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• W2040255846 abstract "To gain insight into the mechanism by which the adapter protein SH2-B promotes nerve growth factor (NGF)-mediated neuronal differentiation and survival, the effect of SH2-B on the serine/threonine kinase Akt/protein kinase B and downstream effector proteins was examined. PC12 cells stably overexpressing SH2-Bβ, which exhibit enhanced NGF-induced neuronal differentiation compared with control cells, showed enhanced and prolonged NGF-induced phosphorylation of Akt on Ser473 and Akt enzymatic activity. Surprisingly, NGF-induced phosphorylation of Akt on Ser473 and Akt activity were not altered in cells overexpressing SH2-Bβ(R555E) with a defective SH2 domain, despite the ability of the overexpressed SH2-Bβ(R555E) to block NGF-induced differentiation. Consistent with SH2-Bβ enhancing the activity of Akt, cells overexpressing SH2-Bβ but not SH2-Bβ(R555E) exhibited increased and/or prolonged phosphorylation of the pro-apoptotic Akt effector proteins, glycogen synthase kinase-3, and forkhead transcription factors, FKHRL1/FOXO3 and FKHR/FOXO1. Immunolocalization studies indicated that, although ectopically expressed FKHR was primarily concentrated in the cytoplasm of control cells and cells transiently overexpressing SH2-Bβ, it was concentrated in the nucleus of cells transiently overexpressing SH2-Bβ(R555E). Similarly, SH2-Bβ stimulated the accumulation of FKHR in the cytoplasm of 293T and COS-7 cells, whereas SH2-Bβ(R555E) enhanced its accumulation in the nucleus. In PC12 cells stably expressing forms of SH2-Bβ, SH2-Bβ mimicked the ability of NGF to promote redistribution of FKHR to the cytoplasm whereas SH2-Bβ(R555E) blocked this effect of NGF. Taken together, these data indicate that SH2-B is a positive regulator of NGF-mediated activation of the Akt/Forkhead pathway. To gain insight into the mechanism by which the adapter protein SH2-B promotes nerve growth factor (NGF)-mediated neuronal differentiation and survival, the effect of SH2-B on the serine/threonine kinase Akt/protein kinase B and downstream effector proteins was examined. PC12 cells stably overexpressing SH2-Bβ, which exhibit enhanced NGF-induced neuronal differentiation compared with control cells, showed enhanced and prolonged NGF-induced phosphorylation of Akt on Ser473 and Akt enzymatic activity. Surprisingly, NGF-induced phosphorylation of Akt on Ser473 and Akt activity were not altered in cells overexpressing SH2-Bβ(R555E) with a defective SH2 domain, despite the ability of the overexpressed SH2-Bβ(R555E) to block NGF-induced differentiation. Consistent with SH2-Bβ enhancing the activity of Akt, cells overexpressing SH2-Bβ but not SH2-Bβ(R555E) exhibited increased and/or prolonged phosphorylation of the pro-apoptotic Akt effector proteins, glycogen synthase kinase-3, and forkhead transcription factors, FKHRL1/FOXO3 and FKHR/FOXO1. Immunolocalization studies indicated that, although ectopically expressed FKHR was primarily concentrated in the cytoplasm of control cells and cells transiently overexpressing SH2-Bβ, it was concentrated in the nucleus of cells transiently overexpressing SH2-Bβ(R555E). Similarly, SH2-Bβ stimulated the accumulation of FKHR in the cytoplasm of 293T and COS-7 cells, whereas SH2-Bβ(R555E) enhanced its accumulation in the nucleus. In PC12 cells stably expressing forms of SH2-Bβ, SH2-Bβ mimicked the ability of NGF to promote redistribution of FKHR to the cytoplasm whereas SH2-Bβ(R555E) blocked this effect of NGF. Taken together, these data indicate that SH2-B is a positive regulator of NGF-mediated activation of the Akt/Forkhead pathway. Nerve growth factor (NGF) 1The abbreviations used are: NGF, nerve growth factor; SH2, Src homology 2; αPY, anti-phosphotyrosine antibody; FKHR, Forkhead in rhabdomyosarcoma; Foxo, Forkhead box, subgroup 0; GFP, green fluorescent protein; GSK, glycogen synthase kinase; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; PKB, protein kinase B; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase. 1The abbreviations used are: NGF, nerve growth factor; SH2, Src homology 2; αPY, anti-phosphotyrosine antibody; FKHR, Forkhead in rhabdomyosarcoma; Foxo, Forkhead box, subgroup 0; GFP, green fluorescent protein; GSK, glycogen synthase kinase; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; PKB, protein kinase B; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase. is a trophic factor essential for the development and survival of sympathetic and sensory neurons. PC12 cells, a well established cell culture model of sympathetic neurons, differentiate into a neuronal-like phenotype in the presence of NGF. In both sympathetic neurons and PC12 cells, removal of trophic support triggers retraction of neurite outgrowths and eventual apoptosis (1.Deckwerth T.L. Johnson Jr, E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar, 2.Batistatou A. Greene L.A. J. Cell Biol. 1993; 122: 523-532Crossref PubMed Scopus (199) Google Scholar). NGF protects PC12 cells from apoptosis induced by trophic factor withdrawal (2.Batistatou A. Greene L.A. J. Cell Biol. 1993; 122: 523-532Crossref PubMed Scopus (199) Google Scholar, 3.Rukenstein A. Rydel R.E. Greene L.A. J. Neurosci. 1991; 11: 2552-2563Crossref PubMed Google Scholar), cytotoxic drugs (4.Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (343) Google Scholar, 5.Lindenboim L. Haviv R. Stein R. J. 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NGF binding activates TrkA, which phosphorylates itself on multiple tyrosines (10.Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (544) Google Scholar). Binding of different signaling proteins to these phosphorylated tyrosines initiates multiple signaling pathways. Several of these TrkA-binding proteins and/or their downstream effector proteins have been implicated in the regulation of neuronal differentiation and/or survival. One of the first pathways shown to be required for NGF-induced neuronal differentiation of PC12 cells is the Ras/Raf/MEK/ERK pathway. The TrkA-binding protein Shc, as well as Ras, Raf, MEK, and ERK have been implicated in neuronal differentiation of PC12 cells (11.Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 12.Cowley S. Paterson H. Kemp P. Marshall C.J. 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Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (656) Google Scholar).A third pathway, involving the TrkA-binding protein phosphatidylinositol 3′-kinase and the serine/threonine kinase Akt/PKB, has been implicated in axon caliber and branching (18.Markus A. Zhong J. Snider W.D. Neuron. 2002; 35: 65-76Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). However, it is primarily emerging as a transducer of survival signals. Akt activity has been linked to cell survival in several cell types (19.Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar, 20.Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2213) Google Scholar) and is both necessary and sufficient for NGF-dependent survival of sympathetic neurons (21.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar). Inhibition of PI 3-kinase by wortmannin or LY294002 blocks the ability of NGF to prevent apoptosis in both PC12 cells and primary sympathetic neurons (21.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar, 22.Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1288) Google Scholar). Activated forms of PI 3-kinase and AKT promote neuronal survival in the absence of external survival factors (20.Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2213) Google Scholar, 21.Crowder R.J. Freeman R.S. J. Neurosci. 1998; 18: 2933-2943Crossref PubMed Google Scholar, 23.D'Mello S.R. Borodezt K. Soltoff S.P. J. Neurosci. 1997; 17: 1548-1560Crossref PubMed Google Scholar, 24.Miller T.M. Tansey M.G. Johnson Jr., E.M. Creedon D.J. J. Biol. 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Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 30.Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1045) Google Scholar, 31.Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (415) Google Scholar). Phosphorylation of both sites is required for full activation of Akt (32.Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). Akt can phosphorylate and modulate the activity of several proteins involved in cellular survival, including members of the FOXO family of forkhead transcription factors (33.Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (252) Google Scholar, 34.Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. 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Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2719) Google Scholar).Phosphorylation of GSK-3 by Akt at Ser21 (α isoform) or Ser9 (β isoform) inactivates GSK-3 and is believed to be the primary mechanism responsible for growth factor inhibition of this protein kinase (42.Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (750) Google Scholar, 43.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Inactivation of GSK-3 is thought to contribute to the survival effects of Akt activation and to reduce neurotrophin factor withdrawal-induced neurite retraction. Inhibition of GSK-3 by LiCl reduces NGF withdrawal-induced apoptosis in PC12 cells (44.Bhat R.V. Shanley J. Correll M.P. Fieles W.E. Keith R.A. Scott C.W. Lee C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11074-11079Crossref PubMed Scopus (354) Google Scholar) and the degree of neurite retraction observed in wortmannin-treated SH-SY5Y neuroblastoma cells (45.Sanchez S. Sayas C.L. Lim F. Diaz-Nido J. Avila J. Wandosell F. J. Neurochem. 2001; 78: 468-481Crossref PubMed Scopus (74) Google Scholar).Multiple forkhead family members are targets of Akt (34.Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 46.Kops G.J.P.L. de Ruiter N.D. de Vries-Smits A.M.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (948) Google Scholar). Three mammalian members of this family, namely FKHR/FOXO1 (47.Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher III, F.J. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (769) Google Scholar), FKHRL1/FOXO3 (48.Hillion J. Le Coniat M. Jonveaux P. Berger R. Bernard O.A. Blood. 1997; 90: 3714-3719Crossref PubMed Google Scholar), and AFX/FOXO4 (49.Borkhardt A. Repp R. Haas O.A. Leis T. Harbott J. Kreuder J. Hammermann J. Henn T. Lampert F. Oncogene. 1997; 14: 195-202Crossref PubMed Scopus (212) Google Scholar), belong to the FOXO subfamily. Proteins in this subfamily have sequence similarity to the nematode homologue Daf-16, which is a downstream target of two Akt homologues in an insulin-related signaling pathway, and have been implicated in apoptosis of neuronal cells (50.Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1195) Google Scholar, 51.Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1525) Google Scholar, 52.Zheng W.H. Kar S. Quirion R. J. Biol. Chem. 2000; 275: 39152-39158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Daf-16 as well as AFX, FKHR, and FKHRL1 contain multiple consensus Akt phosphorylation sites (47.Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher III, F.J. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (769) Google Scholar, 49.Borkhardt A. Repp R. Haas O.A. Leis T. Harbott J. Kreuder J. Hammermann J. Henn T. Lampert F. Oncogene. 1997; 14: 195-202Crossref PubMed Scopus (212) Google Scholar), and Akt can directly phosphorylate the three mammalian forkhead proteins in vitro (34.Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 46.Kops G.J.P.L. de Ruiter N.D. de Vries-Smits A.M.M. Powell D.R. Bos J.L. Burgering B.M.T. Nature. 1999; 398: 630-634Crossref PubMed Scopus (948) Google Scholar).Akt is thought to inhibit the activity of forkhead transcription factors primarily by regulating their subcellular localization (53.Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (938) Google Scholar, 54.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5366) Google Scholar). In this paradigm, FKHR in the nucleus induces the expression of genes critical for cell death, such as the Fas ligand gene. When activated, Akt phosphorylates FKHR proteins in the nucleus, resulting in binding of FKHR proteins to 14-3-3 and subsequent export from the nucleus. This relocation of FKHR proteins to the cytoplasm functionally represses their transcriptional activity (53.Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (938) Google Scholar, 54.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. 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Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), as well as of the receptors for insulin (59.Riedel H. Wang J. Hansen H. Yousaf N. J. Biochem. (Tokyo). 1997; 122: 1105-1113Crossref PubMed Scopus (66) Google Scholar), platelet-derived growth factor (60.Rui L. Carter-Su C. J. Biol. Chem. 1998; 273: 21239-21245Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), fibroblast growth factor (61.Riedel H. Yousaf N. Zhao Y. Dai H. Deng Y. Wang J. Oncogene. 2000; 19: 39-50Crossref PubMed Scopus (39) Google Scholar), insulin-like growth factor-1 (62.Wang J. Riedel H. J. Biol. Chem. 1998; 273: 3136-3139Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), hepatocyte growth factor (61.Riedel H. Yousaf N. Zhao Y. Dai H. Deng Y. Wang J. Oncogene. 