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W2039102208 abstract "After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt−/−). Lysates fromIcmt−/− cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such asN-acetyl-S-geranylgeranyl-l-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt−/− cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt−/− cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt−/− cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells. After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt−/−). Lysates fromIcmt−/− cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such asN-acetyl-S-geranylgeranyl-l-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt−/− cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt−/− cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt−/− cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells. endoplasmic reticulum isoprenylcysteine carboxyl methyltransferase embryonic stem cell phenylmethylsulfonyl fluoride N-acetyl-S-geranylgeranyl-l-cysteine N-acetyl-S-farnesyl-l-cysteine bacterial artificial chromosome green fluorescent protein phosphate-buffered saline kilobase pair(s) neomycin resistance gene established sequence tag Ras proteins, as well as other proteins that terminate with a so-called CaaX sequence, undergo several sequential post-translational processing steps. First, a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprene lipid is added to the thiol group of the cysteine (C) residue by a cytosolic protein prenyltransferase (a process generally referred to as protein prenylation or isoprenylation) (1.Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar). After isoprenylation, the last three amino acids of the protein (i.e., the -aaX) are released by a specific endoprotease associated with the endoplasmic reticulum (ER)1 (2.Schmidt W.K. Tam A. Fujimura-Kamada K. Michaelis S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11175-11180Crossref PubMed Scopus (162) Google Scholar). The newly exposed isoprenylcysteine is then methylated by an ER-associated methyltransferase (3.Clarke S. Vogel J.P. Deschenes R.J. Stock J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4643-4647Crossref PubMed Scopus (292) Google Scholar). In addition, some forms of Ras undergo palmitoylation of cysteines upstream from the isoprenylcysteine residue (4.Fujiyama A. Tsunasawa S. Tamanoi F. Sakiyama F. J. Biol. Chem. 1991; 266: 17926-17931Abstract Full Text PDF PubMed Google Scholar).The post-translational processing steps for the CaaXproteins have attracted considerable scrutiny, in large part because of the key role of activated forms of Ras in the pathogenesis of human cancers. The isoprenylation step is crucial for the proper targeting of the Ras proteins along the plasma membrane (1.Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 5.Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar), and the growth of Ras-induced tumors in mice can be inhibited by pharmacological blockade of the isoprenylation step (6.Tamanoi F. Trends Biochem. Sci. 1993; 18: 349-353Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 7.Gibbs J.B. Graham S.L. Hartman G.D. Koblan K.S. Kohl N.E. Omer C.A. Oliff A. Curr. Opin. Chem. Biol. 1997; 1: 197-203Crossref PubMed Scopus (75) Google Scholar, 8.Kohl N.E. Omer C.A. Conner M.W. Anthony N.J. Davide J.P. deSolms S.J. Giuliani E.A. Gomez R.P. Graham S.L. Hamilton K. Handt L.K. Hartman G.D. Koblan K.S. Kral A.M. Miller P.J. Mosser S.D. O'Neill T.J. Rands E. Schaber M.D. Gibbs J.B. Oliff A. Nat. Med. 1995; 1: 792-797Crossref PubMed Scopus (510) Google Scholar). However, the physiologic importance of the “post-isoprenylation” protein processing steps has remained less certain (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar); this is particularly the case for the carboxyl methylation step (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar).The enzyme responsible for catalyzing the formation of the methyl ester on the carboxyl-terminal isoprenylcysteine is protein-S-isoprenylcysteine O-methyltransferase (EC 2.1.1.100) (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). In Saccharomyces cerevisiae, the enzyme is encoded by STE14. The yeast enzyme (Ste14p) is predicted to have multiple transmembrane-spanning domains (10.Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar), and it resides in the ER (11.Romano J.D. Schmidt W.K. Michaelis S. Mol. Biol. Cell. 1998; 9: 2231-2247Crossref PubMed Scopus (92) Google Scholar). A human orthologue for STE14 was reported recently (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) following the appearance in the data bases of a mouse EST with sequence similarities to yeast STE14. Like the yeast enzyme, the human enzyme resides in the ER and is predicted to have multiple membrane-spanning domains (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The human and mouse genes have been designatedICMT 2The human gene for isoprenylcysteine carboxyl methyltransferase has been designated ICMT, and the mouse gene has been designated Icmt (HUGO Nomenclature Committee and MGD Nomenclature Committee, The Jackson Laboratory). In recent publications, the human protein/cDNA has been referred to alternatively as pcCMT (for prenylcysteine carboxyl methyltransferase) (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), and the rat protein has been referred to as PPMT (for prenylated protein methyltransferase) (40.Desrosiers R. Nguyen Q. Beliveau R. Biochem. Biophys. Res. Commun. 1999; 261: 790-797Crossref PubMed Scopus (17) Google Scholar). 2The human gene for isoprenylcysteine carboxyl methyltransferase has been designated ICMT, and the mouse gene has been designated Icmt (HUGO Nomenclature Committee and MGD Nomenclature Committee, The Jackson Laboratory). In recent publications, the human protein/cDNA has been referred to alternatively as pcCMT (for prenylcysteine carboxyl methyltransferase) (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), and the rat protein has been referred to as PPMT (for prenylated protein methyltransferase) (40.Desrosiers R. Nguyen Q. Beliveau R. Biochem. Biophys. Res. Commun. 1999; 261: 790-797Crossref PubMed Scopus (17) Google Scholar). andIcmt, respectively (for isoprenylcysteine carboxyl methyltransferase).In this study, we sought to determine the influence of carboxyl methylation on the membrane localization of Ras proteins in mammalian cells. One potential means of approaching this issue would be to competitively inhibit the isoprenylcysteine carboxyl methyltransferase with small, cell-permeable, methyl-accepting isoprenylated substrates (e.g.,N-acetyl-S-farnesyl-l-cysteine orN-acetyl-S-geranylgeranyl-l-cysteine) (13.Tan E.W. Pérez-Sala D. Cañada F.J. Rando R.R. J. Biol. Chem. 1991; 266: 10719-10722Abstract Full Text PDF PubMed Google Scholar, 14.Volker C. Lane P. Kwee C. Johnson M. Stock J. FEBS Lett. 1991; 295: 189-194Crossref PubMed Scopus (54) Google Scholar, 15.Volker C. Miller R.A. McCleary W.R. Rao A. Poenie M. Backer J.M. Stock J.B. J. Biol. Chem. 1991; 266: 21515-21522Abstract Full Text PDF PubMed Google Scholar, 16.Pérez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar). The drawback of this approach, however, is that these types of small-molecule competitors probably have multiple effects on cells (see Ref. 9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar for a recent review). For example, the fact that these molecules contain isoprenyl groups could lead them to displace isoprenylated proteins from their cellular binding sites (17.Haklai R. Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (188) Google Scholar). To avoid such problems, we used gene-targeting techniques to produce a mammalian cell line lacking isoprenylcysteine carboxyl methyltransferase. In this report, we document the production of a mouse cell line that is homozygous for an Icmt knockout mutation, and we demonstrate that K-Ras localization is abnormal in those cells.DISCUSSIONIn this study, we inactivated both Icmt alleles in mouse ES cells. One allele was inactivated with a conventional sequence-replacement gene-targeting strategy. The second allele was inactivated by a second round of selection in a high concentration of G418 (6 mg/ml). This procedure, first described by Mortensen et al. (23.Mortensen R.M. Conner D.A. Chao S. Geisterfer-Lowrance A.A.T. Seidman J.G. Mol. Cell. Biol. 1992; 12: 2391-2395Crossref PubMed Scopus (354) Google Scholar), probably works by selecting for the survival of rare cells in which an interchromosomal gene conversion event has “repaired” the wild-type chromosome with the mutant chromosome, thereby providing it with an extra copy of the neo selection marker. In the case of the Icmt locus, the “high G418” selection procedure was extremely efficient and proved to be far simpler than knocking out the other allele with a second gene-targeting vector containing a different drug selection marker (31.te Riele H. Maandag E.R. Clarke A. Hooper M. Berns A. Nature. 1990; 348: 649-651Crossref PubMed Scopus (200) Google Scholar).The homozygous knockout cells lacked all Icmt activity against a protein substrate (farnesyl-K-Ras4B) or against farnesylated and geranylgeranylated small molecules (AFC and AGGC, respectively). Thus, the Icmt−/− cells did not appear to contain any redundant biochemical activity capable of methylating isoprenylated substrates, at least under the conditions of our assays. Interestingly, we had no difficulty in using methylation assays to document an accumulation ofIcmt substrates in the Icmt−/− cells. In the future, it should be possible to methylate the substrates inIcmt−/− cells withS-adenosyl-l-[methyl-14C]methionine, and then use two-dimensional gel electrophoresis to identify the most abundant