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• W2000550050 abstract "The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system. The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system. Ras proteins function as GTP/GDP-regulated switches that cycle between an active GTP-bound and an inactive GDP-bound conformational state to regulate a wide variety of cell functions, including cell proliferation, differentiation, and apoptosis (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2061) Google Scholar). Ras proteins respond to extracellular stimuli by exchanging GTP for bound GDP, thereby triggering intracellular signaling cascades through their interaction with a variety of target proteins (2Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar). The cycle between active and inactive states is tightly controlled, being stimulated by the interaction of Ras proteins with specific guanine nucleotide exchange factors (GEFs) 1The abbreviations used are: GEFguanine nucleotide exchange factorBDRin/Ras-binding domainGFPgreen fluorescent proteinHAhemagglutininDMEMDulbecco's modified Eagle's mediumGSTglutathione S-transferaseGAPGTPase-activating proteinNGFnerve growth factorEGFepidermal growth factorERKextracellular regulated kinaseMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinasePI 3-kinasephosphatidylinositol 3-kinasePBSphosphate-buffered salineRTreverse transcriptaseCMcalcium/magnesiumHEKhuman embryonic kidney that induce the dissociation of GDP to allow GTP association. On the other hand, GTPase-activating proteins (GAPs) induce GTP hydrolysis and serve as negative regulators of the GTPase cycle (3Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1837) Google Scholar, 4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). guanine nucleotide exchange factor Rin/Ras-binding domain green fluorescent protein hemagglutinin Dulbecco's modified Eagle's medium glutathione S-transferase GTPase-activating protein nerve growth factor epidermal growth factor extracellular regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase phosphatidylinositol 3-kinase phosphate-buffered saline reverse transcriptase calcium/magnesium human embryonic kidney Ras proteins also play important roles in many nerve growth factor (NGF)-mediated differentiation events (5Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (905) Google Scholar, 6Greene L.A. Kaplan D.R. Curr. Opin. Neurobiol. 1995; 5: 579-587Crossref PubMed Scopus (288) Google Scholar). The NGF-responsive rat pheochromocytoma PC12 cell line has been used extensively as a model system to study this signal transduction process (7Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4862) Google Scholar). Cultures of PC12 cells undergo rapid changes following NGF stimulation, differentiating to resemble sympathetic neurons (7Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4862) Google Scholar, 8Burstein D.E. Greene L.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 6059-6063Crossref PubMed Scopus (175) Google Scholar). NGF mediates these effects by binding the tyrosine kinase receptor, TrkA, an event that stimulates the activity of multiple signaling proteins, including Ras (5Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (905) Google Scholar, 6Greene L.A. Kaplan D.R. Curr. Opin. Neurobiol. 1995; 5: 579-587Crossref PubMed Scopus (288) Google Scholar, 9Kaplan D.R. Miller F.D. Curr. Opin. Cell Biol. 1997; 9: 213-221Crossref PubMed Scopus (545) Google Scholar). Ras activates additional downstream effectors, including Raf and phosphatidylinositol 3-kinase (PI 3-kinase), which play important roles in the process of neuritogenesis (10Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar, 11Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1853) Google Scholar, 12Fukuda M. Gotoh Y. Tachibana T. Dell K. Hattori S. Yoneda Y. Nishida E. Oncogene. 1995; 11: 239-244PubMed Google Scholar). Indeed, constitutively active Ras mutants can induce morphological differentiation of PC12 cells in the absence of NGF (13Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (569) Google Scholar), whereas inhibition of Ras signaling via the expression of dominant-negative mutant protein or microinjection of anti-Ras inhibitory antibodies represses differentiation induced by NGF (14Szeberenyi J. Cai H. Cooper G.M. Mol. Cell. Biol. 1990; 10: 5324-5332Crossref PubMed Scopus (276) Google Scholar, 15Hagag N. Halegoua S. Viola M. Nature. 