MCP Agilent Technologies
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/mcp.M500021-MCP200 on March 18, 2005.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
M500021-MCP200v1
4/6/785    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Glossary
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Benzinger, A.
Right arrow Articles by Hermeking, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Benzinger, A.
Right arrow Articles by Hermeking, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular & Cellular Proteomics 4:785-795, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Targeted Proteomic Analysis of 14-3-3{varsigma}, a p53 Effector Commonly Silenced in Cancer*,S

Anne Benzinger{ddagger}, Nemone Muster§,, Heike B. Koch{ddagger}, John R. Yates, III§, and Heiko Hermeking{ddagger},||

From the {ddagger} Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany and § Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 Acknowledgments—
 REFERENCES
 
To comprehensively identify proteins interacting with 14-3-3{varsigma} in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3{varsigma} in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3{varsigma}. 25 proteins were not previously assigned to any function and were named SIP2–26 (for 14-3-3{varsigma}-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 14-3-3{varsigma} 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 14-3-3{varsigma} 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 14-3-3{varsigma} expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

Of the seven human 14-3-3 isoforms (designated ß, {epsilon}, {gamma}, {eta}, {varsigma}, {tau}, and {xi}) 14-3-3{varsigma} has been linked to cancer most directly (for a review, see Ref. 1). 14-3-3{varsigma} 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 (1), or due to mutation of p53, which directly induces expression of 14-3-3{varsigma} (2). Epigenetic silencing of 14-3-3{varsigma} has been detected at a high frequency in carcinomas of the breast (3), ovary (4), endometrium (4), prostate (5), skin (6), lung (7), and liver (8). After DNA damage, p53-induced expression of 14-3-3{varsigma} mediates a cell cycle arrest in the G2 phase (2) presumably by cytoplasmic sequestration of CDC2-cyclin B1 complexes (9). Experimental removal of the 14-3-3{varsigma} gene prevents a stable G2/M arrest after DNA damage and sensitizes cells to DNA-damaging treatments commonly used in cancer therapy (5, 9). 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 (10). 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. 1 and 11).

In the past, candidate approaches led to the identification of a few proteins associated with 14-3-3{varsigma}: CDC2 (9), BAX (12), p53 (13), the glucocorticoid receptor (14), WEE1 (15), EFP (16), CDK2 and CDK4 (17), BAD (18), and TBC2 (19) were shown to interact with 14-3-3{varsigma}. So far no analysis attempting to comprehensively detect proteins that interact with 14-3-3{varsigma} has been reported. Interactions identified between other 14-3-3 isoforms and protein ligands do not necessarily apply to 14-3-3{varsigma} as distinct 14-3-3 isoforms show preferential or selective binding of ligands (20–23). Here we describe the identification of 117 possible ligands of 14-3-3{varsigma} that are potentially regulated by direct interaction with 14-3-3{varsigma} and represent putative downstream targets for tumor suppression by p53 in epithelial cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 Acknowledgments—
 REFERENCES
 
Cell Culture Procedures—
Human embryonic kidney (HEK)1 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 x 106 DLD1-tTA cells stably expressing the tTA transactivator were transfected by lipofection (FuGENE 6, Roche Applied Science) with pBI-14-3-3{varsigma}-HA, pBI-14-3-3{varsigma}-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-14-3-3{varsigma}-TAPc, DLD1-tTA-TAPc, and DLD1-tTA-14-3-3{varsigma}-HA.

