Neurofibromatosis Type I Tumor Suppressor Neurofibromin Regulates Neuronal Differentiation via Its GTPase-activating Protein Function toward Ras
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m209413200
ISSN1083-351X
AutoresShunji Yunoue, Hiroshi Tokuo, Kohji Fukunaga, Liping Feng, Tatsuya Ozawa, Toru Nishi, Akira Kikuchi, Seisuke Hattori, Jun‐ichi Kuratsu, Hideyuki Saya, Norie Araki,
Tópico(s)Neuroblastoma Research and Treatments
ResumoNeurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance. Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance. Neurofibromatosis type 1 (NF1) 1The abbreviations used are: NF1, neurofibromatosis type 1; NGF, neural growth factor; EGF, epidermal growth factor; RT, reverse transcriptase; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; Ab, antibody; GRD, GAP-related domain; DN, dominant negative; GFP, green fluorescent protein; CA-Raf, constitutively active Raf; GAP, GTPase-activating protein; PI3K, phosphatidylinositol 3-kinase. is one of the most common autosomal dominantly inherited disorders, with an incidence of about 1 in 3500 individuals (1Stephens K. Riccardi V.M. Rising M. Ng S. Green P. Collins F.S. Rediker K.S. Powers J.A. Parker C. Donis-Keller H. Genomics. 1987; 1: 353-357Crossref PubMed Scopus (23) Google Scholar). The NF1 hallmark is the development of benign tumors of the peripheral nervous system and the increased risk of developing malignancies. The NF1 phenotype is highly variable; it affects several organ systems, including bones, skin, irises, and central nervous system manifested as gliomas and learning disabilities. The NF1 gene lies on chromosome 17q11.2 and encodes neurofibromin, a large 2818-amino acid protein (2Cawthon R.M. Weiss R. Xu G.F. Viskochil D. Culver M. Stevens J. Robertson M. Dunn D. Gesteland R. O'Connell P. White R. Cell. 1990; 62: 193-201Abstract Full Text PDF PubMed Scopus (982) Google Scholar). Since the majority of NF1 gene mutations frequently found in NF1 patients prevent intact neurofibromin expression, functional disruption of neurofibromin is potentially relevant to the expression of some or all of the multiple abnormalities that occur in NF1 patients (3Viskochil D. Buchberg A.M. Xu G. Cawthon R.M. Stevens J. Wolff R.K. Culver M. Carey J.C. Copeland N.G. Jenkins N.A. White R. O'Connell P. Cell. 1990; 62: 187-192Abstract Full Text PDF PubMed Scopus (920) Google Scholar). A region centered around the 360 amino acids encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP) and is also similar to yeast IRA1/2 proteins, which interact with Ras and mediate hydrolysis of Ras-bound GTP to GDP, resulting in Ras protein inactivation. The GAP-related domain of the NF1 gene product (NF1-GRD) also stimulates Ras GTPase and consequently inactivates Ras protein (4Ballester R. Marchuk D. Boguski M. Saulino A. Letcher R. Wigler M. Collins F. Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (689) Google Scholar, 5Xu G.F. Lin B. Tanaka K. Dunn D. Wood D. Gesteland R. White R. Weiss R. Tamanoi F. Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (583) Google Scholar). Two different isoforms, type I and type II, which are formed by alternative splicing, have been identified in the NF1-GRD region. Type II contains an additional 63-bp insertion (exon 23a) that encodes 21 amino acids in the center of NF1-GRD type I (6Nishi T. Lee P.S. Oka K. Levin V.A. Tanase S. Morino Y. Saya H. Oncogene. 1991; 6: 1555-1559PubMed Google Scholar). The specific expression patterns of the two isoforms have been studied in several organs and cells (7Bernards A. Biochim. Biophys. Acta. 1995; 1242: 43-59PubMed Google Scholar, 8Metheny L.J. Skuse G.R. Exp. Cell Res. 1996; 228: 44-49Crossref PubMed Scopus (16) Google Scholar) and provide a basis for implicating differential expression of NF1 type I and type II transcripts in the regulation of neuronal differentiation and development. Recent studies using Nf1 gene targeting animals reported that Drosophila homozygotes with Nf1 null mutation showed significantly decreased olfactory learning performance (9Guo H.F. Tong J. Hannan F. Luo L. Zhong Y. Nature. 