Oncogenic Ki-ras but Not Oncogenic Ha-rasBlocks Integrin β1-Chain Maturation in Colon Epithelial Cells

Abstract

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. 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. PubMed Scopus Google Scholar). The of to transformation include altered and in the and altered cell surface Biol. 1993; PubMed Google Google Scholar). mutations detected in and genes human tumors in the region GTP the genes oncogenic in the in of the three mammalian ras genes with human Ha-ras mutations in of cell of the human mutations in Ki-ras were S. D. 1994; Scopus Google A. C. 1994; PubMed Scopus Google Scholar). the of contain mutations in the Ki-ras gene and not in or Ha-ras A. A. C. PubMed Scopus Google Scholar). of ras mutations is also in colon in which of exhibit mutations in the Ki-ras Only a of exhibit mutations, are in colon S. M. A. B. Nature. PubMed Scopus Google C. M. Nature. PubMed Scopus Google Scholar). in of of mutations in Ki-ras were M. R. B. PubMed Google Scholar). The for the selectivity for a isoform in a tumor is not but may due to the of that isoform in that cell was using gene-specific antisense oligonucleotides that Ki-ras, but not Ha-ras, to the of human oncogenic Ha-ras of human cells S. Biol. PubMed Scopus Google Scholar). and Ki-Ras proteins are in colon epithelial cells C. C. Google but may to mutations in the Ki-ras gene which are in colon cells in to cell through the MAP kinase Ki-ras the of kidney epithelial cells A. D. Biol. PubMed Scopus Google A. D. 1994; PubMed Google the mutated cellular Ki-ras gene may a in colon epithelial in that cellular may both cell to cell and S. PubMed Scopus Google Scholar). is to the of the the of the apicobasal on with the the effects of two ras the Ki-ras which is mutated in colon and the Ha-ras which is rarely, if ever, mutated in colon on colon epithelial cell and integrin expression and The expression are a mini-gene of the cellular Ki-ras4B gene S. F. C. M. Google and an oncogenic cellular Ha-ras gene, both mutated at to The colon epithelial cell used is the HD6-4 which binds to collagen I through the integrin M. S. S. Google Scholar). After to collagen HD6-4 cells then polarize into columnar cells with basal and apical columnar cells into a epithelial cell the colon goblet with a basal and an apical M. D. S. S. S. Google Scholar). HD6-4 cells contain mutations in the tumor genes and but wild-type ras for oncogenic ras genes F. S. S. 1994; Google Scholar). that expression of oncogenic Ki-ras, but not oncogenic Ha-ras, the of columnar cell polarity in cells by blocking glycosylation and maturation of integrin β1-chain, the of this integrin to mediate to extracellular and were protein and which with the of the Ki- and human pan-ras Val-12 which with the forms of the ras mutated at to a for and and not or a for and not Ki-Ras or proteins, and protein were were a of is a to the of and is a of is a to the of Ki-ras sequence is the region of gene into of the a to a protein to amino acids of human integrin β1-chain, a region of the extracellular was and was used for an to integrin β1-chain was and was used for and cell MAP kinase and MAP kinase were and endoglycosidase F also as was I was the and tunicamycin were and was The human colon cell line M. D. S. S. S. Google was the cell line and as the HD6-4 line The and transfectant were in DME or medium, as M. D. S. S. S. Google Scholar). to collagen or was as M. S. S. Google Scholar). encodes the cellular Ki-ras and and of the The mini-gene constructs and S. F. C. M. Google were made which was by and the of this was the first of Ki-ras by with and tumors with mutations in of a gene was into of and of of the gene were of and was with and then to a the was to of to and were into HD6-4 cells by by and in in DME the mutated human Ha-ras gene the human and the gene control of the were at a of by by and in in DME of Ha-ras and Ki-ras transfectants were The is that used S. Biol. 1994; PubMed Google Scholar). were in and in of of and of was of and was for ras were with of protein with for and then two with and with some cells were with of guanine to cells with and as S. Biol. 1994; PubMed Google Scholar). The was and Biol. 1993; PubMed Scopus Google Scholar). were at in to cells into The was then to medium, and cells were After with PBS, cells were in of of PIPES, and at for of in was then and the was for of were and for The was then the cells and Ras proteins as with Ras proteins was detected by a were in of cell S. Biol. 1994; PubMed Google proteins were after on The were in blocking for at for with a of to integrin β1-chain, or of the or pan-ras and detected by After and blocking as and and and were by kinase and MAP kinase and then detected using the Western blotting by the a at with of and of in blocking by a and using a of for by of Ki-ras and Ha-ras transfectants was as S. Biol. PubMed Scopus Google Scholar). were at in the cells were with and then in this with a of to of of for The was then with medium, and was for to reduced ras to in reduced Ras protein S. Biol. PubMed Scopus Google Scholar). were on with of the and the for with and for by with a as M. D. S. S. S. Google Scholar). The is used S. Biol. 1994; PubMed Google Scholar). in each and and were in for and then in a gel and After the gel with four of in for was by the gel for each in gel of The kinases were then with for and then by of each in gel of and After for