Expression Plasmids—
The tandem affinity purification (TAP) tag open reading frame was PCR-amplified from pBS1539 (24) (provided by Cellzome AG, Heidelberg, Germany) using the oligonucleotides 5'-ACGGATCCTGACTGCAAGAGAAGATGGAAAAAGAATTTC-3' and 5'-AGCTGCGGCCGCTCAGGTTGACTTCCCCGCG-3'. The resulting PCR fragment was inserted into the vector pECFP-N1-14-3-3{varsigma}-HA via BamHI and NotI sites. From the resulting plasmid a KpnI-NotI fragment containing 14-3-3{varsigma}-TAPc was isolated and inserted (blunt) into pBI (Clontech) resulting in the vector pBI-14-3-3{varsigma}-TAPc. For pBI-TAPc, pBS1539 was cut using HindIII and NcoI, and the TAPc fragment was inserted (blunt) into pBI. pBI-14-3-3{varsigma}-HA was generated by digestion of pECFP-N1-14-3-3{varsigma}-HA with KpnI and BamHI and ligation (blunt) of the 14-3-3{varsigma}-HA fragment into pBI. For pECFP-N1-14-3-3{varsigma}-HA, 14-3-3{varsigma}-HA was PCR-amplified with the oligonucleotides 5'-ACGGTACCCACCATGGAGAGAGCCAGTCTG-3' and 5'-CCGGATCCTTGCTAGCGTAATCTGGAACATC-3' using pHRCMV-14-3-3{varsigma} (2) as a template. The resulting PCR fragment was cut with KpnI and BamHI and ligated into pECFP-N1 (Clontech). pECFP-C1-14-3-3{varsigma}-HA was generated by insertion of a BglII (blunt) and BamHI fragment derived from pECFP-N1-14-3-3{varsigma}-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-14-3-3{varsigma}-HA was generated by digestion of pEYFP-N1-14-3-3{varsigma}-HA with BamHI and NotI to release the YFP and subsequent religation. All plasmids were confirmed by sequence analysis.

Western Blot 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 ß-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: 14-3-3{varsigma} (9), HA (12CA5), vsv, {alpha}-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.).

Protein Detection by Immunofluorescence and Microscopy—
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.).

Co-immunoprecipitation—
HEK293T cells were transiently transfected with pCMV-HA-14-3-3{varsigma} 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 ß-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).

Tandem Affinity Purification—
3.5 x 108 DLD1-tTA-14-3-3{varsigma}-TAPc or DLD1-tTA-TAPc cells (corresponding to 6 x 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 ß-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 ß-mercaptoethanol) and TCA-precipitated.

Multidimensional Protein Identification Technology (MudPIT)—
Precipitated 14-3-3{varsigma}-TAP-associated protein preparations were dissolved in digestion buffer, digested with trypsin, and analyzed by LC/LC/MS/MS according to published protocols (25). 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 (26). The DTASelect filter settings were: XCorr: +1 ions, 1.8; +2 ions, 2.5; +3 ions, 3.8; {Delta}CN, 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.

Wounding Assay—
DLD1-tTA-14-3-3{varsigma}-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-14-3-3{varsigma}-HA cells were treated with 100 nM doxycycline during the whole experiment to suppress expression of 14-3-3{varsigma}-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).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 Acknowledgments—
 REFERENCES
 
