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• W1969138567 abstract "The lymphoid protein T-cell ubiquitin ligand (TULA)/suppressor of T-cell receptor signaling (Sts)-2 is associated with c-Cbl and ubiquitylated proteins and has been implicated in the regulation of signaling mediated by protein-tyrosine kinases. The results presented in this report indicate that TULA facilitates T-cell apoptosis independent of either T-cell receptor/CD3-mediated signaling or caspase activity. Mass spectrometry-based analysis of protein-protein interactions of TULA demonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously been shown to function as a key factor of caspase-independent apoptosis. Using RNA interference, we demonstrate that AIF is essential for the apoptotic effect of TULA. Analysis of the subcellular localization of TULA and AIF together with the functional analysis of TULA mutants is consistent with the idea that TULA enhances the apoptotic effect of AIF by facilitating the interactions of AIF with its apoptotic co-factors, which remain to be identified. Overall, our results shed new light on the biological functions of TULA, a recently discovered protein, describing its role as one of very few known functional interactors of AIF. The lymphoid protein T-cell ubiquitin ligand (TULA)/suppressor of T-cell receptor signaling (Sts)-2 is associated with c-Cbl and ubiquitylated proteins and has been implicated in the regulation of signaling mediated by protein-tyrosine kinases. The results presented in this report indicate that TULA facilitates T-cell apoptosis independent of either T-cell receptor/CD3-mediated signaling or caspase activity. Mass spectrometry-based analysis of protein-protein interactions of TULA demonstrates that TULA binds to the apoptosis-inducing protein AIF, which has previously been shown to function as a key factor of caspase-independent apoptosis. Using RNA interference, we demonstrate that AIF is essential for the apoptotic effect of TULA. Analysis of the subcellular localization of TULA and AIF together with the functional analysis of TULA mutants is consistent with the idea that TULA enhances the apoptotic effect of AIF by facilitating the interactions of AIF with its apoptotic co-factors, which remain to be identified. Overall, our results shed new light on the biological functions of TULA, a recently discovered protein, describing its role as one of very few known functional interactors of AIF. We recently identified TULA among multiple proteins that co-purified with c-Cbl from T-lymphoblastoid cells (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar). TULA contains an N-terminal UBA domain, a centrally positioned SH3 2The abbreviations used are: SH3, Src homology 3 domain; AIF, apoptosis inducing factor; FBS, fetal bovine serum; LC-ES MS/MS, liquid chromatography-electrospray tandem mass spectrometry; PBS, phosphate-buffered saline; Sts, suppressor of T-cell receptor signaling; TCR, T-cell receptor; TULA, T-cell ubiquitin ligand; UBA, ubiquitin-associated domain; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; shRNA, short hairpin RNA; siRNA, small interfering RNA; HPLC, high pressure liquid chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GFP, green fluorescent protein.2The abbreviations used are: SH3, Src homology 3 domain; AIF, apoptosis inducing factor; FBS, fetal bovine serum; LC-ES MS/MS, liquid chromatography-electrospray tandem mass spectrometry; PBS, phosphate-buffered saline; Sts, suppressor of T-cell receptor signaling; TCR, T-cell receptor; TULA, T-cell ubiquitin ligand; UBA, ubiquitin-associated domain; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; shRNA, short hairpin RNA; siRNA, small interfering RNA; HPLC, high pressure liquid chromatography; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GFP, green fluorescent protein. domain, and a region of homology to phosphoglyceromutases, which was initially termed HCD (Fig. 1) (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar, 2Wattenhofer M. Shibuya K. Kudoh J. Lyle R. Michaud J. Rossier C. Kawasaki K. Asakawa S. Minoshima S. Berry A. Bonne-Tamir B. Shimizu N. Antonarakis S.E. Scott H.S. Hum. Genet. 