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• W2097612997 abstract "To comprehensively identify proteins interacting with 14-3-3ς in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3ς in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3ς. 25 proteins were not previously assigned to any function and were named SIP2–26 (for 14-3-3ς-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3ς expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3ς could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3ς expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes. To comprehensively identify proteins interacting with 14-3-3ς in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3ς in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3ς. 25 proteins were not previously assigned to any function and were named SIP2–26 (for 14-3-3ς-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3ς expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3ς could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3ς expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes. Of the seven human 14-3-3 isoforms (designated β, ε, γ, η, ς, τ, and ξ) 14-3-3ς has been linked to cancer most directly (for a review, see Ref. 1Hermeking H. The 14-3-3 cancer connection.Nat. Rev. Cancer. 2003; 3: 931-943Google Scholar). 14-3-3ς expression is lost in numerous carcinomas either due to epigenetic silencing by CpG methylation, which has been detected in a large number of different tumor types (1Hermeking H. The 14-3-3 cancer connection.Nat. Rev. Cancer. 2003; 3: 931-943Google Scholar), or due to mutation of p53, which directly induces expression of 14-3-3ς (2Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. 14-3-3ς is a p53-regulated inhibitor of G2/M progression.Mol. Cell. 1997; 1: 3-11Google Scholar). Epigenetic silencing of 14-3-3ς has been detected at a high frequency in carcinomas of the breast (3Ferguson A.T. Evron E. Umbricht C.B. Pandita T.K. Chan T.A. Hermeking H. Marks J.R. Lambers A.R. Futreal P.A. Stampfer M.R. Sukumar S. High frequency of hypermethylation at the 14-3-3ς locus leads to gene silencing in breast cancer.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6049-6054Google Scholar), ovary (4Mhawech P. Benz A. Cerato C. Greloz V. Assaly M. Desmond J.C. Koeffler H.P. Lodygin D. Hermeking H. Herrmann F. Schwaller J. Downregulation of 14-3-3ς in ovary, prostate and endometrial carcinomas is associated with CpG island methylation.Mod. Pathol. 2005; 18: 340-348Google Scholar), endometrium (4Mhawech P. Benz A. Cerato C. Greloz V. Assaly M. Desmond J.C. Koeffler H.P. Lodygin D. Hermeking H. Herrmann F. Schwaller J. Downregulation of 14-3-3ς in ovary, prostate and endometrial carcinomas is associated with CpG island methylation.Mod. Pathol. 2005; 18: 340-348Google Scholar), prostate (5Lodygin D. Diebold J. Hermeking H. Prostate cancer is characterized by epigenetic silencing of 14-3-3ς expression.Oncogene. 2004; 23: 9034-9041Google Scholar), skin (6Lodygin D. Yazdi A.S. Sander C.A. Herzinger T. Hermeking H. Analysis of 14-3-3ς expression in hyper-proliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma.Oncogene. 2003; 22: 5519-5524Google Scholar), lung (7Osada H. Tatematsu Y. Yatabe Y. Nakagawa T. Konishi H. Harano T. Tezel E. Takada M. Takahashi T. Frequent and histological type-specific inactivation of 14-3-3ς in human lung cancers.Oncogene. 2002; 21: 2418-2424Google Scholar), and liver (8Iwata N. Yamamoto H. Sasaki S. Itoh F. Suzuki H. Kikuchi T. Kaneto H. Iku S. Ozeki I. Karino Y. Satoh T. Toyota J. Satoh M. Endo T. Imai K. Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3ς gene in human hepatocellular carcinoma.Oncogene. 2000; 19: 5298-5302Google Scholar). After DNA damage, p53-induced expression of 14-3-3ς mediates a cell cycle arrest in the G2 phase (2Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. 