Proteomic Analysis of 14-3-3{varsigma}-associated Ligands—
To comprehensively determine the subset of proteins associated with 14-3-3{varsigma} 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 (27). The TAP tag was fused to the C terminus of 14-3-3{varsigma} (14-3-3{varsigma}-TAPc; Fig. 1A). 14-3-3{varsigma}-TAPc showed a cytoplasmic localization identical to the previously described localization for endogenous 14-3-3{varsigma} protein (9) and to HA-tagged 14-3-3{varsigma} (Fig. 1B). Ectopic 14-3-3{varsigma}-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 14-3-3{varsigma} 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 (28)) stably expressing a conditional allele encoding a 14-3-3{varsigma}-TAPc fusion protein was generated (Fig. 1D). After removal of doxycycline, the expression level of the 14-3-3{varsigma}-TAPc protein was similar to the level of endogenous 14-3-3{varsigma} 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 14-3-3{varsigma}-TAPc was more efficient than in transiently transfected HEK293T cells (data not shown) presumably due to the low levels of competing endogenous 14-3-3{varsigma} protein in DLD1-tTA cells (2), 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 (29). To obtain a comprehensive picture of 14-3-3{varsigma} 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 14-3-3{varsigma}-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 14-3-3{varsigma} 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 14-3-3{varsigma} via other 14-3-3{varsigma}-associated proteins or represent contaminants.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 1. Tandem affinity purification of 14-3-3{varsigma}-associated proteins. A, scheme of the 14-3-3{varsigma}-TAPc fusion protein used (CBP, calmodulin-binding protein; TEV, TEV protease cleavage site). B, confirmation of cellular localization of ectopic 14-3-3{varsigma} proteins. DLD1-tTA cells were transfected with pBI-14-3-3{varsigma}-TAPc (left panel) and pBI-14-3-3{varsigma}-HA (right panel) and analyzed by immunofluorescence with a polyclonal rabbit antibody directed against the HA tag. A representative detection of the ectopic 14-3-3{varsigma} proteins by indirect immunofluorescence (left image) and the corresponding phase-contrast picture (right image) is shown for each transfection. Magnification, 400x. C, 14-3-3{varsigma}-TAPc interacts with proteins phosphorylated at 14-3-3 consensus binding sites. After tandem affinity purification of HEK293T cells transiently transfected with plasmids encoding the indicated proteins, detection of 14-3-3{varsigma}-binding proteins by Western blot analysis was performed using an antibody specific for the consensus 14-3-3-binding motif phosphorylated at the central serine residue. The two left lanes represent the initial lysates (input), and the two right lanes represent the eluates after tandem affinity purification. Purification of transfected TAPc served as a negative control. D, conditional expression of 14-3-3{varsigma}-TAPc and TAPc in DLD1 colorectal cancer cell lines. Stable cell lines were analyzed for inducible expression of 14-3-3{varsigma}-TAPc (upper panel) and TAPc (lower panel) by Western blot analysis after removal of doxycycline for 24 h (left lanes, in the presence of 100 ng/ml doxycycline; right lanes, 24 h after removal of doxycycline). 14-3-3{varsigma}-TAPc was detected by an anti-14-3-3{varsigma} antibody. TAPc was detected by a Protein A-specific antibody. Detection of {alpha}-tubulin served as a loading control.

 

View this table:
[in this window]
[in a new window]
  		TABLE I Potential 14-3-3{varsigma} ligands identified by a combined TAP-MudPIT approach

Proteins identified by MudPIT analysis were grouped into functional classes. The indicated sequence coverage (in percent) was obtained by LC/LC/MS/MS of tandem affinity-purified 14-3-3{varsigma}-associated proteins from DLD1-tTA-14-3-3{varsigma}-TAPc cells left untreated (=exponentially proliferating) (I), treated for 4 h with 0.2 µg/ml doxorubicin for DNA damage (II), serum-starved for 24 h (III), or serum-starved for 24 h and restimulated with 50 ng/ml EGF (Sigma) for 30 min (IV and V). The right column depicts the number of 14-3-3 consensus binding sites identified using the Scansite program at different stringencies: +++, high stringency; ++, medium stringency; +, low stringency; –, no motif found. Here only the sites with the highest scores for each protein are indicated. For all sites found see Supplemental Table 3. Putative protein domains were identified using the conserved domain database (49). RTK, receptor tyrosine kinase; PTP, protein-tyrosine phosphatase; PHD, plant homeodomain; IF, intermediate filaments; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; BR-HLH-ZIP, basic region helix-loop-helix leucine zipper; SH3, Src homology 3; MEKK, MAP kinase/ERK kinase kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MAPKAP, MAP kinase-activated protein; Pol, polymerase; SAM, sterile {alpha} motif; HNRP, heterogeneous nuclear ribonucleoprotein.

