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• W2131482440 abstract "In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions. In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions. phosphatidylinositol 3-kinase hemagglutinin inositol 4-phosphatase detergent-resistant membrane polyacrylamide gel electrophoresis mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase p21-activated kinase phosphatidylinositol Ras proteins are monomeric GTP-binding proteins that operate as inducers of signal transduction cascades regulating cell growth and development (1Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1122) Google Scholar). They cycle between the GDP-bound inactive and the GTP-bound active form. In their active form, Ras proteins interact with and modulate the activity of effector proteins, including Raf kinases, phosphoinositide 3-kinase (PI3-kinase),1 Ral guanine nucleotide dissociation stimulator, and AF6 (2Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1721) Google Scholar, 3Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (497) Google Scholar, 4Klippel A. Escobedo J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Crossref PubMed Scopus (127) Google Scholar, 5Kikuchi A. Williams L.T. J. Biol. Chem. 1996; 271: 588-594Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (582) Google Scholar, 7Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1658) Google Scholar). These proteins initiate multiple signal transduction cascades that must occur cooperatively to produce a full biological response (8Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar). Effector proteins bind to a small region of Ras termed the effector domain (9Katz M.E. McCormick F. Curr. Opin. Gen. Dev. 1997; 7: 75-79Crossref PubMed Scopus (274) Google Scholar), whose core is comprised of residues 32–40. This region is also known as Switch I, as its conformation, along with that of an additional Switch II region (residues 61–77), changes substantially when Ras proteins bind GTP (10Wittinghofer A. Nassar N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar). In addition to GDP/GTP cycling, another requirement for Ras activity involves the correct localization of Ras proteins to the inner surface of the plasma membrane. Plasma membrane binding of Ras is critical for its function because, at least in part, this allows Ras to target its effector proteins to the location where they encounter their substrates or can be activated (11Cox A.D. Der C.J. Biochim. Biophys. Acta Rev. Cancer. 1997; 1333: F51-F71Crossref PubMed Scopus (348) Google Scholar). Newly synthesized Ras proteins are partitioned to the cytoplasmic face of the plasma membrane by a series of post-translational lipid modifications of the C terminus of the protein (12Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1727) Google Scholar). The first lipid to be attached, a farnesyl group, appears to initiate membrane binding. Recent studies indicate that the endoplasmic reticulum is likely to be the site of first contact, followed by further trafficking to the plasma membrane that occurs through as yet unstudied pathways (13Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). For Ha-Ras, farnesylation of Cys186 is followed by the addition of palmitates to two adjacent cysteine residues (Cys181 and Cys184). Native Ha-Ras proteins with all C-terminal cysteines and lipids present are >95% membrane-bound at steady state, whereas Ha-Ras proteins that lack cysteines at 181 and 184 and thus have a farnesyl group as the only lipid are >90% cytosolic (14Cadwallader K.A. Paterson H. MacDonald S.G. Hancock J.F. Mol. Cell. Biol. 1994; 14: 4722-4730Crossref PubMed Scopus (152) Google Scholar, 15Dudler T. Gelb M.H. J. Biol. Chem. 1996; 271: 11541-11547Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 16Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (841) Google Scholar, 17Willumsen B.M. Cox A.D. Solski P.A. Der C.J. Buss J.E. Oncogene. 1996; 13: 1901-1909PubMed Google Scholar). In addition to the lipids' roles in Ha-Ras targeting and stable association with the plasma membrane, the amino acids of the C terminus also appear to be involved in trafficking of Ha-Ras from the endoplasmic reticulum to the cell surface (17Willumsen B.M. Cox A.D. Solski P.A. Der C.J. Buss J.E. Oncogene. 