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• W2069369960 abstract "Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway. Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway. The Ras and Rho families of small GTPases participate in almost all aspects of cell biology by regulating cell survival, proliferation, and migration (1Pruitt K. Der C.J. Cancer Lett. 2001; 171: 1-10Crossref PubMed Scopus (250) Google Scholar, 2Rodriguez-Viciana P. Tetsu O. Oda K. Okada J. Rauen K. McCormick F. Cold Spring Harbor Symp. Quant. Biol. 2005; 70: 461-467Crossref PubMed Scopus (86) Google Scholar, 3Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (278) Google Scholar, 4Karlsson R. Pedersen E.D. Wang Z. Brakebusch C. Biochim. Biophys. Acta. 2009; 1796: 91-98Crossref PubMed Scopus (283) Google Scholar). Some of these small GTPases possess a C-terminal polybasic region (PBR) 3The abbreviations used are: PBRpolybasic regionARMarmadilloDNdominant negativeERendoplasmic reticulumGEFguanine nucleotide exchange factorGFPgreen fluorescent proteinNSCLCnon-small cell lung carcinomaPMplasma membranePTaseprenyltransferaseRce1Ras-converting enzyme 1tettetracyclineWTwild typeAaliphatic amino acid. that regulates their trafficking as they undergo prenylation (5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) and is required as a second signal for membrane localization (7Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (846) Google Scholar). It is not known how the PBR promotes the trafficking of small GTPases through the prenylation pathway. Interestingly, the PBR is required for small GTPases to interact with SmgGDS, a protein consisting almost entirely of predicted Armadillo (ARM) domains (8Peifer M. Berg S. Reynolds A.B. Cell. 1994; 76: 789-791Abstract Full Text PDF PubMed Scopus (549) Google Scholar). Although SmgGDS was previously reported to preferentially interact with small GTPases that possess a PBR, including K-Ras, Rap1, RhoA, and Rac1 (9Takakura A. Miyoshi J. Ishizaki H. Tanaka M. Togawa A. Nishizawa Y. Yoshida H. Nishikawa S. Takai Y. Mol. Biol. Cell. 2000; 11: 1875-1886Crossref PubMed Scopus (17) Google Scholar, 10Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acid Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 11Williams C.L. Cell. Signal. 2003; 15: 1071-1080Crossref PubMed Scopus (158) Google Scholar), the functional significance of these PBR-dependent interactions has not been clearly defined. polybasic region armadillo dominant negative endoplasmic reticulum guanine nucleotide exchange factor green fluorescent protein non-small cell lung carcinoma plasma membrane prenyltransferase Ras-converting enzyme 1 tetracycline wild type aliphatic amino acid. It was recently found that SmgGDS promotes the malignant phenotype in non-small cell lung carcinoma (NSCLC) (12Tew G.W. Lorimer E.L. Berg T.J. Zhi H. Li R. Williams C.L. J. Biol. Chem. 2008; 283: 963-976Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and prostate carcinoma (13Zhi H. Yang X.J. Kuhnmuench J. Berg T. Thill R. Yang H. See W.A. Becker C.G. Williams C.L. Li R. J. Pathol. 2009; 217: 389-397Crossref PubMed Scopus (22) Google Scholar). The participation of SmgGDS in cancer has been attributed to its ability to increase the activities of multiple PBR-possessing small GTPases, many of which promote the malignant phenotype (reviewed in Ref. 12Tew G.W. Lorimer E.L. Berg T.J. Zhi H. Li R. Williams C.L. J. Biol. Chem. 2008; 283: 963-976Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This conclusion is supported by reports that many cellular processes regulated by small GTPases, including NF-κB activity, actin/myosin interactions, contractile responses, and cell migration and proliferation, are inhibited by diminishing SmgGDS expression in cancer cells (12Tew G.W. Lorimer E.L. Berg T.J. Zhi H. Li R. Williams C.L. J. Biol. Chem. 2008; 283: 963-976Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 13Zhi H. Yang X.J. Kuhnmuench J. Berg T. Thill R. Yang H. See W.A. Becker C.G. Williams C.L. Li R. J. Pathol. 2009; 217: 389-397Crossref PubMed Scopus (22) Google Scholar) or other cell types (14Thill R. Campbell W.B. Williams C.L. J. Cell. Biochem. 