2000; 403: 895-898Crossref PubMed Scopus (212) Google Scholar), Nf1 heterozygous mice displayed spatial learning disability (10Silva A.J. Frankland P.W. Marowitz Z. Friedman E. Lazlo G. Cioffi D. Jacks T. Bourtchuladze R. Nat. Genet. 1997; 15: 281-284Crossref PubMed Scopus (302) Google Scholar, 11Costa R.M. Federov N.B. Kogan J.H. Murphy G.G. Stern J. Ohno M. Kucherlapati R. Jacks T. Silva A.J. Nature. 2002; 415: 526-530Crossref PubMed Scopus (480) Google Scholar), and mice lacking alternatively spliced Nf1 exon 23a exhibited specific learning impairment (12Costa R.M. Yang T. Huynh D.P. Pulst S.M. Viskochil D.H. Silva A.J. Brannan C.I. Nat. Genet. 2001; 27: 399-405Crossref PubMed Scopus (158) Google Scholar). Furthermore, abnormal Ras activity in Nf1 knockout mice disrupted learning and memory, indicating that functional modulation of Ras by neurofibromin is essential for learning and memory (11Costa R.M. Federov N.B. Kogan J.H. Murphy G.G. Stern J. Ohno M. Kucherlapati R. Jacks T. Silva A.J. Nature. 2002; 415: 526-530Crossref PubMed Scopus (480) Google Scholar). These observations prompted us to postulate that neurofibromin plays a key role in the Ras signal-dependent pathway as a GAP in neuronal cells and that functional regulation of neurofibromin, such as alternative splicing, could be involved in the neuronal development that may be implicated in the learning disability of NF1 patients. Here, we analyzed alterations in cellular neurofibromin GAP activity (NF1-GAP) using a newly developed NF1-GAP assay system during the neuronal differentiation of PC12 cells. PC12 cells are well known to respond to NGF, followed by activation of a Ras-dependent signal pathway, leading to differentiation into a sympathetic neuron-like phenotype (13Vaudry D. Stork P.J. Lazarovici P. Eiden L.E. Science. 2002; 296: 1648-1649Crossref PubMed Scopus (716) Google Scholar, 14Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4983) Google Scholar). Our results demonstrate that the mutual regulation of Ras and NF1-GAP is implicated in neuronal differentiation. For further confirmation, rat embryonic hippocampal neurons and PC12 cells were analyzed for morphological alterations after overexpression of NF1-GRD type I dominant negative mutants. Finally, we discuss the cellular function of neurofibromin in the normal development of neuronal cells in conjunction with the Ras-related signal modulation via NF1-GAP and implications for the NF1-related neuronal pathogenesis such as learning disability and memory disturbance. Preparation of Recombinant Proteins—Glutathione S-transferase (GST) fusion proteins of human GRD type I (residues 1175–1552), GRD type II (21 amino acids inserted between residues 1345 and 1346 of GRD type I), Ha-Ras, and p120GAP were produced in Escherichia coli under the isopropyl-1-thio-β-d-galactopyranoside induction system, and affinity-purified as described (15Tokuo H. Yunoue S. Feng L. Kimoto M. Tsuji H. Ono T. Saya H. Araki N. FEBS Lett. 2001; 494: 48-53Crossref PubMed Scopus (50) Google Scholar). Construction of Plasmids—pGEX-2TH plasmids harboring NF1-GRD type I and GRD type II cDNAs, which produce GST-GRD type I and GST-GRD type II fusion proteins, respectively, were constructed as described (16Izawa I. Tamaki N. Saya H. FEBS Lett. 1996; 382: 53-59Crossref PubMed Scopus (60) Google Scholar). pGEX-2TH-Ha-Ras and pGEX-2T p120GAP were prepared as described (17Kikuchi A. Sasaki T. Araki S. Hata Y. Takai Y. J. Biol. Chem. 1989; 264: 9133-9136Abstract Full Text PDF PubMed Google Scholar, 18Li S. Nakamura S. Hattori S. J. Biol. Chem. 1997; 272: 