with of and of the gel was by the gel at for in of of and and then the gel in acid the were with tunicamycin for were by in S. S. Google for with endoglycosidase of endoglycosidase F were after the were made in and in and for by at in the of but not the to the The were for and Western After with PBS, the cells were for on in as H. S. Biol. 1993; PubMed Google Scholar). After with a on was to a of and the were at for The was on of a in and at for The membrane was in in and was at for were in so the of cells after were in PBS, and on were with PBS, and then of PBS, two and of were The cells were for with on an at were then with and as were using of to of cell as S. Biol. 1994; PubMed Google Scholar). were by and to in at this and was in each of between the were by was in two using and was as were in then for and with either and or with a of HD6-4 cells were with a mini-gene of the cellular Ki-ras4B gene mutated at to an expression a mini-gene of the wild-type cellular Ki-ras4B gene, and a cellular Ha-ras G12V expression three transfectant expressing the and three transfectant expressing the Ha-ras G12V and and expressing the wild-type Ki-ras4B of the was for a control for expression of of Ki-Ras The were as active in the were by and then to membrane and for the of Ki-Ras proteins by with a for the of proteins by with an Ha-ras for the of Ras proteins by with a pan-ras and for Ras proteins mutated at Val-12 by with a pan-ras Val-12 with mutant Ki-ras and of Ki-Ras with the transfectant and with mutant Ha-ras and of transfectant and cells of protein of the transfectants of Ras proteins Ras proteins were in the transfectant cells of Ras proteins were also by not Each of the three with the Ki-ras4BG12V and each of the three with the Ha-ras G12V mutant Ras proteins, which at the for proteins of and at the same as mutant Ki-ras Val-12 Ras proteins mutated at were detected in the line or in the Ki-ras4B wild-type control transfectant that the 21-kDa protein the Val-12 mutation detected by was Ras proteins were HD6-4 cells, wild-type transfectant cells, and transfectant cells expressing the mutated gene of Ras proteins were each cell line not wild-type and mutant Ki-Ras proteins were to membrane and for the of mutated Ras proteins by with the pan-ras Val-12 antibody. Only the line Ras proteins mutated to at the of the Western The mutant Ras proteins of GTP were The of to Ras proteins was by S. Biol. 1994; PubMed Google Scholar). the line a GDP of cells a of and wild-type transfectants a of The of an activated ras was by a Ras proteins exchange guanine at a that can by of to cells, by of to Ras proteins Biol. 1993; PubMed Scopus Google Scholar). cells three as GTP to Ras proteins in control transfectant cells or in the line not mutated Ras proteins were in both oncogenic Ki-ras and Ha-ras transfectant and the oncogenic Ki-ras and proteins exhibited increased GTP cells, oncogenic Ha-ras transfectants and and oncogenic Ki-ras transfectants and were each into The of cells was used to in tumor cell between the three was in each with of of each of tumor each ras transfectant line in the The Ha-ras transfectants as as the the Ki-ras transfectants as the of oncogenic Ras proteins in each transfectant line was with increased cell in The HD6-4 cell line into to in cells were into F. S. S. 1994; Google Scholar). tumors by the transfectant were after were in each tumor by each of three oncogenic Ki-ras transfectant cell and not the Ha-ras transfectants and wild-type Ki-ras transfectants and that the oncogenic Ki-ras gene disrupted either the cell to of HD6-4 cells or that cell to cell was of each transfectant line were then on to in the tumors by the oncogenic gene was due to a of colon epithelial cell apicobasal of were to differentiation of HD6-4 cells so that the of apicobasal polarity M. D. S. S. S. Google Scholar). The HD6-4 the wild-type transfectant line and the oncogenic Ha-ras transfectant as in on a collagen with two Ha-ras transfectants in not The cells were by through the basal surface as in The cells and so that were at the basal of the cell and was to the apical cells expressing mutated genes not form a but a were not to the basal region of the cell the membrane but at in the cells in into of by apical as the expression of an oncogenic but not an oncogenic gene, HD6-4 colon epithelial cell of either mutated Ki-ras into cells or cells to activation of the MAP kinases and through activation of raf and D. M. S. D. M. 1994; PubMed Google Scholar). 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PubMed Scopus Google Scholar). of HD6-4 cells and Ha-ras transfectant cells exhibited an of the mature forms of integrin as since the mature forms are to the cell surface of the oncogenic Ki-ras transfectant the integrin β1-chain form that the β1-chain was to the cell surface a with an and as a that the aberrant integrin β1-chain was to the cell and cells were and integrin β1-chain were and then by and The β1-chain intermediate form was on the cell surface protein that an integrin was with integrin β1-chain in both and oncogenic Ki-ras transfectant cells but in in the the two by β1-chain integrin in HD6-4 cells and cells, were the β1 mature form and the aberrant the same protein of the integrin β1-chain both β1 integrin and the of integrin was by Western blotting with integrin and the β1 integrin forms the integrin β1-chain faster migrating form was to the cell surface and