14-3-3ς is a p53-regulated inhibitor of G2/M progression.Mol. Cell. 1997; 1: 3-11Google Scholar) presumably by cytoplasmic sequestration of CDC2-cyclin B1 complexes (9Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. 14-3-3ς is required to prevent mitotic catastrophe after DNA damage.Nature. 1999; 401: 616-620Google Scholar). Experimental removal of the 14-3-3ς gene prevents a stable G2/M arrest after DNA damage and sensitizes cells to DNA-damaging treatments commonly used in cancer therapy (5Lodygin D. Diebold J. Hermeking H. Prostate cancer is characterized by epigenetic silencing of 14-3-3ς expression.Oncogene. 2004; 23: 9034-9041Google Scholar, 9Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. 14-3-3ς is required to prevent mitotic catastrophe after DNA damage.Nature. 1999; 401: 616-620Google Scholar). 14-3-3 proteins form dimers that bind to protein ligands following serine/threonine phosphorylation by basophilic kinases, such as cAMP-dependent protein kinase or protein kinase B/AKT, of two canonical 14-3-3-binding motifs, which have been identified as R(S/X)XpSXP and RXXXpSXP where pS represents phosphoserine or phosphothreonine and X represents any amino acid (10Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. The structural basis for 14-3-3:phosphopeptide binding specificity.Cell. 1997; 91: 961-971Google Scholar). Association with 14-3-3 proteins regulates the function of ligands by inter- and intracompartmental sequestration, activation/inactivation of enzymatic activity, and promotion/inhibition of protein interactions. Thereby numerous cellular processes in all multicellular species analyzed are regulated by 14-3-3 proteins (for reviews, see Refs. 1Hermeking H. The 14-3-3 cancer connection.Nat. Rev. Cancer. 2003; 3: 931-943Google Scholar and 11Yaffe M.B. How do 14-3-3 proteins work? —gatekeeper phosphorylation and the molecular anvil hypothesis.FEBS Lett. 2002; 513: 53-57Google Scholar). In the past, candidate approaches led to the identification of a few proteins associated with 14-3-3ς: CDC2 (9Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. 14-3-3ς is required to prevent mitotic catastrophe after DNA damage.Nature. 1999; 401: 616-620Google Scholar), BAX (12Samuel T. Weber H.O. Rauch P. Verdoodt B. Eppel J.T. McShea A. Hermeking H. Funk J.O. The G2/M regulator 14-3-3ς prevents apoptosis through sequestration of Bax.J. Biol. Chem. 2001; 276: 45201-45206Google Scholar), p53 (13Yang H.Y. Wen Y.Y. Chen C.H. Lozano G. Lee M.H. 14-3-3ς positively regulates p53 and suppresses tumor growth.Mol. Cell. Biol. 2003; 23: 7096-7107Google Scholar), the glucocorticoid receptor (14Kino T. Souvatzoglou E. De Martino M.U. Tsopanomihalu M. Wan Y. Chrousos G.P. Protein 14-3-3ς interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway.J. Biol. Chem. 2003; 278: 25651-25656Google Scholar), WEE1 (15Rothblum-Oviatt C.J. Ryan C.E. Piwnica-Worms H. 14-3-3 binding regulates catalytic activity of human Wee1 kinase.Cell Growth Differ. 2001; 12: 581-589Google Scholar), EFP (16Urano T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Efp targets 14-3-3ς for proteolysis and promotes breast tumour growth.Nature. 2002; 417: 871-875Google Scholar), CDK2 and CDK4 (17Laronga C. Yang H.Y. Neal C. Lee M.H. Association of the cyclin-dependent kinases and 14-3-3ς negatively regulates cell cycle progression.J. Biol. Chem. 2000; 275: 23106-23112Google Scholar), BAD (18Subramanian R.R. Masters S.C. Zhang H. Fu H. Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis.Exp. Cell Res. 2001; 271: 142-151Google Scholar), and TBC2 (19Liu M.Y. Cai S. Espejo A. Bedford M.T. Walker C.L. 14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s).Cancer Res. 2002; 62: 6475-6480Google Scholar) were shown to interact with 14-3-3ς. So far no analysis attempting to comprehensively detect proteins that interact with 14-3-3ς has been reported. Interactions identified between other 14-3-3 isoforms and protein ligands do not necessarily apply to 14-3-3ς as distinct 14-3-3 isoforms show preferential or selective binding of ligands (20Stavridi E.S. Chehab N.H. Malikzay A. Halazonetis T.D. Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest.Cancer Res. 2001; 61: 7030-7033Google Scholar, 21Sekimoto T. Fukumoto M. Yoneda Y. 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1).EMBO J. 2004; 23: 1934-1942Google Scholar, 22Mils V. Baldin V. Goubin F. Pinta I. Papin C. Waye M. Eychene A. Ducommun B. Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase.Oncogene. 2000; 19: 1257-1265Google Scholar, 23Uchida S. Kuma A. Ohtsubo M. Shimura M. Hirata M. Nakagama H. Matsunaga T. Ishizaka Y. Yamashita K. Binding of 14-3-3β but not 14-3-3ς controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.J. Cell Sci. 2004; 117: 3011-3020Google Scholar). Here we describe the identification of 117 possible ligands of 14-3-3ς that are potentially regulated by direct interaction with 14-3-3ς and represent putative downstream targets for tumor suppression by p53 in epithelial cells. Human embryonic kidney (HEK) 1The abbreviations used are: HEK, human embryonic kidney; MudPIT, multidimensional protein identification technology; TAP, tandem affinity purification; SIP, 14-3-3ς-interacting protein; HA, hemagglutinin; YFP, yellow fluorescent protein; vsv, vesicular stomatitis virus; MAP, mitogen-activated protein; CCD, charge-coupled device; CFP, cyan fluorescent protein; TEV, tobacco etch virus; ERK, extracellular signal-regulated kinase; APC, adenomatous polyposis coli; EGF, epidermal growth factor. 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with high glucose (Invitrogen) and 5% fetal bovine serum (Hyclone). The cell lines DLD1-tTA and HCT116 were cultured in McCoy’s 5A medium (Invitrogen) with 10% fetal bovine serum (Hyclone) at 37 °C. HEK293T cells were transfected by calcium phosphate precipitation. For the generation of cell lines, 2 × 106 DLD1-tTA cells stably expressing the tTA transactivator were transfected by lipofection (FuGENE 6, Roche Applied Science) with pBI-14-3-3ς-HA, pBI-14-3-3ς-TAPc, or pBI-TAPc vectors in combination with pTK-hygro (Clontech). Single cell clones were obtained by limiting dilution in selective medium containing 100 ng/ml doxycycline and 250 μg/ml hygromycin B (Invitrogen), and induction of ectopic proteins was confirmed by Western blot analysis after removal of doxycycline. The cell lines were designated DLD1-tTA-14-3-3ς-TAPc, DLD1-tTA-TAPc, and DLD1-tTA-14-3-3ς-HA. The tandem affinity purification (TAP) tag open reading frame was PCR-amplified from pBS1539 (24Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. The tandem affinity purification (TAP) method: a general procedure of protein complex purification.Methods. 2001; 24: 218-229Google Scholar) (provided by Cellzome AG, Heidelberg, Germany) using the oligonucleotides 5′-GbHi8bXhDyMP2m1NDUSiYmznAJdJzX36DDATTTC-3′ and 5′-AGCTGCGGCCGCTCAGGTTGACTTCCCCGCG-3′. The resulting PCR fragment was inserted into the vector pECFP-N1-14-3-3ς-HA via BamHI and NotI sites. From the resulting plasmid a KpnI-NotI fragment containing 14-3-3ς-TAPc was isolated and inserted (blunt) into pBI (Clontech) resulting in the vector pBI-14-3-3ς-TAPc. For pBI-TAPc, pBS1539 was cut using HindIII and NcoI, and the TAPc fragment was inserted (blunt) into pBI. pBI-14-3-3ς-HA was generated by digestion of pECFP-N1-14-3-3ς-HA with KpnI and BamHI and ligation (blunt) of the 14-3-3ς-HA fragment into pBI. For pECFP-N1-14-3-3ς-HA, 14-3-3ς-HA was PCR-amplified with the oligonucleotides 5′-ACGGTACCCACCATGGAGAGAGCCAGTCTG-3′ and 5′-CCGGATCCTTGCTAGCGTAATCTGGAACATC-3′ using pHRCMV-14-3-3ς (2Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. 