Rheb and mTOR Regulate Neuronal Polarity through Rap1B
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m802431200
ISSN1083-351X
AutoresYinghua Li, Hendrikje Werner, Andreas W. Püschel,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoThe development of polarized hippocampal neurons with a single axon and multiple dendrites depends on the activity of phosphoinositide 3-kinase (PI3K) and the GTPase Rap1B. Here we show that PI3K regulates axon specification and elongation through the GTPase Rheb and its target mammalian target of rapamycin (mTOR). Overexpression of Rheb induces the formation of multiple axons, whereas its suppression by RNA interference blocks axon specification. mTOR is a central regulator of translation that phosphorylates eIF4E-binding proteins like 4E-BP1. Axon formation was suppressed by inhibition of mTOR and expression of mTOR-insensitive 4E-BP1 mutants. Inhibition of PI3K or mTOR reduced the level of Rap1B, which acts downstream of Rheb and mTOR. The ubiquitin E3 ligase Smurf2 mediates the restriction of Rap1B by initiating its degradation. Suppression of Smruf2 by RNA interference is able to compensate the loss of Rheb. These results indicate that the mTOR pathway is required to counteract the Smurf2-initiated degradation of Rap1B during the establishment of neuronal polarity. The development of polarized hippocampal neurons with a single axon and multiple dendrites depends on the activity of phosphoinositide 3-kinase (PI3K) and the GTPase Rap1B. Here we show that PI3K regulates axon specification and elongation through the GTPase Rheb and its target mammalian target of rapamycin (mTOR). Overexpression of Rheb induces the formation of multiple axons, whereas its suppression by RNA interference blocks axon specification. mTOR is a central regulator of translation that phosphorylates eIF4E-binding proteins like 4E-BP1. Axon formation was suppressed by inhibition of mTOR and expression of mTOR-insensitive 4E-BP1 mutants. Inhibition of PI3K or mTOR reduced the level of Rap1B, which acts downstream of Rheb and mTOR. The ubiquitin E3 ligase Smurf2 mediates the restriction of Rap1B by initiating its degradation. Suppression of Smruf2 by RNA interference is able to compensate the loss of Rheb. These results indicate that the mTOR pathway is required to counteract the Smurf2-initiated degradation of Rap1B during the establishment of neuronal polarity. The formation of a single axon and multiple dendrites is one of the earliest steps during the differentiation of neurons (1Arimura N. Kaibuchi K. Nat. Rev. Neurosci. 2007; 8: 194-205Crossref PubMed Scopus (495) Google Scholar, 2Da Silva J.S. Dotti C.G. Nat. Rev. Neurosci. 2002; 3: 694-704Crossref PubMed Scopus (366) Google Scholar). Primary cultures of dissociated hippocampal neurons are a widely used model system to study the establishment of neuronal polarity and the pathways that direct the specification of axons and dendrites (1Arimura N. Kaibuchi K. Nat. Rev. Neurosci. 2007; 8: 194-205Crossref PubMed Scopus (495) Google Scholar, 2Da Silva J.S. 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It accumulates in the growth cone of a single neurite before neurons are polarized morphologically. Its restriction to a single neurite is initiated by the ubiquitin E3 ligase Smurf2 and mediated by degradation through the ubiquitin/proteasome system (13Schwamborn J.C. Muller M. Becker A.H. Puschel A.W. EMBO J. 2007; 26: 1410-1422Crossref PubMed Scopus (83) Google Scholar). Suppression of Smurf2 results in the persistence of Rap1B in several neurites and the formation of supernumerary axons. PI3K is one of the major regulators of mRNA translation in response to growth factors, nutrient supply, and other signals (14Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar, 15Inoki K. Corradetti M.N. Guan K.L. Nat. Genet. 2005; 37: 19-24Crossref PubMed Scopus (848) Google Scholar, 16Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 17: 158-166Crossref PubMed Scopus (448) Google Scholar, 17Ruggero D. Sonenberg N. Oncogene. 