Icmt substrates.Our principal objective in creating an Icmt-deficient mammalian cell line was to assess the importance of the carboxyl methylation step for the intracellular localization of the Ras proteins. To address this issue, we prepared P100 (membrane) and S100 (cytosolic) fractions from Icmt−/−and Icmt+/+ cells and then analyzed the membrane association of K-Ras by Western blotting. We also transfected Icmt−/−andIcmt+/+ cells with a GFP-K-Ras fusion plasmid in which the GFP coding sequences were ligated in-frame to the carboxyl-terminal 18 amino acids of K-Ras 4B. The results of both lines of investigation were clear. Compared with the Icmt+/+ cells, more of the K-Ras in the Icmt−/− cells was in the S100 fraction. In addition, the K-Ras fusion protein in the wild-type cells was localized almost exclusively along the plasma membrane, whereas a large fraction of the fusion in the Icmt−/− cells was trapped in the cytoplasm and less was at the plasma membrane.These data indicating mislocalization of K-Ras in the absence of carboxyl methylation require several comments. First, these results are clearly consistent with the finding of abnormal Ras2p processing and abnormal Ras2p membrane localization in ste14Δ S. cerevisiae (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). However, despite the concordance of the current data and the yeast data, we are reluctant to extrapolate our results toall Ras isoforms in mammals. In our study, we examined the localization of a K-Ras, which relies in part on a polybasic sequence for plasma membrane localization (32.Hancock J.F. Cadwallader K. Paterson H. Marshall C.J. EMBO J. 1991; 10: 4033-4039Crossref PubMed Scopus (375) Google Scholar, 33.Ghomashchi F. Zhang X. Liu L. Gelb M. Biochemistry. 1995; 34: 11910-11918Crossref PubMed Scopus (106) Google Scholar, 34.Leventis R. Sivinus J. Biochemistry. 1998; 37: 7640-7648Crossref PubMed Scopus (80) Google Scholar). It is possible that the methylation step could be less important for other mammalian Ras isoforms (e.g., H-Ras) that undergo palmitoylation of cysteine residues within the carboxyl-terminal part of the molecule (35.Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1448) Google Scholar, 36.Dunphy J. Linder M. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar, 37.Resh M. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar). Second, the degree of the mislocalization of the K-Ras fusion in Icmt−/− cells, although quite marked, did not appear to be as striking as that observed in Rce1-deficient fibroblasts (where the Ras proteins are neither endoproteolytically processed nor methylated) (25.Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The simplest explanation for this finding is that both the endoproteolysis step and the carboxyl methylation step contribute independently to Ras localization and that eliminating both steps, as occurs in the Rce1 knockout, has a greater impact than eliminating only the methylation step. Once again, however, caution is warranted, because the Rce1experiments were performed in fibroblasts, whereas the Icmtexperiments were performed in ES cells. Third, we emphasize that the mechanism(s) for the K-Ras mislocalization in Icmt−/− cells is not fully understood. The Icmt-mediated conversion of a carboxylate ion to an α-carboxyl methyl ester would be predicted to render the carboxyl terminus of the protein more hydrophobic, facilitating its interaction with the plasma membrane (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). However, that may not be the only explanation for our results. The subcellular trafficking of K-Ras appears to go through a route distinct from that of H-Ras and N-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 39.Choy E. Chiu V. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I. Philips M. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar), and microtubules may be involved in this process for K-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is conceivable that the absence of methylation adversely affects the transport of K-Ras, thereby reducing the level of this protein at the plasma membrane.The Icmt−/− cell line will be a valuable reagent for investigators who are interested in the metabolism of isoprenylated proteins. As noted above, two-dimensional gel experiments with these cells could help to identify Icmt substrates. Perhaps more importantly, the cells could yield clues as to why the isoprenylcysteine methylation step has been so conserved in eukaryotic biology. In yeast, theSTE14-mediated “capping” of a-factor (a farnesylated CaaX protein) with an α-methyl ester improvesa-factor stability by preventing rapid intracellular degradation of the protein (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). Based on that precedent, we hypothesized that the absence of Icmt might adversely affect the stability of mammalian isoprenylated proteins (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). With the production of Icmt−/− cells, it will be possible to test that hypothesis in a direct fashion. In addition, because many of the substrates for Icmt are signaling molecules (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar), it will be possible to assess the influence of methylation on various signal transduction pathways. Finally, we find it difficult to resist speculating that Icmt might function in mammalian cells to facilitate a receptor-mediated transport of an isoprenylated peptide across a membrane. In yeast, methylation of a-factor is crucial for theSTE6-mediated transport of a-factor out of the cell (10.Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar). Although no a-factor orthologue has been identified in higher organisms, it is nevertheless conceivable that transmembrane transport of an as yet unidentified isoprenylated protein occurs in mammals and that the methylation of the isoprenylcysteine is important for this process. The existence of Icmt−/− cells could be quite helpful in examining that possibility. Ras proteins, as well as other proteins that terminate with a so-called CaaX sequence, undergo several sequential post-translational processing steps. First, a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprene lipid is added to the thiol group of the cysteine (C) residue by a cytosolic protein prenyltransferase (a process generally referred to as protein prenylation or isoprenylation) (1.Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar). After isoprenylation, the last three amino acids of the protein (i.e., the -aaX) are released by a specific endoprotease associated with the endoplasmic reticulum (ER)1 (2.Schmidt W.K. Tam A. Fujimura-Kamada K. Michaelis S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11175-11180Crossref PubMed Scopus (162) Google Scholar). The newly exposed isoprenylcysteine is then methylated by an ER-associated methyltransferase (3.Clarke S. Vogel J.P. Deschenes R.J. Stock J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4643-4647Crossref PubMed Scopus (292) Google Scholar). In addition, some forms of Ras undergo palmitoylation of cysteines upstream from the isoprenylcysteine residue (4.Fujiyama A. Tsunasawa S. Tamanoi F. Sakiyama F. J. Biol. Chem. 1991; 266: 17926-17931Abstract Full Text PDF PubMed Google Scholar). The post-translational processing steps for the CaaXproteins have attracted considerable scrutiny, in large part because of the key role of activated forms of Ras in the pathogenesis of human cancers. The isoprenylation step is crucial for the proper targeting of the Ras proteins along the plasma membrane (1.Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 5.Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar), and the growth of Ras-induced tumors in mice can be inhibited by pharmacological blockade of the isoprenylation step (6.Tamanoi F. Trends Biochem. Sci. 1993; 18: 349-353Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 7.Gibbs J.B. Graham S.L. Hartman G.D. Koblan K.S. Kohl N.E. Omer C.A. Oliff A. Curr. Opin. Chem. Biol. 1997; 1: 197-203Crossref PubMed Scopus (75) Google Scholar, 8.Kohl N.E. Omer C.A. Conner M.W. Anthony N.J. Davide J.P. deSolms S.J. Giuliani E.A. Gomez R.P. Graham S.L. Hamilton K. Handt L.K. Hartman G.D. Koblan K.S. Kral A.M. Miller P.J. Mosser S.D. O'Neill T.J. Rands E. Schaber M.D. Gibbs J.B. Oliff A. Nat. Med. 1995; 1: 792-797Crossref PubMed Scopus (510) Google Scholar). However, the physiologic importance of the “post-isoprenylation” protein processing steps has remained less certain (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar); this is particularly the case for the carboxyl methylation step (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). The enzyme responsible for catalyzing the formation of the methyl ester on the carboxyl-terminal isoprenylcysteine is protein-S-isoprenylcysteine O-methyltransferase (EC 2.1.1.100) (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). In Saccharomyces cerevisiae, the enzyme is encoded by STE14. The yeast enzyme (Ste14p) is predicted to have multiple transmembrane-spanning domains (10.Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar), and it resides in the ER (11.Romano J.D. Schmidt W.K. Michaelis S. Mol. Biol. Cell. 1998; 9: 2231-2247Crossref PubMed Scopus (92) Google Scholar). A human orthologue for STE14 was reported recently (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) following the appearance in the data bases of a mouse EST with sequence similarities to yeast STE14. Like the yeast enzyme, the human enzyme resides in the ER and is predicted to have multiple membrane-spanning domains (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The human and mouse genes have been designatedICMT 2The human gene for isoprenylcysteine carboxyl methyltransferase has been designated ICMT, and the mouse gene has been designated Icmt (HUGO Nomenclature Committee and MGD Nomenclature Committee, The Jackson Laboratory). In recent publications, the human protein/cDNA has been referred to alternatively as pcCMT (for prenylcysteine carboxyl methyltransferase) (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), and the rat protein has been referred to as PPMT (for prenylated protein methyltransferase) (40.Desrosiers R. Nguyen Q. Beliveau R. Biochem. Biophys. Res. Commun. 