The Activation of TC10, a Rho Small Gtpase, Contributes to V-Rel-Mediated Transformation

Oncogene (2007) 26, 2318–2329 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE The activation of TC10, a Rho small GTPase, contributes to v-Rel-mediated transformation

S Tong, AS Liss, M You1 and HR Bose Jr

Section of Molecular Genetics and Microbiology and the Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA

v-Rel is the oncogenic member of the Rel/NF-jB family of Introduction transcription factors andtransforms hematopoietic cells andfibroblasts. Differential display was employedto v-rel is an acutely transforming oncogene that belongs identify target genes that exhibit altered expression in to the Rel/NF-kB family of transcription factors v-Rel transformedcells. One of the cDNAs identified (Hrdlickova´ et al., 1999). Rel/NF-kB proteins are encodes the chicken ortholog of TC10, a member of the involved in multiple cellular processes including immune Rho small GTPase family. The expression of TC10 was and inflammatory responses, cell growth control, increasedin v-Rel-transformedchicken embryonic fibro- embryonic development, stress responses and apoptosis blasts (CEFs) 3 to 6-foldrelative to control cells at both (Pahl, 1999; Perkins, 2000).In most cell types, Rel/NF- the RNA andprotein levels. An elevatedlevel of active, kB dimers are sequestered in the cytoplasm by associa- GTP-boundTC10 was also detectedinv-Rel-transformed tion with IkB inhibitory molecules.Appropriate cells relative to control cells. Expression of a dominant- extracellular signaling leads to the proteolysis of IkB negative TC10 mutant (TC10T32N) decreased the colony resulting in the translocation of Rel/NF-kB complexes formation potential of v-Rel-transformedcells. Further- to the nucleus where they bind to decameric kB sites in more, overexpression of wild-type TC10 or a gain-of- the promoters of target genes to regulate their expres- function mutant (TC10Q76L) greatly enhancedthe ability sion.The v- rel oncogene was derived by a nonhomolo- of v-Rel transformedCEFs to form colonies in soft agar. gous recombination event between the turkey c-rel In addition to enhance the transformation potential of v- proto-oncogene and an avian retrovirus.Multiple Rel, the overexpression of wild-type TC10 or the gain-of- mutations in v-Rel contribute to its oncogenicity by function mutant alone enhancedthe saturation densityof altering its DNA binding specificity and transactivation CEFs and was sufficient for their anchorage-independent potential relative to c-Rel (Bose, 1992; Gilmore, 1999). growth in vitro. These results indicate that elevated TC10 These mutations also render v-Rel less sensitive to IkBa activity contributes to v-Rel-mediated transformation of inhibition allowing for its constitutive nuclear access in CEFs anddemonstrate for the first time that a Rho factor the absence of an appropriate exogenous signal alone is capable of inducing the in vitro transformation of (Hrdlickova´ et al., 1995; Schatzle et al., 1995; Nehyba primary cells. et al., 1997; Sachdev and Hannink, 1998). Oncogene (2007) 26, 2318–2329. doi:10.1038/sj.onc.1210023; v-Rel transforms cells of hematopoietic origin and published online 2 October 2006 embryonic fibroblasts in vitro (reviewed by Bose (1992); Gilmore, (1999)).The ability of v-Rel to transform these Keywords: v-Rel; NF-kB; TC10; Rho; GTPase; trans- cells is characterized by their abilities to form colonies in formation soft agar and induce tumors in vivo.In addition, fibroblasts transformed by v-Rel undergo distinct morphological changes and have a prolonged lifespan. Transformation by v-Rel is mediated by the inappropri- ate activation or suppression of genes, which are normally regulated by Rel/NF-kB proteins.Efforts to identify genes induced by v-Rel have led to the identification of more than two dozen genes (You and Correspondence: Dr HR Bose Jr, Section of Molecular Genetics and Bose, 1998; Gilmore, 1999; Hrdlickova´ et al., 1999, Microbiology and the Institute of Cellular and Molecular Biology, 2001; Nehyba et al., 2002). However, only a few of these University of Texas at Austin, 1 University Station A5000, Austin, TX 78712-1095, USA. genes have been shown to contribute to the transformed E-mail: [email protected] phenotype.These include a cell surface adhesion 1Current address: Department of Hematopathology, Division of molecule (DM-GRASP), the chemokine Mip-1b,an Pathology and Laboratory Medicine, UT M.D. Anderson Cancer inhibitor of apoptosis (chIAP-1), transcription factors Center, 1515 Holcombe Boulevard-Unit 72, Houston, TX 77030, USA. belonging to the AP-1 (c-Fos and c-Jun) and interferon Received 15 March 2006; revised 15 June 2006; accepted 11 July 2006; response factor (IRF-4) families, and the reverse published online 2 October 2006 transcriptase of telomerase (TERT) (Zhang et al., TC10 contributes to v-Rel-mediated transformation S Tong et al 2319 1995; Petrenko et al., 1997; You et al., 1997; Kralova Results et al., 1998; Hrdlickova´ et al., 2001, 2006). Here, we report that TC10, a Rho family member, is Isolation and cloning of chicken TC10 (TC10) upregulated in, and contributes to the transformation of Differential display was employed to identify genes with fibroblasts by v-Rel.The Rho family consists of 22 altered expression in cells transformed by the v-rel proteins that are involved in signal transduction path- oncogene.Differentially expressed cDNA fragments ways which control a variety of biological functions were identified by comparing the mRNA expression including actin cytoskeleton remodeling, vesicle traffick- profiles from v-Rel-expressing cells with those of control ing, cell polarity, cell adhesion, cell migration, prolifera- cells.A 467 bp cDNA fragment was identified with tion, differentiation, apoptosis and transformation significantly elevated expression in v-Rel-transformed (reviewed by Symons (1996); Ridley (1997); Tapon and CEFs relative to control cells.Sequencing of this gene Hall (1997); Hall (1998); Mackay and Hall (1998); Zohn fragment revealed that it contained 408 bp of 30UTR et al.(1998); Nobes and Hall (1999); Ellis and Mellor and 59 bp of coding sequence that is highly homologous (2000); Evers et al.(2000); Aznar and Lacal (2001); with the Rho small GTPase, TC10.Previous studies had Ridley (2001a, b); Frame and Brunton (2002)).Rho indicated that certain Rho small GTPases are important GTPases are active when bound to GTP and inactive in the regulation of cell proliferation and transformation when bound to GDP.The exchange of GDP to GTP is (Zohn et al., 1998). Therefore, we isolated the full-length catalysed by guanine nucleotide exchange factors open reading frame (ORF) of chicken TC10 (TC10) for (GEFs) and the hydrolysis of GTP is catlysed by further studies.The 5 0 end of the TC10 ORF was GTPase-activating proteins (GAPs) (reviewed by Ber- isolated by 50RACE from an adapter-ligated cDNA nards and Settleman (2005); Rossman et al.(2005)).The library generated from v-Rel-expressing CEF cultures. GTPase activity of Rho proteins is further regulated by The complete TC10 ORF is 642 nucleotides in length guanine nucleotide dissociation inhibitors (GDIs) (Figure 1a).There are two potential translation start (reviewed by Dransart et al.(2005)).These proteins codons.The predicted protein translated from the first inhibit the activation of RhoGTPases by preventing both start codon has 214 amino acids with a molecular weight the release of GDP as well as their localization to the of 23.5 kDa. The second start codon is located 9 amino plasma membrane where they are activated by GEFs. acids downstream of the first start codon and has a TC10 has been cloned from vertebrates (human, perfect Kozak sequence for translation initiation mouse and rat), but is not present in budding yeast, (Kozak, 1987).The predicted protein translated from suggesting that TC10 only functions in differentiated this codon is 205 amino acids in length with an cells (Drivas et al., 1990; Neudauer et al., 1998; Tanabe estimated molecular weight of 22.8 kDa. Finally, the et al., 2000; Chiang et al., 2002). TC10 is predominantly 408 bp 30UTR contains several potential polyadenyla- localized on the plasma and endosomal membranes tion signals upstream of the poly (A) tract. (Michaelson et al., 2001) and has been implicated in The predicted protein sequence of TC10 (from the various biological functions.TC10 has been reported to first start codon) shares 93% identity with human TC10 function in actin cytoskeleton organization (Neudauer (Figure 1b).TC10 contains four highly conserved GTP et al., 1998; Murphy et al., 1999), nerve regeneration binding domains, a Rho insert domain, effector domain (Tanabe et al., 2000), and the activation of various and the C-terminal CAAX box.It also contains a kinases and transcription factors (Murphy et al., 1999; plasma membrane targeting motif (CXXC) and a Murphy et al., 2001). In addition, the involvement of polybasic region in the hypervariable C-terminus, which TC10 in the translocation of glucose transporter 4 are believed to be important for membrane targeting (GLUT4) upon insulin stimulation in muscle cells and and regulation (Michaelson et al., 2001). The closest adipocytes has been extensively characterized (Chiang related Rho factor to TC10 is TCL, which shares 79% et al., 2001; Jiang et al., 2001; Kanzaki and Pessin, 2001; identity.Compared with other chicken Rho GTPases, Watson et al., 2001). Furthermore, TC10 cooperates TC10 shares 67% identity with Cdc42, 65% with Rac1, with activated Raf to transform fibroblasts and was 63% with Rac2 and Rac3, 53% with RhoA, RhoB and shown to be important in Ras-mediated transformation RhoC and 45% with RhoF. of NIH3T3 cells (Murphy et al., 1999). However, little is known about the transforming potential of TC10. In this report, we describe the identification and RNA levels of TC10 are elevated in Rel transformed cells cloning of the chicken ortholog of TC10.The expression To characterize the altered expression of TC10 in v-Rel and the activity of TC10 are upregulated in fibroblasts transformed cells, Northern blot analysis was performed expressing v-Rel.Both wild-type TC10 and a gain-of- using RNA isolated from CEFs transformed by a function mutant cooperate synergistically with v-Rel to retrovirus expressing v-Rel (DSv-Rel) or cells infected transform fibroblasts.Moreover, a dominant-negative with a control virus (DS3).A TC10-specific probe mutant of TC10 dramatically inhibits the ability of v- detected four RNA species (4.2 kb, 1.9 kb and two high Rel to transform fibroblasts in culture.In addition to its molecular weight species) that were expressed at 3 to 6- role in v-Rel-mediated transformation, we demonstrate fold higher levels in cells transformed by v-Rel relative that the overexpression of either TC10 or a gain-of- to control cells (Figure 2a).These four species of TC10 function mutant of TC10 is sufficient to promote the likely represent alternatively spliced forms of TC10 since transformation of primary fibroblasts in vitro. they were detected with probes encompassing either the