1996; 13: 1901-1909PubMed Google Scholar). An important yet unresolved issue is whether the lipids or residues of the C-terminal region make a further contribution to Ha-Ras function by directly supporting interactions with specific effectors. Recent studies show that farnesyl modification of Ha-Ras is important for high affinity interaction with (18Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 19Luo Z. Diaz B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; 17: 46-53Crossref PubMed Scopus (106) Google Scholar) and full kinase activity of Raf-1 (20Brtva T.R. Drugan J.K. Ghosh S. Terrell R.S. Campbell-Burk S. Bell R.M. Der C.J. J. Biol. Chem. 1995; 270: 9809-9812Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 21Drugan J.K. Khosravi-Far R. White M.A. Der C.J. Sung Y.J. Hwang Y.W. Campbell S.L. J. Biol. Chem. 1996; 271: 233-237Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 22Roy S. Lane A. Yan J. McPherson R. Hancock J.F. J. Biol. Chem. 1997; 272: 20139-20145Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Farnesylation is also reported to increase in vitro binding of Ha-Ras and KRas4B to p110γ, a PI3-kinase catalytic subunit family member (23Rubio I. Wittig U. Meyer C. Heinze R. Kadereit D. Waldmann H. Downward J. Wetzker R. Eur. J. Biochem. 1999; 266: 70-82Crossref PubMed Scopus (50) Google Scholar). Earlier studies employing a constitutively activated yeast Ras2 protein indicated that interaction between Ras2 and adenylyl cyclase is decreased in the absence of the farnesyl lipid modification (24Bhattacharya S. Chen L. Broach J.R. Powers S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2984-2988Crossref PubMed Scopus (71) Google Scholar, 25Kuroda Y. Noburu S. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar). A possible role in effector interaction for the lipids and C-terminal amino acids of Ras proteins is also implied by recent work reporting that Ha- and K-Ras proteins differ in their ability to activate Raf-1 and PI3-kinase (26Voice J. Klemke R. Le A. Jackson J. J. Biol. Chem. 1999; 274: 17164-17170Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 27Yan J. Roy S. Apolloni A. Lane A. Hancock J.F. J. Biol. Chem. 1998; 273: 24052-24056Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The major differences between the Ha-Ras and KRas4B proteins are confined to their post-translational lipid modifications and their last twenty-five amino acids. Although both proteins are farnesylated, Ha-Ras is modified by palmitates that are attached to cysteines 181 and 184, whereas KRas4B contains no palmitates and has instead a polybasic domain (lysine residues 175–180). Taken together, these data suggest that the C terminus of Ras proteins may provide a mechanism (in addition to that of the internal classical effector domain) for influencing effector interactions. In previous work we had characterized Ext61L, a constitutively activated Ha-Ras protein in which the C-terminal residue of the CAAX motif for farnesylation was replaced with six lysines (28Booden M.A. Baker T.L. Solski P.A. Der C.J. Punke S.G. Buss J.E. J. Biol. Chem. 1999; 274: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This design prevented attachment of the farnesyl group but retained the natural sites for palmitoylation at the C terminus. The novel lipidation state of Ext61L presented a new way to determine if the absence of a farnesyl group impaired any signaling pathways, in a protein that was acylated and maintained an interaction with membranes through its C terminus. A shift in Ha-Ras function had already been noted with Ext61L. Expression of the protein in PC12 cells caused an unusual morphological differentiation distinct from that induced by Ha-Ras61L with the native C terminus. Importantly, no changes had been made in the effector binding domain of Ext61L, and its GTP binding properties were preserved (28Booden M.A. Baker T.L. Solski P.A. Der C.J. Punke S.G. Buss J.E. J. Biol. Chem. 1999; 274: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), indicating that these primary requirements for effector interaction were unaltered. Here we describe results that suggest that the accentuated differentiation produced by Ext61L results from heightened activation