2000; 19: 39-50Crossref PubMed Scopus (39) Google Scholar), the cytokine receptor-associated tyrosine kinase JAK2 (63.Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar), and the R1 FcϵR1 receptor (64.Osborne M.A. Dalton S. Kochan J.P. BioTechnology. 1995; 13: 1474-1478Crossref PubMed Scopus (140) Google Scholar). Four SH2-B isoforms (α, β, γ, δ) have now been identified; they differ in their C termini downstream of the SH2 domain (63.Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar, 64.Osborne M.A. Dalton S. Kochan J.P. BioTechnology. 1995; 13: 1474-1478Crossref PubMed Scopus (140) Google Scholar, 65.Nelms K. O'Neill T.J. Li S. Hubbard S.R. Gustafson T.A. Paul W.E. Mamm. Genome. 1999; 10: 1160-1167Crossref PubMed Scopus (66) Google Scholar, 66.Yousaf N. Deng Y. Kang Y. Riedel H. J. Biol. Chem. 2001; 276: 40940-40948Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) (Fig. 1). SH2-B belongs to a family of adapter proteins that include APS and Lnk (67.Yokouchi M. Suzuki R. Masuhara M. Komiya S. Inoue A. Yoshimura A. Oncogene. 1997; 15: 7-15Crossref PubMed Scopus (108) Google Scholar, 68.Huang X. Li Y. Tanaka K. Moore K.G. Hayashi J.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11618-11622Crossref PubMed Scopus (138) Google Scholar).We and others have shown that SH2-B (α or β isoform) is vital for NGF-induced neurite outgrowth in cultured PC12 cells and maintenance of the neuronal phenotype of primary cultured rat sympathetic neurons (57.Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 58.Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). NGF stimulates the association of SH2-Bβ with TrkA via the SH2 domain of SH2-Bβ and the tyrosyl phosphorylation of SH2-Bβ. Mutating the critical Arg to Glu (R555E) within the SH2 domain of SH2-Bβ abolishes both the NGF-induced association of SH2-Bβ with TrkA and the tyrosyl phosphorylation of SH2-Bβ (57.Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Stable overexpression of SH2-Bβ enhances NGF-induced neuronal differentiation of PC12 cells, whereas stable expression of SH2-Bβ(R555E) blocks NGF-induced differentiation of PC12 cells (57.Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Interestingly, cells stably expressing SH2-Bβ(R555E) do not show impaired NGF-induced tyrosyl phosphorylation of TrkA, Shc, phospholipase C-γ, ERK1, or ERK2 (63.Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar), suggesting that impaired activation of these signaling proteins is not responsible for the dramatic overall impairment of neuronal differentiation. SH2-B has also been implicated in neuronal survival. Neuronal sympathetic neurons titrated with anti-SH2-B antibodies exhibit a reduced rate of survival when grown in NGF-containing medium. Similarly, transient expression of an N-terminally truncated form of SH2-B promotes degeneration of axons of sympathetic neurons in explants of superior cervical ganglia grown in the presence of NGF (58.Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In the current work, we extend these earlier studies designed to determine the role of SH2-Bβ in neuronal differentiation and survival by examining the hypothesis that SH2-Bβ positively regulates NGF-induced activation of the protein kinase Akt and its downstream targets.EXPERIMENTAL PROCEDURESCells and Reagents—Parental PC12 cells were obtained from Dr. Ben Margolis (University of Michigan, Ann Arbor, MI) or ATCC. Pools of PC12 cells stably expressing GFP, GFP-SH2-Bβ, and GFP-SH2-Bβ(R555E) were made as described previously (63.Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar) and used for Figs. 2, 3, 4, 5. Fig. 8 used cells made in a similar fashion with the exception that all GFP-positive cells were pooled instead of only those with the top 5% expression level. The stock of 293T cells was obtained from Dr. O. A. MacDougald (University of Michigan, Ann Arbor, MI). Murine NGF was from BD Bioscience. Triton X-100, aprotinin, and leupeptin were purchased from Roche Molecular Biochemicals. The nitrocellulose membranes and enhanced chemiluminescence (ECL) detection system were from Amersham Biosciences. X-ray film came from DuPont or Eastman Kodak Co. (Fig. 8B), horse serum from ICN Biomedicals, fetal bovine serum from Invitrogen, and