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W2037621839 abstract "Polyphosphoinositides are thought to be mediators of cellular signaling pathways as well as regulators of cytoskeletal elements and membrane trafficking events. It has recently been demonstrated that a class of phosphatidylinositol (PI) 3,4,5-P3 5′-phosphatases contains SH2 domains and proline-rich regions, which are present in many signaling proteins. We report here that insulin stimulation of Chinese hamster ovary cells (CHO-T) expressing human insulin receptors causes an 8-10-fold increase in PI 3,4,5-P3 5′-phosphatase activity in anti-phosphotyrosine immunoprecipitates of the cell lysates. This insulin-sensitive polyphosphoinositide 5′-phosphatase did not catalyze dephosphorylation of PI 4,5-P2. No change in 5′-phosphatase activity was detected in insulin receptor or IRS-1 immune complexes in response to insulin. However, insulin treatment of CHO-T cells markedly increased the PI 3,4,5-P3 5′-phosphatase activity associated with Shc and Grb2. The insulin-regulated polyphosphoinositide 5′-phosphatase was not immunoreactive with antibody raised against the recently cloned SHIP 5′-phosphatase reported to associate with Shc and Grb2 in B lymphocytes. These data demonstrate that insulin causes formation of complexes containing a PI 3,4,5-P3 5′-phosphatase, and Shc or Grb2, or both, suggesting an important role of this enzyme in insulin signaling. Polyphosphoinositides are thought to be mediators of cellular signaling pathways as well as regulators of cytoskeletal elements and membrane trafficking events. It has recently been demonstrated that a class of phosphatidylinositol (PI) 3,4,5-P3 5′-phosphatases contains SH2 domains and proline-rich regions, which are present in many signaling proteins. We report here that insulin stimulation of Chinese hamster ovary cells (CHO-T) expressing human insulin receptors causes an 8-10-fold increase in PI 3,4,5-P3 5′-phosphatase activity in anti-phosphotyrosine immunoprecipitates of the cell lysates. This insulin-sensitive polyphosphoinositide 5′-phosphatase did not catalyze dephosphorylation of PI 4,5-P2. No change in 5′-phosphatase activity was detected in insulin receptor or IRS-1 immune complexes in response to insulin. However, insulin treatment of CHO-T cells markedly increased the PI 3,4,5-P3 5′-phosphatase activity associated with Shc and Grb2. The insulin-regulated polyphosphoinositide 5′-phosphatase was not immunoreactive with antibody raised against the recently cloned SHIP 5′-phosphatase reported to associate with Shc and Grb2 in B lymphocytes. These data demonstrate that insulin causes formation of complexes containing a PI 3,4,5-P3 5′-phosphatase, and Shc or Grb2, or both, suggesting an important role of this enzyme in insulin signaling. INTRODUCTIONThe insulin receptor belongs to a family of structurally related transmembrane growth factor receptors that exhibit ligand-activated protein-tyrosine kinase activity (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Rosen O.M. Herrera R. Olowe Y Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (304) Google Scholar, 3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar). The insulin receptor kinase activity is thought to be essential for cellular responses to insulin (4Rosen O.M. Science. 1987; 237: 1452-1458Crossref PubMed Scopus (503) Google Scholar, 5Chou C.K. Dull T.J. Russell D.S. Gherzi R. Lebwohl D. Ullrich A. Rosen O.M. J. Biol. Chem. 1987; 262: 1842-1847Abstract Full Text PDF PubMed Google Scholar, 6Ebina Y. Araki E. Taira M. Shimada F. Mori M. Craik C.S. Siddle K. Pierce S.B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 704-708Crossref PubMed Scopus (267) Google Scholar). Activation of insulin receptor kinase promotes the rapid autophosphorylation of insulin receptor β-subunits as well as tyrosine phosphorylation of several cytoplasmic proteins such as IRS-1, 1The abbreviations used are: IRS-1insulin receptor substrate-1IRinsulin receptorPIphosphatidylinositolSHSrc homologySHIPSH2-containing inositol 5′-phosphataseCHOChinese hamster ovary cellsPBSphosphate-buffered salineBCAbicinchoninic acidPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatography. Shc, and pp60 that appear to be involved in the insulin signaling pathway (3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar, 7Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar, 8Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, 9Lavan B.E. Lienhard G.E. J. Biol. Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). Evidence indicates that a primary function of the insulin receptor kinase is to place tyrosine phosphate docking sites on these proteins for the recruitment of signaling proteins containing Src homology (SH) 2 domains (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 10Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 11Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar). Thus, insulin-induced phosphorylation of IRS-1, Shc, and pp60 promotes their association with specific SH2-containing proteins, which in turn can stimulate the catalytic activity of these SH2 proteins (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 14Kuhne M.R. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, 15Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar). One such SH2-containing protein is the p85 regulatory subunit of the p110 phosphatidylinositol (PI) 3-kinase which catalyzes phosphorylation of the 3-position on PI (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 16Kapeller R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar).Strong evidence supports a pivotal role for signaling complexes containing IRS-1 and the p85/p110-type PI 3-kinases in mediating insulin action on GLUT4 glucose transporter redistribution to the plasma membrane leading to increased glucose uptake as well as glycogen synthesis. Inhibition of PI 3-kinase activity by wortmannin (17Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 18Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (230) Google Scholar, 19Cross D.A.E. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar) or LY294002 (20Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn R.C. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar), microinjection of a fusion protein consisting of an SH2 domain of the p85 regulatory subunit of PI 3-kinase (21Haruta T. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and disruption of PI 3-kinase recruitment to IRS-1 by dominant inhibitory constructs of p85 (22Quon M.J. Chen H. Ing B.L. Liu M.-L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) ablate the stimulation of glucose transport and glycogen synthesis by insulin. Expression of IRS-1 antisense RNA in isolated fat cells also inhibits insulin-mediated translocation of epitope-tagged GLUT4 glucose transporters to the cell surface (23Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). Further, insulin causes the localization of IRS-1·PI 3-kinase complexes to intracellular membrane vesicles containing GLUT4 (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), while other growth factors that stimulate PI 3-kinase activity but fail to activate glucose transport do not. 2R. A. Heller-Harrison, M. Morin, A. Guilherme, E. Skolnik, and M. P. Czech, submitted for publication. These data are consistent with the hypothesis that one or more 3′-phosphoinositide species generated in intracellular membranes in response to insulin regulate cellular components involved in membrane trafficking of GLUT4.It is established that the insulin-sensitive p85/p110 PI 3-kinase activity can catalyze formation of PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 from PI, PI 4-P, and PI 4,5-P2, respectively (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar, 26Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar). However, no information is available about which of these 3′-phosphoinositide species actually participates in the mechanisms of insulin action. Interestingly, interconversion of these species appears to occur through the action of 3′-polyphosphoinositide 4′- and 5′-phosphatases (27Lips D.L. Majerus P.W. J. Biol. Chem. 1989; 264: 19911-19915Abstract Full Text PDF PubMed Google Scholar, 28Norris F.A. Majerus P.W. J. Biol. Chem. 1994; 269: 8716-8720Abstract Full Text PDF PubMed Google Scholar, 29Woscholski R. Waterfield M.D. Parker P.J. J. Biol. Chem. 1995; 270: 31001-31007Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 32Jackson S.P. Schoenwaelder S.M. Matizaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar, 34Kabuyama Y. Nakatsu N. Homma Y. Fukui Y. Eur. J. Biochem. 