of one Ras effector (PI-3 kinase) and suboptimal activation of another (B-Raf). These observations lend support to the idea that Ras-effector interactions may be influenced, in addition to the GTP-sensitive switch regions of the protein, by the C-terminal domain. Construction of Ha-Ras61L and Ext61L in the pcDNA3 vector has been described previously (28Booden M.A. Baker T.L. Solski P.A. Der C.J. Punke S.G. Buss J.E. J. Biol. Chem. 1999; 274: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Amounts of plasmid DNA (in brackets) were titrated so that equivalent amounts of Ha-Ras61L (1 μg of DNA), Ext61L (50 ng of DNA), Ha-Ras (1 μg), or ExtWT (1 μg) proteins were produced in the P100/membrane fraction of transfected cells. Plasmids driving expression of Myc- or hemagglutinin (HA)-epitope-tagged versions of the truncated protein Myc-PAK1-(165–205) (3 μg of DNA) (29Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar) or full-length Myc-Cdc42(17N) (1 μg of DNA), Myc-Rac1(17N) (1 μg or 3 μg of DNA), or HA-Rac1 (7 μg of DNA) were kindly provided by Gary Bokoch (La Jolla, CA). Δp85 (1 μg of DNA) and Myc-RhoA(14V) (1 μg of DNA) in the pEXV3 vector have been described elsewhere (30Miller J. Germain R.N. J. Exp. Med. 1986; 164: 1478-1489Crossref PubMed Scopus (191) Google Scholar) and were gifts from Gideon Bollag (Richmond, CA) and Lawrence Quilliam (Indianapolis, IN). Wild type B-Raf and inositol 4-phosphatase (4-PTase) in the pcDNA3 vector were gifts from Geoff Clark (Bethesda, MD) and F. Anderson Norris (Ames, IA), respectively. The MEK inhibitor PD98059 and the PI3-kinase inhibitor LY294002 were purchased from Calbiochem and used at a concentration of 50 μm. Twenty-four hours before transfection, 1 × 105 PC12 cells were plated onto 60-mm tissue culture dishes coated with laminin (10 μg/ml; Life Technologies, Inc.) and grown overnight. These PC12 cells are derived from an early clone of the original isolate (generously provided by J. H. Pate Skene, Duke University), and if continuously subcultured before reaching high cell density, show high transfection efficiency and very low (<1%) spontaneous neurite extension. Transfections were performed using the LipofectAMINE reagent (Life Technologies, Inc.) as described by the manufacturer. For most experiments the total amount of DNA added to each plate was adjusted to 5 μg using empty pcDNA3 vector DNA. For Rac-GTP binding assays a total of 8 μg of DNA (7 μg of Rac1 + 1 μg of a combination of empty vector and Ha-Ras DNA) was added per dish. Quantitation of the percentage of cells bearing neurites was performed on day 2 when neurites could still be accurately assigned to a particular cell body. Pictures of differentiated cells were taken, and biochemical assays were performed on day 4 when the morphological differences between cells expressing Ha-Ras61L and Ext61L were most distinct and when neurites in both types of Ras-transfected cells were well established. The efficiency of transfection was measured by co-transfection of cells with a β-galactosidase expression plasmid (26Voice J. Klemke R. Le A. Jackson J. J. Biol. Chem. 1999; 274: 17164-17170Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and by counting the percent of cells producing an immunofluorescence signal for Ha-Ras (below). Both methods showed that in cultures that were subjected to transfection, 60–70% of the cells expressed the transfected Ha-Ras protein and that 92–95% of these transfected cells expressed neurites. Expression of either cellular (wild type) forms or the activated Myc-RhoA(19N), Myc-Rac1(12V), or Myc-Cdc42(12V) proteins failed to promote PC12 neurite formation (data not shown) showing, as had been reported previously, that expression of these proteins individually is not sufficient to produce the complex response of neural differentiation (31Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar, 32Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar). Immunoblots using monoclonal antibodies to the HA epitope (Babco, MMS-101R) or to the Myc epitope tag (Santa Cruz Biotechnology, 9E10) confirmed that the dominant negative, cellular, and constitutively activated proteins were expressed (see Figs. 2 B and 3; other data not shown).Figure 3Identification of nucleotide bound