Oncogenic Ki-ras but Not Oncogenic Ha-rasBlocks Integrin β1-Chain Maturation in Colon Epithelial Cells
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.30928
ISSN1083-351X
AutoresZhongfa Yan, Mingxing Chen, Manuel Perucho, Eileen Friedman,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoHuman colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-ras G12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-rasmutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-ras G12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-rastransfectant cells synthesized the mature, fully glycosylated forms of β1 integrin. Instead of the mature integrin β1-chain, a faster migrating β1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered β1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin β1-chain and increased expression of the mature integrin β1-chain. Altered glycosylation generated the new β1 integrin form since integrin core β1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on β1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin β1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin β1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors. Human colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-ras G12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-rasmutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-ras G12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-rastransfectant cells synthesized the mature, fully glycosylated forms of β1 integrin. Instead of the mature integrin β1-chain, a faster migrating β1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered β1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin β1-chain and increased expression of the mature integrin β1-chain. Altered glycosylation generated the new β1 integrin form since integrin core β1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on β1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin β1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin β1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors. The mammalian ras gene family contains three homologous members, Ki-ras, Ha-ras, and N-ras. Each encodes a 21-kDa protein of either 188 or 189 amino acid residues. The first 85 amino acids of each ras isoform are identical, and the next 80 amino acids exhibit an 85% homology. The remaining C-terminal sequence is highly divergent between the isoforms and so is termed the hyper-variable domain. The last four amino acids in the C terminus constitute the CAAX motif, which is required for post-translational modification and subsequent membrane localization after gene-specific lipid modifications (1Lowy D. Willumsen B. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). The C-terminal region can substitute for the entire gene in localizing the downstream effector raf to the plasma membrane (2Leevers S. Paterson H. Marshall C. Nature. 1994; 369: 411-414Crossref PubMed Scopus (886) Google Scholar). Ras proteins switch between an active form that binds GTP and an inactive form that binds GDP (3Boguski M. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). Activated ras in turn activates the serine-threonine kinase raf-1 which then activates MAP 1The abbreviations used are: MAP, mitogen-activated protein; MDCK, Madin-Darby canine kidney cells; PVDF, polyvinylidene difluoride; DME medium, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MBP, myelin basic protein; PIPES, 1,4-piperazinediethanesulfonic acid. kinase kinases (MEKs) which in turn activate the p42/44 MAP kinases (Erks). Ras also activates raf-independent signaling pathways leading to activation of JNK kinases. Although there is evidence linking the raf/MEK/MAP kinase pathway to cellular transformation, ras may mediate some aspects of transformation through raf-independent pathways. Non-raf candidate ras effectors include Rho family proteins, two Ras GTPase-activating proteins (p120 and NF1), guanine nucleotide exchange factors for Ral proteins, and phosphatidylinositol-3-OH kinase (reviewed in Ref. 4Cahill M.A. Janknecht R. Nordheim A. Curr. Biol. 1996; 6: 16-19Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The possible contributions of rasactivation to transformation include altered transcription and translation, alterations in the cytoskeleton, and altered cell surface carbohydrates (5Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 6Dennis J.W. Kosh K. Bryce D.M. Breitman M.L. Oncogene. 