1999; 261: 790-797Crossref PubMed Scopus (17) Google Scholar). 2The human gene for isoprenylcysteine carboxyl methyltransferase has been designated ICMT, and the mouse gene has been designated Icmt (HUGO Nomenclature Committee and MGD Nomenclature Committee, The Jackson Laboratory). In recent publications, the human protein/cDNA has been referred to alternatively as pcCMT (for prenylcysteine carboxyl methyltransferase) (12.Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), and the rat protein has been referred to as PPMT (for prenylated protein methyltransferase) (40.Desrosiers R. Nguyen Q. Beliveau R. Biochem. Biophys. Res. Commun. 1999; 261: 790-797Crossref PubMed Scopus (17) Google Scholar). andIcmt, respectively (for isoprenylcysteine carboxyl methyltransferase). In this study, we sought to determine the influence of carboxyl methylation on the membrane localization of Ras proteins in mammalian cells. One potential means of approaching this issue would be to competitively inhibit the isoprenylcysteine carboxyl methyltransferase with small, cell-permeable, methyl-accepting isoprenylated substrates (e.g.,N-acetyl-S-farnesyl-l-cysteine orN-acetyl-S-geranylgeranyl-l-cysteine) (13.Tan E.W. Pérez-Sala D. Cañada F.J. Rando R.R. J. Biol. Chem. 1991; 266: 10719-10722Abstract Full Text PDF PubMed Google Scholar, 14.Volker C. Lane P. Kwee C. Johnson M. Stock J. FEBS Lett. 1991; 295: 189-194Crossref PubMed Scopus (54) Google Scholar, 15.Volker C. Miller R.A. McCleary W.R. Rao A. Poenie M. Backer J.M. Stock J.B. J. Biol. Chem. 1991; 266: 21515-21522Abstract Full Text PDF PubMed Google Scholar, 16.Pérez-Sala D. Gilbert B.A. Tan E.W. Rando R.R. Biochem. J. 1992; 284: 835-840Crossref PubMed Scopus (58) Google Scholar). The drawback of this approach, however, is that these types of small-molecule competitors probably have multiple effects on cells (see Ref. 9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar for a recent review). For example, the fact that these molecules contain isoprenyl groups could lead them to displace isoprenylated proteins from their cellular binding sites (17.Haklai R. Weisz M. Elad G. Paz A. Marciano D. Egozi Y. Ben-Baruch G. Kloog Y. Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (188) Google Scholar). To avoid such problems, we used gene-targeting techniques to produce a mammalian cell line lacking isoprenylcysteine carboxyl methyltransferase. In this report, we document the production of a mouse cell line that is homozygous for an Icmt knockout mutation, and we demonstrate that K-Ras localization is abnormal in those cells. DISCUSSIONIn this study, we inactivated both Icmt alleles in mouse ES cells. One allele was inactivated with a conventional sequence-replacement gene-targeting strategy. The second allele was inactivated by a second round of selection in a high concentration of G418 (6 mg/ml). This procedure, first described by Mortensen et al. (23.Mortensen R.M. Conner D.A. Chao S. Geisterfer-Lowrance A.A.T. Seidman J.G. Mol. Cell. Biol. 1992; 12: 2391-2395Crossref PubMed Scopus (354) Google Scholar), probably works by selecting for the survival of rare cells in which an interchromosomal gene conversion event has “repaired” the wild-type chromosome with the mutant chromosome, thereby providing it with an extra copy of the neo selection marker. In the case of the Icmt locus, the “high G418” selection procedure was extremely efficient and proved to be far simpler than knocking out the other allele with a second gene-targeting vector containing a different drug selection marker (31.te Riele H. Maandag E.R. Clarke A. Hooper M. Berns A. Nature. 