to HA-Rac1 proteins. PC12 cells were transfected with HA-Rac1 and either Ha-Ras61L or Ext61L, and 4 days later they were incubated with32Pi for 4 h. The HA-Rac1 protein was immunoprecipitated, and bound nucleotides were eluted, separated by thin-layer chromatography, and detected by autoradiography. The amounts of GTP were quantified by phosphoimager analysis. The averages and S.D., from four independent experiments, of the portion of HA-Rac1 that bound GTP were 8 ± 2% in cells without Ras (lane 1), 10 ± 0.7% in cells co-expressing Ha-Ras61L (lane 2), and 17 ± 0.8% in cells expressing Ext61L (lane 3). An immunoblot (lower panel) of replicate plates verified that similar amounts of the HA-Rac1 were present in all immunoprecipitates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PC12 cells were plated on laminin-coated 18-mm coverslips in serum-containing medium. Within 24 h of plating, the cells were transfected as described above and cultured for 4 days. The cells were then washed in phosphate-buffered saline and fixed in fresh 4% paraformaldehyde in 0.1 m phosphate buffer for at least 30 min at room temperature. Cell membranes were permeabilized by incubation of cells in 0.2% Triton X-100, 5% goat serum, and 0.4% bovine serum albumin in phosphate-buffered saline for 20 min at room temperature. To visualize the F-actin cytoskeleton, rhodamine isothiocyanate-phalloidin (Molecular Probes) in phosphate-buffered saline (1 ml of a 1/400 dilution) was added to each coverslip. After a 20-min incubation, coverslips were washed five times in phosphate-buffered saline, and the coverslips were mounted on a glass slide with Vectashield (Vector Laboratories). Detection of cells expressing Ha-Ras was performed by immunofluorescence using monoclonal antibody Y13–238 (Santa Cruz Biotechnology) at a 1/1000 dilution. Fluorescence was detected with a Nikon FXA microscope equipped with a 60× oil objective (1.0 numerical aperture), and images were captured with a Kodak Megaplus 1.4 CCD camera (Kodak Corp.) connected to a Perceptics MegaGrabber framegrabber (Perceptics Corp.) in a Macintosh 8100/80AV computer using NIH Image. Figures were prepared using Adobe Photoshop 4.0 and Macromedia Freehand Version 8.0 for the Macintosh. Cytosol and crude membrane fractions were separated by hypotonic lysis and high speed centrifugation as described (33Buss J.E. Solski P.A. Schaeffer J.P. MacDonald M.A. Der C.J. Science. 1989; 243: 1600-1603Crossref PubMed Scopus (129) Google Scholar). For detection of interaction between Ha-Ras and PI3-kinase, the P100 membrane-containing pellets were dissolved in buffer A (20 mm Tris (pH 8.0), 100 mm NaCl, 1 mmEDTA, 0.3 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride, 1% Triton X-100, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The S100 cytoplasmic fractions were adjusted to the composition of buffer A and Ha-Ras or PI3-kinase immunoprecipitates formed as described (34Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar) with 150 μg of total protein from each fraction, as determined using the DC Protein Assay kit (Bio-Rad). To learn if detergent-resistant membranes (DRMs) were preserved in the chilled 1% Triton X-100 buffer used for immunoprecipitation, any domains were solubilized, and proteins were released by warming the dissolved lysates for 2 min at 37°, followed by normal formation and washing of the immunoprecipitates (35Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). In addition, the protein concentration in these samples was 150 or 300 μg/ml, far less than the 5 mg/ml needed to retain DRM integrity. To detect interaction between Ha-Ras and B-Raf, P100 membrane pellets were dissolved in buffer B (70 mm β-glycerophosphate (pH 7.2), 100 μm Na3VO4, 2 mmMgCl2, 1 mm EGTA, 0.5% Triton X-100, 1 mm dithiothreitol, 5 μg/ml leupeptin, and 20 μg/ml aprotinin). The S100 fractions were adjusted to the composition of buffer B (36Vaillancourt R.R. Gardner A.M. Johnson G.L. Mol. Cell. Biol. 1994; 14: 6522-6530Crossref PubMed Scopus (148) Google Scholar), and 150 μg of total protein from each fraction were used to form either Ha-Ras or B-Raf immunoprecipitates. Ha-Ras, B-Raf, or PI3-kinase proteins were immunoprecipitated using anti-Ha-Ras rat monoclonal antibody (3E4-146, Quality