2008; 104: 1760-1770Crossref PubMed Scopus (5) Google Scholar). The mechanism by which SmgGDS promotes the activities of PBR-containing small GTPases is unclear. SmgGDS was previously reported to be a weak guanine nucleotide exchange factor (GEF) because of its ability to promote the dissociation of GDP and uptake of GTP by its small GTPase partners (9Takakura A. Miyoshi J. Ishizaki H. Tanaka M. Togawa A. Nishizawa Y. Yoshida H. Nishikawa S. Takai Y. Mol. Biol. Cell. 2000; 11: 1875-1886Crossref PubMed Scopus (17) Google Scholar, 10Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acid Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 11Williams C.L. Cell. Signal. 2003; 15: 1071-1080Crossref PubMed Scopus (158) Google Scholar). However, SmgGDS does not possess any of the known catalytic domains found in other GEFs, leading to the suggestion that SmgGDS regulates small GTPases by a novel, unknown mechanism that might not involve classical guanine nucleotide exchange. In this study, we provide evidence of a previously undescribed mechanism utilized by SmgGDS to regulate small GTPases, involving their entrance and trafficking through the prenylation pathway. Prenylation is necessary for optimal activity of members of the Ras and Rho families of small GTPases, increasing their hydrophobic character and thereby promoting their participation in membrane-localized signaling pathways and other biological interactions (3Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (278) Google Scholar, 5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The newly synthesized GTPases first interact with cytosolic prenyltransferases (PTases) (15Casey P.J. Solski P.A. Der C.J. Buss J.E. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 8323-8327Crossref PubMed Scopus (780) Google Scholar, 16Reiss Y. Goldstein J.L. Seabra M.C. Casey P.J. Brown M.S. Cell. 1990; 62: 81-88Abstract Full Text PDF PubMed Scopus (703) Google Scholar, 17Farnsworth C.C. Gelb M.H. Glomset J.A. Science. 1990; 247: 320-322Crossref PubMed Scopus (163) Google Scholar, 18Joly A. Popják G. Edwards P.A. J. Biol. Chem. 1991; 266: 13495-13498Abstract Full Text PDF PubMed Google Scholar, 19Finegold A.A. Johnson D.I. Farnsworth C.C. Gelb M.H. Judd S.R. Glomset J.A. Tamanoi F. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 4448-4452Crossref PubMed Scopus (120) Google Scholar) that add a farnesyl isoprenoid or geranylgeranyl isoprenoid to the cysteine in the C-terminal CAAX (where A indicates aliphatic amino acid) motif of the GTPases (20Katayama M. Kawata M. Yoshida Y. Horiuchi H. Yamamoto T. Matsuura Y. Takai Y. J. Biol. Chem. 1991; 266: 12639-12645Abstract Full Text PDF PubMed Google Scholar, 21Kinsella B.T. Erdman R.A. Maltese W.A. J. Biol. Chem. 1991; 266: 9786-9794Abstract Full Text PDF PubMed Google Scholar, 22Buss J.E. Quilliam L.A. Kato K. Casey P.J. Solski P.A. Wong G. Clark R. McCormick F. Bokoch G.M. Der C.J. Mol. Cell. Biol. 1991; 11: 1523-1530Crossref PubMed Scopus (78) Google Scholar, 23Hancock J.F. Magee A.I. Childs J.E. Marshall C.J. Cell. 1989; 57: 1167-1177Abstract Full Text PDF PubMed Scopus (1460) Google Scholar). The prenylated GTPases then move to the endoplasmic reticulum (ER) to interact with the Ras-converting enzyme I (Rce1) and the isoprenylcysteine carboxylmethyltransferase for post-prenylation processing (3Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (278) Google Scholar, 5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The presence of a PBR influences which pathway a Ras or Rho family member will take to reach the plasma membrane (PM) after post-prenylation processing is completed in the ER. Small GTPases such as H-Ras and N-Ras, which lack a PBR, move from the ER to the Golgi, where they are palmitoylated, and then move by vesicular transport to the PM (5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). In contrast, small GTPases that possess a PBR, such as K-Ras, Rap1, RhoA, and Rac1, move directly from the ER to the PM by an uncharacterized mechanism that most likely involves unidentified chaperone proteins (5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Several crucial events in this prenylation pathway remain a mystery. It is not known how a newly synthesized small GTPase initially finds its PTase in the cytosol. In the absence of any known regulatory mechanisms, it is often assumed that small GTPases are prenylated