1989; 4: 853-860PubMed Google Scholar). Oncogenic mutations detected in Ha-, Ki-, and N-ras genes isolated from different human tumors localize in the N-terminal region controlling GTP binding, suggesting the genes become oncogenic when they remain in the GTP-bound state. Mutations in predominantly one of the three mammalian ras genes have been found associated with specific human cancers. For example, Ha-ras mutations have been reported in 18% of transitional cell carcinomas of the human urinary bladder, whereas very few mutations in Ki-ras were observed (7Burchill S. Neal D. Lunec J. J. Urol. 1994; 73: 516-521Crossref Scopus (52) Google Scholar, 8Sandberg A. Berger C. J. Urol. 1994; 151: 545-560Crossref PubMed Scopus (162) Google Scholar). In contrast, the vast majority, over 90%, of pancreatic adenocarcinomas contain mutations in the Ki-ras gene and not in N-ras or Ha-ras (9Smit V. Boot A. Smits A. Flueren G. Cornelisse C. Bos J. Nucleic Acids Res. 1988; 16: 7773-7782Crossref PubMed Scopus (544) Google Scholar). Specificity of ras mutations is also seen in colon cancers, in which roughly 40–50% of cases exhibit activating mutations in the Ki-ras gene. Only a few percent of cases exhibit N-ras mutations, whereas Ha-rasmutations are very uncommon in colon cancers (10Bos J. Fearon E. Hamilton S. Verlann-de Vries M. van Boom J. van der Eb A. Vogelstein B. Nature. 1987; 327: 293-297Crossref PubMed Scopus (1610) Google Scholar, 11Forrester K. Almoguera C. Han K. Grizzle W.E. Perucho M. Nature. 1987; 327: 298-303Crossref PubMed Scopus (911) Google Scholar). N-rasmutations have been found in approximately 20–25% of cases of acute myeloid leukemia, although mutations in Ki-ras were infrequent (12Bos J. Verlann-de Vries M. van der Eb A.J. Janssen J. Delwel R. Lowenberg B. Colly L. Blood. 1987; 69: 1237-1241Crossref PubMed Google Scholar). The reason for the selectivity for a specific mutatedras isoform in a tumor type is not known but may be due to the importance of that isoform in controlling proliferation within that cell type. Recently it was shown using gene-specific antisense oligonucleotides that Ki-ras, but not Ha-ras, contributes to the proliferation of normal human lung fibroblasts (MCR-5 cells), whereas oncogenic Ha-ras drives proliferation of T24 human bladder carcinoma cells (13Chen G. Oh S. Monia B.P. Stacey D.W. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Both Ha-Ras and Ki-Ras proteins are expressed in colon epithelial cells (14Augenlicht L. Augeron C. Yander G. LaBoisse C. Cancer Res. 1987; 47: 3763-3765PubMed Google Scholar) but may have different functions. We wished to determine whether mutations in the Ki-ras gene which are found in colon carcinoma cells induce another function in addition to cell growth through the MAP kinase cascade. Viral Ki-ras prevents the polarization of MDCK kidney epithelial cells (15Schoenenberger C.-A. Zuk A. Kendall D. Matlin K. J. Cell Biol. 1991; 112: 873-889Crossref PubMed Scopus (87) Google Scholar, 16Schoenenberger C.-A. Zuk A. Zinkl G. Kendall D. Matlin K. J. Cell Sci. 1994; 107: 527-541Crossref PubMed Google Scholar) suggesting the mutated cellular Ki-ras gene may have a similar function in colon epithelial cells. Studies in tissue culture have shown that cellular polarization may require both E-cadherin-mediated cell to cell contact and integrin-mediated cell-substratum interactions (17Eaton S. Simons K. Cell. 1995; 82: 5-8Abstract Full Text PDF PubMed Scopus (166) Google Scholar). E-cadherin is critical to the formation of the basolateral domain, whereas the orientation of the apicobasal axis depends on integrin-mediated associations with the substratum. We now compare the effects of two ras genes, the Ki-ras which is often mutated in colon cancer and the Ha-ras which is rarely, if ever, mutated in colon cancer, on colon epithelial cell polarity, integrin-mediated adhesion, and integrin expression and function. The expression plasmids utilized are a mini-gene construct of the large cellular Ki-ras4B gene (18Kahn S. Yamamoto F. Almoguera C. Winter E. Forrester K. Jordano J. Perucho M. Anticancer Res. 1987; 7: 639-652PubMed Google Scholar) and an oncogenic cellular Ha-ras gene, both mutated at codon 12 from Gly to Val. The colon epithelial cell used is the HD6-4 line, which binds strongly to collagen I through the integrin heterodimer α2β1 (19Hafez M. Hsu S. Yan Z. Winawer S. Friedman E. Cell Growth Differ. 