin (in cells expressing wild-type insulin receptors). The data are mean + S.E. from two independent experiments, each performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, at concentrations of insu" @default.
• W2161573035 created "2016-06-24" @default.
• W2161573035 creator A5005300373 @default.
• W2161573035 creator A5006661087 @default.
• W2161573035 creator A5013261212 @default.
• W2161573035 creator A5019388256 @default.
• W2161573035 creator A5027175930 @default.
• W2161573035 creator A5029349104 @default.
• W2161573035 creator A5038362347 @default.
• W2161573035 creator A5046558354 @default.
• W2161573035 creator A5067510273 @default.
• W2161573035 creator A5068081328 @default.
• W2161573035 creator A5068690836 @default.
• W2161573035 creator A5069914075 @default.
• W2161573035 date "1997-11-01" @default.
• W2161573035 modified "2023-09-26" @default.
• W2161573035 title "Two Naturally Occurring Insulin Receptor Tyrosine Kinase Domain Mutants Provide Evidence That Phosphoinositide 3-Kinase Activation Alone Is Not Sufficient for the Mediation of Insulin's Metabolic and Mitogenic Effects" @default.
• W2161573035 cites W1488724125 @default.
• W2161573035 cites W1493815818 @default.
• W2161573035 cites W1496640920 @default.
• W2161573035 cites W1505075493 @default.
• W2161573035 cites W1517759896 @default.
• W2161573035 cites W1523684384 @default.
• W2161573035 cites W1541173869 @default.
• W2161573035 cites W1582699368 @default.
• W2161573035 cites W1862633417 @default.
• W2161573035 cites W1966083436 @default.
• W2161573035 cites W1971498717 @default.
• W2161573035 cites W1989324173 @default.
• W2161573035 cites W1992513963 @default.
• W2161573035 cites W1997460544 @default.
• W2161573035 cites W2008532096 @default.
• W2161573035 cites W2015707895 @default.
• W2161573035 cites W2016910304 @default.
• W2161573035 cites W2033429522 @default.
• W2161573035 cites W2039180413 @default.
• W2161573035 cites W2066052044 @default.
• W2161573035 cites W2068891266 @default.
• W2161573035 cites W2075326322 @default.
• W2161573035 cites W2081698610 @default.
• W2161573035 cites W2091131600 @default.
• W2161573035 cites W2095416319 @default.
• W2161573035 cites W2099817971 @default.
• W2161573035 cites W2106329420 @default.
• W2161573035 cites W2109300167 @default.
• W2161573035 cites W2344973376 @default.
• W2161573035 cites W42380194 @default.
• W2161573035 doi "https://doi.org/10.1074/jbc.272.48.30208" @default.
• W2161573035 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9374504" @default.
• W2161573035 hasPublicationYear "1997" @default.
• W2161573035 type Work @default.
• W2161573035 sameAs 2161573035 @default.
• W2161573035 citedByCount "89" @default.
• W2161573035 countsByYear W21615730352015 @default.
• W2161573035 countsByYear W21615730352017 @default.
• W2161573035 countsByYear W21615730352018 @default.
• W2161573035 countsByYear W21615730352021 @default.
• W2161573035 crossrefType "journal-article" @default.
• W2161573035 hasAuthorship W2161573035A5005300373 @default.
• W2161573035 hasAuthorship W2161573035A5006661087 @default.
• W2161573035 hasAuthorship W2161573035A5013261212 @default.
• W2161573035 hasAuthorship W2161573035A5019388256 @default.
• W2161573035 hasAuthorship W2161573035A5027175930 @default.
• W2161573035 hasAuthorship W2161573035A5029349104 @default.
• W2161573035 hasAuthorship W2161573035A5038362347 @default.
• W2161573035 hasAuthorship W2161573035A5046558354 @default.
• W2161573035 hasAuthorship W2161573035A5067510273 @default.
• W2161573035 hasAuthorship W2161573035A5068081328 @default.
• W2161573035 hasAuthorship W2161573035A5068690836 @default.
• W2161573035 hasAuthorship W2161573035A5069914075 @default.
• W2161573035 hasBestOaLocation W21615730351 @default.
• W2161573035 hasConcept C101544691 @default.
• W2161573035 hasConcept C112446052 @default.
• W2161573035 hasConcept C126322002 @default.
• W2161573035 hasConcept C134018914 @default.
• W2161573035 hasConcept C170493617 @default.
• W2161573035 hasConcept C180361614 @default.
• W2161573035 hasConcept C184235292 @default.
• W2161573035 hasConcept C2775960820 @default.
• W2161573035 hasConcept C2777391703 @default.
• W2161573035 hasConcept C2779306644 @default.
• W2161573035 hasConcept C2781322055 @default.
• W2161573035 hasConcept C42362537 @default.
• W2161573035 hasConcept C55493867 @default.
• W2161573035 hasConcept C62478195 @default.
• W2161573035 hasConcept C71924100 @default.
• W2161573035 hasConcept C79186569 @default.
• W2161573035 hasConcept C86554907 @default.
• W2161573035 hasConcept C86803240 @default.
• W2161573035 hasConcept C95444343 @default.
• W2161573035 hasConceptScore W2161573035C101544691 @default.
• W2161573035 hasConceptScore W2161573035C112446052 @default.
• W2161573035 hasConceptScore W2161573035C126322002 @default.
• W2161573035 hasConceptScore W2161573035C134018914 @default.
• W2161573035 hasConceptScore W2161573035C170493617 @default.
• W2161573035 hasConceptScore W2161573035C180361614 @default.
• W2161573035 hasConceptScore W2161573035C184235292 @default.
• W2161573035 hasConceptScore W2161573035C2775960820 @default.

• next