19328-19332Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). pEGFPc1-GRD type I and type II vectors were constructed by insertion of a HindIII fragment from pGEX-2TH-GRD type I or type II, respectively, into a HindIII site in the pEGFPc-1 multicloning sites and cloned. To construct expression plasmids for FLAG-tagged NF1-GRD type I and type II, NF1-GRD type I and type II DNA fragments were prepared by double digestion of the BglII and XbaI sites of the pEGFPc1-GRD type I and type II plasmids. Each fragment was ligated into the BamHI and XbaI sites of pcDNA3-FLAG, which contained the FLAG epitope DNA sequence in the BamHI-EcoRI sites of pcDNA3 followed by EcoRI-BamHI linker DNA (AATTCGCGGCCGCCCGGG). To construct pcDNA3-Myc-cRaf-1CAAX, a Myc-tagged cRaf-1CAAX fragment was prepared from the pmt-SM-Myc-cRaf-1CAAX plasmid (19Okazaki M. Kishida S. Hinoi T. Hasegawa T. Tamada M. Kataoka T. Kikuchi A. Oncogene. 1997; 14: 515-521Crossref PubMed Scopus (56) Google Scholar) by double digestion of the NotI and EcoRI sites. The fragment was blunt-ended on the EcoRI site and inserted into the NotI and blunt-ended XbaI sites of pcDNA3 vector. The entire sequences of the plasmids were confirmed using a BigDye Terminator DNA sequence kit (Applied Biosystems) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Site-directed Mutagenesis of NF1-GRD Type I—Mutants of pcDNA3-FLAG-GRD type I (R1276K (FLAG-R1276K)) and pcDNA3-FLAG-GRD type I (R1276A/R1391K (FLAG-R1276A/R1391K)) were prepared as described (20Picard V. Ersdal-Badju E. Lu A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (217) Google Scholar). Briefly, pcDNA3-FLAG-GRD type I cDNA was used as a template for PCR amplification with Pfu DNA polymerase (Stratagene). For the R1276K and R1276A mutations, the primers used were upstream and downstream of the mutation site in R1276 (R1276A-sense, 5′-ACTCTCTTCGCCGGCAACAGCTTGGCCAGTAAAATA-3′; R1276A-antisense, 5′-GCTGTTGCCGGCGAAGAGAGTCTGCATGGAGTCTGC-3′; R1276K-sense, 5′-CAGACTCTCTTCAAAGGCAACAGCTTGGCCAGTAAAATA-3′; R1276K-antisense, 5′-CAAGCTGTTGCCTTTGAAGAGAGTCTGCATGGAGTCTGC-3′). For R1276A/R1391K double mutations, pcDNA3-FLAG R1276A was used as a template, and the primers used were as follows: R1391K-sense, 5′-GCCATGTTCCTCAAATTTATCAATCCTGCCATTGTCTCACCg-3′; R1391K-antisense, 5′-AGGATTGATAAATTTGAGGAACATGGCACTTCCTACTGCACC-3′. The sequences of the mutant plasmids were confirmed by sequencing as described above. Cell Culture, Stimulation, Transfection, and Preparation of Cell Lysates—PC12 cells were cultured under 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal bovine serum. COS7 cells were cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham (Sigma) with 10% fetal bovine serum. Transfection into cells was performed with LipofectAMINE 2000 (Invitrogen) or Fugene 6 (Roche Applied Science) according to the manufacturer's protocol. PC12 cells expressing RasN17 (M-M17–26 cells; RIKEN, Saitama, Japan) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 5% horse serum, and 400 μg/ml G418 (Invitrogen). PC12 cells were stimulated with 50 ng/ml 2.5 S NGF (WAKO) or EGF (WAKO) for up to 48 h. For preparation of cellular lysates, cells were solubilized with NF1 lysis buffer (20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 150 mm NaCl, 1 mm EDTA, 0.1% Nonidet P-40, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 μg/ml each aprotinin, pepstatin A, and leupeptin) and passed through a 25-gauge syringe 15 times. Lysates were centrifuged at 20,000 × g for 20 min at 4 °C, and the protein concentrations of the supernatants were determined using the BCA protein assay (Pierce). GAP Activity Assay—To prepare the [γ-32P]GTP-bound form of Ras (Ras-[γ32P]GTP), 40 μl of GST-Ha-Ras (5 μm) was preincubated with 6 μl of [γ-32P]GTP diluted with 5 μm GTP (0.2 mCi/ml) for 2.5 min at 37 °C in 42 μl of Ras loading buffer A (20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 20 mm EDTA, 100 mm NaCl, 10% glycerol, 2.5 mg bovine serum albumin). After the incubation, 8 μlof0.5 m MgCl2 and 240 μl of ice-cold Ras loading buffer B (Ras loading buffer A without EDTA) were added. The indicated amounts of GST fusion proteins or cell lysates in 40 μl of GAP assay buffer (50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 2.5 mg of bovine serum albumin, 5% glycerol, 0.5 mm GTP) in the presence or absence of 3 μg of anti-NF1-GAP IgG were incubated with 10 μl of Ras-[γ-32P]GTP for 10 min at 30 °C. After a second incubation, the reactions were quenched by keeping the sample on ice and adding 0.8 ml of ice-cold stop buffer (25 mm Tris-HCl, pH 7.6, 100 mm NaCl, 5 mm MgCl2). The sample was passed through a nitrocellulose filter (0.45 μm; Schleicher & Schuell), and the filter was washed three times with 0.8 ml of ice-cold stop buffer. After drying the filter, the radioactivity was counted. Total GAP activities were obtained from the differences between the total Ras-[γ-32P]GTP radioactivity and the sample radioactivity hydrolyzed after the GAP reaction. Specific NF1-GAP activity was obtained from the difference between the sample radioactivities after the reaction with and without anti-NF1-GAP IgG, and the activity of other GAPs was obtained by subtraction of NF1-GAP activity from the total GAP activity. Ras Activity Assay—The Ras activity assay was performed using the Ras-binding domain of Raf-1 bound to agarose in a Ras activation assay kit (Upstate Biotechnology) according to the manufacturer's instructions and as described (21de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). RT-PCR Analysis of NF1-GRD—For NF1-GRD amplification by RT-PCR, primers 5′-CAGAGTTCCCCTCGCAGCTTCG-3′ (NF1rGRD-sense) and 5′-CTCCGTGCCAAGTCGGAGTTGC-3′ (NF1rGRD-antisense) were used. First-strand cDNA, which served as the PCR template, was synthesized from 2 μg of total RNA purified using an RNeasy minikit (Qiagen) from PC12 cells or M-M17–26 cells. Reverse transcription was performed using oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen), and the PCR conditions were as described (8Metheny L.J. Skuse G.R. Exp. Cell Res. 1996; 228: 44-49Crossref PubMed Scopus (16) Google Scholar). After PCR, each sample was separated in a 7.5% polyacrylamide gel and stained with ethidium bromide. To quantify the relative expression levels of NF1 type I and type II transcripts, the bands were scanned and analyzed with MacBas2000 (Fuji Film). Antibodies—An anti-NF1-GAP antibody was generated in female Japanese white rabbits immunized against GST-NF1-GRD (amino acids 1175–1552) and affinity-purified on a formylcellulofine column (Seikagaku Co.) covalently coupled with GST-p120GAP protein, followed by a protein G-Sepharose column. A monoclonal anti-FLAG antibody (M5) was purchased from Sigma, and anti-phospho-p44/42 MAPK and anti-p44/42 MAPK antibodies were purchased from Cell Signaling Technology. Neuronal Culture, Transfection, and Immunocytochemistry—Rat hippocampal cell cultures were prepared as described (22Fukunaga K. Soderling T.R. Miyamoto E. J. Biol. Chem. 1992; 267: 22527-22533Abstract Full Text PDF PubMed Google Scholar). Neurons separated from rat embryonic day 18–19 hippocampus were plated at high density on coverslips coated with polyethyleneimine (IWAKI). FLAG-R1276A/R1391K was transfected into the neurons with LipofectAMINE 2000 at 12 h after plating. As a control for the morphological analysis, FLAG and GFP vectors were co-transfected and expressed. For morphological studies, neurons were fixed in 4% paraformaldehyde for 15 min at room temperature, aldehyde was quenched in 50 mm ammonium chloride for 5 min, and cells were permeabilized with 0.1% Triton X-100. To identify transfected neurons, cells were immunostained with the monoclonal anti-FLAG antibody