1990; 348: 649-651Crossref PubMed Scopus (200) Google Scholar).The homozygous knockout cells lacked all Icmt activity against a protein substrate (farnesyl-K-Ras4B) or against farnesylated and geranylgeranylated small molecules (AFC and AGGC, respectively). Thus, the Icmt−/− cells did not appear to contain any redundant biochemical activity capable of methylating isoprenylated substrates, at least under the conditions of our assays. Interestingly, we had no difficulty in using methylation assays to document an accumulation ofIcmt substrates in the Icmt−/− cells. In the future, it should be possible to methylate the substrates inIcmt−/− cells withS-adenosyl-l-[methyl-14C]methionine, and then use two-dimensional gel electrophoresis to identify the most abundant Icmt substrates.Our principal objective in creating an Icmt-deficient mammalian cell line was to assess the importance of the carboxyl methylation step for the intracellular localization of the Ras proteins. To address this issue, we prepared P100 (membrane) and S100 (cytosolic) fractions from Icmt−/−and Icmt+/+ cells and then analyzed the membrane association of K-Ras by Western blotting. We also transfected Icmt−/−andIcmt+/+ cells with a GFP-K-Ras fusion plasmid in which the GFP coding sequences were ligated in-frame to the carboxyl-terminal 18 amino acids of K-Ras 4B. The results of both lines of investigation were clear. Compared with the Icmt+/+ cells, more of the K-Ras in the Icmt−/− cells was in the S100 fraction. In addition, the K-Ras fusion protein in the wild-type cells was localized almost exclusively along the plasma membrane, whereas a large fraction of the fusion in the Icmt−/− cells was trapped in the cytoplasm and less was at the plasma membrane.These data indicating mislocalization of K-Ras in the absence of carboxyl methylation require several comments. First, these results are clearly consistent with the finding of abnormal Ras2p processing and abnormal Ras2p membrane localization in ste14Δ S. cerevisiae (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). However, despite the concordance of the current data and the yeast data, we are reluctant to extrapolate our results toall Ras isoforms in mammals. In our study, we examined the localization of a K-Ras, which relies in part on a polybasic sequence for plasma membrane localization (32.Hancock J.F. Cadwallader K. Paterson H. Marshall C.J. EMBO J. 1991; 10: 4033-4039Crossref PubMed Scopus (375) Google Scholar, 33.Ghomashchi F. Zhang X. Liu L. Gelb M. Biochemistry. 1995; 34: 11910-11918Crossref PubMed Scopus (106) Google Scholar, 34.Leventis R. Sivinus J. Biochemistry. 1998; 37: 7640-7648Crossref PubMed Scopus (80) Google Scholar). It is possible that the methylation step could be less important for other mammalian Ras isoforms (e.g., H-Ras) that undergo palmitoylation of cysteine residues within the carboxyl-terminal part of the molecule (35.Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1448) Google Scholar, 36.Dunphy J. Linder M. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar, 37.Resh M. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar). Second, the degree of the mislocalization of the K-Ras fusion in Icmt−/− cells, although quite marked, did not appear to be as striking as that observed in Rce1-deficient fibroblasts (where the Ras proteins are neither endoproteolytically processed nor methylated) (25.Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The simplest explanation for this finding is that both the endoproteolysis step and the carboxyl methylation step contribute independently to Ras localization and that eliminating both steps, as occurs in the Rce1 knockout, has a greater impact than eliminating only the methylation step. Once again, however, caution is warranted, because the Rce1experiments were performed in fibroblasts, whereas the Icmtexperiments were performed in ES cells. Third, we emphasize that the mechanism(s) for the K-Ras mislocalization in Icmt−/− cells is not fully understood. The Icmt-mediated conversion of a carboxylate ion to an α-carboxyl methyl ester would be predicted to render the carboxyl terminus of the protein more hydrophobic, facilitating its interaction with the plasma membrane (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). However, that may not be the only explanation for our results. The subcellular trafficking of K-Ras appears to go through a route distinct from that of H-Ras and N-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 39.Choy E. Chiu V. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I. Philips M. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar), and microtubules may be involved in this process for K-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is conceivable that the absence of methylation adversely affects the transport of K-Ras, thereby reducing the level of this protein at the plasma membrane.The Icmt−/− cell line will be a valuable reagent for investigators who are interested in the metabolism of isoprenylated proteins. As noted above, two-dimensional gel experiments with these cells could help to identify Icmt substrates. Perhaps more importantly, the cells could yield clues as to why the isoprenylcysteine methylation step has been so conserved in eukaryotic biology. In yeast, theSTE14-mediated “capping” of a-factor (a farnesylated CaaX protein) with an α-methyl ester improvesa-factor stability by preventing rapid intracellular degradation of the protein (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). Based on that precedent, we hypothesized that the absence of Icmt might adversely