1996; 238: 350-356Crossref PubMed Scopus (12) Google Scholar). Recent reports have described PI 3,4,5-P3 5′-phosphatases that contain SH2 and proline-rich domains characteristic of signaling proteins (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Recent reports demonstrate the association of PI 3,4,5-P3 5′-phosphatase with Shc (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Also, a PI 3,4,5-P3 5′-phosphatase which forms a complex with p85/p110 PI 3-kinase in platelets has been described (32Jackson S.P. Schoenwaelder S.M. Matizaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar). The combination of actions of this stimulated PI 3-kinase activity and 5′-phosphatase activity is expected to produce cellular PI 3,4-P2 which may be uniquely important in triggering downstream cellular effects. These considerations prompted us to investigate whether insulin action might also modulate polyphosphoinositide 5′-phosphatase activity. We report here a marked insulin-mediated recruitment of 5′-phosphatase activity specific for PI 3,4,5-P3 to complexes containing Shc and Grb2. This novel action of insulin is likely to play an important role in one or more downstream cascades important to this hormone's function.RESULTS AND DISCUSSIONInitial experiments were conducted to establish assay conditions for quantifying polyphosphoinositide 5′-phosphatase activity in lysates of CHO-T cells expressing human insulin receptors. Dephosphorylation of [3′-32P]PI 3,4,5-P3 to di- and monophosphoinositides in cell lysates was readily observed in the absence of vanadate upon thin layer chromatography analysis of the reaction products (not shown). However, inclusion of vanadate in the assay buffer almost completely blocked PI 3,4-P2 phosphatase activity with little effect on the PI 3,4,5-P3 5′-phosphatase activity. This is consistent with a previous report that 3′-phosphatase is inhibited by vanadate (27Lips D.L. Majerus P.W. J. Biol. Chem. 1989; 264: 19911-19915Abstract Full Text PDF PubMed Google Scholar). Thus, in the presence of vanadate, nearly quantitative conversion of PI 3,4,5-P3 to PI 3,4-P2 was observed, measured either as the disappearance of the former or appearance of the latter (not shown). All subsequent assays were therefore performed under these conditions.To determine whether insulin regulates PI 3,4,5-P3 5′-phosphatase, lysates of CHO-T cells incubated with or without insulin were immunoprecipitated with anti-Tyr(P) antibody, and the precipitates were assayed as described above. The results revealed a marked increase in PI 3,4,5-P3 5′-phosphatase activity in the anti-Tyr(P) precipitates due to insulin action (Fig. 1, A and B). Tyrosine-phosphorylated insulin receptor and IRS-1 were immunoprecipitated when cells were treated with insulin under these conditions, as evidenced by Western blot analysis (Fig. 1C). However, when either insulin receptors or IRS-1 were specifically immunoprecipitated with anti-insulin receptor or anti-IRS-1 antibody (Fig. 1C), no insulin-stimulated PI 3,4,5-P3 5′-phosphatase activity could be detected in the immune complexes (Fig. 1, A and B). This was also the case when whole cell lysates from control versus insulin-treated CHO-T cells were assayed (data not shown).The insulin-regulated PI 3,4,5-P3 5′-phosphatase activity present in the anti-tyrosine phosphate immunoprecipitates was dependent on MgCl2 in the assay buffer (data not shown). The anti-Tyr(P) immunoprecipitates did not contain insulin-regulated phosphatase activity when PI 4,5-P2 was used as substrate (data not shown). High pressure liquid chromatography of the deacylated 32P-labeled phosphoinositide reaction products was performed to identify the diphosphoinositide formed in the presence of tyrosine phosphate immune complexes from control and insulin-treated CHO-T cells (Fig. 2). This analysis demonstrated virtually quantitative conversion of PI 3,4,5-P3 to PI 3,4-P2 in this assay as well as a marked increase in PI 3,4-P2 formation catalyzed by the immune complexes derived from insulin-treated cells (Fig. 2).Fig. 2Insulin-regulated PI 3,4,5-P3 phosphatase dephosphorylates PI 3,4,5-P3 at the 5′-position of the inositol ring. Lysates from CHO-T cells treated with or without insulin (control) were immunoprecipitated with anti-Tyr(P), and PI 3,4,5-P3 5′-phosphatase activity was assayed as described in Fig. 1. Shown are the HPLC profiles of 32P-labeled deacylated polyphosphoinositides (gPIs) after the phosphatase assays. Top panel, assay containing PI 3,4,5-P3 with no addition of immune complexes (NA). Bottom panels, assays with immune complexes from control cells (CON) and insulin-treated (INS) cells, as indicated. Dashed arrows indicate retention time of the deacylated [3H]PI 4,5-P2 standard.