Biotech), anti-p85α rabbit polyclonal serum (06–195, UBI), or anti-B-Raf rabbit polyclonal serum (C19, Santa Cruz Biotechnology). Immunoprecipitates were captured on protein G-agarose beads (Life Technologies, Inc.), washed three times in their respective lysis buffer, and analyzed for co-immunoprecipitating proteins by immunoblotting with a p85α-specific mouse monoclonal antibody (UBI), Ha-Ras mouse monoclonal (3E4–146, Quality Biotech), or B-Raf goat polyclonal antibody (SC166, Santa Cruz Biotechnology). Peroxidase-labeled secondary antibodies (anti-mouse or goat, Pierce) were used with development by ECL (Pierce) using the manufacturer's protocol. PI3-kinase lipid kinase activity was measured using the in vitro assay described by Ref. 34Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar. Anti-p85 immune complexes were prepared from 300 μg of whole cell lysates or from 150 μg (each) of S100 and P100 fractions and incubated with [γ-32P]ATP (10 μCi/reaction, ICN) and phosphatidylinositol (10 μg/reaction, Sigma) for 10 min. The phospholipids were extracted in CHCl3:CH3OH (1:1) and separated by thin layer chromatography on potassium oxalate-coated silica plates (Analtech) developed in propanol:2 m acetic acid (65:35). Radioactive32P-phosphatidylinositol-3 phosphate was detected by autoradiography, and the film images were scanned and quantified using the program ImageQuant (Molecular Dynamics). The radioactive phosphatidylinositol-3 phosphate product was identified on the basis of co-migration with an unlabeled phosphatidylinositol 4-phosphate standard visualized by iodine staining. Duplicate p85 immunoprecipitates were resolved by SDS-PAGE, and p85 was detected by immunoblotting and quantified by scanning to determine the amounts of p85 captured in the immunoprecipitates. The activity of the endogenous B-Raf kinase was measured with a coupledin vitro kinase assay (37Li X. Lee J.W. Graves L.M. Earp H.S. EMBO J. 1998; 17: 2574-2583Crossref PubMed Scopus (144) Google Scholar). B-Raf immune complexes from whole cell lysates (300 μg) were collected on protein A-conjugated agarose beads and incubated with nonradioactive ATP and purified recombinant MEK protein (100 μg/ml; provided by Dr. Lee Graves, Chapel Hill, NC). After 10 min, recombinant ERK2 (250 μg/ml; provided by Dr. Lee Graves) was added, and after another 10 min, 250 μg/ml myelin basic protein (Fisher) and [γ-32P]ATP (5 μCi/assay) were added. The reactions were finally terminated 10 min later by the addition of 100 mm EDTA; reaction products were spotted onto P-81 phosphocellulose paper (Whatman) and washed in 10% phosphoric acid, and incorporation of 32P into the precipitated myelin basic protein was quantitated by scintillation counting. At 4 days post-transfection, PC12 cells co-expressing HA-tagged Rac1 (7 μg DNA) and either Ha-Ras61L or Ext61L were incubated overnight in medium containing 1% dialyzed calf serum. Cells were then radiolabeled with 0.5–1 mCi/ml 32P inorganic phosphate (NEN Life Science Products) for 4 h in phosphate-free medium containing 1% dialyzed calf serum. Cells were lysed; samples were precleared of Ha-Ras by immunoprecipitation with antibody 3E4–146, and then the HA-Rac1 proteins were isolated by immunoprecipitation, as described above, using an anti-HA antibody (Babco, MMS-101R). GTP and GDP were separated by thin layer chromatography (28Booden M.A. Baker T.L. Solski P.A. Der C.J. Punke S.G. Buss J.E. J. Biol. Chem. 1999; 274: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) with the following important changes. Cells were lysed in a buffer containing 50 mm Tris-HCl, pH 7.4, 250 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20 μg/ml aprotinin, 1 mm EGTA, 1 mmNa3VO4. For more quantitative elution of Rac-bound nucleotides it was necessary to heat the samples to 70 °C for 10 min in 20 μl of buffer containing 20 mm Tris-HCl (pH 7.4), 2 mm EDTA, 2% (w/v) SDS, 2 mm GDP, and 2 mm GTP. PC12 cells respond to the expression of activated Ras proteins by the cessation of growth and the extension of neurites (38Greene L.A. Tischler A.S. Adv. Cell. Neurobiol. 1982; 3: 373-414Crossref Google Scholar, 39Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (568) Google Scholar, 40Guerrero I. Wong H. Pellicer R. Burstein D.E. J. Cell. Physiol. 