1992; 3: 753-762PubMed Google Scholar). After binding to collagen I, HD6-4 cells then polarize into columnar cells with distinctive basal and apical compartments. These columnar cells differentiate into a specialized epithelial cell type, the colon goblet cell, with a basal nucleus and an apical theca containing mucin granules (20Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar). HD6-4 cells contain inactivating mutations in the tumor suppressor genes p53 and APC but have wild-type ras genes, making them suitable recipients for oncogenic ras genes (21Huang F. Hsu S. Yan Z. Winawer S. Friedman E. Oncogene. 1994; 9: 3701-3706PubMed Google Scholar). We now find that constitutive expression of oncogenic Ki-ras, but not oncogenic Ha-ras, prevents the establishment of columnar cell polarity in HD6 cells by blocking glycosylation and maturation of integrin β1-chain, thus reducing the capacity of this integrin to mediate binding to extracellular matrix components. [α-32P]GTP, 125I, [32P]H3PO4, and [35S]methionine were obtained from NEN Life Science Products, protein A-Sepharose from Pharmacia Biotech Inc., PVDF transfer paper Immobulin-P from Millipore, and PEI-cellulose-F TLC plates from EM Separations. Pan-ras rat monoclonal antisera Y13-259 which reacts with the p21 translational products of the Ha-, Ki- and N-ras human oncogenes; monoclonal pan-ras Val-12 antibody which reacts with only the forms of the ras oncogenes mutated at codon 12 to valine; c-Ki-ras Ab1 (clone 234-4.2) a mouse monoclonal antibody specific for c- and v-Ki-ras p21 and not recognizing c-Ha-ras or c-Ha-ras p21; c-Ha-ras (Ab-1) clone 235–1.7.1 a mouse monoclonal antibody specific for Ha-Ras and not Ki-Ras or N-Ras p21 proteins, and purified p21 recombinant protein Ki-RasVal-12 were purchased from Oncogene Science. Phosphorothioate oligodeoxynucleotides were a gift of Dr. Brett Monia, Isis Pharmaceuticals, Carlsbad, CA. Isis-2503 is a 20-mer targeted to the initiation codon (AUG) of c-Ha-rasmRNA (TCC-GTC-ATC-GCT-CCT-CAG-GG), and Isis-13177 is a 20-mer of random sequence; Isis-6957 is a 20-mer targeted to the 5′-UTR of Ki-ras (CAG-TGC-CTG-CGC-CGC-GCT-CG). This sequence is found within the promoter region of c-Ki-ras4B gene cloned into pMiKVal-12 (see below), about 60 base pairs upstream of the translational start. Anti-integrin β1, a polyclonal mouse IgG-clone 18 raised to a protein fragment corresponding to amino acids 76–256 of human integrin β1-chain, a region of the extracellular domain, was purchased from Transduction Laboratories and was used for immunoblotting. Monoclonal antibody clone P4C10, an IgG1 isotype directed to integrin β1-chain was purchased from Life Technologies, Inc. and was used for immunoprecipitation and cell binding studies. Phospho-specific MAP kinase antibody and p44/42 MAP kinase antibody were rabbit polyclonal IgGs from New England BioLabs, and endoglycosidase F (N-glycosidase F-free), also known as endo-β-N-acetylglucosidase F, was purchased from Boehringer Mannheim. Collagen I was purchased from the Collagen Corp., Palo Alto, CA. Fibronectin and tunicamycin were purchased from Sigma, and laminin was from Life Technologies, Inc. The HD6 human colon carcinoma cell line (20Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar) was subcloned from the HT29 cell line and recloned as the HD6-4 line immediately before transfection. The parental and transfectant lines were maintained in DME medium containing 7% fetal bovine serum or ITS-DME medium, as described (20Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar). Binding to 30 μg/ml collagen I-coated, 10 μg/ml fibronectin-coated, or 40 μg/ml laminin-coated 24-well plates was performed exactly as described (19Hafez M. Hsu S. Yan Z. Winawer S. Friedman E. Cell Growth Differ. 