14-3-3ς is a p53-regulated inhibitor of G2/M progression.Mol. Cell. 1997; 1: 3-11Google Scholar) as a template. The resulting PCR fragment was cut with KpnI and BamHI and ligated into pECFP-N1 (Clontech). pECFP-C1-14-3-3ς-HA was generated by insertion of a BglII (blunt) and BamHI fragment derived from pECFP-N1-14-3-3ς-HA into pECFP-C1 (Clontech). pEYFP-C1-MIG-6 was generated by PCR amplification of the MIG-6 open reading frame with the oligonucleotides 5′-ATCGGTACCTCAATAGCAGGAGTTGCTG-3′ and 5′-ATCGGTACCCTAAGGAGAAACCACATAGG-3′ using the RZPD clone IRALp962G0742Q2 as a template. After restriction with KpnI, the resulting fragment was ligated into pEYFP-C1 (Clontech). For pCDNA3-AJUBA-vsv, the AJUBA open reading frame was PCR-amplified from the RZPD clone IRATp970D0227D using the oligonucleotides 5′-ATCAAGCTTCAGAGCGGTTAGGAGAGAAAGC-3′ and 5′-ATCGAATTCGATCTCGTTGGCAGGGGGTTG-3′. The PCR fragment was cut with HindIII and EcoRI and ligated into pCDNA3vsv. pCMV-14-3-3ς-HA was generated by digestion of pEYFP-N1-14-3-3ς-HA with BamHI and NotI to release the YFP and subsequent religation. All plasmids were confirmed by sequence analysis. Cells were harvested in lysis buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, and 1 mm DTT supplemented with protease (Complete Mini EDTA-free, Roche Applied Science) and phosphatase inhibitors (2 mm sodium orthovanadate, 100 nm okadaic acid, 1 mm NaF, 1 mm β-glycerophosphate, and Cocktail 1 (Sigma)). Protein amounts were quantified using Bradford reagents. Proteins were separated by SDS-PAGE and transferred onto PVDF filters. The membranes were incubated overnight at 4 °C with antibodies against the following proteins/epitopes: 14-3-3ς (9Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. 14-3-3ς is required to prevent mitotic catastrophe after DNA damage.Nature. 1999; 401: 616-620Google Scholar), HA (12CA5), vsv, α-tubulin (TU-02; Santa Cruz Biotechnology), Protein A (ab6659, Abcam), 14-3-3-phospho-binding motif (4E2, Cell Signaling Technologies), p44/p42 MAP kinase (Cell Signaling Technologies), and phospho-p44/p42 MAP kinase (Thr-202/Tyr-204; Cell Signaling Technologies). Enhanced chemoluminescence generated by secondary antibodies (Promega) conjugated with horseradish peroxidase was detected with a CCD camera (440CF imaging system, Eastman Kodak Co.). DLD1-tTA cells were grown on glass coverslips and transfected with the indicated plasmids. After 24 h cells were fixed in 3.7% paraformaldehyde, PBS; permeabilized with PBS, 0.2% Triton X-100; blocked with fetal bovine serum; and stained with primary (rabbit anti-HA) and with secondary anti-rabbit IgG-Cy3 antibodies (Jackson Immunoresearch Laboratories). Images of immunofluorescence and green fluorescent protein or CFP fusion proteins were generated with an inverted microscope (Axiovert 200M, Zeiss) equipped with a CCD camera (Coolsnap HQ, Photometrics) and Metamorph software (Universal Imaging Corp.). HEK293T cells were transiently transfected with pCMV-HA-14-3-3ς and pEYFP-C1-MIG-6 or pCDNA3-AJUBA-vsv using calcium phosphate precipitation. 24 h after transfection, cells were lysed on ice for 15 min with lysis buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, and 1 mm DTT) supplemented with protease (Complete Mini EDTA-free, Roche Applied Science) and phosphatase inhibitors (2 mm sodium orthovanadate, 100 nm okadaic acid, 1 mm NaF, 1 mm β-glycerophosphate, and Cocktail 1 (Sigma)). Lysates were centrifuged at 13,000 rpm for 20 min. 3 mg of lysate were used for incubation with a mouse anti-HA antibody (Covance) for 2 h. Subsequently 25 μl of Protein G-Sepharose beads (Amersham Biosciences) were added for 2 h. After washing five times in 10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.1% Nonidet P-40, 2 mm sodium orthovanadate, the proteins were separated by SDS-PAGE and subjected to Western blot analysis with antibodies against green fluorescent protein (Santa Cruz Biotechnology) or the vsv or the HA tag (Covance). 