as soon as they are synthesized and advance through the prenylation pathway unimpeded in a nonregulated manner. Recently, several models have emerged to define how the Rab escort protein Rep might regulate the interactions of newly synthesized Rab small GTPases with their PTase, geranylgeranyltransferase-II (24Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 25Wu Y.W. Tan K.T. Waldmann H. Goody R.S. Alexandrov K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12294-12299Crossref PubMed Scopus (78) Google Scholar, 26Baron R.A. Seabra M.C. Biochem. J. 2008; 415: 67-75Crossref PubMed Scopus (20) Google Scholar). However, the mechanisms that might regulate the interactions of newly synthesized Ras and Rho family members with their PTases have not been characterized. Also unknown is how PBR-possessing small GTPases, once modified by the addition of a hydrophobic prenyl group, move through the cytosol to the ER for final processing by Rce1 and isoprenylcysteine carboxylmethyltransferase nor is it known how the fully processed PBR-possessing small GTPases move from the ER to the PM. It has been suggested that unidentified chaperone proteins might escort PBR-containing small GTPases between the cytosol, ER, and PM during the prenylation pathway (5Hancock J.F. Nat. Rev. Mol. Cell. Biol. 2003; 4: 373-384Crossref PubMed Scopus (692) Google Scholar, 6Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), but these suspected chaperones have not been identified. Here, we report that the prenylation and trafficking of PBR-containing small GTPases is regulated by GDP/GTP exchange and by interactions with two splice variants of SmgGDS, which we have named SmgGDS-558 and SmgGDS-607. Our results demonstrate that SmgGDS-607 specifically interacts with nonprenylated small GTPases and regulates their entry into the prenylation pathway, whereas SmgGDS-558 specifically associates with prenylated small GTPases and regulates trafficking to the PM. These findings suggest that, in contrast to the previous view of unregulated entry of small GTPases into the prenylation pathway, prenylation of PBR-containing small GTPases is regulated in multiple ways. Interactions of PBR-containing small GTPases with SmgGDS splice variants in conjunction with GDP/GTP exchange presents, for the first time, a specific cellular mechanism to regulate the prenylation and subsequent membrane localization of these GTPases. All cell lines were obtained from the American Type Tissue Collection (Manassas, VA). The NSCLC cell lines NCI-H1703 and NCI-H23 were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum and antibiotics; the HEK-293T cell line was maintained in DMEM with 10% heat-inactivated fetal bovine serum and antibiotics. All siRNA duplexes were purchased from Dharmacon (Lafayette, CO). Sequences of the siRNAs are presented in supplemental Table S1. Nontargeting siRNAs (Dharmacon siControl 3) were designed not to target any human mRNA. Cells were transfected with 25 nm siRNA using DharmaFECT-3 transfection reagent (Dharmacon, Lafayette, CO) according to the manufacturer's instruction. All cDNAs were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. A construct encoding human SmgGDS-607 (cDNA clone FLJ30470) was obtained from the National Institute of Technology and Evaluation Biological Resource Center (Chiba, Japan) and was subcloned into the pcDNA3.1(+) vector with a single C-terminal hemagglutinin (HA) epitope tag (YPYDVPDYA). Two amino acids in the original construct that did not agree with the sequences in the NCBI Database were changed by site-directed mutagenesis to agree with the sequences in the NCBI Database (accession number NP_001093897). These changes were T197C to produce V66A and C1154T to produce F385L. SmgGDS-558-HA was generated by overlap PCR from SmgGDS-607-HA in the pcDNA3.1(+) vector. The Myc-tagged and GFP-tagged small GTPase constructs were generated as described previously (27Lanning C.C. Ruiz-Velasco R. Williams C.L. J. Biol. Chem. 2003; 278: 12495-12506Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The SAAX mutants of the small GTPases described in supplemental Table S2 were generated by site-directed mutagenesis to change the cysteine in the CAAX motif to a serine. Site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocols, using