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2320

a 1 ATGCCCGGGGGAGGGCGGGCTCCGGCCATGGCCCATGGCAGCGCCGCGGTCATGCTGAAGTGCGTGGTGGTGGGGGACGGCGCCGTGGGCAAGACGTGC M P G G G R A P A M A H G S A A V M L K C V V V G D G A V G K T C 33 100 CTGCTGATGAGCTACGCCAACGACGCCTTCCCTGAGGAGTACGTGCCCACCGTCTTCGACCACTACGCAGTCAGCGTTACCGTGGAGGGCAAGCAGTAC L L M S Y A N D A F P E E Y V P T V F D H Y A V S V T V E G K Q Y 66 200 CTGCTGGGGCTCTACGACACCGCCGGGCAGGAAGACTATGACCGTCTGAGACCTTTATCTTATCCTATGACCGATGTCTTCCTTATCTGCTTCTCAGTG L L G L Y D T A G Q E D Y D R L R P L S Y P M T D V F L I C F S V 99 300 GTAAACCCTGCTTCATTTCAAAACGTGAAGGAAGAATGGGTACCGGAGTTGAAGGAATATGCACCTAATGTTCCTTTTTTACTAGTAGGAACACAGATT V N P A S F Q N V K E E W V P E L K E Y A P N V P F L L V G T Q I 132 400 GATCTTCGTGATGACCCCAAAACTCTGGCAAGATTGAATGATATGAAAGAGAAGCCTTTATCTGTGGAACAAGGACAGAAATTAGCAAAAGAGATAGGA D L R D D P K T L A R L N D M K E K P L S V E Q G Q K L A K E I G 165 500 GCCTACTGCTATGTGGAGTGTTCAGCTTTAACACAGAAAGGACTGAAGACTGTTTTTGATGAAGCTATTATAGCCATTCTAACTCCAAAGAAACACACG A Y C Y V E C S A L T Q K G L K T V F D E A I I A I L T P K K H T 198 600 GTGAAGAAGAGAATAGGTTCGAGATGCATAAACTGCTGTTTGATCACGTGAGATACATTTGACAATGGCCAGGCTTACTGTGAAATTCTACTGAATTTA V K K R I G S R C I N C C L I T * 231 700 AATACAACGCGTTAAGTACACAGCAAACTTGTTTGAAAGCTAAAATAATGATTCTGCCTTCTACAACCAGTGAAATCCAGAGGAATACAATCAAAATCA 800 ATGAACTTTGTAATAAGAAGCCACCACATCTGTTACAAATATTTTATTCAGACTGGAAGGTACAATCTCTCAGGGTTACATAGTCTAGAGGAAGCAAAA 900 ACTGCACCATGTTGAAACAGCATCTGTACGATGCCATTGAGTGGAGGTGCTTGGCCTGTAATACTCTAAATGAATTTGGATAGTCTTCTGCTATGACTA 1000 CACATATACATATATATCTAGAGAAAAAACAACAACAACAACAACAAAAAAAAAAAGCTT