 
In Silico Detection of 14-3-3{varsigma}-binding Motifs—
The presence and location of putative 14-3-3 consensus binding motifs were determined for the 117 identified 14-3-3{varsigma}-associated proteins using the software Scansite (30). 55.6% of these proteins contained at least one 14-3-3-binding consensus motif (RSXpSXP) when a query with the highest stringency was performed (Supplemental Table 3). When a set of proteins identified by a combined TAP-MudPIT approach using a non-14-3-3 bait was analyzed using the same settings only 15% of the identified proteins contained a RSXpSXP motif (data not shown). When medium and low stringency settings of the Scansite algorithm were applied, ~90% of the identified, putative 14-3-3{varsigma} ligands showed 14-3-3-binding sites (Supplemental Table 3). However, the sites identified by the Scansite program using medium and low stringency settings have to be treated cautiously because ~90% of the proteins isolated with a non-14-3-3 bait also showed putative 14-3-3-binding sites at these settings.

Several reported interactions, which are not phosphorylation-dependent, were not detected in our approach (CDC2, CDK2, CDK4, and BAX (9, 12, 17)). Presumably these interactions are less stable than the high affinity binding of 14-3-3 proteins mediated by phosphorylated consensus 14-3-3-binding motifs (10). The low frequency (~10%) of 14-3-3{varsigma}-associated proteins not harboring a 14-3-3-binding motif found here is in agreement with the low number of 14-3-3 ligands reported to associate in a phosphorylation-independent manner (31).

Confirmation of 14-3-3{varsigma} Interactions—
Exemplary confirmations of selected interactions between 14-3-3{varsigma} and newly identified interacting proteins were performed. 14-3-3{varsigma} was ectopically expressed in combination with MIG-6 or AJUBA in HEK293T cells. The interaction between 14-3-3{varsigma} and MIG-6 as well as between 14-3-3{varsigma} and AJUBA was confirmed by co-immunoprecipitation (Fig. 2A). For this analysis a 14-3-3{varsigma} construct N-terminally tagged with an HA tag was used. This suggests that the C-terminal TAP tag does not lead to artificial associations of proteins with 14-3-3{varsigma}-TAPc. In agreement with these results, MIG-6 and 14-3-3{varsigma} showed a cytoplasmic co-localization (Fig. 2B). These results show that the MudPIT results obtained here may serve as the basis for detailed functional analysis.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 2. Confirmation of interactions detected by proteomic analysis of 14-3-3{varsigma}. A, co-immunoprecipitation of novel 14-3-3{varsigma} interactors. Upper panel, HEK293T cells were transfected with plasmids encoding N-terminally HA-tagged 14-3-3{varsigma} and YFP-tagged MIG-6 (left lanes in pictures of lysates and of immunoprecipitates) or as a control YFP-tagged MIG-6 alone (right lanes in pictures of lysates and of immunoprecipitates). Lower panel, transfection with plasmids encoding N-terminally HA-tagged 14-3-3{varsigma} and vsv-tagged AJUBA (left lanes in pictures of lysates and of immunoprecipitates) or vsv-tagged AJUBA alone (right lanes in pictures of lysates and of immunoprecipitates). To confirm expression of the ectopic proteins, the lysates were subjected to Western blot analysis (WB) with the indicated antibodies (left panels). The right panel shows immunoprecipitations (IP) with an HA-specific antibody and subsequent detection of the (co-)precipitated proteins with the indicated antibodies. B, co-localization of MIG-6 and 14-3-3{varsigma}. DLD1-tTA cells were cotransfected with plasmids encoding CFP-14-3-3{varsigma} and YFP-MIG-6. Pictures were taken from living cells 24 h after transfection. For detection of YFP and CFP exposure time of 100 ms was used. The right panel shows co-localization as indicated by yellow color. The left panel shows the corresponding phase-contrast representation. Magnification, 400x.