M5 (Sigma), followed by a fluorescent dye-conjugated secondary antibody, and analyzed using confocal microscopy (Fluoview, FV500; Olympus). To define the axons and dendrites of neurons, simultaneous immunostaining was performed with a rabbit polyclonal anti-tau antibody (Sigma) and anti-mitogen-activated protein antibody (Sigma), respectively, and the morphological criteria of axons (thin, uniform caliber, nontapering processes) or dendrites (thick, tapering with distant processes) were used. Morphological analysis was performed using the measuring software (Mac SCOPE, Mitani Co.). Measurement of the Specific Ras GAP Activity of Neurofibromin—To analyze the specific GAP function of neurofibromin in neuronal cells, we first established a method for measuring NF1-GAP activity using the anti-NF1-GAP IgG (NF1-GAP Ab), prepared from a rabbit antiserum against GST-NF1-GRD type I depleted of fractions reactive to p120GAP. Immunoblotting using NF1-GAP Ab indicated no cross-reactivity with any cellular proteins other than neurofibromin (Fig. 1F), suggesting its specificity for NF1-GAP. The dose-dependent GAP activities of both GST-NF1-GRD and p120GAP (Fig. 1, A and B) detected by the original filtration assay (23Hattori S. Maekawa M. Nakamura S. Oncogene. 1992; 7: 481-485PubMed Google Scholar) were completely blocked by NF1-GAP Ab, whereas GST-p120GAP was not inhibited (Fig. 1, C and D). We also analyzed the specific effects of NF1-GAP Ab on cellular lysates of Nf1–/– mouse embryo fibroblasts, which had GST-NF1-GRD, p120GAP, or GST proteins added. The GAP activity of GST-NF1-GRD in the Nf1–/– mouse embryo fibroblast extracts was completely inhibited by NF1-GAP Ab to the basal level, but that of p120GAP was not inhibited (Fig. 1E). These results indicate that NF1-GAP Ab specifically inhibits the GAP activity of neurofibromin in cellular extracts, but not p120GAP, and is therefore useful for measurement of cellular specific NF1-GAP. The percentages for the hydrolyzed Ras-[γ-32P]GTP with the reaction of GST-NF1-GRD or the cellular lysates without NF1-GAP Ab were subtracted by those with NF1-GAP Ab and plotted as the specific NF1-GAP activity (Fig. 2, A and B).Fig. 2GAP activities of NF1-GRD type I and type II. A, Ras-[γ-32P]GTP was incubated at 30°C for 10 min with the indicated amounts of GST-NF1-GRD type I (closed circle) or GST-NF1-GRD type II (open circle), and their GAP activities were analyzed. Data are plotted as the GTP hydrolyzed relative to the total GTP bound to Ras in the control without GAP addition. B, GAP activities of cellular lysates (25 μg) from COS7 cells expressing pcDNA3-FLAG vector alone (closed triangle), FLAG-NF1-GRD type I (closed circle), or FLAG-NF1-GRD type II (open circle). The indicated amounts of cell lysates were incubated with NF1-GAP Ab or preimmune IgG for 2 h on ice and analyzed for GAP activity. C, the FLAG protein expression levels in 25 μg of whole cell lysates were detected by immunoblotting using NF1-GAP Ab.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GAP Activities of NF1 Type I and Type II in Mammalian Cells—The GAP activities of the two alternatively spliced NF1 isoforms, type I and type II, have previously been studied, and it was reported that NF1-GRD type II resulted in a 50% reduction of the GAP activity compared with that of NF1-GRD type I (24Nur E.K.M.S. Varga M. Maruta H. J. Biol. Chem. 1993; 268: 22331-22337Abstract Full Text PDF PubMed Google Scholar). However, when we analyzed the GST-NF1-GRD type I and type II GAP activities using our system, GRD type I GAP activity was more than 10-fold higher than that of GRD type II at their lower protein concentrations (ng level of protein) (Fig. 2A). This difference between NF1-GRD type I and type II was reproduced in the cellular GAP analysis of FLAG-tagged NF1-GRD type I (FLAG-NF1-GRD type I) and FLAG-NF1-GRD-type