primers purchased from Operon. The FLAG-tagged Rap1 GEF constructs MR-GEF, C3G, GRP3, and PDZ-GEF were the kind gifts from Dr. Lawrence Quilliam, Indiana University School of Medicine and Walther Oncology Center (28Rebhun J.F. Castro A.F. Quilliam L.A. J. Biol. Chem. 2000; 275: 34901-34908Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). NCI-H1703 cells were transduced with lentiviral vectors encoding the luciferase protein (a kind gift from Dr. Michael Dwinell, Medical College of Wisconsin) and the tetracycline repressor protein (Invitrogen) and selected for stable expression of the vectors. Lentiviral vectors expressing inducible shRNA for SmgGDS-558 (shRNA BD), SmgGDS-607 (shRNA C2), or a nontargeting control shRNA were generated using the “Block-iT” inducible H1 lentiviral RNAi system (Invitrogen, K4925-00). Cells were transduced with the lentiviral vectors containing the inducible shRNAs and selected for stable expression of the vectors. Cells were maintained in RPMI 1640 triple selection media (200 μg/ml Zeocin, 12 μg/ml blasticidin, 400 ng/ml puromycin). Expression of shRNA was induced using 2 μg/ml tetracycline (Invitrogen). Oligonucleotide sequences encoding the shRNA were purchased from Operon. The forward sequences were as follows: nontargeting shRNA, 5′-CACCATGGTTTACATGTTTTCTGATTGAGAAATCAGAAAACATGTAAACCA-3′; C2 shRNA, 5′-CACCATTCTCATTGCTATAGTTCCGAAGAACTATAGCAATGAGAA-3′; and BD shRNA, 5′-CACCATGAAGCGAATGGCTATCGTGAGAACGATAGCCATTCGCTTCA-3′. Reverse complements of the sequences were also purchased for formation of the shRNA double-stranded DNA complexes. Equal numbers of transfected cells were boiled in Laemmli sample buffer and subjected to SDS-PAGE. The proteins were transferred to polyvinylidene difluoride and immunoblotted using antibody to SmgGDS (BD Transduction Laboratories 612511), GAPDH (Santa Cruz Biotechnology sc-32233), mouse HA antibody (Covance, MMS-101P), rabbit HA antibody (Covance, PRB-101P), mouse Myc antibody (Santa Cruz Biotechnology, sc-40), rabbit Myc antibody (Covance, PRB-150P), mouse antibody to FLAG (Sigma, F3165), rabbit antibody to FLAG (Sigma, F7425), mouse antibody to Actin (Santa Cruz Biotechnology, sc-47778), rabbit antibody to tet-repressor (Chemicon, AB3541), rabbit antibody to lamin B1 (Abcam, ab16048), or goat antibody to nonprenylated Rap1 (29Peterson Y.K. Kelly P. Weinbaum C.A. Casey P.J. J. Biol. Chem. 2006; 281: 12445-12450Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) (Santa Cruz Biotechnology, sc-1482). Bound antibodies were visualized using horseradish peroxidase-linked anti-mouse IgG, anti-rabbit IgG (Amersham Biosciences), or anti-goat IgG (Santa Cruz Biotechnology, sc-2056) and ECL reagents (PerkinElmer Life Sciences), as described previously (30Lanning C.C. Daddona J.L. Ruiz-Velasco R. Shafer S.H. Williams C.L. J. Biol. Chem. 2004; 279: 44197-44210Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). HEK-293T cells were transiently transfected with cDNAs encoding HA-tagged SmgGDS splice variants and Myc-tagged small GTPases. After 24 h, equal numbers of cells were lysed in buffer containing 0.5% Nonidet P-40 with protease and phosphatase inhibitors, and the lysates were centrifuged (2500 × g, 5 min, 4 °C). A portion of the resulting supernatants was reserved for total cell lysates, and the remainder was immunoprecipitated using mouse HA antibody (Covance, MMS-101P) or mouse Myc antibody (Santa Cruz Biotechnology, sc-40). Immunoprecipitates and total cell lysates were subjected to ECL-Western blotting as described above. Using an approved Institutional Review Board protocol (Medical College of Wisconsin Institutional Review Board PRO5554), lysates of archival lung tumors and their matched normal lung tissue were homogenized in 50 mm HEPES, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40, and 10% glycerol with protease inhibitors (Complete Mini Tablets, Roche Applied Science) and then centrifuged (18,000 × g for 10 min at 4 °C). The resulting supernatants were then normalized to a protein concentration of 1 mg/ml protein, boiled in Laemmli sample buffer, and subjected to ECL-Western blotting using antibody to SmgGDS or actin. The expression of mRNAs for SmgGDS-558 and SmgGDS-607 in a panel of archival lung tumors and matched normal tissue