poly-l-lysine from Sigma.Fig. 2SH2-Bβ enhances and prolongs NGF-induced phosphorylation of Ser473 in Akt. PC12 cells stably overexpressing GFP alone (GFP), GFP-SH2-Bβ, or GFP-SH2-Bβ(R555E) as indicated were stimulated with 100 ng/ml NGF for the indicated times. In parallel experiments, an equal amount of protein in the lysates was immunoblotted with αpAkt(Ser473) (panel A), αAkt (panel B), or αSH2-B (panel C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3SH2-Bβ enhances and prolongs NGF-induced activity of Akt. PC12 cells stably overexpressing GFP alone and GFP-SH2-Bβ were stimulated with 100 ng/ml NGF for the indicated times. Akt was immunoprecipitated, and kinase activity was determined. The results show the mean and range of two separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4SH2-Bβ enhances and prolongs NGF-induced phosphorylation of GSK-3α/β on Ser21/Ser9. PC12 cells stably overexpressing GFP alone, GFP-SH2-Bβ, and GFP-SH2-Bβ(R555E) were stimulated with 100 ng/ml NGF for the indicated times. An equal amount of protein in the lysates was immunoblotted with αpGSK-3(Ser21/9) (panel A) or αGSK-3 (panel B). The resulting films from three separate experiments using αpGSK-3 were scanned, and the relative amounts of bound pGSK-3 were calculated (panel C). The results show the mean and standard error of the mean for three separate experiments, normalized to the 15-min control (GFP). An asterisk (*) denotes a p value <0.05 (two-tailed, paired Student's t test) between experimental (GFP-SH2-Bβ and GFP-SH2-Bβ(R555E)) and control (GFP) values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5SH2-Bβ enhances and prolongs NGF-induced phosphorylation of FKHRL1. PC12 cells stably overexpressing GFP alone, GFP-SH2-Bβ, and GFP-SH2-Bβ(R555E) were stimulated with 100 ng/ml NGF for the indicated times. An equal amount of protein in the lysates was immunoblotted with αFKHRL1 (panel A). The films in panel A were scanned, and the relative amounts of FKHRL1 were calculated (panel B). The results show the mean intensity of the phosphorylated FKHRL1 band (upper band) divided by the unphosphorylated FKHRL1 band (lower band).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8SH2-Bβ mimics and SH2-Bβ(R555E) blocks NGF-induced redistribution of FKHR from the nucleus to the cytoplasm. PC12 cells stably expressing GFP, GFP-SH2-Bβ, or GFP-SH2-Bβ(R555E) were transiently transfected with cDNA encoding Flag-FKHR. A, cells were incubated in serum free medium for 24 h and fixed (white bars) or treated with 100 ng/ml NGF for 1 h and fixed (gray bars). Units are defined as the percentage of cells with Flag-FKHR excluded from the nucleus (defined C for cytoplasmic) divided by the total number of cells counted of which expressed Flag-FKHR (defined N+C). 103, 100, 114, 192, 126, and 107 cells expressing GFP alone (minus and plus NGF), GFP-SH2-Bβ (minus and plus NGF), and GFP-SH2-Bβ(R555E) (minus and plus NGF), respectively, were assessed in two trials. B, cells were deprived for 24 h and then treated with 100 ng/ml NGF for 0, 0.25, 0.5, 2, and 3 h. An equal amount of protein in the lysates was immunoblotted with αFKHR. It is important to note that this antibody recognizes only unphospho-rylated FKHR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Plasmids, Antibodies, and Fluorescent Probes—pcDNA-Flag-FKHR was a gift of Drs. E. Tang and K. Guan (34.Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). cDNAs encoding GFP-tagged wild-type SH2-Bβ and SH2-Bβ(R555E) have been described previously (57.Rui L. Herrington J.B. Carter-Su C. J. Biol. Chem. 1999; 274: 10590-10594Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Anti-SH2-Bβ antibody (αSH2-Bβ) was prepared as described previously (63.Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar) and used at a dilution of 1:15,000 for Western blotting. Antibodies that recognize the following proteins were used fo" @default.
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• W2040255846 date "2004-01-01" @default.
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• W2040255846 title "SH2-B Is a Positive Regulator of Nerve Growth Factor-mediated Activation of the Akt/Forkhead Pathway in PC12 Cells" @default.
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• W2040255846 doi "https://doi.org/10.1074/jbc.m310040200" @default.
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