2001; 108: 140-147Crossref PubMed Scopus (39) Google Scholar). TULA binds to c-Cbl through its SH3 domain and to ubiquitin and ubiquitylated proteins through its UBA domain (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar, 3Kowanetz K. Crosetto N. Haglund K. Schmidt M.H. Heldin C.H. Dikic I. J. Biol. Chem. 2004; 279: 32786-32795Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Dimerization of TULA through its phosphoglyceromutase domain has also been shown (3Kowanetz K. Crosetto N. Haglund K. Schmidt M.H. Heldin C.H. Dikic I. J. Biol. Chem. 2004; 279: 32786-32795Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Analysis of cell and tissue expression of TULA demonstrates that this protein is expressed primarily in T and B lymphocytes and is localized both in the cytoplasm and nucleus (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar, 4Carpino N. Turner S. Mekala D. Takahashi Y. Zang H. Geiger T.L. Doherty P. Ihle J.N. Immunity. 2004; 20: 37-46Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).A mouse orthologue of TULA (Sts-2) was recently identified (4Carpino N. Turner S. Mekala D. Takahashi Y. Zang H. Geiger T.L. Doherty P. Ihle J.N. Immunity. 2004; 20: 37-46Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), as was a second member of the family, Sts-1 (5Carpino N. Kobayashi R. Zang H. Takahashi Y. Jou S.T. Feng J. Nakajima H. Ihle J.N. Mol. Cell. Biol. 2002; 22: 7491-7500Crossref PubMed Scopus (54) Google Scholar). Unlike TULA, Sts-1 is expressed ubiquitously (4Carpino N. Turner S. Mekala D. Takahashi Y. Zang H. Geiger T.L. Doherty P. Ihle J.N. Immunity. 2004; 20: 37-46Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 5Carpino N. Kobayashi R. Zang H. Takahashi Y. Jou S.T. Feng J. Nakajima H. Ihle J.N. Mol. Cell. Biol. 2002; 22: 7491-7500Crossref PubMed Scopus (54) Google Scholar). (In this report we will use the term TULA for consistency.)TULA has been implicated in the regulation of cell signaling mediated by protein-tyrosine kinases. On the one hand, TULA was reported to increase activity of receptor protein-tyrosine kinases by inhibiting c-Cbl-driven down-regulation of their activated forms. This appears to be mediated by preventing interactions between ubiquitylated forms of activated protein-tyrosine kinases and proteins recruiting them to the degradation pathway and, possibly, by decreasing the level of c-Cbl (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar, 3Kowanetz K. Crosetto N. Haglund K. Schmidt M.H. Heldin C.H. Dikic I. J. Biol. Chem. 2004; 279: 32786-32795Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). On the other, the lack of both proteins of the TULA/Sts family resulted in hyper-reactivity of T lymphocytes correlated with an increase in the activity of Zap-70, the molecular basis of which remained unclear (4Carpino N. Turner S. Mekala D. Takahashi Y. Zang H. Geiger T.L. Doherty P. Ihle J.N. Immunity. 2004; 20: 37-46Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These results implied that the effect of TULA on protein-tyrosine kinases might not be the only mechanism through which TULA exerts its biological effect. Indeed, the presence in TULA of multiple functional domains and extensive stretches of amino acid sequences with unknown functions suggested that TULA might exert effects unrelated to either c-Cbl or protein-tyrosine kinases.In an effort to discover novel functions of TULA, we purified proteins that interact with TULA and identified among them apoptosis-inducing factor (AIF). AIF is a key factor of caspase-independent apoptosis (6Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3429) Google Scholar, 7Daugas E. Susin S.A. Zamzami N. Ferri K.F. Irinopoulou T. Larochette N. Prevost M.C. Leber B. Andrews D. Penninger J. Kroemer G. FASEB J. 2000; 14: 729-739Crossref PubMed Scopus (701) Google Scholar, 8Loeffler M. Daugas E. Susin S.A. Zamzami N. Metivier D. Nieminen A.L. Brothers G. Penninger J.M. Kroemer G. FASEB J. 2001; 15: 758-767Crossref PubMed