was determined by quantitative real time PCR using methods described previously (31Regala R.P. Thompson E.A. Fields A.P. Cancer Res. 2008; 68: 5888-5895Crossref PubMed Scopus (70) Google Scholar). Anchorage-independent proliferation of the NSCLC cells NCI-H1703 and NCI-H23 was analyzed by their ability to form colonies in soft agar. 7 × 104 cells were transfected with siRNA or treated with transfection reagents only (mock transfection). After 24 h, cells were lifted, and 9.9 × 103 cells were suspended in 0.3% low-melt agarose (Bio-Rad) in complete RPMI. Triplicate wells were plated with 1 ml of the cell suspension over 1 ml of solidified 0.6% low-melt agarose in RPMI. After the 0.3% agarose layer set, each well was overlaid with 2 ml of complete RPMI. Digital images of the cells were collected 4 weeks (NCI-H1703) or 5 weeks (NCI-H23) later, and the numbers of visible colonies in the agar were counted using ImageJ. As described previously (32Hancock J.F. Methods Enzymol. 1995; 255: 237-245Crossref PubMed Scopus (33) Google Scholar), cells were lysed in 1% Triton X-114 in TBS (50 mm Tris, 150 mm NaCl, pH 7.5), incubated 15 min on ice, and then centrifuged at 25,000 × g at 4 °C to remove insoluble debris. An aliquot of the cleared lysate was retained as total cell lysate. The remaining lysate was subjected to temperature-dependent separation of the aqueous and detergent phases (2 min of incubation at 37 °C followed by room temperature centrifugation at 1500 × g). After separation of the aqueous and detergent phase, the aqueous phase was transferred to a separate microcentrifuge tube, and 11% Triton X-114 was added to a final concentration of 1%. Any remaining aqueous sample was then scavenged from the detergent phase and discarded. This step resulted in a loss of both detergent and aqueous sample relative to total cell lysate, visible as a disproportionate isolation of lamin B1 in the detergent fractions and GAPDH in aqueous fractions compared with the lamin B1 and GAPDH in the total cell lysate. TBS was then added to the detergent phase to a final concentration 1% Triton X-114. The detergent fractions, aqueous fractions, and total cell lysate were combined 1:1 with 2× Laemmli sample buffer, and equal volumes of each fraction and the total cell lysate were separated by SDS-PAGE and examined by ECL-Western blotting. Proteins were detected with mouse HA antibody to detect SmgGDS isoforms and small GTPases, GAPDH antibody (an aqueous phase loading control), and rabbit antibody to lamin B1 (a detergent phase loading control). OD values of the GTPases detected in the aqueous, detergent, and total cell lysate samples were measured on an AlphaImager HP and analyzed using AlphaView SA software (Alpha Innotech, San Leandro, CA). As discussed above, we observed in some experiments that there was disproportionate isolation of the detergent fraction (containing lamin B1) compared with the aqueous fraction (containing GAPDH). Therefore, all OD values for the detergent fraction were normalized by the factor (OD lamin in detergent phase/OD lamin in total cell lysates). Similarly, all OD values in the aqueous phase were normalized by the factor (OD GAPDH in aqueous phase/OD of GAPDH in the total cell lysate). NCI-H1703 cells stably expressing tet-inducible shRNAs were cultured with or without tetracycline for 72 h, transfected with cDNA encoding GFP-tagged Rap1A, and 24 h later examined by fluorescence microscopy as described previously (30Lanning C.C. Daddona J.L. Ruiz-Velasco R. Shafer S.H. Williams C.L. J. Biol. Chem. 2004; 279: 44197-44210Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The sample identities were masked, and two investigators independently ranked the membrane localization of GFP-Rap1A on a scale of 1–3, a score of 1 representing undetectable, 2 moderate, and 3 extensive membrane localization. All results are the means ± S.E. Symbols above a column indicate a statistical comparison between the bracketed sample by analysis of variance with Dunnett's post hoc multiple comparison's test, by Student's t test, or by χ2 analysis, as indicated in the figure legends. p values less than 0.05 were considered significant. SmgGDS (also known as smgGDS and Rap1GDS1) was originally isolated from bovine brain (33Kaibuchi K. Mizuno T. Fujioka H. Yamamoto T. Kishi K. Fukumoto Y. Hori Y. Takai Y. Mol. Cell. Biol. 1991; 11: 2873-2880Crossref PubMed Scopus (80) Google Scholar) and human brain (34Kikuchi A. Kaibuchi K. Hori Y. Nonaka H. Sakoda T. Kawamura M. Mizuno T. Takai Y. Oncogene. 