1992; 3: 753-762PubMed Google Scholar). pMiKCys encodes the endogenous cellular Ki-ras promoter, exons 1, 2, 3, and 4B, and 4.2 kilobase pairs of the 3′-untranslated sequence. The mini-gene constructs pMiKGly and pMiKVal-12 plasmids (18Kahn S. Yamamoto F. Almoguera C. Winter E. Forrester K. Jordano J. Perucho M. Anticancer Res. 1987; 7: 639-652PubMed Google Scholar) were made from pMiKCys, which was cut by PstI and self-ligated. Into the StuI-NsiI sites of this plasmid was ligated the first exon of Ki-ras amplified by polymerase chain reaction with primers KIUSX and KIDAX from tumors with determined mutations in codon 12 of Ki-ras. AKpnI fragment carrying a lacI-Z gene was ligated into KpnI-SmaI sites of pBluescriptIKs+ (Stratagene) and designated pML1. AKpnI-BamHI fragment of pML1 andXhoI-BamHI fragment of pMClneopoly(A) (Stratagene) containing the neo gene were ligated intoKpnI-XhoI sites of pBluescriptIKs+ and designated pMLN. pMLN was linearized with BssH2 and then cut bySalI to obtain a fragment carrying the neo gene. This was ligated to BssSalI sites of pMiKVal-12and pMiKGly-12 to give plasmids pMLVal-12 and pMLGly-12. These plasmids were transfected into HD6-4 cells by calcium phosphate-mediated transfection followed by osmotic shock and selected in 600 μg/ml G418 in DME medium. pUC EJ 6.6 encoding the mutated human Ha-ras gene cloned from the human EJ bladder carcinoma and pSV2neo encoding the neomycin resistance gene under control of the SV40 late promoter were co-transfected at a ratio of 9:1 by calcium phosphate-mediated transfection followed by osmotic shock and selected in 600 μg/ml G418 in DME medium. Individual clones of Ha-ras and Ki-ras transfectants were isolated. The method is that essentially used previously (22Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar). Cells were cultured in 100-mm dishes until subconfluent and lysed in 0.5 ml of buffer consisting of 0.5% Nonidet P-40, 50 mm Tris-HCl, pH 7.5, 20 mmMgCl2, 150 mm NaCl, 1 mmNa2P04, pH 7.4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 mm benzamidine, 10 μg/ml soybean trypsin inhibitor, and 1 mm phenylmethylsulfonyl fluoride. 50 μg of monoclonal antibody Y13-259 was added per mg of protein, and incubation was continued for 1 h. p21 ras ·Y13-259 complexes were precipitated with 60 μl of protein A-Sepharose previously coupled with rabbit anti-rat IgG (Cappel) for 1 h and then washed two times with lysis buffer and 3 × with TBST (25 mm Tris, pH 8, 125 mm NaCl, 0.025% Tween 20) before analysis. For some experiments cells were prelabeled overnight with [35S]methionine (625 μCi/ml, 1220 Ci/mmol). For detection of guanine nucleotides bound to ras, cells had been prelabeled with [32P]orthophosphate and assayed as detailed (22Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar). The method was adapted from Buday and Downward (23Buday L. Downward J. Mol. Cell. Biol. 1993; 13: 1903-1910Crossref PubMed Scopus (106) Google Scholar). Cells were cultured 2 days post-plating at 4 × 105/cm2 in 10-cm tissue culture dishes to bring cells into log phase. The medium was then changed to serum-free ITS-DME medium, and cells were cultured overnight. After 1 × wash with PBS, cells were placed in 2.4 ml of permeabilization buffer consisting of 150 mm KCl, 37.5 mm NaCl, 6.25 mm MgCl2, 0.8 mm EGTA, 1 mm CaCl2, 1.25 mm ATP, 12.5 mm PIPES, pH 7.4, and incubated at 37 °C for 10 min. 0.6 ml of 2 international units/ml streptolysin O (Sigma) in permeabilization buffer was then added, and the incubation was continued for 5 min. 15 μCi of [α-32P]GTP (300 Ci/mmol) were added and incubated for 10 min. The buffer was then removed, the cells lysed, and Ras proteins immunoprecipitated as above. Total specific radioactivity associated with immunoprecipitated Ras proteins was detected by a Beckman 2000 counter. Samples were assayed in triplicate. 50 μg of cell lysate (22Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar) proteins were blotted onto PVDF membranes after separation on 8% SDS-PAGE. The blots were blocked in blocking buffer: 25 mm Tris, pH 8, 125 mm NaCl, 0.1% Tween 20, 4% bovine serum albumin for 1 h at room temperature, incubated for 2 h with a 1:2500 dilution of mouse IgG clone 18 antibody to integrin β1-chain, or 5 μg/ml of the Ki-ras-specific, Ha-ras-specific, or pan-ras antibodies and detected by enhanced chemiluminescence. After SDS-PAGE and blocking as above, tyrosine-phosphorylated erk1 and erk2 and total erk1 and erk2 were detected, respectively, by 1 μg/ml phospho-MAP kinase and 1 μg/ml total MAP kinase antibodies and then detected using the Western blotting detection system provided by the manufacturer: a 1-h incubation at room temperature with 1:1000 dilution of alkaline phosphatase-conjugated anti-rabbit secondary antibody and 1:1000 dilution of alkaline phosphatase-conjugated anti-biotin antibody in blocking buffer, followed by a wash, and detection using a 1:500 dilution of CDP-Star for 5 min, followed by autoradiography. Treatment of Ki-ras and Ha-ras transfectants was essentially as described (13Chen G. Oh S. Monia B.P. Stacey D.W. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cells were seeded at 2 × 105 per well in 6-well plates. 48 h later the cells were washed with pre-warmed serum-free ITS-DME medium and then incubated in this medium with a fixed ratio of oligonucleotide to Lipofectin (2.4 μl of Lipofectin per 40 pmol of oligonucleotides) for 4 h. The oligonucleotide-containing medium was then replaced with normal growth medium, and growth was continued for 48 h to allow rasturnover plus reduced ras mRNA levels to result in reduced Ras protein level (13Chen G. Oh S. Monia B.P. Stacey D.W. J. Biol. Chem. 1996; 271: 28259-28265Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cells were cultured on Costar transwells with 2 ml of medium under the layer and 1.5 ml above the layer for 2 weeks post-confluence with 3 times weekly media changes, fixed, and stained for mucin detection by Alcian blue dye with a nuclear fast red counterstain, exactly as described (20Hafez M. Infante D. Winawer S. Friedman E. Yan Z. Hsu S. Winawer S. Friedman E. Cell Growth Differ. 1990; 1: 617-626PubMed Google Scholar). The method is adapted from one used previously (22Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar). Cell lysates in high salt EBC buffer (50 mm Tris-HCl, pH 8.0, 120 mmNaCl, 100 mm NaF, 1% Nonidet P-40, 200 μmsodium orthovanadate, 10 μg/ml each aprotinin and leupeptin, and 1 mm phenylmethylsulfonyl fluoride) were boiled in Laemmli sample buffer for 5 min and then electrophoresed in a 7.5% SDS-PAGE gel (0.5 mm thick and 5 cm long) containing 0.5 mg/ml MBP (Sigma). After fixing the gel with four changes of 20% 2-propanol in 50 mm Tris-HCl buffer, pH 8.0, for 2 h, SDS was removed by washing the gel twice for 2 h each in several gel volumes of 50 mm Tris-HCl, pH 8.0, containing 5 mm2-mercaptoethanol. The MBP kinases were then re-denatured with 6m guanidine HCl for 2 h and then renatured by 10 washes of 20 min each in several gel volumes of 50 mmTris-HCl, pH 8.0, containing 0.04% Tween 40 and 5 mm2-mercaptoethanol. After preincubation for 1 h with 5 ml of 40 mm HEPES, pH 8.0, containing 2 mm2-mercaptoethanol and 10 mm MgCl2, phosphorylation of MBP within the gel was carried out by incubating the gel at room temperature for 1 h in 5 ml of 40 mmHEPES, pH 8.0, containing 25 μCi of [γ-32P]ATP, 40 μm ATP, 0.5 mm EGTA, and 10 mmMgCl2, and then washing the gel in 5% (w/v) trichloroacetic acid containing 1% sodium pyrophosphate several times until the radioactivity reached background levels. Cells were treated with 3 μg/ml tunicamycin for 24 h before lysis. Cell lysates were denatured by boiling in 2% SDS-Laemmli sample loading buffer (24Yan Z. Hsu S. Winawer S. Friedman E. Oncogene. 1992; 7: 801-805PubMed Google Scholar) for 5 min. For digestion with endoglycosidase F, 0.5 units of endoglycosidase F were added after the lysates were made 1% in Nonidet P-40 and 0.1% in SDS and boiled for 5 min. Denaturation by heating at 100 °C in the presence of SDS, but not Nonidet P-40, increases the deglycosylation rate considerably according to the vendor. The lysates were incubated for 36 h before SDS-PAGE and Western blot analysis. After washing with PBS, the cells were swollen for 5 min on ice in hypotonic buffer A (20 mm Tris-HCl, pH 7.5, containing 1 mm NaF, 100 μm sodium orthovanadate, 2 mm EDTA, 1 mm EGTA) containing protease inhibitors exactly as described (25Lee H. Ghose-Dastidar J. Winawer S. Friedman E. J. Biol. Chem. 1993; 268: 5255-5263Abstract Full Text PDF PubMed Google Scholar). After 20–25 strokes with a Dounce homogenizer on ice, sucrose was added to a final concentration of 0.25 m, and the nuclei were pelleted at 1000 × g for 5 min. The post-nuclear supernatant was layered on top of a 15% sucrose cushion in buffer A and centrifuged at 150,000 × g for 1 h. The sedimentable membrane pellet was suspended in 1% Nonidet P-40 in buffer A, and any Nonidet P-40-insoluble material was removed at 10,000 × g for 60 min. Cells were plated in 6-cm dishes so the majority of cells would be doublets 2 days post-plating, after doubling once. IODO-BEADS (Pierce) were washed twice in freshly prepared PBS, pH 6.5, and dried on filter paper just before using. Cells were rinsed with PBS, pH 6.5, twice, and then 1 ml of PBS, pH 6.5, two washed IODO-BEADS, and 10 μl (1 mCi) of 125I were added per dish. The cells were incubated for 20 min with rotation on an