1986; 319: 680-682Crossref PubMed Scopus (302) Google Scholar). Recently, the Rin/Rit/dRIC branch of the Ras subfamily has been described (16Lee C.H.J. Della N.G. Chew C.E. Zack D.J. J. Neurosci. 1996; 16: 6784-6794Crossref PubMed Google Scholar, 17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar, 18Wes P.D., Yu, M. Montell C. EMBO J. 1996; 15: 5839-5848Crossref PubMed Scopus (72) Google Scholar). These Ras-related proteins lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for the subcellular localization and biological function of the majority of Ras-like GTPases. Additional unique features of this Ras subfamily include a distinct but conserved G2 core effector domain and a C-terminal calmodulin-binding domain for Rin and RIC (16Lee C.H.J. Della N.G. Chew C.E. Zack D.J. J. Neurosci. 1996; 16: 6784-6794Crossref PubMed Google Scholar, 18Wes P.D., Yu, M. Montell C. EMBO J. 1996; 15: 5839-5848Crossref PubMed Scopus (72) Google Scholar). As an initial step in the biological characterization of Rit and Rin, we investigated their ability to regulate signaling pathways used by other Ras family proteins to control cell growth and transformation. These studies demonstrated that Rit signaled to Ras-responsive elements and transformed NIH3T3 cells to tumorigenicity (19Rusyn E.V. Reynolds E.R. Shao H. Grana T.M. Chan T.O. Andres D.A. Cox A.D. Oncogene. 2000; 19: 4685-4694Crossref PubMed Scopus (42) Google Scholar). However, Rin had little or no activity in these assays; thus, no characterization of its cellular function has yet been performed. In this study, we investigated the involvement of Rin in NGF-induced signaling cascades in pheochromocytoma cells. Using an affinity activation assay, we demonstrate that growth factor stimulation of neuronal cell lines results in the rapid activation of Rin. Experiments characterizing this signaling cascade reveal that Rin activation appears to rely upon Ras and additional, perhaps neuronal-specific, regulatory factors. The role of Rin in NGF-induced neurite outgrowth was also examined. Although the expression of wild type or constitutively active Rin had no effect on neurite extension in PC6 cells, dominant-negative Rin suppressed NGF-mediated neurite outgrowth. Furthermore, constitutively active H-Ras-induced neurite outgrowth was also suppressed by dominant-negative Rin. These studies suggest that Rin may play a critical role in transducing growth factor-dependent signals that are involved in maintaining normal nervous system function. PC6 is a subline of PC-12 cells that produces neurites in response to NGF but grows as well isolated cells rather than in clumps. The PC6 line used in these studies was the parental line described by Pittman et al.(20Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar) and was the generous gift of Dr. Thomas Vanaman (University of Kentucky, Lexington, KY). PC6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) containing 10% (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum (Invitrogen), and 50 μg/ml gentamicin at 37 °C in a humidified atmosphere of 5% CO2. HEK293 cells were grown in DMEM supplemented with 5% (v/v) fetal bovine serum and 50 μg/ml gentamicin, whereas Vero and MCIXC cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum and 50 μg/ml gentamicin. These lines were also maintained at 37 °C in a humidified atmosphere of 5% CO2. NGF (Becton Dickinson) and epidermal growth factor (EGF; Sigma) were administered at a dose of 100 ng/ml. Transfection of PC6, Vero, and MCIXC cells was performed using Effectene (Qiagen). For Rin and H-Ras activation assays, PC6 cells (7.5 × 104 cells/60-mm plate) were transiently transfected with 1 μg of the indicated mammalian expression plasmid mixed with 8 μl of enhancer and 10 μl of Effectene according to the manufacturer's protocol. HEK293 cells were transfected using the calcium phosphate method as described previously (21Shao H. Andres D.A. J. Biol. Chem. 2000; 275: 26914-26924Abstract Full Text Full Text PDF PubMed Google Scholar). Mammalian expression vectors for wild type and mutant H-Ras and Rin have been described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar, 21Shao H. Andres D.A. J. Biol. Chem. 2000; 275: 26914-26924Abstract Full Text Full Text PDF PubMed Google Scholar). pCGN-HA-RasWT and pDCR-Ras(V12) effector domain mutant vectors were the kind gift of Dr. Adrienne Cox (University of North Carolina, Chapel Hill, NC), whereas pCDNA-Sos1-C AAX and pCDNA-GRF1-C AAX were the gift of Dr. Lawrence Quilliam (University of Indiana School of Medicine, Indianapolis, IN). 