Scopus (210) Google Scholar). In the absence of cellular stress signals, AIF is localized to the internal mitochondrial membrane, where it functions as a FAD-dependent NADH oxidase, which is required for normal oxidative phosphorylation (9Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (519) Google Scholar) and maintenance of mitochondrial structure (10Cheung E.C. Joza N. Steenaart N.A. McClellan K.A. Neuspiel M. McNamara S. MacLaurin J.G. Rippstein P. Park D.S. Shore G.C. McBride H.M. Penninger J.M. Slack R.S. EMBO J. 2006; 25: 4061-4073Crossref PubMed Scopus (157) Google Scholar). Under conditions inducing apoptosis, AIF is released from mitochondria (11Otera H. Ohsakaya S. Nagaura Z. Ishihara N. Mihara K. EMBO J. 2005; 24: 1375-1386Crossref PubMed Scopus (291) Google Scholar, 12Uren R.T. Dewson G. Bonzon C. Lithgow T. Newmeyer D.D. Kluck R.M. J. Biol. Chem. 2005; 280: 2266-2274Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 13Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 14Yuste V.J. Moubarak R.S. Delettre C. Bras M. Sancho P. Robert N. d'Alayer J. Susin S.A. Cell Death Differ. 2005; 12: 1445-1448Crossref PubMed Scopus (110) Google Scholar) and translocated to the nucleus, where it induces caspase-independent apoptotic events through binding to DNA (15Ye H. Cande C. Stephanou N.C. Jiang S. Gurbuxani S. Larochette N. Daugas E. Garrido C. Kroemer G. Wu H. Nat. Struct. Biol. 2002; 9: 680-684Crossref PubMed Scopus (299) Google Scholar). These two functions of AIF are mediated by distinct structural domains (15Ye H. Cande C. Stephanou N.C. Jiang S. Gurbuxani S. Larochette N. Daugas E. Garrido C. Kroemer G. Wu H. Nat. Struct. Biol. 2002; 9: 680-684Crossref PubMed Scopus (299) Google Scholar, 16Mate M.J. Ortiz-Lombardia M. Boitel B. Haouz A. Tello D. Susin S.A. Penninger J. Kroemer G. Alzari P.M. Nat. Struct. Biol. 2002; 9: 442-446Crossref PubMed Scopus (151) Google Scholar) and can be dissociated (6Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3429) Google Scholar, 10Cheung E.C. Joza N. Steenaart N.A. McClellan K.A. Neuspiel M. McNamara S. MacLaurin J.G. Rippstein P. Park D.S. Shore G.C. McBride H.M. Penninger J.M. Slack R.S. EMBO J. 2006; 25: 4061-4073Crossref PubMed Scopus (157) Google Scholar, 17Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar).Overall, the molecular mechanism of the apoptotic effect of AIF remains poorly understood, and in particular, few functional interaction partners of AIF have been identified (18Ravagnan L. Gurbuxani S. Susin S.A. Maisse C. Daugas E. Zamzami N. Mak T. Jaattela M. Penninger J.M. Garrido C. Kroemer G. Nat. Cell. Biol. 2001; 3: 839-843Crossref PubMed Scopus (743) Google Scholar, 19Wang X. Yang C. Chai J. Shi Y. Xue D. Science. 2002; 298: 1587-1592Crossref PubMed Scopus (328) Google Scholar, 20Gurbuxani S. Schmitt E. Cande C. Parcellier A. Hammann A. Daugas E. Kouranti I. Spahr C. Pance A. Kroemer G. Garrido C. Oncogene. 2003; 22: 6669-6678Crossref PubMed Scopus (238) Google Scholar, 21Cande C. Vahsen N. Kouranti I. Schmitt E. Daugas E. Spahr C. Luban J. Kroemer R.T. Giordanetto F. Garrido C. Penninger J.M. Kroemer G. Oncogene. 2004; 23: 1514-1521Crossref PubMed Scopus (224) Google Scholar). Our work, presented here, demonstrates that TULA and AIF are interaction partners and establishes a functional link between them in inducing caspase-independent apoptosis. These results shed new light on the mechanism of the apoptotic effect of AIF and reveal a novel biological function of TULA.EXPERIMENTAL PROCEDURESDNA Constructs and Mutagenesis—cDNA encoding the full-length TULA or its N-terminal half (TULA-N1/2) was subcloned into the pFLAG 5a vector (Sigma) using the Advantage-Hf2 polymerase (Clontech). The forward primer (5′-CAGGATATCATGGCAGCGGGGGAG-3′) annealed to nucleotides at the N-terminal end of TULA and included a unique EcoRV restriction site. The reverse primers (5′-TAGGGTACCATCCGTGTAGTTTTCC-3′ and 5′-TAGGGTACCGTTGCCTGAGATCCAGTT-3′) annealed to nucleotides 893 to 908 (TULA-N1/2) or 1863 to 1880 (full-length TULA) within the TULA short (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar) protein sequence and included a unique KpnI restriction site. These restriction sites were included to create compatible ends for ligating the fragments into the pFLAG 5a vector. The obtained constructs were confirmed by sequencing.To introduce mutations, two synthetic oligonucleotides complementary to the opposite strands of double-stranded DNA containing the sequence to be mutated were designed to contain 15-18 nucleotides on either side of the mutation site. The oligonucleotides were gel purified (IDT Technologies, Coralville, IA). The mutagenesis reactions were performed using the QuikChange site-directed mutagenesis kit according to the manufacturer's recommendations (Stratagene, La Jolla, CA).Cells—HEK293T and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS) (complete medium). HEK293T cells were plated 24 h before transfection to be 80% confluent on the day of transfection in antibiotic-free medium. Purified plasmid DNA was transfected into HEK293T cells (10-20 μg/2 × 106 cells) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. After a total of 48 h, transfected cells were harvested and washed with phosphate-buffered saline (PBS). Cells were lysed in CelLytic buffer (Sigma) for 15 min at room temperature, and cell debris was removed by centrifugation. HeLa cells were transfected in the same fashion, but using Lipofectin (Invitrogen) or FuGENE 6 (Roche Applied Science).Jurkat tag cells were cultured in RPMI1640 supplemented with 20 mm HEPES, 2 mm l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% FBS (complete medium). The cells were grown in antibiotic-free medium for 24 h prior to electroporation. Cells were centrifuged and resuspended at a final density of 2 × 107 cells/ml in antibiotic-free medium. DNA (10 μg) was added to a 4-mm cuvette followed by addition of 1 × 107 cells in 500 μl of medium. The mixture was pulsed at 310 V for 10 ms in an electroporator (ECM 830 from BTX, Holliston, MA). After electroporation, cells were cultured in complete medium for 48 h. The efficiency of electroporation was ∼70%.In several experiments Jurkat tag cells were transfected using DMRIE-C (3 μg of DNA/5 × 106 cells) according to the manufacturer's recommendations. Because the efficiency of DMRIE-C-mediated transfection did not exceed 10%, a GFP-encoding expression plasmid (pEGFP-C2, Clontech) was co-transfected in each sample at a ratio of 1:15 to the total DNA, and only GFP+ cells were analyzed using flow cytometry. Stable Jurkat cells with a reduced TULA expression level and the corresponding control cells were generated using the shRNA-encoding or empty control lentiviral vector (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar).Z-VAD-fmk and Z-IETD-fmk (Biomol, Plymouth Meeting, PA) and camptothecin and etoposide (Sigma) were added to final concentrations of 100, 4, 5, and 10 μm, respectively. Growth factor withdrawal of Jurkat tag cells was carried out in medium supplemented with 0.5% FBS. For anti-CD3 stimulation, wells of a 24-well plate were pre-coated with the mouse monoclonal antibody OKT3 at 10 μg/ml in PBS overnight at 4 °C.Isolation of TULA-associated Proteins—1-3 mg of total protein from FLAG-TULA-expressing or vector-transfected HEK293T cells was incubated with 20 μl of anti-FLAG M2 affinity gel (Sigma) and incubated at 4 °C for 4 h. The beads were washed three times with lysis buffer, and anti-FLAG-bound proteins were eluted from the beads with 0.1 m glycine (pH 3). Proteins eluted from the anti-FLAG beads were separated on a one-dimensional BisTris minigel and stained in Simply Blue Coomassie (Invitrogen). Each gel lane was divided and cut into 10 equal-sized gel slices. Proteins contained within each slice were equilibrated in 100 mm ammonium bicarbonate and reduced, alkylated, and digested with trypsin as previously described (22Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). One-tenth of each unfractionated tryptic digest was analyzed by LC-ES MS/MS using a micro-column (Zorbax C18, 75 mm × 12 cm) reverse-phased HPLC interfaced with an Agilent LC-MSD Ion