affect the stability of mammalian isoprenylated proteins (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). With the production of Icmt−/− cells, it will be possible to test that hypothesis in a direct fashion. In addition, because many of the substrates for Icmt are signaling molecules (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar), it will be possible to assess the influence of methylation on various signal transduction pathways. Finally, we find it difficult to resist speculating that Icmt might function in mammalian cells to facilitate a receptor-mediated transport of an isoprenylated peptide across a membrane. In yeast, methylation of a-factor is crucial for theSTE6-mediated transport of a-factor out of the cell (10.Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar). Although no a-factor orthologue has been identified in higher organisms, it is nevertheless conceivable that transmembrane transport of an as yet unidentified isoprenylated protein occurs in mammals and that the methylation of the isoprenylcysteine is important for this process. The existence of Icmt−/− cells could be quite helpful in examining that possibility. In this study, we inactivated both Icmt alleles in mouse ES cells. One allele was inactivated with a conventional sequence-replacement gene-targeting strategy. The second allele was inactivated by a second round of selection in a high concentration of G418 (6 mg/ml). This procedure, first described by Mortensen et al. (23.Mortensen R.M. Conner D.A. Chao S. Geisterfer-Lowrance A.A.T. Seidman J.G. Mol. Cell. Biol. 1992; 12: 2391-2395Crossref PubMed Scopus (354) Google Scholar), probably works by selecting for the survival of rare cells in which an interchromosomal gene conversion event has “repaired” the wild-type chromosome with the mutant chromosome, thereby providing it with an extra copy of the neo selection marker. In the case of the Icmt locus, the “high G418” selection procedure was extremely efficient and proved to be far simpler than knocking out the other allele with a second gene-targeting vector containing a different drug selection marker (31.te Riele H. Maandag E.R. Clarke A. Hooper M. Berns A. Nature. 1990; 348: 649-651Crossref PubMed Scopus (200) Google Scholar). The homozygous knockout cells lacked all Icmt activity against a protein substrate (farnesyl-K-Ras4B) or against farnesylated and geranylgeranylated small molecules (AFC and AGGC, respectively). Thus, the Icmt−/− cells did not appear to contain any redundant biochemical activity capable of methylating isoprenylated substrates, at least under the conditions of our assays. Interestingly, we had no difficulty in using methylation assays to document an accumulation ofIcmt substrates in the Icmt−/− cells. In the future, it should be possible to methylate the substrates inIcmt−/− cells withS-adenosyl-l-[methyl-14C]methionine, and then use two-dimensional gel electrophoresis to identify the most abundant Icmt substrates. Our principal objective in creating an Icmt-deficient mammalian cell line was to assess the importance of the carboxyl methylation step for the intracellular localization of the Ras proteins. To address this issue, we prepared P100 (membrane) and S100 (cytosolic) fractions from Icmt−/−and Icmt+/+ cells and then analyzed the membrane association of K-Ras by Western blotting. We also transfected Icmt−/−andIcmt+/+ cells with a GFP-K-Ras fusion plasmid in which the GFP coding sequences were ligated in-frame to the carboxyl-terminal 18 amino acids of K-Ras 4B. The results of both lines of investigation were clear. Compared with the Icmt+/+ cells, more of the K-Ras in the Icmt−/− cells was in the S100 fraction. In addition, the K-Ras fusion protein in the wild-type cells was localized almost exclusively along the plasma membrane, whereas a large fraction of the fusion in the Icmt−/− cells was trapped in the cytoplasm and less was at the plasma membrane. These data indicating mislocalization of K-Ras in the absence of carboxyl methylation require several comments. First, these results are clearly consistent with the finding of abnormal Ras2p processing and abnormal Ras2p membrane localization in ste14Δ S. cerevisiae (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). However, despite the concordance of the current data and the yeast data, we are reluctant to extrapolate our results toall Ras isoforms in mammals. In our study, we examined the localization of a K-Ras, which relies in part on a polybasic sequence for plasma membrane localization (32.Hancock J.F. Cadwallader K. Paterson H. Marshall C.J. EMBO J. 1991; 10: 4033-4039Crossref PubMed Scopus (375) Google Scholar, 33.Ghomashchi F. Zhang X. Liu L. Gelb M. Biochemistry. 1995; 34: 11910-11918Crossref PubMed Scopus (106) Google Scholar, 34.Leventis R. Sivinus J. Biochemistry. 