b G1 effector domain G2 chTC10 1 ------MPGGGRAPAMAHGSA---AVMLKCVVVGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVSVTVEGKQYLLGLYDTAGQEDYDRLRPLSYPMTDVFLICFSVVNP hTC10 1 ------MPGAGRS-SMAHGPG---ALMLKCVVVGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVSVTVGGKQYLLGLYDTAGQEDYDRLRPLSYPMTDVFLICFSVVNP mTC10 1 ------MAHGPG---ALMLKCVVVGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVSVTVGGKQYLLGLYDTAGQEDYDRLRPLSYPMTDVFLICFSVVNP TCL 1 ------MSCKAEGDDCTDGSSNSTKKMLKCVVVGDGAVGKTCLLMSYANDAFPEEYVPTVFDHYAVTVTVGGQQHLLGLYDTAGQEDYNQLRPLSYPNTDVFLICFSVVNP CDC42 1 ------MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSP RhoA 1 ------MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSP RhoB 1 ------MAAIRKKLVVVGDGACGKTCLLIVFSKDEFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSVDSP RhoC 1 ------MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYIADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSP RhoF 1 MEAANGAVPDGAAGGKGGSAAAPSGKKEVKMVIVGDGGCGKTSLLMVYAKGAFPEQYAPSVFEKYATSITIGKKEVILNLYDTAGQEDYDRLRPLSYQNTNVVLICYDVMNP hRhoG 1 ------MQSIKCVVVGDGAVGKTCLLICYTTNAFPKEYIPTVFDNYSAQSAVDGRTVNLNLWDTAGQEEYDRLRTLSYPQTNVFVICFSIASP Rac1 1 ------MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSP Rac2 1 ------MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDSKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSP Rac3 1 ------MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSP

Rho insert G4 CAAX G3 chTC10 103 ASFQNVKEEWVPELKEYAPNVPFLLVGTQIDLRDDPKTLARLNDMKEKPLSVEQGQKLAKEIGAYCYVECSALTQKGLKTVFDEAIIAILTPKKHTVKKRIGSRCINCCLIT hTC10 102 ASFQNVKEEWVPELKEYAPNVPFLLIGTQIDLRDDPKTLARLNDMKEKPICVEQGQKLAKEIGACCYVECSALTQKGLKTVFDEAIIAILTPKKHTVKKRIGSRCINCCLIT mTC10 94 ASFQNVKEEWVPELKEYAPNVPFLLIGTQIDLRDDPKTLARLNDMKEKPVCVEQGQKLAKEIGACCYVECSALTQKGLKTVFDEAIIAILTPKKHTVKKRIGSRCINCCLIT TCL 106 ASYHNVQEEWVPELKVCMPNVPYVLIGTQIDLRDDPKTLARLLYMKEKPLTYEHGVKLAKEIGAQCYLECSALTQKGLKTVFDEAILTIFHPKK---KKKHCAKCHRCCTIV CDC42 88 SSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQKGLKNVFDEAILAALEPPE------PKKTRRCVLL- RhoA 90 DSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARR----GKKK---SGCLLL- RhoB 90 DSLENIPEKWVPEVKHFCPNVPIILVANKKDLRNDEHVRNELARMKQEPVRTEDGRAMAIRIQAYDYLECSAKTKEGVREVFETATRAALQKRY----GTQNGCINCCKVL- RhoC 90 DSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRINAFGYLECSAKTKEGVREVFEMATRAGLQVRK----NKKR---RGCPLL- RhoF 113 TSYDNVADKWYPEVNHFCQGVPLVLIGCKTDLRKDKEQLRKLRASKQEPITYNQGEAACQQINAEIYLECSAKCRENIENVFKEATTIALSAMK---KAKRQKKQRACSVL- hRhoG 88 PSYENVRHKWHPEVCHHCPDVPILLVGTKKDLRAQPDTLRRLKEQGQAPITPQQGQALAKQIHAVRYLECSALQQDGVKEVFAEAVRAVLNPTP------IK-RGRSCILL- Rac1 88 ASFENVRAKWYPEVRHHCPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRAVLCPPP------VKKRKRKCLLL- Rac2 88 ASYENVRAKWFPEVRHHCPSTPIILVGTKLDLRDDKDTIEKLKEKKLSPITYPQGLALAKEIDSVKYLECSALTQRGLKTVFDEAIRAVLCPQP------TRTKKRACSLL- Rac3 88 ASFENVRAKWYPEVRHHCPNTPIILVGTKLDLRDDKDTIERLRDKKLAPITYPQGLAMAREIGSVKYLECSALTQRGLKTVFDEAIRAVLCPPP------VKKPGKKCTVF- Figure 1 Sequence of TC10 cDNA.( a) Nucleotide and predicted amino-acid sequence of the TC10 ORF and 30UTR.Two potential translation start codons (ATG) are shaded.Underlined nucleotides indicate the location of the sequences complimentary to the primers used to obtain the full-length ORF.Highlighted amino acids indicate the location of amino acids that were mutated to construct the gain-of-function (TC10Q76L) and dominant-negative (TC10T32N) mutants.The polyadenylation signal is underlined and in italics. (b) Homology of TC10 with other Rho small GTPases.The protein sequence of human TC10 (hTC10) and RhoG (hRhoG), mouse TC10 (mTC10) and chicken TCL, Cdc42, RhoA, RhoB, RhoC, RhoF, Rac1, Rac2 and Rac3 are compared.The black shadings represent amino acids that are identical in at least half of these proteins.The gray shadings represent conserved amino acids.The locations of the predicted GTP binding/hydrolysis domains (G1-4), Rho effector domain and Rho insert domain of TC10 are indicated.The locations of the C-terminal CXXC membrane-targeting motif (boxed) and CAAX box are also indicated.