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The recent molecular cloning of polyphosphoinositide 5′-phosphatases that associate with Shc in response to myeloid cell activation (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and that associate with the adapter protein Grb2 in response to B cell activation, prompted experiments to test such possible associations with the insulin-regulated 5′-phosphatase(s) (Fig. 3). Immune complexes obtained from lysates of insulin-treated CHO-T cells using anti-Shc or anti-Grb2 antibodies exhibited PI 3,4,5-P3 5′-phosphatase activity that was severalfold higher than that derived from control cells. However, the insulin-stimulated 5′-phosphatase activity in the anti-Shc and anti-Grb2 immunoprecipitates was about half that associated with the anti-Tyr(P) antibody (Fig. 3). This probably reflects, at least in part, the fact that the anti-Shc and anti-Grb2 antibodies used do not quantitatively precipitate Shc and Grb2 from the cell lysates (data not shown). Taken together, the data in Figs. 1, 2, 3 demonstrate a marked effect of insulin to cause association of Shc and Grb2 with one or more 5′-phosphatases able to dephosphorylate PI 3,4,5-P3 but not PI 4,5-P2.Fig. 3Insulin increases association of PI 3,4,5-P3 5′-phosphatase activity with Shc and Grb2. Lysates were prepared from CHO-T cells treated with (+) or without (−) 100 nM insulin for 15 min. A, cell lysates were immunoprecipitated with anti-Tyr(P), anti-Shc, or anti-Grb2, and immunoprecipitates were assayed for PI 3,4,5-P3 5′-phosphatase by incubation with [32P]PI 3,4,5-P3. NA indicates assay, without immune complex. B, graph shows the effect of insulin on PI 3,4,5-P3 5′-phosphatase activity in immune complexes. Spots corresponding to PI 3,4-P2 in the thin layer chromatography plate were cut out and quantitated using a β-counter. The data presented are average values from 3 independent experiments ± S.E., normalized by equating each value in the absence of insulin to 1. The actual counts/min values in basal conditions were: 1014, anti-Tyr(P); 313, anti-Shc; and 890, anti-Grb2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The characteristics of this insulin-regulated polyphosphoinositide 5′-phosphatase resembles those of SHIP, the recently cloned 5′-phosphatase that binds to the C-terminal SH3 domain of Grb2 in vitro and associates with Shc in response to cytokines in hemopoietic cells (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar). This enzyme also requires Mg2+ for activity. The availability of anti-SHIP antibody (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar) allowed us to test whether SHIP was the insulin-regulated 5′-phosphatase detected in the present experiments. CHO-T cell lysates were immunoprecipitated with anti-SHIP antibody, and both the precipitate and supernatant were assayed for PI 3,4,5-P3 5′-phosphatase activity. As shown in Fig. 4, only the latter catalyzed formation of labeled PI 3,4-P2 in this assay. Parallel immunoprecipitations of hamster lung lysates with anti-SHIP revealed easily detectable PI 3,4,5-P3 5′-phosphatase in the precipitates as well as in the supernatants (Fig. 4). Similarly, the anti-SHIP precipitates from hamster lung displayed a 150-kDa band upon SDS-PAGE and immunoblotting that was not present in anti-Tyr(P) imunoprecipitates of insulin-treated CHO-T cells lysates (data not shown). These latter immune complexes exhibited significant PI 3,4,5-P3 5′-phosphatase activity as demonstrated in Figs. 1, 2, 3. Thus, these data indicate that the insulin-regulated polyphosphoinositide 5′-phosphatase is not SHIP itself. However, it is possible that the CHO-T cell 5′-phosphatase is an isoform of this enzyme that is not recognized by anti-SHIP antibody. Resolving the identity of the presently described insulin-regulated PI 3,4,5-P3 5′-phosphatase is an important future objective.Fig. 4The insulin-regulated PI 3,4,5-P3 5′-phosphatase is not reactive with anti-SHIP. Lysates were prepared from CHO-T cell or hamster lung tissue. A, cell lysates were immunoprecipitated with anti-SHIP antibody, and the immunoprecipitates were assayed for PI 3,4,5-P3 5′-phosphatase activity as described. NA indicates no addition, and Sup indicates supernatant after immunoprecipitation. B, graph shows PI 3,4,5-P3 5′-phosphatase in anti-SHIP immunoprecipitates and supernatant (Sup.) from immunoprecipitates from CHO-T cell or hamster lung lysates. Spots corresponding to PI 3,4-P2 in thin layer chromatography plate were cut out and quantified using a β-counter.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is significant that the polyphosphoinositide 5′-phosphatases described above (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and reported here require phosphorylation of the 3′-position of the phosphoinositol head group to catalyze dephosphorylation. Phosphorylation of this 3′-position is catalyzed by four known classes of PI 3-kinases that exhibit diverse regulatory mechanisms (16Kapeller R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, 36Virbasius J.V. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 13304-13307Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 38Volinia S. Dhand R. Vanhaesbroeck B. MacDougall L.K. Stein R. Zvelebil M.J. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (306) Google Scholar, 39MacDougall L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, the regulated phosphorylation of PI 4,5-P2 by PI 3-kinases theoretically provides substrate for the 5′-phosphatases specific for PI 3,4,5-P3. These two reactions catalyzed by PI 3-kinase and PI 3,4,5-P3 5′-phosphatase in combination promote the conversion of PI 4,5-P2 to PI 3,4-P2. The fact that insulin and other growth factor receptor tyrosine kinases regulate both of these enzymes indicates that at least part of the cellular PI 3,4-P2 generated in response to these signaling pathways derives from newly formed PI 3,4,5-P3. Further, these considerations strongly suggest an important role for PI 3,4-P2 in signaling by these receptors. PI 3,4-P2 may be an effector that is unique and specific for one or more downstream signaling pathways such as the cAkt/Rac protein kinase (40Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1869) Google Scholar). Thus, the PI 3,4,5-P3 5′-phosphatase reaction may serve as a branch point for polyphosphoinositide signaling in which PI 3,4,5-P3 activates a set of events distinct from those activated by PI 3,4-P2. It will be important to search for cellular proteins that specifically bind each of these phosphoinositides.The association of 5′-phosphatase with Shc and Grb2 in response to insulin reported here (Fig. 3) suggests a potential role of this phosphatase in regulating the p21ras pathway. Both Shc and Grb2 are components of multiprotein complexes containing the guanine nucleotide exchange factor son of sevenless that catalyzes GTP loading of p21ras (8Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, 11Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, 41Pronk G.J. de Vries-Smits A.M. Buday L. Downward J. Maassen J.A. Medema R.H. Bos J.L. Mol. Cell. Biol. 1994; 14: 1575-1581Crossref PubMed Google Scholar, 42Waters S.B. Chen D. Kao A.W. Okada S. Holt K.H. Pessin J.E. J. Biol. Chem. 1996; 271: 18224-18230Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Interactions between p21ras and the p110 PI 3-kinase have been reported (43Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar), and in some cell types a downstream effect of p21ras, mitogen-activated protein kinase activation, in response to insulin or growth factors is blocked by wortmannin, a potent inhibitor of PI 3-kinases (44Welsh G.I. Foulstone E.J. Young S.W. Tavare J.M. Proud C.G. Biochem. J. 1994; 303: 15-20Crossref PubMed Scopus (182) Google Scholar). Futher studies on this issue are clearly warranted.In addition to a potential positive signaling function of the insulin-sensitive PI 3,4,5-P3 5′-phosphatase described above, a negative role in signal transmission is also possible. If PI 3,4,5-P3 generated by PI 3-kinases is a positive effector of downstream signaling events, as appears likely (16Kapeller R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar), its concentration is expected to be reduced by the action of the 5′-phosphatase. Thus, the signaling potential of PI 3,4,5-P3 may be reduced or desensitized by insulin-regulated PI 3,4,5-P3 5′-phosphatase. Perhaps it is desirable to control the cellular localization of PI 3,4,5-P3 and to restrict it from regions containing Shc·Grb2 complexes. This hypothesis requires rigorous testing subsequent to identification of additional cellular targets of PI 3,4,5-P3. In any case, the data reported here indicate an important function of PI 3,4,5-P3 5′-phosphatase activity in one or more signaling pathways emanating from the insulin receptor. INTRODUCTIONThe insulin receptor belongs to a family of structurally related transmembrane growth factor receptors that exhibit ligand-activated protein-tyrosine kinase activity (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Rosen O.M. Herrera R. Olowe Y Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (304) Google Scholar, 3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar). The insulin receptor kinase activity is thought to be essential for cellular responses to insulin (4Rosen O.M. Science. 1987; 237: 1452-1458Crossref PubMed Scopus (503) Google Scholar, 5Chou C.K. Dull T.J. Russell D.S. Gherzi R. Lebwohl D. Ullrich A. Rosen O.M. J. Biol. Chem. 1987; 262: 1842-1847Abstract Full Text PDF PubMed Google Scholar, 6Ebina Y. Araki E. Taira M. Shimada F. Mori M. Craik C.S. Siddle K. Pierce S.B. Roth R.A. Proc. Natl. Acad. Sci. U. S. 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Thus, insulin-induced phosphorylation of IRS-1, Shc, and pp60 promotes their association with specific SH2-containing proteins, which in turn can stimulate the catalytic activity of these SH2 proteins (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 14Kuhne M.R. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, 15Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar). 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Inhibition of PI 3-kinase activity by wortmannin (17Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 18Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (230) Google Scholar, 19Cross D.A.E. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar) or LY294002 (20Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn R.C. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar), microinjection of a fusion protein consisting of an SH2 domain of the p85 regulatory subunit of PI 3-kinase (21Haruta T. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and disruption of PI 3-kinase recruitment to IRS-1 by dominant inhibitory constructs of p85 (22Quon M.J. Chen H. Ing B.L. Liu M.-L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) ablate the stimulation of glucose transport and glycogen synthesis by insulin. Expression of IRS-1 antisense RNA in isolated fat cells also inhibits insulin-mediated translocation of epitope-tagged GLUT4 glucose transporters to the cell surface (23Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). Further, insulin causes the localization of IRS-1·PI 3-kinase complexes to intracellular membrane vesicles containing GLUT4 (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), while other growth factors that stimulate PI 3-kinase activity but fail to activate glucose transport do not. 2R. A. Heller-Harrison, M. Morin, A. Guilherme, E. Skolnik, and M. P. Czech, submitted for publication. These data are consistent with the hypothesis that one or more 3′-phosphoinositide species generated in intracellular membranes in response to insulin regulate cellular components involved in membrane trafficking of GLUT4.It is established that the insulin-sensitive p85/p110 PI 3-kinase activity can catalyze formation of PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 from PI, PI 4-P, and PI 4,5-P2, respectively (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar, 26Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar). However, no information is available about which of these 3′-phosphoinositide species actually participates in the mechanisms of insulin action. Interestingly, interconversion of these species appears to occur through the action of 3′-polyphosphoinositide 4′- and 5′-phosphatases (27Lips D.L. Majerus P.W. J. Biol. Chem. 1989; 264: 19911-19915Abstract Full Text PDF PubMed Google Scholar, 28Norris F.A. Majerus P.W. J. Biol. Chem. 1994; 269: 8716-8720Abstract Full Text PDF PubMed Google Scholar, 29Woscholski R. Waterfield M.D. Parker P.J. J. Biol. Chem. 1995; 270: 31001-31007Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 32Jackson S.P. Schoenwaelder S.M. Matizaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar, 34Kabuyama Y. Nakatsu N. Homma Y. Fukui Y. Eur. J. Biochem. 1996; 238: 350-356Crossref PubMed Scopus (12) Google Scholar). Recent reports have described PI 3,4,5-P3 5′-phosphatases that contain SH2 and proline-rich domains characteristic of signaling proteins (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Recent reports demonstrate the association of PI 3,4,5-P3 5′-phosphatase with Shc (30Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 31Kavanaugh W.M. Pot D.A. Chin S.M. Reinhard M.D. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes & Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar). Also, a PI 3,4,5-P3 5′-phosphatase which forms a complex with p85/p110 PI 3-kinase in platelets has been described (32Jackson S.P. Schoenwaelder S.M. Matizaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar). The combination of actions of this stimulated PI 3-kinase activity and 5′-phosphatase activity is expected to produce cellular PI 3,4-P2 which may be uniquely important in triggering downstream cellular effects. These considerations prompted us to investigate whether insulin action might also modulate polyphosphoinositide 5′-phosphatase activity. We report here a marked insulin-mediated recruitment of 5′-phosphatase activity specific for PI 3,4,5-P3 to complexes containing Shc and Grb2. This novel action of insulin is likely to play an important role in one or more downstream cascades important to this hormone's function." @default.
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W2037621839 title "Regulation of Phosphatidylinositol 3,4,5-Trisphosphate 5′-Phosphatase Activity by Insulin" @default.
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