 
Functional Classification of 14-3-3{varsigma}-associated Proteins—
The 14-3-3{varsigma} interactors were grouped according to the processes in which they had been implicated previously (Table I). 25 of the identified proteins represented putative open reading frames without functional annotation and were designated as SIP2–26 (for 14-3-3{varsigma}-interacting protein). The 14-3-3{gamma} isoform was co-purified with 14-3-3{varsigma}, which is in accordance with previous reports showing heterodimerization between different 14-3-3 isoforms (32). However, the sequence coverage for 14-3-3{gamma} was relatively low in the two states where it was detected, indicating that the majority of the 14-3-3{varsigma}-TAPc protein was present as a 14-3-3{varsigma} homodimer. This might, at least in part, be due to the abundance of the ectopic 14-3-3{varsigma}-TAPc. At lower concentrations endogenous 14-3-3{varsigma} may heterodimerize with additional 14-3-3 isoforms.

The largest functional class among the 14-3-3{varsigma}-associated proteins was represented by 36 proteins that affect cytoskeletal dynamics and organization (Table I). Among other processes, the proteins represented in this class control adhesion, polarity, and actin polymerization. All these processes are involved in cellular migration (for a review, see Ref. 33). To evaluate whether induced levels of 14-3-3{varsigma} expression may have an effect on these processes, we analyzed cellular migration in a wounding assay. Ectopic expression of 14-3-3{varsigma} inhibited closure of a wound in a confluent monolayer of DLD1 cells, whereas the cells not expressing ectopic 14-3-3{varsigma} completely closed a similar wound by migration within 24 h as determined by time lapse microscopy (Fig. 3). The minimal narrowing of the wound observed after induction of ectopic 14-3-3{varsigma} was due to enlargement and flattening of the cells (Fig. 3, left panel). In this experiment cell proliferation was blocked by the addition of mitomycin C. Complete inhibition of cell proliferation by mitomycin C was confirmed with and without ectopic 14-3-3{varsigma} expression (data not shown). The increased number of rounded cells observed after ectopic 14-3-3{varsigma} expression may be due to inhibition of cytokinesis, which has been observed previously (2). Therefore, the observed wound closure was not due to cellular proliferation in the absence of ectopic expression of 14-3-3{varsigma}. These results show that elevated 14-3-3{varsigma} expression may influence processes involved in cellular migration.



View larger version (116K):
[in this window]
[in a new window]
  		FIG. 3. Ectopic 14-3-3{varsigma} expression inhibits wound healing. Ectopic 14-3-3{varsigma} expression was induced in DLD1-tTA-14-3-3{varsigma}-HA cells by removal of doxycycline for 48 h. The left panel represents cells expressing induced levels of ectopic 14-3-3{varsigma}-HA, and the right panel represents cells with repressed expression of 14-3-3{varsigma}-HA due to the presence of 100 ng/ml doxycycline. For inhibition of cell proliferation 10 µg/ml mitomycin C was added 3 h before wounding of the confluent cell monolayer. Cells were monitored by time lapse microscopy. Pictures taken at identical locations at the indicated time points are shown. For details see "Experimental Procedures."

 
23 interactors were assigned to functions in mitogenic signaling (Table I). As a general readout for mitogenic signaling we analyzed MAP kinase activity after activation of a conditional 14-3-3{varsigma} allele (Fig. 4). In cells ectopically expressing 14-3-3{varsigma} we observed a moderate increase in basal MAP kinase activity and a significant increase in stimulated MAP kinase activity as detected by phosphorylation of phospho-ERK1/2 (Fig. 4).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4. Influence of 14-3-3{varsigma} expression on the MAP kinase pathway. The phosphorylation status of ERK1/ERK2 was determined by Western blot analysis. DLD1-tTA-14-3-3{varsigma}-HA cells expressing 14-3-3{varsigma}-HA under control of the Tet system were cultivated in the absence of serum and doxycycline for 24 h and then restimulated with serum (10% fetal bovine serum) for 10 min. As a control the cells were kept in the presence of 100 ng/ml doxycycline. Total cell lysates were subjected to SDS-PAGE and Western blotting. Phosphorylated ERK1/ERK2 (p-ERK1 and p-ERK2) was detected with a phospho-p44/42 ERK1/ERK2 (Thr-202/Tyr-204)-specific antibody. The membrane was reprobed with an antibody specific for ERK1/ERK2 and a mouse monoclonal anti-HA antibody to detect 14-3-3{varsigma}-HA.

