FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1991074752> ?p ?o ?g. }

• W1991074752 endingPage "2499" @default.
• W1991074752 startingPage "2487" @default.
• W1991074752 abstract "We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways. We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d0/d4) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d0/d4 values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d0/d4 scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser19 of ZNRF2 (RTRAYpS19GS), phospho-Ser90 of SASH1 (RKRRVpS90QD), and phospho- Ser493 of lipolysis-stimulated lipoprotein receptor (RPRARpS493LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways. Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1 1The abbreviations used are:IGF1insulin-like growth factor 1CCDC6coiled coil domain-containing 6IRSinsulin receptor substrateLSRlipolysis-stimulated lipoprotein receptor (also known as LISCH7, liver-specific basic helix-loop-helix leucine zipper transcription factor)PASphospho-Akt substratePIphosphatidylinositolPKBprotein kinase B, also known as AktPMAthe phorbol ester phorbol 12-myristate 13-acetateSASH1sterile α motif- and SH3 domain-containing protein 1VASPvasodilator-stimulated phosphoproteinYAPYes-associated proteinZNRFzinc and ring fingerMAPKmitogen-activated protein kinaseHAhemagglutininErkextracellular signal-regulated kinaseSCXstrong cation exchangeLTQlinear trap quadrupoleMGFMascot generic formatKLCkinesin light chainSILACstable isotope labeling with amino acids in cell cultureE3ubiquitin-protein isopeptide ligaseE2ubiquitin carrier proteinAGCcAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases G family of protein kinases1The abbreviations used are:IGF1insulin-like growth factor 1CCDC6coiled coil domain-containing 6IRSinsulin receptor substrateLSRlipolysis-stimulated lipoprotein receptor (also known as LISCH7, liver-specific basic helix-loop-helix leucine zipper transcription factor)PASphospho-Akt substratePIphosphatidylinositolPKBprotein kinase B, also known as AktPMAthe phorbol ester phorbol 12-myristate 13-acetateSASH1sterile α motif- and SH3 domain-containing protein 1VASPvasodilator-stimulated phosphoproteinYAPYes-associated proteinZNRFzinc and ring fingerMAPKmitogen-activated protein kinaseHAhemagglutininErkextracellular signal-regulated kinaseSCXstrong cation exchangeLTQlinear trap quadrupoleMGFMascot generic formatKLCkinesin light chainSILACstable isotope labeling with amino acids in cell cultureE3ubiquitin-protein isopeptide ligaseE2ubiquitin carrier proteinAGCcAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases G family of protein kinases promotes survival, growth, and proliferation of many cell types (1Cohen P. The twentieth century struggle to decipher insulin signalling.Nat. Rev. Mol. Cell Biol. 2006; 7: 867-873Crossref PubMed Scopus (178) Google Scholar, 2Randhawa R. Cohen P. The role of the insulin-like growth factor system in prenatal growth.Mol. Genet. Metab. 2005; 86: 84-90Crossref PubMed Scopus (182) Google Scholar). insulin-like growth factor 1 coiled coil domain-containing 6 insulin receptor substrate lipolysis-stimulated lipoprotein receptor (also known as LISCH7, liver-specific basic helix-loop-helix leucine zipper transcription factor) phospho-Akt substrate phosphatidylinositol protein kinase B, also known as Akt the phorbol ester phorbol 12-myristate 13-acetate sterile α motif- and SH3 domain-containing protein 1 vasodilator-stimulated phosphoprotein Yes-associated protein zinc and ring finger mitogen-activated protein kinase hemagglutinin extracellular signal-regulated kinase strong cation exchange linear trap quadrupole Mascot generic format kinesin light chain stable isotope labeling with amino acids in cell culture ubiquitin-protein isopeptide ligase ubiquitin carrier protein cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases G family of protein kinases insulin-like growth factor 1 coiled coil domain-containing 6 insulin receptor substrate lipolysis-stimulated lipoprotein receptor (also known as LISCH7, liver-specific basic helix-loop-helix leucine zipper transcription factor) phospho-Akt substrate phosphatidylinositol protein kinase B, also known as Akt the phorbol ester phorbol 