1992; 7: 289-293PubMed Google Scholar) as a cDNA encoding a 558-residue protein (NCBI accession NP_001093899, isoform 5). In addition to this previously described protein, which we refer to as SmgGDS-558, the NCBI Database also lists a 607-residue SmgGDS splice variant (NCBI accession NP_001093897, isoform 3), which we designate as SmgGDS-607. Both SmgGDS-607 and SmgGDS-558 consist mainly of predicted ARM repeats, with SmgGDS-607 differing from SmgGDS-558 by the addition of only one ARM repeat (Fig. 1A; supplemental Fig. S1). Western blotting of NSCLC cell lysates using a monoclonal SmgGDS antibody detects two proteins with apparent molecular masses of 55 and 60 kDa (Fig. 1B). We suspected that the 55-kDa protein corresponds to SmgGDS-558 and the 60-kDa protein corresponds to SmgGDS-607. To confirm the identities of these immunoreactive proteins, we designed siRNA duplexes to target either both SmgGDS splice variants simultaneously (siRNAs I1 and I2), only SmgGDS-607 (siRNAs C1 and C2), or only SmgGDS-558 (siRNA BD) (Fig. 1A; supplemental Table S1). Transfection of two NSCLC cell lines, NCI-H1703 and NCI-H23 (Fig. 1B), with siRNAs I1 and I2 decreases expression of both proteins detected by the SmgGDS antibody. siRNA BD decreases only the 55-kDa protein, and siRNAs C1 and C2 decrease only the 60-kDa protein. These results confirm the identities of the 55- and 60-kDa immunoreactive proteins and demonstrate that both SmgGDS-558 and SmgGDS-607 are expressed in NSCLC cell lines (Fig. 1B). In our previous immunohistochemical analysis of lung tumors and tissues, we found that SmgGDS protein is significantly elevated in NSCLC tumors compared with normal lung tissue (12Tew G.W. Lorimer E.L. Berg T.J. Zhi H. Li R. Williams C.L. J. Biol. Chem. 2008; 283: 963-976Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To determine whether both SmgGDS splice variants are expressed in lung tumors and tissues, we conducted Western blotting of lung tumors and matched normal lung tissue from nine patients (Fig. 1C). Two proteins are detected by the SmgGDS antibody in most of these immunoblotted tissue samples, and the migration of the immunoreactive proteins corresponds to the migration of SmgGDS-607 and SmgGDS-558 (Fig. 1C). The SmgGDS antibody also detects an unidentified 42-kDa protein (p42) in some of the tumor or lung tissue samples (Fig. 1C, samples 2–8), which may be an SmgGDS degradation product or potentially another uncharacterized SmgGDS splice variant. We examined the expression of mRNA transcripts for SmgGDS-558 and SmgGDS-607 using quantitative RT-PCR in a panel of 50 matched samples of lung tumors and normal lung tissues (Fig. 1D). In both the normal tissue samples and in the tumor samples, quantitative RT-PCR analysis detected more abundant SmgGDS-607 mRNA than SmgGDS-558 mRNA (Fig. 1D). This detection of more SmgGDS-607 mRNA than SmgGDS-558 mRNA is consistent with the immunoblotting results, which indicated more detectable SmgGDS-607 protein than SmgGDS-558 protein in the majority of the tumors and normal tissue samples examined (Fig. 1C) as well as in the cultured cells (Fig. 1B). Somewhat surprisingly, we observed that neither SmgGDS-558 mRNA expression nor SmgGDS-607 mRNA expression is elevated in the tumors compared with the matched normal lung tissues (Fig. 1D). The finding that SmgGDS mRNA is not significantly elevated in the tumors was somewhat unexpected, based on our earlier report that SmgG" @default.
• W2069369960 created "2016-06-24" @default.
• W2069369960 creator A5009935794 @default.
• W2069369960 creator A5031310762 @default.
• W2069369960 creator A5039652334 @default.
• W2069369960 creator A5043876626 @default.
• W2069369960 creator A5055139554 @default.
• W2069369960 creator A5081306238 @default.
• W2069369960 creator A5081449694 @default.
• W2069369960 date "2010-11-01" @default.
• W2069369960 modified "2023-10-01" @default.
• W2069369960 title "Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization" @default.
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