1986; 129: 71-76Crossref PubMed Scopus (108) Google Scholar). This differentiation process is characterized by rearrangements of the actin cytoskeleton at the plasma membrane, leading to the formation of small lamellipodia, growth cones, and the subsequent extension of axon-like processes (41Gallo G. Letourneau P.C. Curr. Biol. 1998; 8: 80-82Abstract Full Text Full Text PDF PubMed Google Scholar). Previously, the Ext61L protein had been shown to cause morphologic changes in PC12 cells that were easily distinguishable from those caused by Ha-Ras61L, including an increased number and accelerated rate of outgrowth of neuron- like structures and large lamellipodia with ruffles (28Booden M.A. Baker T.L. Solski P.A. Der C.J. Punke S.G. Buss J.E. J. Biol. Chem. 1999; 274: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This exaggerated response suggested that the Ext61L protein produced differentiation signals that were either exceptionally strong or that utilized different signaling pathways than Ha-Ras with a native C terminus and lipid modifications. To examine if the dramatic external morphological changes caused by Ext61L were accompanied by exaggerated changes in the actin filaments that are normally rearranged during neurite outgrowth, the distribution of filamentous actin was examined by immunofluorescence using rhodamine-conjugated phalloidin. Control cells treated with nerve growth factor formed actin-rich neurites after 4 days, with growth cones visible at the ends of the extending neurites (Fig.1 , NGF panel). Cell bodies displayed little flattening and low levels of filamentous actin. Similar changes in the actin cytoskeleton accompanied differentiation triggered by expressing Ha-Ras61L (Fig. 1 , 61L panel). Expression of Ext61L led to more extensive changes in actin structures. Neurites, often longer than 100 μm, developed within 24 h of transfection and at 4 days post-transfection cells exhibited marked somal flattening and very long, thin, actin-rich neurites displaying extensive branching (Fig. 1, Ext61L 50 μm panel). At higher magnification, cortical actin and actin-containing microspikes could be seen in the large membrane ruffles, with short perpendicular filament bundles leading to the edge of the cells and truncated and poorly organized actin filaments throughout the cell interior (Fig. 1 , Ext61L 25 μm panel). Thus the morphological changes caused by Ext61L involved actin cytoskeletal changes. More importantly, the exaggerated features of the changes caused by Ext61L indicated that Ext61L, although identical to Ha-Ras in its switch I (designated effector domain) and switch II regions, affected the actin cytoskeleton in a distinct way. Many aspects of actin cytoskeleton regulation, in both fibroblasts and PC12 cells, are controlled by the Rho family of GTPases, which include several Rho, Rac, and Cdc42 proteins (42Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar, 43Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (384) Google Scholar, 44Qiu R. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar). Each of these proteins has also been reported to function downstream of Ha-Ras and to contribute to the effects of Ha-Ras on the actin cytoskeleton (45Joneson T. White M.A. Wigler M.H. Bar-Sagi D. Science. 1996; 271: 810-812Crossref PubMed Scopus (356) Google Scholar, 46Qiu R. Chen J. McCormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (486) Google Scholar, 47Khosravi-Far R. Solski P.A. Clark G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (639) Google Scholar). The Rac proteins in particular appear to be involved in remodeling of the actin cytoskeleton during formation of ruffles or lamellipodia. The large flattened and ruffled cell bodies of the cells expressing Ext61L suggested that a pathway involving Rac proteins might be strongly" @default.
• W2131482440 created "2016-06-24" @default.
• W2131482440 creator A5017933625 @default.
• W2131482440 creator A5049406843 @default.
• W2131482440 creator A5003558649 @default.
• W2131482440 date "2000-08-01" @default.
• W2131482440 modified "2023-09-29" @default.
• W2131482440 title "Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization" @default.
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