12-myristate 13-acetate sterile α motif- and SH3 domain-containing protein 1 vasodilator-stimulated phosphoprotein Yes-associated protein zinc and ring finger mitogen-activated protein kinase hemagglutinin extracellular signal-regulated kinase strong cation exchange linear trap quadrupole Mascot generic format kinesin light chain stable isotope labeling with amino acids in cell culture ubiquitin-protein isopeptide ligase ubiquitin carrier protein cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases G family of protein kinases Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1Cohen P. The twentieth century struggle to decipher insulin signalling.Nat. Rev. Mol. Cell Biol. 2006; 7: 867-873Crossref PubMed Scopus (178) Google Scholar, 3Manning B.D. Cantley L.C. AKT/PKB signaling: navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4692) Google Scholar). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4Hawkins P.T. Anderson K.E. Davidson K. Stephens L.R. Signalling through Class I PI3Ks in mammalian cells.Biochem. Soc. Trans. 2006; 34: 647-662Crossref PubMed Scopus (459) Google Scholar, 5Yuan T.L. Cantley L.C. PI3K pathway alterations in cancer: variations on a theme.Oncogene. 2008; 27: 5497-5510Crossref PubMed Scopus (1472) Google Scholar). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations. Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3Manning B.D. Cantley L.C. AKT/PKB signaling: navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4692) Google Scholar). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar), PRAS40 (7Kovacina K.S. Park G.Y. Bae S.S. Guzzetta A.W. Schaefer E. Birnbaum M.J. Roth R.A. Identification of a proline-rich Akt substrate as a 14-3-3 binding partner.J. Biol. Chem. 2003; 278: 10189-10194Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), AS160 (8Kane S. Sano H. Liu S.C. Asara J.M. Lane W.S. Garner C.C. Lienhard G.E. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain.J. Biol. Chem. 2002; 277: 22115-22118Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9Berwick D.C. Dell G.C. Welsh G.I. Heesom K.J. Hers I. Fletcher L.M. Cooke F.T. Tavaré J.M. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles.J. Cell Sci. 2004; 117: 5985-5993Crossref PubMed Scopus (115) Google Scholar) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10Rubio M. Pozuelo Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (382) Google Scholar, 11Rubio M. Pozuelo Peggie M. Wong B.H. Morrice N. MacKintosh C. 14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase.EMBO J. 2003; 22: 3514-3523Crossref PubMed Scopus (75) Google Scholar), BimEL (12Qi X.J. Wildey G.M. Howe P.H. Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function.J. Biol. Chem. 2006; 281: 813-823Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), β-catenin (13Tian Q. Feetham M.C. Tao W.A. He X.C. Li L. Aebersold R. Hood L. Proteomic analysis identifies that 14-3-3zeta interacts with beta-catenin and facilitates its activation by Akt.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 15370-15375Crossref PubMed Scopus (124) Google Scholar), p27(Kip1) (14Sekimoto T. Fukumoto M. Yoneda Y. 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1).EMBO J. 2004; 23: 1934-1942Crossref PubMed Scopus (132) Google Scholar), PRAS40 (7Kovacina K.S. Park G.Y. Bae S.S. Guzzetta A.W. Schaefer E. Birnbaum M.J. Roth R.A. Identification of a proline-rich Akt substrate as a 14-3-3 binding partner.J. Biol. Chem. 2003; 278: 10189-10194Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), FOXO1 (15Rena G. Prescott A.R. Guo S. Cohen P. Unterman T.G. Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting.Biochem. J. 2001; 354: 605-612Crossref PubMed Scopus (219) Google Scholar), Miz1 (16Wanzel M. Kleine-Kohlbrecher D. Herold S. Hock A. Berns K. Park J. Hemmings B. Eilers M. Akt and 14-3-3eta regulate Miz1 to control cell-cycle arrest after DNA damage.Nat. Cell Biol. 2005; 7: 30-41Crossref PubMed Scopus (72) Google Scholar), TBC1D4 (AS160 (17Geraghty K.M. Chen S. Harthill J.E. Ibrahim A.F. Toth R. Morrice N.A. Vandermoere F. Moorhead G.B. Hardie D.G. MacKintosh C. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.Biochem. J. 2007; 407: 231-241Crossref PubMed Scopus (140) Google Scholar, 18Ramm G. Larance M. Guilhaus M. James D.E. A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160.J. Biol. Chem. 2006; 281: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), and TBC1D1 (19Chen S. Murphy J. Toth R. Campbell D.G. Morrice N.A. Mackintosh C. Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators.Biochem. J. 2008; 409: 449-459Crossref PubMed Scopus (162) Google Scholar). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10Rubio M. Pozuelo Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (382) Google Scholar) and PKB-dependent cell survival (20Masters S.C. Fu H. 14-3-3 proteins mediate an essential anti-apoptotic signal.J. Biol. Chem. 2001; 276: 45193-45200Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21Jacinto E. Lorberg A. TOR regulation of AGC kinases in yeast and mammals.Biochem. J. 2008; 410: 19-37Crossref PubMed Scopus (163) Google Scholar). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17Geraghty K.M. Chen S. Harthill J.E. Ibrahim A.F. Toth R. Morrice N.A. Vandermoere F. Moorhead G.B. Hardie D.G. MacKintosh C. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.Biochem. J. 2007; 407: 231-241Crossref PubMed Scopus (140) Google Scholar, 18Ramm G. Larance M. Guilhaus M. James D.E. A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160.J. Biol. Chem. 2006; 281: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 19Chen S. Murphy J. Toth R. Campbell D.G. Morrice N.A. Mackintosh C. Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators.Biochem. J. 2008; 409: 449-459Crossref PubMed Scopus (162) Google Scholar). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22Yip M.F. Ramm G. Larance M. Hoehn K.L. Wagner M.C. Guilhaus M. James D.E. CaMKII-mediated phosphorylation of the myosin motor Myo1c is required for insulin-stimulated GLUT4 translocation in adipocytes.Cell Metab. 2008; 8: 384-398Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), are also convergence points for phosphorylation by more than one AGC and/or Ca2+/calmodulin-dependent protein kinase. Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10Rubio M. Pozuelo Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (382) Google Scholar, 23Angrand P.O. Segura I. Völkel P. Ghidelli S. Terry R. Brajenovic M. Vintersten K. Klein R. Superti-Furga G. Drewes G. Kuster B. Bouwmeester T. Acker-Palmer A. Transgenic mouse proteomics identifies new 14-3-3-associated proteins involved in cytoskeletal rearrangements and cell signaling.Mol. Cell. Proteomics. 2006; 5: 2211-2227Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 24Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 25Meek S.E. Lane W.S. Piwnica-Worms H. Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.J. Biol. Chem. 2004; 279: 32046-32054Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 26Ichimura T. Wakamiya-Tsuruta A. Itagaki C. Taoka M. Hayano T. Natsume T. Isobe T. Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein.Biochemistry. 2002; 41: 5566-5572Crossref PubMed Scopus (47) Google Scholar, 27Benzinger A. Muster N. Koch H.B. Yates 3rd, J.R. Hermeking H. Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.Mol. Cell. Proteomics. 2005; 4: 785-795Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling. Synthetic peptides were from Graham Bloomberg (University of Bristol). Oligonucleotides were from MWG-Biotech. IGF1 was from BIOSOURCE. Microcystin-LR was from Linda Lawton (Robert Gordon's University, Aberdeen, Scotland, UK). Vivaspin concentrators were from Vivascience. Tissue culture reagents were from Invitrogen. Protease inhibitor mixture tablets (catalog number 1697498) and sequencing grade trypsin were from Roche Applied Science. Precast SDS-polyacrylamide gels were from Invitrogen. Protein G-Sepharose and chromatographic matrices were from GE Healthcare. Formaldehyde-d0 and -d2 were from Sigma-Aldrich, and unless stated other chemicals were from BDH Chemicals or Sigma-Aldrich. To our knowledge, HeLa S3 and HEK293 cells do not express insulin receptors, although they have IGF1 receptors that bind insulin with lower affinity than the cognate receptor (28Entingh-Pearsall A. Kahn C.R. Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I.J. Biol. Chem. 2004; 279: 38016-38024Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Insulin, which is less expensive than IGF1, was therefore used for large scale experiments with HeLa S3 cells, which were cultured in suspension in Dulbecco's modified Eagle's medium containing 10% FCS, 1% glutamine, 1% penicillin-streptomycin, and 1% sodium pyruvate. Trial experiments indicated that serum starvation of HeLa S3 cells for 4 h followed by 20-min stimulation with 50 milliunits/ml (300 nm) insulin gave maximal phosphorylation of PKB (Thr(P)308 and Ser(P)473) and AS160 (Thr(P)642). Cells were harvested by centrifugation and snap frozen. For Fig. 5, HEK293 cells (European Collection of Cell Cultures) were cultured in 15-cm dishes in Dulbecco's modified Eagle's medium containing 10% (v/v) FCS, 2 mm l-glutamine, non-essential amino acids, and 1% penicillin-streptomycin. At ∼60% confluency, cells were transfected using 30 µl of 1 mg/ml polyethylenimine for 5 µg of DNA. After 36 h, cells were rinsed with warm PBS and serum-starved in Dulbecco's modified Eagle's medium for 8 h. Where indicated, cells were preincubated with PI-103 (1 µm for 30 min) and BI-D1870 (10 µm for 30 min) and stimulated for 20 min with 50 ng/ml IGF1 and 100 ng/ml PMA. Cells were lysed in 0.5 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1% Triton X-100, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.27 m sucrose, 0.1% (by volume) 2-mercaptoethanol, “Complete” proteinase inhibitor mixture (one tablet/50 ml). Cell lysates were clarified by centrifugation at 4 °C for 20 min at 15,000 rpm. 14-3-3 affinity chromatography involved binding of protein to 14-3-3-Sepharose (mixed BMH1 and BMH2, the 14-3-3 isoforms from Saccharomyces cerevisiae) and elution of specifically bound proteins by competition with the 14-3-3-binding synthetic ARAApSAPA phosphopeptide (Fig. 2) as in Pozuelo Rubio et al. (10Rubio M. Pozuelo Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking.Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (382) Google Scholar) except that the high salt wash was only 500 ml and the mock peptide elution was omitted. Sheep anti-HA was raised against the peptide YPYDVPDYA, and sheep anti-AS160 was raised against KAKIGNKP (17Geraghty K.M. Chen S. Harthill J.E. Ibrahim A.F. Toth R. Morrice N.A. Vandermoere F. Moorhead G.B. Hardie D.G. MacKintosh C. Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR.Biochem. J. 2007; 407: 231-241Crossref PubMed Scopus (140) Google Scholar). Anti-phospho-Erk1/2 (Thr(P)202/Tyr(P)204), anti-phospho-Thr308 PKB, and anti-PKB/Akt were from Cell Signaling Technology. For Western blots the indicated antibodies were used at 1 µg/ml. Western blots and 14-3-3 overlays (using digoxigenin-labeled 14-3-3s in place of primary antibody) were visualized by ECL reagent or the Odyssey Infrared Imaging System (LI-COR, Inc.) as indicated. For immunoprecipitations with anti-HA, 4 µg of antibody/mg of lysate was mixed at 4 °C for 1 h, and then Protein G-Sepharose (30 µl of a 50% suspension in lysis buffer) was added and mixed for a further 1 h. The suspension was centrifuged at 12,000 × g for 1 min between washes. 14-3-3-binding proteins that had been purified from unstimulated or insulin-stimulated HeLa cells were denatured in lithium dodecyl sulfate sample buffer (Invitrogen) containing 10 mm DTT at 95 °C for 5 min, cooled, and alkylated with 50 mm iodoacetamide for 30 min in the dark at room temperature. The protein samples were loaded on adjacent lanes of a NuPAGE 4–12% gradient gel (Invitrogen) and electrophoresed at 160 V for 60 min, and the gel was stained with colloidal Coomassie Blue (Invitrogen). The gel lanes were each cut into seven equal sections (with band 1 at the top of the gel) that were washed successively with 50 mm triethylammonium bicarbonate; 50% acetonitrile, 50 mm triethylammonium bicarbonate (twice); and acetonitrile (15 min each wash) before drying in a SpeedVac (Eppendorf). Trypsin (5 µg/ml trypsin gold; Promega) in sufficient 25 mm triethylammonium bicarbonate to cover the gel pieces was added for 12 h at 30 °C. The supernatant was transferred to a fresh tube to which two 50% acetonitrile washes of the gel pieces were also added. The digested samples were split into two equal fractions and dried in a SpeedVac. One half was enriched for phosphopeptides using titanium dioxide, and the other half was dimethylated with formaldehyde using a modified version of the procedure described previously (29Hsu J.L. Huang S.Y. Chen S.H. Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.Electrophoresis. 2006; 27: 3652-3660Crossref PubMed Scopus (59) Google Scholar). Individual tryptic digests were redissolved in 2 µl of 25 mm sodium acetate buffer, pH 5.5, 30 mm sodium cyanoborohydride containing 0.2% (v/v) formaldehyde (d0 for the preparation from insulin-stimulated cells and d2 for the preparation from unstimulated cells) and incubated at room temperature for 15 min. The dimethylated digests were mixed pairwise for corresponding gel sections, diluted 25-fold with strong cation exchange (SCX) loading buffer (25% acetonitrile, 0.2% formic acid), and loaded onto 5 µl of Poros 50 HS beads equilibrated in the same buffer. The slurry was loaded on a Millipore SCX ZipTip and washed three times with 60 µl of SCX loading buffer. Peptides were eluted with 2 × 40 µl of 50% isopropanol, 0.2 m ammonium hydroxide and dried under vacuum. For phosphopeptide enrichment, tryptic digests of the preparations from insulin-stimulated cells were dissolved in 200 mg/ml 2,3-dihydrobenzoic acid (Sigma-Aldrich) in 80% (v/v) acetonitrile, 5% (v/v) TFA (loading buffer). Titanspheres (5 mg of 5-µm spheres; Hichrom Ltd.) equilibrated in loading buffer were added to each digest and agitated for 10 min. The slurry was loaded in a C18 StageTip (Proxeon); washed three times with 80% (v/v) acetonitrile, 5% (v/v) TFA; eluted with 40 µl of 1 m ammonium hydroxide, 50% (v/v) acetonitrile; 40 µl of 50% (v/v) acetonitrile; and 40 µl of 0.5% (v/v) formic acid, 50% (v/v) acetonitrile; combined; and dried under vacuum. Tryptic digests were analyzed using Ultimate 3200 nanoflow chromatography (LC Packings) coupled to an LTQ-Orbitrap (Thermo Finnigan) mass spectrometer equipped with a dynamic NanoSpray source (Optron). The dimethylated peptide mixtures were separated using an LC Packings Integrated System (Dionex, Camberley, UK) consisting of a WPS3000T microautosampler, FLM3200 microcolumn switching module, UltiMate LPG3600 micropump, a PepMap C18 column (75 µm, 15 cm; LC Packings), and mobile phases of 2% acetonitrile, 0.1% formic acid in water (A) and 90% acetonitrile, 0.085% formic acid in water (B). The column was equilibrated in 2% B at a flow rate of 300 nl/min. The dried digests were resolubilized in 50% (v/v) acetonitrile, 0.1% (v/v) TFA; diluted 10-fold with 0.1% (v/v) TFA; and loaded onto a C18 capillary trap (Michrom Bioresources, Auburn, CA) equilibrated in buffer A at a flow rate of 20 µl/min. After 8 min, the capillary cartridge was switched in line with the analytical column and eluted with the following gradient: 2–50% buffer B (8–80 min), 50–85% B (80–85 min), 85–2% B (85–90 min), and 2% B (90–100 min). The column eluate was electrosprayed with a voltage of 1200 V applied to a Picotip (FS360-50-15-N, New Objective, Woburn, MA). Mass spectra were acquired using two different acquisition methods. For protein identification, the LTQ-Orbitrap was programmed to perform two FT scans (60,000 resolution) on 300–800- and 800–1800-amu mass ranges with the top five ions from each scan selected for LTQ-MS/MS. FT spectra were internally calibrated using a single lock mass (445.1200 atomic mass units). For phosphopeptide analysis, the same two FT scans were performed, but multistage activation was performed on the selected ions with a neutral loss of 97.98, 48.99, 32.66, and 24.50. For these two methods, target ion numbers were 500,000 for FT full scan on the orbitrap and 10,000 MSn on the LTQ. Raw files were converted to peak lists in Mascot generic format (MGF) files using raw2msm v1.7 software (Matthias Mann) using default parameters and without any filtering, charge sta" @default.
• W1991074752 created "2016-06-24" @default.
• W1991074752 creator A5013906638 @default.
• W1991074752 creator A5018713678 @default.
• W1991074752 creator A5028009861 @default.
• W1991074752 creator A5049317673 @default.
• W1991074752 creator A5060856971 @default.
• W1991074752 creator A5067524265 @default.
• W1991074752 creator A5085148897 @default.