2005; 24: 7426-7434Crossref PubMed Scopus (161) Google Scholar). It stimulates the phosphorylation of the tumor suppressor Tsc2 (also called tuberin) by Akt (18Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar, 19Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 20Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (782) Google Scholar) (Fig. 1A). The complex of Tsc1 (also known as hamartin) and Tsc2 acts as a GTPase activating protein for the GTPase Rheb (21Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 22Inoki K. Li Y. Xu T. Guan K.L. 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Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar, 21Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 22Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1439) Google Scholar, 23Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 24Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (720) Google Scholar). Rheb is a direct activator of mTOR (18Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar, 21Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. 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Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1188) Google Scholar). PI3K, Akt, Tsc1/2, and Rheb act upstream of mTOR and mediate the activation of mTOR by insulin and other growth factors (14Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar, 31Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (761) Google Scholar, 34Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1070) Google Scholar). PI3K/Akt, Tsc1/2, and mTOR have been also implicated in dendritic morphogenesis, axon guidance, synaptic plasticity, and regeneration (35Jaworski J. Spangler S. Seeburg D.P. Hoogenraad C.C. Sheng M. J. Neurosci. 2005; 25: 11300-11312Crossref PubMed Scopus (474) Google Scholar, 36Kelleher R.J. 3rd, Govindarajan A. Tonegawa S. Neuron. 2004; 44: 59-73Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 37Kumar V. Zhang M.X. Swank M.W. Kunz J. Wu G.Y. J. 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Reagents, Plasmids, and Antibodies—Insulin (Sigma) was dissolved in 0.01 m hydrochloric acid at 5 mg/ml, rapamycin, and LY294002 (Calbiochem) in DMSO (AppliChem). pCGN-Rheb (67Clark G.J. Kinch M.S. Rogers-Graham K. Sebti S.M. Hamilton A.D. Der C.J. J. Biol. Chem. 1997; 272: 10608-10615Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), 4E-BP1-myc, Δ24-4E-BP1-myc (40Tee A.R. Proud C.G. Mol. Cell. Biol. 2002; 22: 1674-1683Crossref PubMed Scopus (112) Google Scholar), HA-4E-BP1, and HA-4E-BP1-AA (33Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1188) Google Scholar) were kindly provided by Drs. Clark (National Institutes of Health, NCI, Rockville, MD), Proud (University of British Columbia, Vancouver), and Sonenberg (McGill University, Montreal), respectively. The coding sequence for mouse Rheb (Mus musculus) was amplified from E12 spinal cord cDNA and cloned into a modified pEGFP-C1 vector (Clontech) (68Rohm B. Rahim B. Kleiber B. Hovatta I. Püschel A.W. FEBS Lett. 2000; 486: 68-72Crossref PubMed Scopus (161) Google Scholar). For small interfering RNA constructs, oligonucleotides (Oligoengine, Seattle) containing the 19-nucleotide targeting sequences of Rheb transcript were cloned into the pSuper vector to generate short hairpin RNAs (69Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3971) Google Scholar): GATCC CCTAC GATCC AACCA TAGAA ATTCA AGAGATTTCT ATGGT TGGAT CGTAT TTTTC and TCGAG AAAAA TACGA TCCAA CCATA GAAAT CTCTT GAATT TCTAT GGTTG GATCG TAGGG for pSb102; GATCC CCTAA GAAGG ACCTG CATAT GTTCA AGAGA CATAT GCAGG TCCTT CTTAT TTTTC and TCGAG AAAAA TAAGA AGGAC CTGCA TATGT CTCTT GAACA TATGC AGGTC CTTCT TAGGG for pSb357. The underlined target region corresponds to positions 102-120 and 357-375, respectively, of the rat Rheb open reading frame. The shRNA vectors directed against Rap1B and Smurf2 have been described before (11Schwamborn J.C. Püschel A.W. Nat. Neurosci. 