3’UTR or the entire ORF.The kinetics of TC10 expressed in three histologically distinct v-Rel trans- expression during v-Rel transformation was also exam- formed cell lines, B-cell lines DT95 and DT40 and a ined.CEFs were infected with DS3 or DSv-Rel and total macrophage cell line BM2.By contrast, TC10 was RNA was isolated at different times after infection.Two poorly expressed in the Marek’s disease virus trans- days after infection, the expression of TC10 was formed T-cell lines, MSB-1 and RP-1. increased two-fold in cells expressing v-Rel relative to The elevated expression of other Rho family members control cultures (Figure 2b).This is much earlier than has been correlated with oncogenesis in a variety of cells become morphologically transformed by v-Rel systems.To determine whether the upregulation of (8–10 days after infection).The mRNA levels continued TC10 mRNA in v-Rel transformed cells is unique, the to increase until the 10th day after infection and expression of avian Rho GTPases was evaluated in maintained the same level at least 45 days after infection ﬁbroblasts transformed by the expression of v-Rel or the (data not shown).As v-Rel also transforms lymphoid overexpression of c-Rel (Figure 3).The expression of cells, we examined the expression of TC10 in several TC10 was induced almost three-fold in cells transformed hematopoietic cell lines by Northern blot analysis by v-Rel and c-Rel relative to control cells.The (Figure 2c).TC10 was most highly expressed in AEV- expression of RhoC was elevated three-fold in v-Rel- 1, a transformed erythroid cell line.TC10 was also transformed cells but was not signiﬁcantly altered in

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2321 (KGPKTLARLNDMKEKPLS) which contains the Rho insert domain (underlined, amino acids 138–153) of TC10.To verify the specificity of the antisera, immunoprecipitation experiments were performed with in vitro translated chicken TC10 and the closely related RhoGTPase, TCL.The in vitro translated proteins were heat denatured in the presence of sodium dodecyl sulfate (SDS) to expose all available epitopes, and immunopre- cipitated with antisera against the TC10-specific peptide (Figure 4a).Only the in vitro translated TC10 was detected in the immunopreciptiates from these experiments.This antisera was employed to determine the steady-state level of TC10 protein in c-Rel and v-Rel transformed cells by Western blot analyses (Figure 4b, top panel).A 20–25 kDa protein was detected in whole- cell lysates from CEF cultures, which corresponds to the predicted size of TC10.The endogenous level of TC10 was very low in control CEFs, and increased approximately 3 to 6-fold in both c-Rel and v-Rel transformed CEFs.The increase in the level of TC10 corresponded with the increase in the steady-state level of TC10 mRNA in these transformed cells (Figure 4c). The GTP-bound form of TC10 is biologically active and able to bind downstream effectors to exert its function.To determine if the activity of TC10 was increased in response to the increase in expression of Figure 2 Expression of TC10 RNA in CEFs.( a) Total RNA was isolated from v-Rel transformed CEFs (V) or CEFs infected with TC10 in Rel-transformed cells, a GST-CRIB (Cdc42 control DS3 virus (DS).RNA (10 mg) was analysed for TC10 and Rac interactive binding) domain pull-down assay expression by Northern blot.The four mRNA species hybridizing was performed.The CRIB domain is a conserved with the TC10 probe are indicated on the right of the top panel. sequence identified in a variety of downstream effectors Methylene blue stained 18S ribosomal RNA is shown in the lower panel as a control for loading.( b) Time course expression of TC10 of Cdc42 and Rac and the CRIB domain of PAK1 was in CEFs infected with DS3 or DSv-Rel viruses.Total RNA (15 mg) demonstrated to specifically bind to the GTP-bound was isolated 2, 3 and 4 days after infection.The hybridization of forms of human TC10, Cdc42 and Rac (Benard et al., viral genomic RNA with a v-rel specific probe is shown in the 1999; Murphy et al., 1999). GST-CRIB domain agarose middle panel.Ethidium bromide stained 18S ribosomal RNA is beads were incubated with cell extracts prepared from shown in the lower panel as a control for loading.( c) Expression of TC10 in transformed cell lines.Total RNA was isolated from control CEF cultures or those transformed by c-Rel or transformed cell lines of hematopoietic origin including the v-Rel and the amount of GTP-bound TC10 pulled- erythroblastoid cell line AEV-1 (AEV), S2A3v-Rel transformed down by the beads was detected by immunoblotting splenic B-cell line C4-1, B-cell lines DT40 and DT95, myeloblastoid using TC10-specific antiserum.Significantly more GTP- cell line BM2, T-cell lines MSB-1 (MSB) and RP-1, v-Rel transformed B-cell line 123/12, T-cell line 160/2 and non-B/non- bound TC10 was detected in both c-Rel and v-Rel T-cell line 123/6 T.Ethidium bromide stained 18S ribosomal RNA transformed cells relative to control cells, indicating that is shown in the lower panel as a control for loading. the activity of TC10 is increased in Rel-transformed CEF cultures (Figure 4, middle panel).Two forms of TC10 were observed in these assays and the lower molecular weight form was the same size as the TC10 cells transformed by c-Rel.The expression of other Rho protein detected in whole-cell extracts.Treatment of family members was not dramatically altered in cells lysates with protein phophatase did not prevent the transformed by v-Rel, although TCL and RhoG appearance of the higher molecular weight form (data exhibited reduced expression in c-Rel transformed cells not shown).TC10 is one of a few RhoGTPases that can relative to control cells.These results demonstrate that be modified by isoprenylation (either geranylgeranyla- the elevated expression of TC10 in both v-Rel- and c- tion or farnesylation) at its C-terminal CAAX motif as Rel-transformed cells is unique among the Rho small well as palitoylation of the polybasic sequence upstream GTPases and suggests a role for it in transformation. of this motif (Michaelson et al., 2001). Therefore, it is likely that the two forms of TC10 represent different C-terminal post-traslational modifications. The expression and activityof TC10 protein is upregulated in Rel-transformed CEFs To determine whether the increase in TC10 RNA levels Expression of TC10, TC10Q76L, TC10T32N and in v-Rel-transformed cells results in a corresponding coexpression with v-Rel in CEFs increase at the protein level, a rabbit polyclonal The increased levels of mRNA, protein, and activity of antiserum was generated against a synthetic peptide TC10 in Rel-transformed CEF cultures suggests that

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2322

Figure 3 Expression of Rho family members in c-Rel- and v-Rel-transformed CEFs.( a) CEFs were infected with control DS3 virus (DS), DS3 expressing c-Rel (C), or DS3 expressing v-Rel (V).Total RNA was isolated after cultures infected with viruses expressing c- Rel and v-Rel became transformed.RNA (10 mg) was analysed for the expression of TC10, TCL, cdc42, rhoA, rhoB, rhoC, rhoG, rac1, rac2 and rac3 by Northern blot (top panels).Hybridization of an oligonucleotide to the 18S ribosomal RNA is shown in the lower panels as a control for loading.The probe used in each hybridization is indicated to the right of each panel.Only the 4.2kb mRNA hybridizing to the TC10 probe is shown.The relative abundance of each mRNA was determined by phosphorimager analysis after standardizing for RNA loading with 18S rRNA and is indicated below the 18S rRNA panels.