3.5 × 108 DLD1-tTA-14-3-3ς-TAPc or DLD1-tTA-TAPc cells (corresponding to 6 × 500 cm2 plates) were lysed by incubation on ice for 15 min in lysis buffer (see “Western Blot Analysis”). Samples were cleared by centrifugation at 13,000 rpm for 2 min, and pellets were frozen in liquid nitrogen, thawed at room temperature, centrifuged at 13,000 rpm for 2 min, and combined with the first supernatant. Cell lysates corresponding to 100 mg of protein were incubated with 400 μl of IgG-Sepharose beads (Amersham Biosciences) overnight at 4 °C. Beads were collected using chromatography columns (Polyprep, Bio-Rad) and washed extensively in 50 mm Tris/HCl, pH 8.0, 150 mm NaCl, and 0.1% Triton X-100 and in tobacco etch virus (TEV) cleavage buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm DTT, 0.1% Triton X-100, and 0.5 mm EDTA). TEV cleavage was performed in 1 ml of TEV cleavage buffer with 150 units of TEV protease (Invitrogen) for 3 h at 10 °C. The eluate of the TEV cleavage was transferred onto a column containing 400 μl of calmodulin affinity resin (Stratagene) in calmodulin binding buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm magnesium acetate, 2 mm CaCl2, 0.1% Triton X-100, 1 mm imidazole, and 10 mm β-mercaptoethanol) and incubated for 4 h at 10 °C. After washing repeatedly with calmodulin binding buffer, proteins were eluted twice with 1 ml of elution buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm magnesium acetate, 1 mm imidazole, 2 mm EGTA, 0.1% Triton X-100, and 10 mm β-mercaptoethanol) and TCA-precipitated. Precipitated 14-3-3ς-TAP-associated protein preparations were dissolved in digestion buffer, digested with trypsin, and analyzed by LC/LC/MS/MS according to published protocols (25Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar). Approximately 100 μg of protein were used for a 12-step LC/LC/MS/MS analysis. The obtained MS/MS spectra were analyzed by SEQUEST 2.7 using a non-redundant mammalian data base (May 2003 release, NCBI). The SEQUEST outputs were then analyzed by DTASelect (26Tabb D.L. McDonald W.H. Yates III, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.J. Proteome. Res. 2002; 1: 21-26Google Scholar). The DTASelect filter settings were: XCorr: +1 ions, 1.8; +2 ions, 2.5; +3 ions, 3.8; ΔCN, 0.08; only half or full tryptic peptides were considered, and all subset proteins were removed (the “-o” option in DTASelect). Proteins with four to five peptides that passed the DTASelect filter were considered real hits. Proteins with one to three peptides that passed the DTASelect filter were manually validated. DLD1-tTA-14-3-3ς-HA cells were seeded at 80% confluency into 6-well plates 24 h after removal of doxycycline and cultivated for an additional 24 h. As a control, DLD1-tTA-14-3-3ς-HA cells were treated with 100 nm doxycycline during the whole experiment to suppress expression of 14-3-3ς-HA. To prevent proliferation, cells were treated with 10 μg/ml mitomycin C (Sigma) for 3 h. Subsequently the cell monolayer was scratched with a Pasteur pipette. Cells were washed, and fresh medium with or without doxycycline was added. Cell migration was monitored for 24 h using an Axiovert 200M microscope (Zeiss) integrated into a CO2 37 °C incubator (Life Imaging Services) and equipped with a CCD camera (Coolsnap HQ, Photometrics) and Metamorph software (Universal Imaging Corp.). Pictures were taken every 6 min in two different wells with 50-ms exposure time using a motorized XY precision stage (LEP). To comprehensively determine the subset of proteins associated with 14-3-3ς in vivo we used a TAP tag approach, which allows the isolation of native protein complexes from cells ectopically expressing the tagged protein of interest (27Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. A generic protein purification method for protein complex characterization and proteome exploration.Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). The TAP tag was fused to the C terminus of 14-3-3ς (14-3-3ς-TAPc; Fig. 1A). 14-3-3ς-TAPc showed a cytoplasmic localization identical to the previously described localization for endogenous 14-3-3ς protein (9Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. 14-3-3ς is required to prevent mitotic catastrophe after DNA damage.Nature. 1999; 401: 616-620Google Scholar) and to HA-tagged 14-3-3ς (Fig. 1B). Ectopic 14-3-3ς-TAPc transiently expressed in HEK293T cells was co-purified with proteins that contain phosphorylated 14-3-3-binding consensus motifs as determined by detection with an antibody raised against the motif (Fig. 1C). Fusion of the TAP tag to the N terminus of the 14-3-3ς protein did not result in efficient co-purification of associated proteins presumably due to interference of the TAP domain with dimerization (data not shown). Subsequently a colorectal cancer cell line expressing the tTA repressor (DLD1-tTA (28Yu J. Zhang L. Hwang P.M. Rago C. Kinzler K.W. Vogelstein B. Identification and classification of p53-regulated genes.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14517-14522Google Scholar)) stably expressing a conditional allele encoding a 14-3-3ς-TAPc fusion protein was generated (Fig. 1D). After removal of doxycycline, the expression level of the 14-3-3ς-TAPc protein was similar to the level of endogenous 14-3-3ς expression detected after DNA damage of the wild-type p53-expressing colorectal cancer cell line HCT116 (data not shown). Stably transfected DLD-1-tTA cells were used for the subsequent protein identifications because purification of proteins associated with 14-3-3ς-TAPc was more efficient than in transiently transfected HEK293T cells (data not shown) presumably due to the low levels of competing endogenous 14-3-3ς protein in DLD1-tTA cells (2Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. 14-3-3ς is a p53-regulated inhibitor of G2/M progression.Mol. Cell. 1997; 1: 3-11Google Scholar), which express mutant p53. As a control, we generated a cell line stably expressing the TAPc protein derived from the same parental DLD1-tTA cells (Fig. 1D). To identify the proteins present in the final TAP-tagged eluates, MudPIT analyses were performed (29Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Google Scholar). To obtain a comprehensive picture of 14-3-3ς interactions under different cellular conditions, DLD1-tTA cells either exponentially proliferating (untreated), treated with doxorubicin for induction of DNA damage, serum-starved, or EGF-stimulated after serum starvation were analyzed after induction of 14-3-3ς-TAPc expression. In each case 100 mg of protein extract were used for tandem affinity purification with the EGF-stimulated cells being analyzed in duplicate. A number of highly abundant proteins (e.g. ribosomal proteins and keratins) were also detected in the eluates obtained after purification of the TAPc tag protein (for a complete list see Supplemental Table 1). These proteins and further proteins regarded as contaminants in previously published TAP tag purifications were excluded from Table I (for a list of all excluded proteins see Supplemental Table 2). After subtraction of contaminants, 117 protein identifications representing potential ligands of 14-3-3ς were obtained (listed in Table I). 