2004; 7: 923-929Crossref PubMed Scopus (329) Google Scholar, 13Schwamborn J.C. Muller M. Becker A.H. Puschel A.W. EMBO J. 2007; 26: 1410-1422Crossref PubMed Scopus (83) Google Scholar). The Tau-1 and polyclonal anti-MAP2 antibodies were obtained from Chemicon, antibodies specific for 4E-BP1, 4E-BP2, Tsc2 phosphorylated at Thr1462 (phospho-tuberin Thr1462), and phospho-4E-BP1 (Ser65) from Cell Signaling Technology, anti-RhoA, -Rheb, and -Tsc2 (tuberin) from Santa Cruz Biotechnology, monoclonal anti-MAP2, anti-β-tubulin, and rabbit anti-HA from Sigma, and mouse monoclonal anti-Rap1 from BD Transduction Labs. The Alexa 594-, Alexa 488-, and Alexa 350-conjugated secondary antibodies and phalloidin-rhodamine were obtained from Molecular Probes. The specificity of the anti-phospho-4E-BP1 antibody was confirmed by Western blot (supplemental Fig. S5A). Cell Culture, Pharmacological Treatment, and Transfection of Hippocampal Neurons—Primary cultures of hippocampal neurons from embryonic day 18 rat (Rattus norvegicus) embryos were prepared as described previously (70Schwamborn J.C. Li Y. Püschel A.W. Methods Enzymol. 2006; 406: 715-727Crossref PubMed Scopus (20) Google Scholar). Briefly, dissected hippocampi were incubated with papain (Sigma) for 15 min at 37 °C and dissociated by pipetting in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 50 units/ml penicillin/streptomycin (Invitrogen). Cells were seeded onto glass coverslips coated with polyornithine (Sigma) and cultured at 37 °C and 5% CO2. Neurons were plated at a higher density (30-50,000 cells per 14-mm coverslip) for transfection and lower density (10-20,000 cells per coverslip) for protein localization and treatment with rapamycin. After neurons attached to the substrate (around 3-5 h after plating) the medium was changed to Neurobasal medium with B27 supplement, 2 mm glutamine, and 50 units/ml penicillin/streptomycin (Invitrogen). Rapamycin, LY294002, and insulin were added directly to neuronal cultures after the medium was changed to Neurobasal medium. Neurons were transfected by calcium phosphate coprecipitation 6-8 h after plating or by electroporation using the Rat Neuron Nucleofector Kit (Amaxa). For electroporation, 2.5 × 106 cells were resuspended in 100 μl of Nucleofectamine solution, transfected according to the manufacturer's recommendations, and plated at 100,000 cells per coverslip. Cells were fixed with 4% paraformaldehyde and 15% sucrose in phosphate-buffered saline for 20 min on ice. Immunofluorescence and Data Analysis—Neuronal morphology was analyzed using a Zeiss Axioskop 40 microscope (Carl Zeiss MicroImaging, Inc.) equipped with a Plan-Apochromat ×63/1.4 NA oil objective (Carl Zeiss MicroImaging, Inc.) and a Universal Imaging SPOT CCD camera (SPOT Insight 4; Diagnostic Instruments) and the SPOT Advanced Imaging software 4.1 (Diagnostic Instruments), and Adobe Photoshop. The same settings (exposure time, gain, and binning) and the same post-processing operations were always used to analyze the distribution of a specific protein. During post-processing with Adobe Photoshop, the brightest pixel in a picture was set as white, the darkest pixel as black, and intermediate values were distributed proportionally. The stage of neuronal differentiation was determined following published criteria (3Dotti C.G. Sullivan C.A. Banker G.A. J. Neurosci. 1988; 8: 1454-1468Crossref PubMed Google Scholar). To analyze the establishment of neuronal polarity, neurons were stained with the Tau-1 (as a marker for axons) and anti-MAP2 antibodies (minor neurites) as described previously (11Schwamborn J.C. Püschel A.W. Nat. Neurosci. 2004; 7: 923-929Crossref PubMed Scopus (329) Google Scholar, 70Schwamborn J.C. Li Y. Püschel A.W. Methods Enzymol. 