5′-EcoRI and 3′-BamHI sites were introduced to wild type H-Ras by polymerase chain reaction (PCR)-mediated DNA amplification. The PCR product was subcloned to the corresponding sites in pEGFP-C1 to generate the GFP-tagged wild type H-Ras mammalian expression vector. Oligonucleotide site-directed mutagenesis was used to generate dominant-negative H-Ras (Ras(N17)) using pEGFP-RasWT as described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). All PCR products were verified by DNA sequence analysis. HA epitope-tagged proteins were detected by immunoblotting using anti-HA monoclonal antibody (12CA5) followed by incubation with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Zymed Laboratories Inc.). GFP fusion proteins were detected using anti-GFP Living Colors antibody (CLONTECH) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.) as described (22Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (89) Google Scholar, 23Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar). Total RNA was isolated using a STAT-60 kit (Tel-Test B, Friendswood, TX) according to the manufacturer's protocol. A riboprobe plasmid for mouse Rin was generated by polymerase chain reaction (PCR) amplification of a 228-bp fragment from the N terminus of Rin (amino acids 1–76) and subcloned into pZero-2™ (Invitrogen). The plasmid containing an 89-bp fragment of the mouse ribosomal protein L32 (19Rusyn E.V. Reynolds E.R. Shao H. Grana T.M. Chan T.O. Andres D.A. Cox A.D. Oncogene. 2000; 19: 4685-4694Crossref PubMed Scopus (42) Google Scholar) was a gift of Dr. Daniel Noonan (University of Kentucky, Lexington, KY). Antisense radiolabeled riboprobes were prepared using linearized templates and a Maxiscript™ (Ambion) kit according to the manufacturer's protocol. RNase protection assays were performed as described (24Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Protected fragments were resolved by electrophoresis in a 5% acrylamide, 8 murea sequencing gel. The gel was dried and exposed to X-Omat AR film (Eastman Kodak Co.) for the indicated time. The gel was quantitated using a Molecular Dynamics PhosphorImager SF (model 455A). Simultaneous measurement of the rpL32 transcripts, which encode the L32 ribosomal protein (24Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), served as an internal control for housekeeping gene levels. Primary dissociated cultures of sympathetic neurons were prepared from the superior cervical ganglia of embryonic day 21 rats as described previously (25Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson E.M., Jr. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar), except that the non-neuronal cells were minimized by incubating the dissociated ganglia for 3 h on plastic culture dishes prior to plating onto laminin-coated 35-mm dishes (∼5,000 cells/dish). Cultures were maintained in culture medium containing 90% minimal essential medium (Invitrogen), 10% fetal calf serum (HyClone, Logan, UT), 2 mm l-glutamine, 20 μm uridine, and 20 μm fluorodeoxyuridine in the presence of 50 ng/ml 2.5 S NGF for either 1 (young) or 4 weeks (old). Poly(A+) RNA was isolated from the cultured neurons and converted to cDNA, and specific cDNAs were amplified by subjecting 2% of the cDNA to 25 PCR cycles, which is well within the linear range of PCR amplification for these specific genes and primer pairs as described previously (25Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson E.M., Jr. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar, 26Furukawa K. Estus S., Fu, W. Mark R.J. Mattson M.P. J. Cell Biol. 1997; 136: 1137-1149Crossref PubMed Scopus (112) Google Scholar, 27Estus S. Tucker H.M. van Rooyen C. Wright S. Brigham E.F. Wogulis M. Rydel R.E. J. Neurosci. 