• W1991074752 creator A5087930535 @default.
• W1991074752 creator A5090500699 @default.
• W1991074752 date "2009-11-01" @default.
• W1991074752 modified "2023-10-11" @default.
• W1991074752 title "Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling" @default.
• W1991074752 cites W1528986553 @default.
• W1991074752 cites W1575276040 @default.
• W1991074752 cites W1581618913 @default.
• W1991074752 cites W1963634449 @default.
• W1991074752 cites W1965483278 @default.
• W1991074752 cites W1966193117 @default.
• W1991074752 cites W1968031368 @default.
• W1991074752 cites W1968948640 @default.
• W1991074752 cites W1969744108 @default.
• W1991074752 cites W1975537939 @default.
• W1991074752 cites W1975718576 @default.
• W1991074752 cites W1979270142 @default.
• W1991074752 cites W1979827725 @default.
• W1991074752 cites W1980823210 @default.
• W1991074752 cites W1981343702 @default.
• W1991074752 cites W1981531386 @default.
• W1991074752 cites W1982745900 @default.
• W1991074752 cites W1984169187 @default.
• W1991074752 cites W1984621921 @default.
• W1991074752 cites W1988224683 @default.
• W1991074752 cites W1988439811 @default.
• W1991074752 cites W1990283596 @default.
• W1991074752 cites W1990425248 @default.
• W1991074752 cites W1994238739 @default.
• W1991074752 cites W1994289453 @default.
• W1991074752 cites W1998796032 @default.
• W1991074752 cites W2002692130 @default.
• W1991074752 cites W2004331701 @default.
• W1991074752 cites W2008096080 @default.
• W1991074752 cites W2010752097 @default.
• W1991074752 cites W2014669689 @default.
• W1991074752 cites W2020995084 @default.
• W1991074752 cites W2025612803 @default.
• W1991074752 cites W2049930674 @default.
• W1991074752 cites W2052122921 @default.
• W1991074752 cites W2052511418 @default.
• W1991074752 cites W2053987947 @default.
• W1991074752 cites W2054683873 @default.
• W1991074752 cites W2055512533 @default.
• W1991074752 cites W2056021994 @default.
• W1991074752 cites W2056112154 @default.
• W1991074752 cites W2059664914 @default.
• W1991074752 cites W2059907698 @default.
• W1991074752 cites W2062819794 @default.
• W1991074752 cites W2070624893 @default.
• W1991074752 cites W2071075259 @default.
• W1991074752 cites W2073672336 @default.
• W1991074752 cites W2076075378 @default.
• W1991074752 cites W2078142614 @default.
• W1991074752 cites W2081920342 @default.
• W1991074752 cites W2094941511 @default.
• W1991074752 cites W2095301158 @default.
• W1991074752 cites W2095820546 @default.
• W1991074752 cites W2097612997 @default.
• W1991074752 cites W2098929356 @default.
• W1991074752 cites W2105494151 @default.
• W1991074752 cites W2113288593 @default.
• W1991074752 cites W2113617470 @default.
• W1991074752 cites W2117262983 @default.
• W1991074752 cites W2119035325 @default.
• W1991074752 cites W2119123594 @default.
• W1991074752 cites W2122624068 @default.
• W1991074752 cites W2124013343 @default.
• W1991074752 cites W2127746314 @default.
• W1991074752 cites W2136626081 @default.
• W1991074752 cites W2140864364 @default.
• W1991074752 cites W2142161829 @default.
• W1991074752 cites W2143786004 @default.
• W1991074752 cites W2144491728 @default.
• W1991074752 cites W2145094824 @default.
• W1991074752 cites W2146414563 @default.
• W1991074752 cites W2154204112 @default.
• W1991074752 cites W2159310898 @default.
• W1991074752 cites W2159856430 @default.
• W1991074752 cites W2161838282 @default.
• W1991074752 cites W2171363796 @default.
• W1991074752 cites W4231442978 @default.
• W1991074752 doi "https://doi.org/10.1074/mcp.m800544-mcp200" @default.
• W1991074752 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2773716" @default.
• W1991074752 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19648646" @default.
• W1991074752 hasPublicationYear "2009" @default.
• W1991074752 type Work @default.
• W1991074752 sameAs 1991074752 @default.
• W1991074752 citedByCount "65" @default.

• next