TC10 may be important in Rel-mediated transforma- gain-of-function mutant or wild-type TC10 in cells tion.To evaluate the role of TC10 in transformation, expressing v-Rel resulted in higher levels of GTP-bound mutants of TC10 with altered biochemical activities TC10 than observed in cells expressing v-Rel alone. were constructed.Gain-of-function (TC10Q75L) and Coexpression of the dominant-negative mutant pre- dominant-negative (TC10T31N) mutants of human vented the induction of endogenous, activated TC10 by TC10 have been previously described (Murphy et al., v-Rel.Due to the high levels of activated TC10 present 1999; Neudauer et al., 1998). TC10Q75L is unable to in cells overexpressing the gain-of-function TC10, only hydrolyze bound GTP, resulting in a constitutively one fourth the amount of cell extract used in Figure 4 active protein.TC10T31N does not bind GTP and only was used in these assays and therefore, the elevated inefficiently binds GDP.The expression of this domi- levels of activated TC10 in extracts from v-Rel nant-negative mutant is believed to block endogenous transformed cells are not evident. TC10 function by sequestering GEFs.The corresponding mutations in chicken TC10 (TC10Q76L and TC10T32N) were made by site-directed mutagenesis. TC10 induces in vitro transformation characteristics in These two mutants and wild-type TC10 were cloned into primaryfibroblasts and contributes to v-Rel-mediated an SNV-based retroviral vector, which allowed the transformation expression of a gene encoding neomycin resistance and To evaluate whether TC10 contributes to transforma- each of the TC10 proteins.To verify the biochemical tion of fibroblasts by v-Rel, TC10 activity was either activities of these TC10 proteins, CEF cultures were increased or decreased by the overexpression of wild- coinfected with the viruses expressing TC10, TC10Q76L type TC10, the gain-of-function mutant, or the domi- or TC10T32N, and a control virus (DS3) or a virus nant-negative mutant in v-Rel expressing fibroblasts. expressing v-Rel (DSv-Rel).Cells were grown under CEF cultures were coinfected with both SNV-based G418 selection to maintain expression of the viruses viruses expressing the various TC10 genes and DS3 encoding TC10 proteins.Cell lysates were prepared 20 retroviruses expressing v-Rel as described above.Stable days after infection, and the expression of TC10 and v- cell cultures were selected with G418 for 15 days and Rel was determined by Western blot analysis their ability to form colonies in soft agar was evaluated (Figure 5a).Each of the forms of TC10 was expressed (Figure 5b).Both TC10 and the gain-of-function mutant at levels higher than the endogenous protein (top panel). synergistically cooperated with v-Rel to increase colony The expression of TC10 proteins did not alter the levels formation in soft agar (four- and seven-fold, respec- of v-Rel (bottom panel).The relative amount of GTP- tively).Moreover, coexpression of the dominant-nega- bound TC10 in each of these cells was determined by tive mutant of TC10 with v-Rel dramatically decreased GST-CRIB domain pull-down assays (middle panel).As (78%) the number of colonies relative to cells expressing expected, cells expressing the gain-of-function TC10 had v-Rel alone.These results indicate that TC10 contri- high levels of the GTP-bound protein, while cells butes to v-Rel-mediated transformation of fibroblasts. expressing the dominant-negative mutant did not Previous studies have shown that the activated form contain detectable levels of the GTP-bound form.Cells of human TC10, while not transforming alone, can overexpressing wild-type TC10 had intermediate cooperate with activated Raf to transform NIH3T3 levels of GTP-bound protein.Overexpression of the cells (Murphy et al., 1999). In contrast, our studies

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2323 of the overexpression of TC10 on primary fibroblast cultures, saturation density experiments were performed (Figure 5c).The overexpression of TC10 increased the saturation density of CEFs by more than 40%, while the expression of the gain of function mutant of TC10 enhanced the saturation density of these cells by two- fold.The combined results of the soft agar colony formation and saturation density experiments demonstrate that overexpression of TC10 is sufficient for the in vitro transformation of primary fibroblasts.

Discussion

In this report, we describe the isolation and characterization of the chicken homolog of TC10.The expression and activity of TC10 is upregulated by the v-Rel oncoprotein and the overexpression of the proto- oncoprotein c-Rel.We further demonstrated that the elevated activity of TC10 is important for v-Rel- mediated transformation of CEFs and that the overexpression of TC10 alone induces the in vitro transformation of primary ﬁbroblasts.

Increases in TC10 expression and activitycontribute to v-Rel-mediated transformation v-Rel transforms cells by inappropriately altering the expression of genes regulated by the Rel/NF-kB family of transcription factors.More than two dozen genes have been identified that exhibit altered expression in response to v-Rel (Gilmore, 1999; Hrdlickova´ et al., 1999).However, only a few of these genes have been shown to contribute to the phenotype of v-Rel- transformed cells.In this report, we demonstrate that TC10 is unique among the Rho family of small GTPases in that it exhibits elevated expression in cells transformed by both the expression of v-Rel and by the Figure 4 The expression and activation status of TC10 in c-Rel- overexpression of c-Rel (Figure 3).Further, the elevated and v-Rel-transformed CEFs.( a) In vitro translated TC10 and TCL levels of TC10 mRNA correspond with an increase in were heat-denatured in the presence of SDS and immunoprecipti- TC10 protein and activity in Rel-transformed cells tated with normal rabbit serum (NRS) or TC10-specific antisera (anti-TC10).Translated proteins (Input) and immunoprecipitates relative to control cells (Figure 4).The coexpression of were resolved by SDS–PAGE and visualized by radiography.( b) TC10 or a gain-of-function mutant of TC10 The levels of TC10 in whole-cell lysates (75 mg) from CEFs (TC10Q75L) synergistically cooperated with v-Rel to transformed by v-Rel, c-Rel or infected with DS3 empty vector enhance the colony forming ability of CEFs (Figure 5). were determined by Western blot analysis using the TC10-specific antiserum ID1 (top panel).The levels of GTP-bound TC10 in these By contrast, the expression of a dominant-negative cells were determined by a GST-CRIB pull-down assay followed by mutant of TC10 (TC10T32N) greatly decreased the Western blot analysis (middle panel).The expression of v-Rel and ability of v-Rel-expressing fibroblasts to form colonies c-Rel in these cell lysates were detected with the monoclonal in soft agar.These results demonstrate that TC10 plays antibody HY87 (bottom panel).( c) Total RNA was isolated from an important role in v-Rel-mediated transformation. the cultures described above.RNA (10 mg) was analysed for the expression of TC10 by Northern blot (top panel).Methylene blue The mechanisms by which TC10 contribute to cell staining of the 18S ribosomal RNA is shown in the lower panel as a transformation, have yet to be defined.Potential down- control for loading. stream effectors of TC10 include p21-activated kinase (PAK), mixed lineage kinase 2, myotonic dystrophy- related Cdc42 kinase, Borgs, N-WASP (Wisktt-Aldrich syndrome protein), and the mammalian partition- demonstrated that the overexpression of wild-type TC10 defective homolog Par6 and CAL (cystic fibrosis alone weakly promoted colony formation in soft agar transmembrane conductance regulator-associated li- (Figure 5b).In addition, the gain-of-function mutant gand) (Neudauer et al., 1998; Joberty et al., 1999; induced colony formation four-fold more efficiently Cheng et al., 2005). In addition, TC10 mediates multiple than wild-type TC10.To further characterize the effects signaling pathways leading to the activation of kinases