1997; 17: 7736-7745Crossref PubMed Google Scholar). After amplification, cDNAs were separated by polyacrylamide gel electrophoresis on 12% gels, stained with SYBR® Gold (Molecular Probes) and visualized by phosphorimaging technology (Fuji Medical Systems, Stamford, CT). Primer sequences were Rin sense primer, 5′-CTCTTGCTCGAGACTACAAC-3′, and Rin antisense primer, 5′-CCTTCCTGCGTATTTCTCTC-3′ (105-bp product); neurofilament M sense primer, 5′-ACGCTGGACTCGCTGGGCAA-3′, and neurofilament M antisense primer, 5′-GCGAGCGCGCTGCGCTTGTA-3′ (156-bp product). The identity of the amplified cDNAs was confirmed by DNA sequencing. A similar approach was used to examine Rin expression from a series of neuronal cell lines. Poly(A+) RNA was isolated and converted to cDNA, and specific cDNAs were amplified by subjecting 3% of the cDNA to 20–25 PCR cycles in reactions containing 0.5 ml of [α-32P]dCTP. Reaction conditions were well within the linear range of PCR amplification for these gene/primer combinations. Following amplification, PCR products were separated on 12.5% gels and exposed to film for 24 h. Autoradiograms were used to excise the amplified products and the radioactivity in each band determined by scintillation counting. Measurement of the RPS16 transcripts, which encode the ribosomal protein S16, served as an internal control for housekeeping gene levels. A glutathione S-transferase (GST) fusion of the Rin and Ras binding domain (BD) of Raf (residues 1–140) was expressed and purified as described (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar, 21Shao H. Andres D.A. J. Biol. Chem. 2000; 275: 26914-26924Abstract Full Text Full Text PDF PubMed Google Scholar). Rin and Ras activation was assessed essentially as described previously (28Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar) with minor modifications. Rin activation was monitored in cells transiently transfected with the mammalian expression vector pKH3-RinWT alone or in combination with pKH3-Ras(L61), pDCR-Ras(V12), and the indicated pDCR-Ras(V12) effector mutants, pCDNA-Sos1, pCDNA-GRF1, or pEGFP-Ras(N17), and incubated for an additional 36 h to allow maximal gene expression. Cells were then starved in serum-free DMEM for an additional 12 h and, where indicated, stimulated with growth factors for the indicated times. Cell monolayers were washed once in ice-cold PBS and lysed for 1 min on the plate with ice-cold lysis buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 20 mm MgCl2, 10% glycerol, 1% Nonidet P-40, and 1× protease inhibitor mix (Calbiochem)). Lysate was transferred to a 1.5-ml microcentrifuge tube and clarified for 10 min at 14,000 rpm. Protein concentrations were determined by the Bradford assay (Bio-Rad) using bovine serum albumin as standard. GST-RafBD precoupled to glutathione beads (10 μg of protein/30 μl of resin beads) were added to 200 μg of total cell lysate in a final volume of 400 μl and incubated with rotation for 1 h at 4 °C to initiate the pull-down assay. Following three washes in ice-cold lysis buffer, bound proteins were eluted by incubation for 5 min at 100 °C in 20 μl of SDS-PAGE sample buffer. Bound proteins and 20 μg of total cell lysate from each sample were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) using a 12.5% polyacrylamide gel, transferred to nitrocellulose membranes, and subjected to immunoblotting using anti-HA or anti-GFP antibodies. To analyze the role of MEK/ERK signaling in Ras-mediated Rin stimulation, PC6 cells were transfected with pKH3-RinWT, pKH3-Ras(L61), or co-transfected with both expression vectors and incubated for an additional 36 h to allow for maximal gene expression. 24 h after transfection, cells were cultured in serum-free DMEM (± 10 μm PD-98059 as indicated) for 24 h. Fresh serum-free DMEM (± 50 μm PD-98059) was added 2 h prior to the preparation of total cell lysates in ice-cold lysis buffer containing phosphatase inhibitors (50 mm Tris (pH 7.5), 150 mm NaCl, 20 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 20 mm β-glycerol phosphate, 1 mm vanadate, 50 mm KF, and 1× protease inhibitor mix (Calbiochem)). HA-Rin-GTP was pulled down using GST-RafBD and analyzed by immunoblot analysis as described above. In vivo guanine nucleotide binding assays were performed essentially as described (29Quilliam L.A. Rebhun J.F. Zong H. Castro A.F. Methods Enzymol. 