Trap MS. ES MS/MS-based sequencing was performed on-line in a data-dependent manner, and two tandem mass spectra were taken per survey scan as peptides eluted from the HPLC (23McCormack A.L. Schieltz D.M. Goode B. Yang S. Barnes G. Drubin D. Yates 3rd, J.R. Anal. Chem. 1997; 69: 767-776Crossref PubMed Scopus (443) Google Scholar). Uninterpreted mass spectra from each of the 10 individual liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs were collated and searched as a single file against a human nonredundant protein data base using the Mascot search engine (Matrix Science) (24Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6706) Google Scholar). Errors used were 2.0 Da on MS data and 0.8 Da on MS/MS data.Immunoprecipitation and Immunoblotting—1-3 mg of total protein from whole cell lysate was immunoprecipitated with 1-3 μg of anti-TULA-N (GETQLYAKVSNKLKSRSSPS) (Proteintech Group Inc., Chicago, IL) in a total volume of 1 ml as described previously (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar). Then proteins were separated using SDS-PAGE, transferred to nitrocellulose, and probed with 1:1000 anti-FLAG M2 (Sigma), 1:1000 anti-TULA-N, or 1:500 anti-AIF (Santa Cruz Biotechnology, Santa Cruz, CA). After blots were washed, the appropriate peroxidase-conjugated secondary antibody was added, and proteins were visualized using the ECL Plus Kit and the Typhoon Fluorescent Imager (GE Healthcare).Annexin-V Staining—Electroporated Jurkat tag cells were washed and resuspended in 100 μl of annexin-V binding buffer (10 mm HEPES, 140 mm NaCl, 2.5 mm CaCl2, pH 7.4). Then 5 μl of 0.1 mg/ml propidium iodide and 5 μl annexin-V allophycocyanin conjugate (Molecular Probes, Eugene, OR) were added to the cells. After cells were incubated for 15 min at room temperature, 400 μl of annexin binding buffer was added, and cells were analyzed using flow cytometry. DMRIE-C-transfected Jurkat tag cells and TULA-knockdown Jurkat cells were analyzed using an annexin V-Cy5 apoptosis kit from Biovision (Mountain View, CA).Transfection of Small Interfering RNAs (siRNAs)—To deplete endogenous AIF and simultaneously overexpress TULA, a 21-mer annealed AIF-targeting siRNA and scrambled control (Ambion, Austin, TX) were resuspended in water at a final concentration of 100 μm. The sense sequence of the AIF-specific siRNA corresponded to nucleotides 1540-1558 in the AIF sequence. (Several AIF-specific siRNAs were tested in pilot experiments, and this siRNA was selected as the most efficient one.) siRNA was electroporated into Jurkat tag cells (100 nm siRNA and 1 × 105 cells in 75 μl of Opti-MEM (Invitrogen)) using 1-mm cuvettes in the BTX Electroporator at 150 V for 100 μs. To simultaneously electroporate siRNA and DNA, FLAG-TULA expression or control plasmid (2 μg) was added to siRNA. After recovery in complete medium for 48 h, transfected cells were either cultured in complete RPMI1640 medium or subjected to serum deprivation in RPMI1640 supplemented with 0.5% FBS for an additional 24 h. At that time overall cell death was measured using trypan blue exclusion. To deplete endogenous TULA, the same electroporation procedure was done using TULA-specific siRNA SMARTpool L-008616-00 from Dharmacon (Lafayette, CO).Subcellular Distribution—To obtain immunofluorescence images, HeLa cells were seeded onto fibronectin-coated coverslips (BD Biocoat) at a confluence of 50% in Dulbecco's modified Eagle's medium containing 10% FBS without antibiotics. On the following day, the cells were transfected to express FLAG-TULA and/or Myc-AIF (3 μg of each construct per coverslip) using FuGENE 6 as per the manufacturer's recommendations. Forty-eight hours post-transfection the cells were washed, fixed with 4% paraformaldehyde in PBS, washed again, and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. Cells were blocked with 1% bovine serum albumin and washed twice with PBS. Fluorescein isothiocyanate-conjugated anti-FLAG (5-10 μg/ml) and Cy3-conjugated anti-Myc (1 μg/ml) (Sigma) were added as appropriate. The antibodies were incubated with the cells overnight at 4 °C