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2324 such as c-Jun N-terminal kinase (JNK) and PAK, and have been shown to be required for the ability of TC10 the transcription of reporter constructs containing the to participate in the transformation of cells. cyclin D1 promoter or binding sites for serum response Some known functions of TC10 are enticing possibi- factor (SRF) or NF-kB (Murphy et al., 1999; Murphy lities for its role in v-Rel-mediated transformation. et al., 2001). Although a number of these pathways and Activated TC10 has been shown to induce JNK activity proteins have been implicated in transformation, none and we have previously demonstrated that AP-1 activity is important for anchorage-independent growth of v-Rel-transformed fibroblasts (Kralova et al., 1998). A dominant-negative mutant of c-Jun greatly decreased colony formation of v-Rel-transformed CEFs in soft agar.The demonstrated importance of c-Jun activity suggests that increased activity of JNK, the major kinase for activating c-Jun, may contribute to v-Rel- mediated transformation.However, the expression of TC10Q76L and TC10T32N in v-Rel-transformed CEFs does not alter the activity of JNK in a manner that is consistent with it being activated by TC10 during transformation (data not shown). TC10 has been shown to facilitate the translocation of the glucose transporter GLUT4 from the endosome to the plasma membrane in muscle cells and adipocytes stimulated with insulin (Kanzaki and Pessin, 2001; Watson et al., 2001; Chang et al., 2002). Interestingly, increased glucose uptake is a common characteristic of tumor cells and v-Rel transformed CEFs take up glucose more rapidly than normal fibroblasts (Warburg, 1930; Franklin et al., 1977). Therefore, TC10 may contribute to cell transformation by increasing glucose transport.However, the expression of the dominant- negative mutant of TC10 did not alter the ability of v- Rel transformed cells to uptake 3H-glucose from the culture media (data not shown).Future studies will focus on defining the pathways through which TC10 participates in the transformation of CEFs by v-Rel.

Figure 5 In vitro transformation potential of TC10 in CEFs.( a) The protein levels and the activation status of TC10 in CEFs.Cells were coinfected with the SNV-based viruses (empty vector, vector expressing TC10 (WT), TC10Q76L (GOF) or TC10T32N (DN)) and DS3 or DSv-Rel viruses for 20 days.The TC10 protein levels were determined by Western blot analysis using TC10 (ID1) antiserum (top panel).Proteins (40 mg) in whole-cell extracts were loaded in each lane.Endogenous TC10 could not be detected in cells expressing DS3 or DSv-Rel alone when use this amount of cell extract.The middle panel shows the amount of GTP-bound TC10 as determined by GST-CRIB pull-down assays.Whole-cell extracts (1 mg of protein) from each cell type was incubated with 30 mg GST-CRIB fusion protein for each assay.The bottom panel shows the expression of v-Rel detected by a monoclonal antibody HY87. (b) Soft agar colony formation ability of CEFs.CEF cultures coinfected with the SNV-based viruses (empty vector, vector expressing TC10 (WT), TC10Q76L (GOF) or TC10T32N (DN)) and DS3 (or DSv-Rel) viruses for 15 days were used for this assay. Cells (1 Â 105) were seeded in 0.37% soft agar media colonies were scored 4 weeks after seeding.The mean and s.e.of six independent experiments are shown.( c) Saturation density of CEF cultures. CEF cultures (1 Â 105) infected with control SNV-based viruses or those expressing TC10 or GOF TC10 were seeded on 60 mm culture dishes.Two days after plating, three plates of each infection were counted daily until cells detached from the plate.The average and s.d. of from one of two independent experiments are shown.