 
Many other interesting putative 14-3-3{varsigma} interactors were identified (Table I). For example the detection of the adenomatous polyposis coli (APC) protein as a 14-3-3{varsigma}-associated protein indicates that 14-3-3{varsigma} may influence the Wnt signaling pathway (Table I). We found four perfect 14-3-3-binding sites in the APC protein (Supplemental Table 3). Which aspect of APC function is affected by 14-3-3{varsigma} remains to be shown.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
Acknowledgments—
 REFERENCES
 
As the tandem affinity purification approach allows the purification of protein complexes, it is possible that some of the 14-3-3{varsigma}-associated proteins identified in this study do not directly interact with 14-3-3{varsigma} but with other proteins directly binding to 14-3-3{varsigma}. Whether the identified 14-3-3{varsigma}-associated proteins directly bind to the amphipathic groove of 14-3-3{varsigma} in a phosphorylation-dependent manner will require the experimental confirmation of the putative 14-3-3 consensus binding sites identified here by mutational analysis.

Recently, the identification of ligands associated with 14-3-3 proteins in vitro using affinity columns coupled to the yeast 14-3-3 proteins (BMH1 and BMH2) or human 14-3-3{zeta} was reported (34, 35). The overlap with the proteins identified in vivo in this study was only 6 and 17%, respectively (indicated in Table I). This disparity is presumably due to differences between the 14-3-3-ligand associations in intact cells versus those that occur between recombinant 14-3-3 and protein ligands on columns. In another recent study four tagged 14-3-3 isoforms ({gamma}, ß, {tau}, and {zeta}) were isolated after transient transfection of HEK293 cells, and ligands were identified after PAGE separation (36). 38 of the proteins identified by Jin et al. (36) were also detected as potential 14-3-3{varsigma} ligands in our study (indicated in Table I). The relatively small overlap may result from the different experimental approaches or from isoform-specific differences in ligand binding. In the future, parallel proteomic studies comparing the ligands associated with different 14-3-3 isoforms under identical conditions may be used to address these issues.

At present it is unclear whether the observed inhibition of wound closure is due to effects of elevated 14-3-3{varsigma} expression on cellular adhesion, actin polymerization, or polarity. Inhibition of any of these processes may prevent directed migration. For example down-regulation of PAR3 was shown to prevent migration in Drosophila (37). Interestingly binding of ectopic 14-3-3{zeta} to PAR3 is known to disrupt polarity of mammalian epithelial cells (38). The inhibition of migration by 14-3-3{varsigma} suggests that loss of 14-3-3{varsigma} expression by epigenetic silencing or mutation of p53 in cancer cells may potentially lead to increased motility and thereby promote invasion. Recently conditional ectopic expression of 14-3-3ß was shown to enhance cellular migration in a wound closure assay (39). This opposing result suggests that inhibition of wound closure by 14-3-3{varsigma} is not due to an increase in overall 14-3-3 concentration but rather due to isoform-specific interactions. Isoform-specific differences in the association with certain motifs within one 14-3-3 ligand were recently shown for the positive cell cycle regulator CDC25B, which contains five 14-3-3 consensus binding sites (23). Interestingly association of CDC25B with different 14-3-3 isoforms results in different functional outcomes (23). We recently determined the crystal structure of 14-3-3{varsigma} (Protein Data Bank code 1YZ5) and detected structural differences between the 14-3-3 isoforms {varsigma}, {tau}, and {zeta} that are likely to contribute to selective ligand interactions and specific dimerization patterns (40).