in the dark. The cells were washed three times with PBS before mounting the coverslips onto a slide with anti-fade mounting solution including 4′,6-diamidino-2-phenylindole stain (Molecular Probes). Cell images were obtained using the Leica DM IRE2 confocal microscope with a ×100 objective.For subcellular fractionation, 293T cells were transfected with either empty or TULA expression vector (10 μg/75-cm2 flask) using Lipofectamine 2000. Subcellular fractions were obtained from transfected cells at 48 h post-transfection using a Qproteome Cell Compartment kit (Qiagen).RESULTSAIF Is a Novel TULA Interacting Protein—To search for novel functions of TULA we sought to identify TULA interaction partners via a proteomics approach. For this purpose, FLAG-tagged full-length TULA and TULA-N1/2 (1-299), a truncation mutant lacking the C-terminal half, but containing both binding domains of TULA (UBA and SH3) (see Fig. 1), were transiently overexpressed in HEK293T cells and immunoprecipitated with anti-FLAG antibody. The eluted immune complexes were separated by SDS-PAGE and proteins associated with these forms of TULA were identified using LC-ES MS/MS. Several proteins were identified in the TULA and TULA-N1/2 immunoprecipitates and not in the vector control, and one of these was c-Cbl (8 unique peptides), a previously characterized TULA-interacting protein (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar, 3Kowanetz K. Crosetto N. Haglund K. Schmidt M.H. Heldin C.H. Dikic I. J. Biol. Chem. 2004; 279: 32786-32795Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). A second protein identified with 15 unique peptides was AIF. Originally, we identified AIF only in the TULA-N1/2 immunoprecipitates. However, the molecular mass of AIF suggested that it co-migrates with full-length TULA, which is a very large band on the SDS-PAGE gel. Because co-migration with TULA was likely to hinder identification of AIF in this system, we targeted four unique AIF peptides for mass spectrometry-based sequencing in the gel band corresponding to full-length TULA and identified AIF from all four peptide sequencing events. We performed these experiments using TULA and TULA-N1/2 in triplicate, and AIF was identified each time with more than 10 peptides in each trial (supplemental Table S1). Interestingly, c-Cbl was only identified in the immune complexes with full-length TULA.To verify association of TULA and AIF and to identify the region of TULA involved in AIF binding, we transiently overexpressed TULA and TULA mutants in HEK293T cells, immunoprecipitated them, and analyzed the obtained immune complexes using Western blotting. Consistent with the mass spectrometry results, co-immunoprecipitation of AIF was clearly detectable (Fig. 2A). Immunoblotting also showed that TULA-N1/2 binds to AIF better than full-length TULA. To assure that the difference in the amount of co-immunoprecipitated AIF was not due to differences in the cellular levels of AIF in cells overexpressing full-length TULA and TULA-N1/2 (as well as other TULA mutant forms, see below), we immunoblotted AIF in whole cell lysates and demonstrated that its level did not vary significantly between samples (Fig. 2B). Because the TULA-N1/2 mutant contains an SH3 domain and because AIF has several putative SH3-binding motifs (PXXP) including 545PSTPAVPQAP554, we hypothesized that TULA binds to AIF through the SH3 domain. However, the mutant form of TULA lacking a functional SH3 domain as a result of the W279L point mutation bound AIF with the same efficiency as wild-type TULA did (Fig. 2A). Furthermore, the SH3-deleted forms of both TULA-N1/2 and full-length TULA bound AIF to the extent characteristic of the binding of AIF by TULA-N1/2 (data not shown). Likewise, deletion of the UBA domain had no effect on AIF binding (Fig. 2A). Finally, the TULA-C1/2 truncated form (amino acids 300-623) did not bind to AIF (data not shown). Taken together these findings indicate that the N-terminal half of TULA is necessary for AIF binding, but that neither the SH3 nor the UBA domain is critical.FIGURE 