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2325 TC10 transforms primaryfibroblasts in culture Hrdlickova´ et al., 2001). Therefore, the ability of TC10 Human TC10 has been implicated in the Ras-mediated to induce the in vitro transformation of CEFs suggests transformation of fibroblast cell lines (Murphy et al., that TC10 may also serve as a marker for tumor 1999).The overexpression of the gain-of-function progression. mutant, TC10Q75L, cooperated with the active form of Raf-CAAX to induce foci formation of NIH3T3 cells, but overexpression of TC10Q75L alone did not. Materials andmethods The dominant-negative mutant, TC10T31N, suppressed the focus formation ability of these cells.These Cloning and mutagenesis of TC10 observations are similar to those obtained for Cdc42, Differential display was performed using total RNA from v- Rac1, RhoA and RhoB (Prendergast et al., 1995; Qiu Rel transformed and control cells as described previously (You et al., 1995, 1997; Osada et al., 1997; Westwick et al., et al., 1997; You and Bose, 1998). A repertoire of cDNA 1997; Whitehead et al., 1998). Moreover, previous fragments was amplified from each cell type using an RNAimage kit (GenHunter Corp., Nashville, TN, USA) with studies indicated that the constitutively active mutants the H-T11G primer and a random primer set (H-AP1 to H- of Cdc42 (G12V) and Rac (G12V) increased the colony AP8).The full-length ORF of TC10 was obtained by 5 0 formation ability of mammalian fibroblast cell lines in RACE.Two adaptor-ligated cDNA libraries were generated soft agar.Although there are a large number of reports with RNA isolated from v-Rel transformed CEFs using the describing the oncogenic potential of various RhoGT- Marathon cDNA amplification kit (Clontech, Palo Alto, CA, Pases, all of these studies were performed with USA) and the SMART cDNA synthesis kit (Clontech) immortalized cell lines, such as NIH3T3 cells (Perona according to the manufacturer’s protocol.A partial sequence et al., 1993; van Leeuwen et al., 1995; del Peso et al., of TC10 was amplified from the Marathon cDNA library by polymerase chain reaction (PCR) using the 50 PCR primer (50- 1997; van Golen et al., 2000; Singh et al., 2004). In 0 contrast to these studies, the experiments described here AAGCAGTGGTATCAACGCAGAGT-3 , Clontech) and the TC10-specific primer CB22 (50-TCACAGTAAGCCTGGC employed primary fibroblast cultures and indicate that CATTGTCA-30).The remaining sequences encoding the N- TC10 alone is sufficient to transform primary fibroblasts terminal domain were isolated from the SMART cDNA in culture.The overexpression of TC10 or TC10Q76L library using the UPMS primer (Clontech) and the TC10- from retroviral vectors allowed for the anchorage- specific primer CB23 (50-CACTCCACATAGCAGTAGGC independent growth of CEFs (Figure 5b).Furthermore, TCCT-30) by touchdown PCR.PCR was performed as follows: the overexpression of TC10Q76L enhanced the satura- 941C for 1 min, five cycles of 941C for 30 s and 721C for 4 min, tion density of CEFs (Figure 5c).This is the first report five cycles of 941C for 30 s and 701C for 4 min, followed by 20– of the overexpression of a Rho small GTPase alone 25 cycles of 941C for 20 s and 681C for 4 min.PCR products inducing the in vitro transformation of primary fibro- were sequenced by an automated Perkin-Elmer ABI PRISM blast cultures. 377 sequencer at the DNA Core Facility at the University of Texas at Austin.The sequence of TC10 reported in this paper Previous studies demonstrated that aberrant expres- was submitted to GenBank with accession number AF372468. sion and activation of Rho small GTPases are important TC10 was cloned into the NotI and SacII sites in pBluescript in tumor generation and progression, although mutation II SK (À).Point mutations in TC10 were made with a of Rho GTPase genes rarely occurs in tumors (Keely, Transformer site-directed mutagenesis kit (Clontech) accord- 2001; Boettner and Van Aelst, 2002).Elevated expres- ing to the manufacturer’s protocol.To make the Q76L and sion of RhoA, RhoC, Rac1B and Rac2 were found in a T31N mutants, mutagenic primers M1 (50-CACCGCCGG variety of cancers including head and neck squamous- GCTGGAAGACTAT-30) and M2 (50-GTGGGCAAGAAC cell cancers, ductal adenocarcinomas of the pancreas, TGCCTGCTGA-30) as well as a selection primer SP2 (50- 0 inflammatory breast cancer cells, colon cancers and lung GGCCCCCCCTTAAGGTCGACGG-3 ) were used.The cancers (Suwa et al., 1998; van Golen et al., 2000; underlined nucleotides define the point mutations. Abraham et al., 2001). Aberrant expression of Rho and Rac have been found in even moderately differentiated Cell lines and tissue culture tumors, suggesting that Rho proteins may be early CEF cultures were prepared from 10 to 11-day-old SPAFAS (Charles River SPAFAS, North Franklin, CT, USA) or SPF- markers of tumor progression (Moscow et al., 1994; SC (Hy-Vac, Adel, IA) embryos and grown in Dulbecco’s Suwa et al., 1998). This report provides the first evidence modified Eagle’s medium (DMEM) supplemented with 5% that the overexpression of TC10 results in transforma- newborn calf serum (Atlanta Biologicals, Norcross, GA, tion of primary cells in culture.The ability of proteins to USA), 5% chicken serum (Invitrogen, Carlsbad CA, USA), transform CEFs has been a powerful prognosticator of 100 U/ml penicillin and 50 mg/ml streptomycin in 8% CO2 at their involvement in the development of human cancer. 371C.The DT40, DT95, BM2, 123/12, 160/2, 123/6 T, MSB-1, Although CEFs bear little resemblance to the cell types RP-1 and AEV-1 cell lines were maintained in the same of most human tumors, the orthologs of a large number conditions as CEFs.AEV-1 is an avian erythroblastosis virus- of wild-type and mutant proteins (such as c-Rel, c-Src, transformed erythroid cell line (Rao et al., 1990). DT95 and c-Myc and IRF-4) that transform CEFs are involved in DT40 are c-myc-transformed chicken B-cell lines (Baba et al., 1985).MSB-1 and RP-1 are T-cell lines derived from the development of a variety of histologically distinct lymphomas induced by Marek’s disease virus (Akiyama and neoplasias, including lymphomas, myelomas, colon and Kato, 1974; Nazerian et al., 1977). BM2 is a macrophage-like breast cancers (Kato et al., 1986; Frykberg et al., 1987; cell line derived from avian myeloblastosis virus (AMV) Kralova et al., 1994; Iida et al., 1997; Nass and Dickson, transformed yolk sac cells (Moscovici et al., 1982). 123/12 1997; Irby et al., 1999; Rayet and Gelinas, 1999; (B-cell), 160/2 (T-cell) and 123/6T (non-B/non-T) are