2006; 406: 715-727Crossref PubMed Scopus (20) Google Scholar). Cells were embedded in Fluorescent Mounting Medium (DakoCytomation). Processes showing Tau-1 immunoreactivity in their distal segments were counted as axons, MAP2-positive neurites longer than one cell diameter as dendrites. Neurons, which did not extend any axon, were classified as unpolarized and those that extended a single axon as polarized. The length of neurites was determined using the SPOT Advanced Imaging software, fluorescence intensity with Imagetool. The Student's t test was used to determine statistical significance. Western Blot—Human embryonic kidney 293T cells and neurons were washed in phosphate-buffered saline and lysed in 2× SDS buffer. Lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences). Blots were incubated with primary antibodies overnight at 4 °C and with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Peroxidase activity was visualized by the Enhanced Chemiluminescence Detection system (Pierce). Rheb and mTOR Activity Are Required for Axon Formation—The activity of mTOR is regulated by the small GTPase Rheb (18Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar, 19Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 20Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (782) Google Scholar, 21Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 22Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1439) Google Scholar, 23Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 27Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar, 28Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell Biol. 2003; 5: 566-571Crossref PubMed Scopus (537) Google Scholar). PI3K activates Rheb by stimulating the phosphorylation of Tsc2 at Thr1462 (p-Tsc2) by Akt (Fig. 1A). Rheb, Tsc2, and p-Tsc2 were detectable in the cell body and all processes in stage 2 neurons (supplemental Fig. S1). At stage 3, Rheb and p-Tsc2 became enriched in the axon. To test if Rheb is required for the specification of axons, we suppressed endogenous Rheb by RNAi in cultures of hippocampal neurons from E18 rat embryos. The effectiveness of the shRNA vector pSb102 directed against Rheb was confirmed by Western blot after expression in human embryonic kidney 293T cells and immunofluorescence using primary neurons (supplemental Fig. S2, A and B). Suppression of Rheb by RNAi significantly reduced the number of polarized neurons. 42 ± 3% (mean ± S.E.) of the neurons did not extend an axon (control (pSuper): 9 ± 4%) and only 52 ± 2% formed a single axon compared with 82 ± 5% in controls (Fig. 1, B and C). The length of those axons that were formed was significantly reduced to 117 ± 13 μm compared with 186 ± 8 μm in controls (Fig. 1D). Suppression of Rheb had no effect on minor neurites (Fig. 1E). The loss of Rheb could be rescued by expression of mouse Rheb that is resistant to suppression by the RNAi vector pSb102 because of differences to the sequence of rat Rheb (supplemental Fig. S2, C-I). These results show that Rheb is required for the specification and elongation of axons. To investigate if Rheb acts through mTOR, we treated hippocampal neurons shortly after plating (0 days in vitro, d.i.v.) with rapamycin, a specific inhibitor of mTOR. Rapamycin blocked the establishment of neuronal polarity and inhibited the specification of axons in a dose-dependent manner (Fig. 1, F and G). Only a minority of neurons extended an axon in the presence of rapamycin, possibly because it was already specified at the time when rapamycin was added to the culture. However, the length of these axons was significantly reduced compared with controls. In the continuous presence of rapamycin, axon formation was blocked even at 5 d.i.v. (supplemental Fig. S2J). The number and length of minor neurites were not changed significantly (data not shown). Treatment with rapamycin did not increase the number of neurons undergoing apoptosis (data not shown). These results show that mTOR activity is required for axon specification and elongation during the establishment of neuronal polarity. Rheb Acts Downstream of PI3K and Upstream of mTOR to Specify Axons—To test if activation of mTOR has an effect on axon formation, we transfected hippocampal neurons with an expression vector for Rheb. Overexpression of Rheb increased the number of neurons with multiple axons compared with controls (Fig. 2, A and B). In addition, it also enhanced axon extension (Fig. 2C, EGFP, 194 ± 18 μm; Rheb, 344 ± 20 μm). Both the number and length of minor neurites were not changed, showing that the effects of Rheb overexpression are specific for axons and do not result a general stimulation of neurite growth (Fig. 2, D and E). PI3K activity is required for neuronal polarity and the PI3K inhibitor LY294002 blocks axon formation (Fig. 3, A and B) (9Shi S.H. Jan L.Y. Jan Y.N. Cell. 2003; 112: 63-75Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 11Schwamborn J.C. Püschel A.W. Nat. Neurosci. 