2001; 333: 187-202Crossref PubMed Scopus (10) Google Scholar). Briefly, PC6 cells were transiently transfected with pKH3-RinWT or pKH3-Rin(N34) in 60-mm dishes. After 36 h, cells were incubated in serum- and phosphate-free media for 30 min followed by similar medium supplemented with 150 μCi of 32P-labeled orthophosphate for an additional 4 h. Cells were washed once with ice-cold PBS, lysed on the plate for 1 min using ice-cold lysis buffer 2 (50 mm Tris-HCl (pH 7.5), 500 mm NaCl, 6 mm MgCl2, 1 mm EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS, and 1× protease inhibitor mix (Calbiochem)), clarified by microcentrifugation (10 min at 14,000 rpm), and added to a fresh tube containing 30 μl of glutathione beads precoupled with 10 μg of GST-RafBD. This mixture was incubated with rotation at 4 °C for 1 h, after which the beads were collected and washed three times with ice-cold lysis buffer. Bound protein was released by the addition of 20 μl of denaturation buffer (0.2% SDS and 2 mm EDTA), the beads incubated at 68 °C for 15 min, and 1 mm GTP and 1 mm GDP added to serve as nucleotide standards. An aliquot of the sample (10 μl) was spotted to 20-cm poly(ethylene)imine-cellulose TLC plates (EM Separations) for nucleotide separation. The chromatogram was developed using 1 m LiCl2, 1 m formic acid and analyzed as described (22Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (89) Google Scholar). PC6 cells were seeded at 5 × 104 cells on coverslips placed in six-well dishes. Coverslips were precoated with 5 μg/ml laminin (Sigma) and 25 μg/ml poly-l-lysine (Sigma) in PBS for 2 h. PC6 cells were transiently transfected using Effectene (Qiagen) with one of the following plasmids: pEGFP-C1, pEGFP-Rin, pEGFP-Rin(L78), pEGFP-H-Ras(L61), pEGFP-Ras(N17), or pEGFP-Rin(N34), and examined by epifluorescence microscopy. Counting the day of transfection as day 0, cells were either not subject to growth factor stimulation or were treated with NGF (100 ng/ml) on day 2 and then fixed on day 5. Cells were then washed three times in CM-PBS (1.26 mmCaCl2, 0.49 mm MgCl2, 0.91 mm MgSO4) and fixed with 3.7% formaldehyde in CM-PBS for 20 min at room temperature. Cells were permeabilized in 0.1% Triton X-100 in CM-PBS for 5 min, blocked for 30 min with 1% bovine serum albumin in CM-PBS, incubated with Texas Red-X-phalloidin (Molecular Probes) for 20 min, and washed extensively prior to mounting. For studies using HA-tagged H-Ras(L61), slips were fixed and blocked as above, incubated with 2 μg/ml anti-HA antibody for 20 min, washed with CM-PBS, and incubated with Texas Red-labeled anti-mouse (1:1000 dilution, Vector Laboratories). Fixed and stained slips were mounted on glass slides with 12 μl of Vectashield (Vector Laboratories) and examined under the appropriate illumination with a 40× objective lens on an E600 microscope (Nikon). Cells were scored positive for neurite outgrowth if one or more neurites exceeded 1 cell body diameter in length. At least 200 cells were counted per experiment with each experiment performed in triplicate. PC6 cells were transiently transfected as described above with the empty pKH3 vector control, pKH3-Rin(V29), or pKH3-Ras(L61). Transfected cells were incubated for 48 h to allow maximal gene expression and serum-starved overnight in DMEM. Cells were then washed twice with ice-cold PBS and lysed on the plate with phospholysis buffer (20 mm Tris (pH 7.6), 250 mm NaCl, 2.5 mm EDTA, 3 mmEGTA, 20 mm β-glycerol phosphate, 1 mmvanadate, 50 mm KF, and 1× protease inhibitor mixture (Calbiochem)). Lysates were cleared by centrifugation, equal amounts of protein as determined by Bradford assay (Bio-Rad), and separated on 10% SDS-polyacrylamide gels, using a standard SDS-PAGE protocol. After electrophoresis, the gels were transferred to nitrocellulose membranes and probed with either polyclonal anti-ERK (New England Biolabs) or a phosphospecific ERK antibody (Promega) and developed using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (ECL,Amersham Biosciences). ERK activity was also monitored by immunoblot analysis following immunoprecipitation. PC6 cells were transfected as above with an expression vector encoding HA epitope-tagged ERK (the kind gift of Dr. Ginell Post, University of Kentucky, Lexington, KY) in combination with GFP-tagged Rin(L78), GFP-Ras(L61), or control plasmids. Transfected cells were incubated for 48 h to allow maximal gene expression, serum-starved overnight in DMEM, and lysates prepared in phospholysis