orbital shaker at room temperature. Cells were then washed 3 times with cold PBS and lysates prepared as above. Immunoprecipitations were performed using 5 μg of monoclonal antibody clone P4C10 to 400 μg of cell lysate exactly as described (22Yan Z. Winawer S. Friedman E. J. Biol. Chem. 1994; 269: 13231-13237Abstract Full Text PDF PubMed Google Scholar). Cells were collected by trypsinization and adjusted to 106/ml, pelleted, resuspended in PBS at this volume, and 0.1 ml (105 cells) was injected in each of 5 male BALB/c nu/nu athymic mice between the shoulder blades. Cells were >99% viable by trypan blue exclusion. Tumor size was measured in two dimensions every 2–3 days using calipers, and volume was calculated as (width)2 × length/2. Sections were fixed in formalin, then processed for routine histology, and stained with either hematoxylin and eosin or Alcian blue with a nuclear fast red counterstain. Transfections of HD6-4 cells were performed with a mini-gene construct of the cellular Ki-ras4B gene mutated at codon 12 to valine (Ki-ras G12V), an expression plasmid encoding a mini-gene construct of the wild-type cellular Ki-ras4B gene, and a cellular Ha-ras G12V expression plasmid. We isolated three independent transfectant clones expressing the Ki-ras2G12V oncogene: 4V, 4V1 and 4V2; three independent transfectant clones expressing the Ha-ras G12V oncogene: H15, H18, and H25; and several independent clones expressing the transfected wild-type Ki-ras4B gene. One of the latter, 4G1, was arbitrarily selected for a control for expression of excess copies of Ki-Ras protein. The transfected oncogenes were expressed as active proteins. Proteins in lysates from the transfected lines were size-fractionated by SDS-PAGE and then transferred to PVDF membrane and analyzed for the presence of Ki-Ras proteins by immunoblotting with a Ki-ras-specific antibody (Fig.1 A, top panel), analyzed for the presence of Ha-Ras proteins by immunoblotting with an Ha-ras specific antibody (Fig. 1 A, second panel), analyzed for the presence of total Ras proteins by immunoblotting with a pan-ras antibody (Fig. 1 A, third panel), and analyzed for Ras proteins mutated at Val-12 by immunoblotting with a pan-ras Val-12 antibody (Fig. 1 A, bottom panel). Cells transfected with mutant Ki-ras (4V1, 4V2, and 4V) expressed elevated levels of Ki-Ras protein, presumably mutant, compared with the other transfectant and parental lines (Fig. 1 A, top panel). Cells transfected with mutant Ha-ras (H15, H18, and H25) expressed elevated levels of Ha-Ras protein, presumably mutant, whereas other transfectant lines and parental cells displayed lower levels of Ha-Ras protein (Fig. 1 A, second panel). All of the transfectants displayed similar levels of total Ras proteins (Fig. 1 A, third panel) suggesting endogenous Ras proteins were down-regulated in the transfectant cells (Fig. 1 A, third panel). Similar levels of total Ras proteins were also observed by immunoprecipitation experiments (data not shown). Each of the three lines transfected with the Ki-ras4BG12V oncogene and each of the three lines transfected with the Ha-ras G12V oncogene expressed mutant Ras proteins, which migrated at the expected mobility for proteins of 21 kDa and at the same position as recombinant mutant Ki-ras Val-12 (Fig. 1 A). No Ras proteins mutated at valine 12 were detected in the parental line or in the Ki-ras4B wild-type control transfectant (Fig. 1 A, bottom panel). To confirm that the 21-kDa protein bearing the Val-12 mutation detected by immunoblotting was indeed ras, Ras proteins were immunoprecipitated from 35S-prelabeled HD6-4 parental cells, wild-type c-Ki-ras 4G1 transfectant cells, and 4V transfectant cells expressing the mutated c-Ki-ras gene (Fig. 1 B). Similar amounts of total Ras proteins were immunoprecipitated from each cell line (data not shown). Immunoprecipitated wild-type and mutant Ki-Ras proteins were transferred to PVDF membrane and analyzed for the presence of mutated Ras proteins by immunoblotting with the pan-ras Val-12 antibody. Only the 4V line expressed Ras proteins mutated to valine at codon 12 (Fig.1 B), confirming the results of the Western blot analysis (Fig. 1 A). The mutant Ras proteins bound elevated levels of GTP showing they were functional. The ratio of GTP/GDP bound to immunoprecipitated Ras proteins was determined by thin layer chromatography
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