14 proteins were previously shown to associate with other 14-3-3 isoforms in detailed case-by-case studies (indicated in Table I). The detection of these 14-3-3 ligands implies that the conditions used for the TAP tag purification allow the identification of bona fide 14-3-3 ligands. However, it is possible that some of the proteins detected here indirectly associate with 14-3-3ς via other 14-3-3ς-associated proteins or represent contaminants.Table IPotential 14-3-3ς ligands identified by a combined TAP-MudPIT approachGene symbolDescriptive nameFunction/homology domainSequence coverageMotifIIIIIIIVV%SFNStratifin, 14-3-3ς85.977.884.783.978.6YWHAG14-3-3γ7.719.415.4+ 2Cytoskeletal organization and dynamicsABLIMaLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).ABLIMActin-binding protein6.27.89.57.88.7+++ 1APXL2aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).Apical protein 2Actin-binding protein11.3+++ 2ARHERhoERho-binding protein14.314.314.35.3++ 1ARHGAP11AARHGAP11ARho inhibition by GTP hydrolysis4.4++ 2ARHGAP21bLigands recently identified as binding to affinity columns coupled to human 14-3-3ζ (34).RhoGAP10Rho inhibition by GTP hydrolysis2.54.82.94.33.3+++ 1ARHGEF5TIM oncogeneRho activation by GDP/GTP exchange20.411.013.54.66.4+ 8ARHGEF17Rho GEF 17Rho activation by GDP/GTP exchange3.45.83.9+++ 2ARHGEF16bLigands recently identified as binding to affinity columns coupled to human 14-3-3ζ (34).,cLigands recently identified as binding to affinity columns coupled to the two 14-3-3 isoforms from Saccharomyces cerevisiae BMH1 and BMH2 (35).Rho GEF 16Rho activation by GDP/GTP exchange6.9+++ 1BAIAP1aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).BAI1-associated protein 1Scaffold for GEFs at tight junctions6.36.7++ 1BCAR1cLigands recently identified as binding to affinity columns coupled to the two 14-3-3 isoforms from Saccharomyces cerevisiae BMH1 and BMH2 (35).,dInteractions with 14-3-3 isoforms previously characterized in detail.p130CasActin reorganization; SRC transformation4.7+++ 1CGNaLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).,bLigands recently identified as binding to affinity columns coupled to human 14-3-3ζ (34).CingulinTight junction regulation4.83.3+++ 1CTENC-terminal tensin-likeTensin-like with focal adhesion function12.9+++ 2GANcLigands recently identified as binding to affinity columns coupled to the two 14-3-3 isoforms from Saccharomyces cerevisiae BMH1 and BMH2 (35).GigaxoninIntermediate filament organization21.111.422.118.617.8+ 5GRB7GRB7RTK substrate; invasion5.65.87.0+++ 1LAD1Ladinin 1Anchoring filament of basement membrane17.29.113.312.0++ 2MARK1aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).MARK1Phosphorylation of microtubule proteins11.15.85.95.8++ 3MARK2aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).MARK2Phosphorylation of microtubule proteins28.128.69.17.7+++ 2M-RIPaLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).–cLigands recently identified as binding to affinity columns coupled to the two 14-3-3 isoforms from Saccharomyces cerevisiae BMH1 and BMH2 (35).Rho-interacting protein 3PHD domain13.311.8++ 2PAK4aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).,bLigands recently identified as binding to affinity columns coupled to human 14-3-3ζ (34).PAK 4Actin cytoskeleton reorganization; filopodia16.89.514.75.4++ 2PAR3LPartitioning-defective 3-like proteinCellular polarity2.41.94.3++ 3PAR3aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).,dInteractions with 14-3-3 isoforms previously characterized in detail.Partitioning-defective 3 proteinCellular polarity3.22.13.9++ 2PKP2aLigands recently identified as binding to either ectopic 14-3-3β, 14-3-3γ, 14-3-3τ or 14-3-3ζ in HEK293 cells (36).,dInteractions with 14-3-3 isoforms previously characterized in detail.Plakophilin 2aConnects desmosomal plaque to IF21.318.9" @default.
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• W2097612997 title "Targeted Proteomic Analysis of 14-3-3ς, a p53 Effector Commonly Silenced in Cancer" @default.
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