buffer. HA-ERK was immunoprecipitated from 500 μg of whole cell lysate using anti-HA antibody (5 μg) prebound to a slurry of protein G-Sepharose/protein A-Sepharose (80:20 mix). Bound proteins were eluted by incubation for 5 min at 100 °C in 20 ml of SDS-PAGE sample buffer. Immunoprecipitated protein and 50 μg of total cell lysate were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and subjected to immunoblot analysis using polyclonal anti-ERK (New England Biolabs), phosphospecific mitogen-activated protein kinase/ERK antibody (Promega), or polyclonal anti-GFP antibody (CLONTECH). To address the tissue distribution of Rin, an exhaustive ribonuclease protection assay was performed (Fig. 1 A). In contrast to the ubiquitous expression pattern seen with the closely related Rit GTPase and majority of Ras family proteins (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2061) Google Scholar, 16Lee C.H.J. Della N.G. Chew C.E. Zack D.J. J. Neurosci. 1996; 16: 6784-6794Crossref PubMed Google Scholar, 18Wes P.D., Yu, M. Montell C. EMBO J. 1996; 15: 5839-5848Crossref PubMed Scopus (72) Google Scholar), the mouse Rin gene was expressed exclusively in neuronal tissues. Rin mRNA was most abundant in brain and spinal cord, with detectable levels found in the eye, but not in an extensive series of additional tissues. In the adult mouse brain, Rin was expressed in all of the subregions examined, with highest expression in the mid-hind region of the brain. Ribonuclease protection analysis was also used to examine Rin expression during murine development. As seen in Fig. 1 B, Rin expression was initially detected in total RNA prepared from the heads of 14-day mouse embryos, and expression levels continued to steadily increase within the brain until ∼20–25 days after birth, at which time Rin expression reached a plateau (Fig. 1 B). This high level of expression continued in adult neuronal tissues. Although the restricted and developmentally controlled expression of Rin in neural tissues suggested that Rin was expressed in neurons, it is possible that Rin is expressed in other cell types, such as supporting cells or fibroblasts. To determine whether Rin is expressed in neurons, primary cultures of superior cervical ganglia were prepared from embryonic day 21 rats and incubated in the presence of NGF for 6 days (immature sympathetic neurons) or 30 days (mature neurons) and subjected to RT-PCR analysis. As shown in Fig. 2 A (upper panel), Rin mRNA was not detected in immature neuronal cultures, but in mature superior cervical ganglia robust Rin expression was observed. No significant changes were seen in the mRNA level of the constitutively expressed gene neurofilament M under these same conditions (Fig. 2 A, lower panel). To extend these results, and to identify a cultured cell system in which to examine the biological function of Rin, we examined Rin expression in a series of neuronal cell lines. RT-PCR analysis found Rin to be expressed in all of neuronal cell lines tested (Fig. 2 B); Northern blot analysis was used to confirm Rin expression in PC6 cells (data not shown). Because the biological function of Rin is largely unknown, we wished to investigate the signaling pathways that lead to Rin activation in neuronal cells. To this end we developed a pull-down assay system to detect the levels of GTP-bound Rin in cells. This assay is based on the proven ability of Ras family effector proteins to interact specifically with the activated, GTP-bound form of their target GTPase. This type of assay has been used previously to assay for activation of a variety of Ras GTPases including Ras, Rap, Ral, and Rho family members (21Shao H. Andres D.A. J. Biol. Chem. 2000; 275: 26914-26924Abstract Full Text Full Text PDF PubMed Google Scholar, 28Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar, 30Wolthuis R.M. Zwartkruis F" @default.
• W2000550050 created "2016-06-24" @default.
• W2000550050 creator A5032368967 @default.
• W2000550050 creator A5037908220 @default.
• W2000550050 creator A5042723212 @default.
• W2000550050 creator A5089285767 @default.
• W2000550050 date "2002-05-01" @default.
• W2000550050 modified "2023-10-18" @default.
• W2000550050 title "Nerve Growth Factor-dependent Activation of the Small GTPase Rin" @default.
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