2Co-immunoprecipitation of TULA and AIF. 293T cells were transfected with wild-type (WT) and various mutant forms of TULA (10 μg of each construct per 75-cm2 flask, except for TULA-N1/2, which was transfected at a dose of 20 μg/75-cm2 flask) using Lipofectamine 2000. Control cells were transfected with the empty vector at a dose equal that of the expression constructs. Empty vector was added to transfections to make total amounts of DNA equal. Cells were lysed 48 h after transfection. Cell lysates were subjected to immunoprecipitation (IP) with an indicated antibody (NRS, normal rabbit serum) (A) or analyzed as whole cell lysates (WCL) (B). Antibodies used for Western blotting (WB) are indicated. The proteins detected are indicated by arrowheads at the right. The molecular weight markers are indicated at the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because c-Cbl is well characterized as a TULA binding partner in T cells, we examined its binding to TULA relative to AIF. Interestingly, high binding of c-Cbl to various forms of TULA was invariably linked to the low AIF binding to them and vice versa (Fig. 3), thus being in agreement with the findings of our mass spectrometry based experiments (see above). This mutual exclusion is unlikely to be due to a direct competition of c-Cbl and AIF for the same binding site, because c-Cbl binds to the SH3 domain of TULA (1Feshchenko E.A. Smirnova E.V. Swaminathan G. Teckchandani A.M. Agrawal R. Band H. Zhang X. Annan R.S. Carr S.A. Tsygankov A.Y. Oncogene. 2004; 23: 4690-4706Crossref PubMed Scopus (62) Google Scholar), which is dispensable for AIF binding (see Fig. 2). It is more likely that c-Cbl and AIF bind to alternative conformation states of TULA or induce such states upon binding.FIGURE 3Binding of TULA to AIF and c-Cbl. 293T cells were transfected with various mutants of TULA as described in the legend to Fig. 2. Cells were lysed 48 h after transfection. Cell lysates were subjected to immunoprecipitation with anti-TULA. Immunoprecipitates were analyzed using Western blotting (WB) with the antibodies indicated. The proteins detected are indicated by arrowheads at the right. The molecular weight markers are indicated at the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also eval" @default.
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• W1969138567 cites W1965383843 @default.
• W1969138567 cites W1965975711 @default.
• W1969138567 cites W1966846303 @default.
• W1969138567 cites W1967368177 @default.
• W1969138567 cites W1970297715 @default.
• W1969138567 cites W1976695228 @default.
• W1969138567 cites W1981593008 @default.
• W1969138567 cites W1982555380 @default.
• W1969138567 cites W1988728998 @default.
• W1969138567 cites W1990232129 @default.
• W1969138567 cites W1998734127 @default.
• W1969138567 cites W2003530465 @default.
• W1969138567 cites W2007827117 @default.
• W1969138567 cites W2011123535 @default.
• W1969138567 cites W2014947131 @default.
• W1969138567 cites W2020670611 @default.
• W1969138567 cites W2027196702 @default.
• W1969138567 cites W2028925334 @default.
• W1969138567 cites W2032444047 @default.
• W1969138567 cites W2048982233 @default.
• W1969138567 cites W2059901120 @default.
• W1969138567 cites W2060507522 @default.
• W1969138567 cites W2068890239 @default.
• W1969138567 cites W2070920868 @default.
• W1969138567 cites W2071908913 @default.
• W1969138567 cites W2073118386 @default.
• W1969138567 cites W2074573663 @default.
• W1969138567 cites W2082458260 @default.
• W1969138567 cites W2085090715 @default.
• W1969138567 cites W2088988576 @default.
• W1969138567 cites W2121082164 @default.
• W1969138567 cites W2123671636 @default.
• W1969138567 cites W2139410513 @default.
• W1969138567 cites W2148414191 @default.
• W1969138567 cites W2151898419 @default.
• W1969138567 cites W2155089409 @default.
• W1969138567 cites W2155891625 @default.
• W1969138567 cites W2159650080 @default.
• W1969138567 cites W2161378034 @default.
• W1969138567 cites W4243368124 @default.
• W1969138567 cites W6731686 @default.
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