2004; 7: 923-929Crossref PubMed Scopus (329) Google Scholar). Rheb stimulates cell growth downstream of PI3K and upstream of mTOR (14Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar, 26Inoki K. Ouyang H. Li Y. Guan K.L. Microbiol. Mol. Biol. Rev. 2005; 69: 79-100Crossref PubMed Scopus (285) Google Scholar). To investigate whether the same pathway is active during neuronal polarization, we tested if expression of Rheb can rescue the loss of neuronal polarity observed after treatment with LY294002. In the presence of LY294002, 69 ± 2% of the neurons transfected with a vector for EGFP failed to form an axon (Fig. 3). Expression of Rheb rescued these polarity defects and induced the formation of a single axon in 70 ± 3% of the neurons in the presence of LY294002. The number and length of minor neurites was not affected (data not shown). Rheb induced multiple axons in only 16 ± 1% of the cells when PI3K was inhibited compared with 61 ± 3% in controls (Fig. 3). This effect is similar to that of Rap1BV12 expression in neurons treated with LY294002, which also induces primarily a single axon and requires PI3K to induce supernumerary axons (8Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 11Schwamborn J.C. Püschel A.W. Nat. Neurosci. 2004; 7: 923-929Crossref PubMed Scopus (329) Google Scholar, 12Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). To confirm that the effects of Rheb expression are mediated by mTOR, we tested whether they can be blocked by rapamycin. Rapamycin suppressed the induction of multiple axons and the increase in axon length by Rheb (Fig. 3). Although 61 ± 3% of the neurons formed multiple axons after expression of Rheb, only 1 ± 0.4% of the neurons had supernumerary axons when rapamycin was added. The number and length of minor neurites was not affected (data not shown). These results demonstrate that Rheb regulates axon specification and extension downstream of PI3K and upstream of mTOR. mTOR-insensitive 4E-BP1 Mutants Block Axon Formation and Suppress the Effects of Rheb—One of the targets of mTOR is 4E-BP1 that regulates translation through its interaction with eIF4E (17Ruggero D. Sonenberg N. Oncogene. 2005; 24: 7426-7434Crossref PubMed Scopus (161) Google Scholar, 31Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (761) Google Scholar). Phosphorylation of 4E-BP1 at Thr37 and Thr46 by mTOR serves as a priming event for phosphorylation at other sites such as Ser65. 4E-BP1 hyperphosphorylation results in the stimulation of translation (29Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1138) Google Scholar, 32Gingras A.C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (730) Google Scholar, 33Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1188) Google Scholar). Hippocampal neurons express both 4E-BP1 and 4E-BP2 (supplemental Fig. S3A). At 24 h in culture (early stage 2), 4E-BP1 phosphorylated at Ser65 (p-4E-BP1) was present throughout the cell body and all neurites, but became enriched in the growth cone of a single neurite in the majority of neurons at 36 h (late stage 2) and to the axon tip at stage 3 (supplemental Fig. S3, B-H). The axonal enrichment of p-4E-BP1 in stage 3 neurons was not detectable after inhibition of PI3K or mTOR (supplemental Fig. S4, A and B). An antibody to analyze the distribution of phosphorylated 4E-BP2 by immunofluorescence was not available. Consistent with the function of Rheb as an activator of mTOR, the supernumerary axons induced by Rheb were positive for p-4E-BP1 (data not shown). Thus, 4E-BP1 phosphorylation is stimulated through the PI3K/mTOR pathway during neuronal polarization. To examine the function of 4E-BP1, two mTOR-insensitive 4E-BP1 mutants were expressed
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