II overexpressed in COS7 cells (Fig. 2B). The expression levels of each FLAG-GRD in 25 μg of cell lysate were almost equal in intensity (Fig. 2C). The activity of GRD type I expressed in COS7 cells in 5 μg of cell lysate was more than 10-fold higher than that of GRD type II. These results suggest that the alternative splicing of NF1 type I and type II regulates cellular NF1-GAP activity and may control the Ras-related signals, such as those for cellular proliferation and differentiation. Relationship of Neurite Outgrowth, Ras Activity, and Endogenous NF1-GAP Activity in PC12 Cells—To study the GAP function of neurofibromin in neuronal cell differentiation, we examined NF1-GAP in PC12 cells after stimulation by several different growth factors. After NGF stimulation, neurite outgrowth occurred in PC12 cells within 24 h, followed by significant neurite extension (Fig. 3B). Neurite outgrowth bearing PC12 cells that carried processes longer than the cell body diameter represented up to 50% of the total cells after 48 h of NGF stimulation (Fig. 3A). In contrast, PC12 cells treated with EGF showed little morphological change, scored less than 2% of neurite outgrowth-bearing cells, and were similar to nontreated cells (Fig. 3, A and B). In the same experiment, the specific NF1-GAP activities of PC12 cells were shown to increase significantly during 12–48 h after NGF stimulation (Fig. 3C). After 48 h, the NF1-GAP activity in NGF-stimulated cells reached 2.5-fold that before stimulation, whereas that of nonstimulated or EGF-stimulated cells only increased 1.25-fold. The activity of other GAPs obtained after subtraction of the specific NF1-GAP activity from the total GAP activity showed a small increase after NGF stimulation but stayed at a lower level compared with the NF1-GAP activity (50% increase in other GAPs versus 180% increase in NF1-GAP compared with each GAP in the nonstimulated state). The effect of EGF stimulation on the activity of other GAPs was also insignificant and as low as the effect on NF1-GAP (Fig. 3D). Cellular NF1-GAP activity also increased in a dose-dependent manner with NGF stimulation (data not shown), indicating that NF1-GAP activity is responsive to NGF stimulation. Cellular Ras activity was analyzed using an affinity technique with Raf-1RBD (Ras-binding domain)-agarose. After NGF stimulation, Ras activity quickly reached the maximum level within 5 min and was down-regulated during 15–60 min (Fig. 4, A and B), as reported previously (25Kao S. Jaiswal R.K. Kolch W. Landreth G.E. J. Biol. Chem. 2001; 276: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Surprisingly, upon further stimulation with NGF, Ras activity gradually increased up to more than 10-fold the original level, reached a plateau at around 24 h, and was then down-regulated over 24–72-h stimulation (Fig. 4, A and C). EGF stimulation increased Ras activity quickly and down-regulated it during an early phase in the same manner as NGF stimulation. However, later than 60 min after EGF stimulation, Ras activity regulated at the basal level did not change over 72-h stimulation (Fig. 4, A–C). The Ras activity regulation in PC12 cells in the later phase of NGF stimulation was well correlated with the time-dependent increase in cellular NF1-GAP activity as well as with the increase in neurite-bearing PC12 cells (Fig. 3, A and C). Thus, these results prompted us to speculate that the increase in endogenous NF1-GAP activity in PC12 cells may regulate cellular Ras activity and control neuronal outgrowth of PC12 cells during longer NGF stimulation. Effects of NGF and EGF on NF1 Type I and Type II Alternative Splicing in PC12 Cells—To elucidate how the NF1-GAP activity was increased by NGF stimulation in PC12 cells, we examined the alternative splicing pattern of NF1 type I and type II in PC12 cells by RT-PCR at the later