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2326 v-Rel-transformed cell lines (Hrdlickova´ et al., 1994b). C4-1 is staining, or hybridization with a probe specific for the 18S a B-cell line transformed by S2A3v-Rel and was cultured in rRNA.Membranes were hybridized with [ 32P]dCTP-labeled DMEM supplemented with 6% newborn calf serum, 2% DNA probes using UltraHyb solution (Ambion) at 42 or 551C. chicken serum (Zhang and Bose, 1989; Romero and Humph- Probes were prepared by random priming (Prime-A-Gene kit, ries, 1995).D17 is a dog osteosarcoma cell line and was Promega, Madison, WI, USA) and purified using Micro Bio- maintained in DMEM containing 8% newborn calf serum spin 30 columns (Bio-Rad Laboratories, Hercules, CA).The (Dougherty and Temin, 1988). probes for detecting TC10 were the 467 bp cDNA identified by differential display or the whole TC10 ORF.Probes for chicken Rho family member were derived from the cDNAs Retroviral vectors of CDC42 (GI:45384261), RhoA (GI:45382666), RhoC DS3, DSc-Rel and DSv-Rel are replication competent Rous (GI:71143153) and Rac1(GI:3184509) and the ESTs of TCL sarcoma-based retroviruses that were generated using the (ChEST66d11), RhoB (chEST66P8), RhoG (ChEST455i12), pDS3, pDSc-Rel, pDSv-Rel and pREP vectors as described Rac2 (ChEST231i16) and Rac3 (chEST989E16). previously (Okuno et al., 1991; Kralova et al., 1998). The spleen necrosis virus (SNV)-based bicistronic retroviral vector pMJ612 was derived from pJD216neoHy (Dougherty and Western blot analysis and antisera Temin, 1986) and contains two long terminal repeats (LTR), For Western blot analysis, cell lysates were separated by 12% neomycin and hygromycin resistance genes, and a gene SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and encoding the green fluorescence protein (GFP).The sequences subsequently transferred to an Optitran nitrocellulose mem- between the SmaI site and the SalI site of pMJ612 encode brane (Schleicher and Schuell, Keene NH).A rabbit anti-TC10 the hygromycin resistance gene and were replaced with serum, TC10 (ID1) was generated against an 18 amino acid double-stranded adapters (50-GCCACCATGCCCGGGG peptide (KGPKTLARLNDMKEKPLS) containing the spe- GAGGGCGGGCT-30 and 50-CATTGTCGACTCACGTGA cific Rho insert domain of TC10.The peptide was synthesized TCAAACAGCAGT-30) to facilitate the cloning of the ORFs in the Protein Facility of the University of Texas at Austin. of TC10, the gain-of-function mutant TC10Q76L, or the Monoclonal antibody HY87 was used for detecting both v-Rel dominant-negative mutant TC10T32N.A Kozak sequence and c-Rel (Hrdlickova´ et al., 1994a). Secondary antibodies (GCCACC) was added to the 50 end of the start codon of used for this study were horseradish peroxidase-conjugated TC10 to enhance translation initiation.The pSW253 plasmid goat anti-rabbit IgG and goat anti-mouse IgG antibodies contains a genomic clone of a replication competent helper (Jackson ImmunoResearch Laboratories, West Grove, PA, virus, REV-A, which was employed for packaging the SNV- USA).The signal was detected with the Western lightning based viruses (Watanabe and Temin, 1983). chemiluminescence reagent plus (Perkin-Elmer Life and The SuperFect transfection reagent (Qiagen, Valencia, CA, Analytical Sciences, Wellesley, MA, USA). USA) was used for the introduction of the retroviral constructs into CEF cultures.CEF cultures (7 Â 105) were seeded into In vitro translations and immunopreciptiations 60 mm plates and were transfected when cells were 50–70% In vitro translations were performed using the T7 polymerase confluent (approximately 18–24 h later).DS3, DSc-Rel and based TNT Coupled Reticulocyte Lysate System (Promega, DSv-Rel viral supernatant fluids were prepared and titered by Madison, WI, USA) with [35S]-methionine according to the an immunochemical titration assay as described previously manufacturer’s directions.Genes encoding TC10 and TCL (Kralova et al., 1998). To prepare recombinant SNV-based were expressed from the pBluescript or pGEM-T-EZ expres- viruses, the constructs were introduced into CEF cultures as sion vectors, respectively.Approximately equal amounts of described above using 5 mg of the pMJ612 construct, 200 ng of translated TC10 and TCL were diluted in 20 ml of low the pSW253 construct and 20 ml of SuperFect reagent for each immunoprecipitation buffer (IPB) (25 mM Tris pH7.5, 60 mm culture plate.Twenty-four hours after transfection, 150 mM NaCl, 2 mM ethylenediamine-N,N,N0,N0-tetraacetic cells were placed under selection in media containing 500 mg/ml acid (EDTA), 0.5% NP-40) containing 10% SDS and of G418.Viral supernatant fluids were harvested 7–12 days denatured at 1001C for 5 min.The denatured proteins were after drug selection.The infectious units (IU) of the MJ612 diluted with low IPB to a final volume of 200 ml and used in virus were determined by infection of D17 cells and quantita- immunopreciptiation experiments with 5 ml of either normal tion of neomycin-resistant colonies as described previously rabbit serum or serum specific to TC10.Reactions were (Dougherty and Temin, 1986). rotated for 1 h at 41C.Protein A Sepharose beads were added To infect CEF cultures with retroviruses, 1.8 Â 106 cells were to the precipitations and rotated at 41C for 1 h.Immune seeded into 100 mm tissue culture plates and grown for 18 h or complexes bound to the Protien A Sepharose beads were until they reached 50–70% confluence.Cells were coinfected at washed three times with 1 ml of low IPB and removed from a multiplicity of infection of 1.5 with viral supernatant fluids the beads by boiling the beads in 25 ml of loading buffer. containing DS3- and SNV-based viruses.Forty-eight hours Immunoprecipitates were resolved by 12% SDS–PAGE. after infection, cultures were maintained in medium containing 500 mg/ml G418. GST-CRIB domain pull-down assay A DH-10B strain of Escherichia coli transformed by a pGEX- Northern blot analysis 4T construct containing a GST-CRIB domain fusion gene was Total RNA was isolated from CEF cultures or chicken a gift from Dr Suranganie Dharmawadahane (University of transformed cell lines using RNAwiz (Ambion, Austin, TX, Texas at Austin).This CRIB domain corresponds to amino USA).RNA was separated in a 1% denaturing agarose gel acids 74–89 of PAK1.The protocols for purification of the (20 mM MOPS (3-[N-morpholino]propanesulfonic acid) and fusion protein and the pull-down assay were previously 2% formaldehyde) in 20 mM MOPS and transferred to a described (Benard and Bokoch, 2002).The expressed fusion Hybond N þ nylon membranes (Amersham Pharmacia Bio- proteins were purified with glutathione-agarose beads (Sigma, technology, Piscataway, NJ, USA).The relative amount of St Louis, MO, USA), and the beads were incubated with RNA on the membrane was determined by quantitating the lysates from CEF cultures.Proteins pulled down with the intensity of ethidium bromide staining, methylene blue beads were separated on 12% SDS–PAGE, transferred to a

Oncogene TC10 contributes to v-Rel-mediated transformation S Tong et al 2327 nitrocellulose membrane, and detected with TC10 (ID1) Saturation density experiments were performed using CEF antiserum. cultures that were infected with SNV-based retroviruses and selected in G418 for 20 days.Multiple 60 mm culture plates 5 In vitro transformation assays were seeded with 1 Â 10 cells of each infection in 4 ml of media Soft agar colony formation assays were performed using CEF containing G418.The number of cells from three plates of each cultures that were coinfected with DS3 and pMJ612 viruses infection was determined with a hemocytometer 48 h after and selected in G418 for 15 days.CEFs (1 Â 105) were placed in seeding.Culture media was changed everyday.The cell 4 ml of plating media (DMEM containing 15% fetal bovine number from three plates from each infection was determined serum, 5% chicken serum, 100 U/ml penicillin, 50 mg/ml every day until the cells detached from the plate. streptomycin, 500 mg/ml G418 and 0.37% Noble agar) and plated on a bed of medium containing 0.75% agar in 60 mm Acknowledgements tissue culture plates.Cells were fed with 3 ml of plating medium every 10 days.The number of colonies was scored This study was supported by Public Health Service grants microscopically 4 weeks after seeding. CA33192 from the National Cancer Institute.

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