in with the aberrant β1 form was due to of HD6-4 cells and cells were with endoglycosidase F Western for β1 the same of were with the line not F of and the mature β1 integrin forms in the control the and the line were to a form the same as the β1 integrin in cells not but not that the aberrant β1 integrin was an are in to the of the aberrant β1 integrin by tunicamycin of cells and Western also that the β1 integrin core protein was in both cells and oncogenic Ki-ras transfectant cells the aberrant β1 integrin in oncogenic cells was the of and of the oncogenic Ki-ras gene in cells of the β1 integrin and S. Biol. PubMed Scopus Google used phosphorothiolated antisense oligonucleotides to the region of the Ki-ras gene to the for proteins in the of human treatment with oligonucleotides was by a to of the Ki-ras and protein S. Biol. PubMed Scopus Google Scholar). used the same to cells with antisense oligonucleotides to the Ki-ras gene exhibited no mutated Ki-Ras protein with cells with sequence oligonucleotides or or each of two in a migrating integrin β1-chain form was in cells with antisense oligonucleotides to the Ki-ras gene with cells with sequence oligonucleotides The glycosylated integrin β1-chain in cells was not by the mature because the of the glycosylated integrin β1-chain. that gene by antisense the of maturation of integrin β1-chain. the in β1 integrin maturation in each of three transfectant is not due to some in the of but is due to the of the oncogenic HD6-4 cells to collagen as a first in apicobasal and HD6-4 cells integrin β1-chain to mediate to both collagen I and as of to a cell to and to collagen I was by in the oncogenic cells, to was were by expression of oncogenic proteins or of wild-type Ki-Ras proteins not of or cells, to either collagen I or laminin. Ki-Ras proteins of β1 at an intermediate The glycosylated integrin β1-chain is to the cell surface a of cells to extracellular apicobasal and cell to cell are a family of that mediate cell adhesion to extracellular proteins, plasma proteins, or to cells R. PubMed Scopus Google Scholar). The integrin form that mediate to and a cell adhesion control between maturation of and β1 integrin by cells of surface proteins by of either a integrin or β1 integrin that the mature forms of or integrin with mature β1 integrin R. M. C. Biol. PubMed Google Biol. PubMed Scopus Google Scholar). an in of β1 integrin to the mature form to an in the of β1 with mature of The integrin β1 and each with the to of the mature integrin to in human kidney epithelial cells M. D. Biol. 1994; PubMed Google or human A. F. Biol. PubMed Scopus Google Scholar). the maturation of β1 integrin in each of three oncogenic and in of three oncogenic Ha-ras transfectants. expression of the Ki-ras gene by a of an antisense the maturation that expression of the oncogenic Ki-ras gene inhibited β1 integrin glycosylation. The β1 integrin forms in oncogenic Ki-ras transfectants and the mature β1 integrin in control cells yielded of the same with endoglycosidase F or cells were with the that oncogenic of β1 integrin at some intermediate in of the β1 integrin in the After to the forms of integrin may by as and and as and in β1 integrin. of both the and of the integrin in the is for the of the and for M. H. S. Biol. 1994; PubMed Google Scholar). of residues integrin or cells the of and β1 integrin of in the of is an of at the surface of integrin β1 by the that a are M. H. S. Biol. 1994; PubMed Google Scholar). differentiation of the maturation of the β1 and the was by blocking the glycosylation of both integrin Biol. 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PubMed Google Scholar). is a protein highly homologous to Ha-ras Ha-ras, cell of a active into cells in activated by a cell PubMed Scopus Google Scholar). and ras genes either the of which β1 integrin maturation in cell in the of in cells, integrin β1-chain in the of isoforms is also the following Ki-ras the apical of kidney epithelial cells A. D. Biol. PubMed Scopus Google by expression of β1 integrin no in the of mature integrin β1-chain was A. D. 1994; PubMed Google Scholar). effects on glycosylation of integrin β1-chain were in to the with colon epithelial cells, and an aberrant β1 integrin intermediate was not in the cells A. D. 1994; PubMed Google Scholar). of a ras gene, oncogenic Ha-ras, into epithelial cell colon cells, not to of control or to of cell but to of of differentiation and the for differentiation M. D. 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PubMed Scopus Google Scholar). of colorectal with of β1 integrin with of in β1 integrin the was in with in the of β1 integrin to mature The for the in β1 integrin protein in colorectal is not the of the that the mutation in the cellular Ki-ras gene in a of colon may the of β1 integrin and to of cell or Expression of the oncogenic Ki-ras gene in colon tumors is also of cells form D. M. 1993; PubMed Scopus Google Scholar). the the oncogenic Ki-ras HD6-4 cell transfectants also in of both Ki-ras and Ha-ras genes may the the of the effects of oncogenic Ki-ras on colon epithelial cell through of β1 integrin maturation may to selection of cells Ki-ras mutations. of colon cells with oncogenic Ki-ras mutations is the of tumors D. M. 1993; PubMed Scopus Google and this selection is in the to and of the cells to highly and S. R. A. 1993; PubMed Scopus Google B. C. H. 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