phase of NGF treatment, compared with the pattern with EGF treatment. Interestingly, the transcriptional levels of both the type I and type II isoforms increased after NGF exposure in a time-dependent fashion, but the rate of increase of type I mRNA was significantly higher than that of type II (Figs. 5A and 6, A and B). Before exposure to NGF, the type I/type II transcript ratio was 22%, but after NGF stimulation, it gradually increased in a time-dependent manner, and after 48 h, the ratio was up to 110% (Figs. 5B and 6C). In contrast, EGF stimulation did not increase the type I/type II ratio, even after 48-h stimulation (Fig. 5, A and B), suggesting that the specific splicing of NF1 type I/type II mRNA could be induced by cellular signals via NGF stimulation in particular. The pattern of the time-dependent increase for type I/type II transcription was very similar to that of the NF1-GAP activity in PC12 cells after NGF stimulation (Fig. 3C). These results indicate that NGF stimulation up-regulates the cellular expression of NF1 type I, and since it possesses a much higher GAP activity than type II, this causes the increase in cellular NF1-GAP activity in PC12 cells.Fig. 6Effects of NGF on NF1 type I and type II alternative splicing in PC12 and M-M17–26 cells. Expression levels of NF1 type I and type II transcripts induced in PC12 (A–C) or M-M17–26 (D–F) cells during treatment with NGF were analyzed by RT-PCR at the indicated times after NGF stimulation. β-Actin transcripts were used as an internal standard. Relative intensities of NF1 type I/actin (closed circle) and type II/actin (closed triangle) were calculated as follows: (LAU-BG of GRD/mm2)/(LAU-BG of actin/mm2) (B and E). NF1 type I/type II intensity ratios (closed square) were calculated as follows: (LAU-BG of type I/mm2)/(LAU-BG of type II/mm2) × 100 (C and F). Data represent means ± S.D. from four experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NF1-GRD Alternative Splicing Requires Ras Activity—It has been shown that the initial activation of Ras by NGF stimulation acts as a trigger for differentiation of PC12 cells via the transcriptional up-regulation of several genes. To investigate the mechanism for the transcriptional increase of NF1 type I after NGF stimulation, we analyzed the regulation of NF1-GRD alternative splicing in a subline of PC12 cells (M-M17–26) that stably overexpress a dominant inhibitory Ras mutant, Ha-Ras (S17N). Previous analysis of this subline has shown that the absence of Ras activity completely blocks neurite outgrowth after NGF stimulation (26Szeberenyi J. Cai H. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (292) Google Scholar). As shown in Fig. 6, NF1 type I and type II mRNA levels in untreated M-M17–26 cells were similar to those detected in wild-type PC12 cells when the cells were maintained in the absence of NGF. However, the specific time-dependent increase in NF1 type I mRNA normally observed in response to NGF stimulation was significantly inhibited in M-M17–26 cells (Fig. 6, D–F). These results suggest that Ras activity regulates the alternative splicing of NF1 mRNA after NGF stimulation in PC12 cells, resulting in increasing cellular NF1-GAP activity. Mechanism of NGF-Ras-induced NF1-GRD Alternative Splicing—To understand which Ras-mediated signals induced by NGF stimulation in PC12 cells are involved in NF1 type I/type II alternative splicing, we tested the effects of several kinds of pharmacological inhibitors against signals upstream or downstream of Ras, such as MEK1 inhibitor (PD98059), PI3K inhibitor (LY294002), MEK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), and an inhibitor of the NGF receptor tyrosine kinase (K252a), on the PC12 cells after NGF stimulation. As shown in Fig. 7, A and B, the inhibitors for both the N
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