1998; 37: 7640-7648Crossref PubMed Scopus (80) Google Scholar). It is possible that the methylation step could be less important for other mammalian Ras isoforms (e.g., H-Ras) that undergo palmitoylation of cysteine residues within the carboxyl-terminal part of the molecule (35.Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1448) Google Scholar, 36.Dunphy J. Linder M. Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar, 37.Resh M. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar). Second, the degree of the mislocalization of the K-Ras fusion in Icmt−/− cells, although quite marked, did not appear to be as striking as that observed in Rce1-deficient fibroblasts (where the Ras proteins are neither endoproteolytically processed nor methylated) (25.Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The simplest explanation for this finding is that both the endoproteolysis step and the carboxyl methylation step contribute independently to Ras localization and that eliminating both steps, as occurs in the Rce1 knockout, has a greater impact than eliminating only the methylation step. Once again, however, caution is warranted, because the Rce1experiments were performed in fibroblasts, whereas the Icmtexperiments were performed in ES cells. Third, we emphasize that the mechanism(s) for the K-Ras mislocalization in Icmt−/− cells is not fully understood. The Icmt-mediated conversion of a carboxylate ion to an α-carboxyl methyl ester would be predicted to render the carboxyl terminus of the protein more hydrophobic, facilitating its interaction with the plasma membrane (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). However, that may not be the only explanation for our results. The subcellular trafficking of K-Ras appears to go through a route distinct from that of H-Ras and N-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 39.Choy E. Chiu V. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I. Philips M. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar), and microtubules may be involved in this process for K-Ras (38.Thissen J. Gross J. Subramanian K. Meyer T. Casey P. J. Biol. Chem. 1997; 272: 30362-30370Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is conceivable that the absence of methylation adversely affects the transport of K-Ras, thereby reducing the level of this protein at the plasma membrane. The Icmt−/− cell line will be a valuable reagent for investigators who are interested in the metabolism of isoprenylated proteins. As noted above, two-dimensional gel experiments with these cells could help to identify Icmt substrates. Perhaps more importantly, the cells could yield clues as to why the isoprenylcysteine methylation step has been so conserved in eukaryotic biology. In yeast, theSTE14-mediated “capping” of a-factor (a farnesylated CaaX protein) with an α-methyl ester improvesa-factor stability by preventing rapid intracellular degradation of the protein (18.Hrycyna C.A. Sapperstein S.K. Clarke S. Michaelis S. EMBO J. 1991; 10: 1699-1709Crossref PubMed Scopus (190) Google Scholar). Based on that precedent, we hypothesized that the absence of Icmt might adversely affect the stability of mammalian isoprenylated proteins (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar). With the production of Icmt−/− cells, it will be possible to test that hypothesis in a direct fashion. In addition, because many of the substrates for Icmt are signaling molecules (9.Young S.G. Ambroziak P. Kim E. Clarke S. Tamanoi F. Sigman D.G. Enzymes for Lipid Metabolism. Academic Press, New York2000Google Scholar), it will be possible to assess the influence of methylation on various signal transduction pathways. Finally, we find it difficult to resist speculating that Icmt might function in mammalian cells to facilitate a receptor-mediated transport of an isoprenylated peptide across a membrane. In yeast, methylation of a-factor is crucial for theSTE6-mediated transport of a-factor out of the cell (10.Sapperstein S. Berkower C. Michaelis S. Mol. Cell. Biol. 1994; 14: 1438-1449Crossref PubMed Scopus (96) Google Scholar). Although no a-factor orthologue has been identified in higher organisms, it is nevertheless conceivable that transmembrane transport of an as yet unidentified isoprenylated protein occurs in mammals and that the methylation of the isoprenylcysteine is important for this process. The existence of Icmt−/− cells could be quite helpful in examining that possibility. We thank Drs. Edward Kim of the Gladstone Institutes and Steven Clarke of the University of California, Los Angeles for helpful discussions. We thank Dr. David Sanan for assistance with confocal microscopy." @default.
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W2039102208 title "Targeted Inactivation of the Isoprenylcysteine Carboxyl Methyltransferase Gene Causes Mislocalization of K-Ras in Mammalian Cells" @default.
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