As activation of the MAP kinase pathway protects against programmed cell death induced by DNA damage (41), the previously observed sensitization toward DNA-damaging agents after loss of 14-3-3{varsigma} expression (5, 9) may result, at least in part, from decreased MAP kinase activity. The increase in MAP kinase activity observed after 14-3-3{varsigma} expression may be due to interactions with multiple ligands. Among others, A-RAF, B-RAF, and c-RAF seem to be good candidates as previous studies of the interaction between RAF proteins and other 14-3-3 isoforms have shown that 14-3-3 proteins are critical modulators of RAF activity (42).

This analysis revealed multiple new candidate interactors of 14-3-3{varsigma} that may mediate its negative effects on cell cycle progression. The AJUBA protein has recently been reported to be essential for entry into mitosis as it is required for activation of nuclear Aurora A kinase (43). Cytoplasmic sequestration of AJUBA by 14-3-3{varsigma} might therefore prevent Aurora A kinase activation. However, the LIM domain protein AJUBA may be multifunctional as it also contributes to the formation or strengthening of cadherin-mediated cell-cell adhesion (44). 14-3-3{varsigma} was found to associate with WEE1. The activity of WEE1 kinase is required for inhibition of CDC2 by phosphorylation of Tyr-15 and thereby prevents entry into mitosis. WEE1 kinase activity is stimulated by association with 14-3-3 proteins (15, 45). Another regulator of mitosis, c-TAK1, was found to associate with 14-3-3{varsigma}: c-TAK1 phosphorylates CDC25C on Ser-216 and thereby prevents its nuclear localization and activation of CDC2 (46). How c-TAK1 is regulated by binding to 14-3-3{varsigma} protein has to be determined in the future. The WEE1, c-TAK1, and AJUBA proteins represent attractive candidates for mediating the inhibition of G2/M progression that has been described for 14-3-3{varsigma} previously (2). The activity of the kinesin KIF23/MKLP1 is necessary for completion of cytokinesis (47). Interestingly overexpression of 14-3-3{varsigma} results in abortive cytokinesis with subsequent formation of tetraploid cells (2). Potentially this effect may be related to an association between 14-3-3{varsigma} and MKLP1. PTOV-1 is a mitogenic factor that migrates to the nucleus during S phase entry (48). It is therefore conceivable that association with 14-3-3{varsigma} slows G1/S progression by cytoplasmic sequestration of PTOV-1.

Taken together the results suggest that the p53 inducibility of the 14-3-3{varsigma} gene may have evolved to allow p53 to simultaneously regulate a large number of different signaling processes at the protein level (Fig. 5). Future research can now focus on the identification of signaling pathways mediating the phosphorylation of novel 14-3-3{varsigma} ligands. Some of the identified 14-3-3{varsigma}-associated proteins may be components of multiprotein complexes and therefore may not directly interact with 14-3-3{varsigma}. Furthermore the functional relevance of the 14-3-3{varsigma}-ligand associations or 14-3-3{varsigma} complex formations for 14-3-3{varsigma}- and p53-mediated tumor suppression may now be analyzed. Pharmacological inhibition of enzymes identified in this study may be a useful therapeutic approach as they represent activities that are presumably deregulated after loss of p53 and/or 14-3-3{varsigma}.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5. Model of the p53–14-3-3{varsigma} pathway: induction of 14-3-3{varsigma} as a nodal point of regulation, which mediates modulation of multiple cellular processes by protein-protein interactions. Expression of 14-3-3{varsigma} is induced after DNA damage and during differentiation and senescence (1). p53 is a known mediator of the induction of 14-3-3{varsigma} expression (2). In cancer cells expression of 14-3-3{varsigma} is often lost due to mutation of p53 or CpG methylation. In normal cells, the induced 14-3-3{varsigma} protein may affect a variety of cellular functions by associating with the ligands identified in this report. Whether 14-3-3{varsigma} associates with these proteins is determined by their appropriate phosphorylation. Positive and negative regulation of the respective processes, which were inferred from the identity of the proteins identified by the TAP-MudPIT analysis (Table I), is indicated. Dotted lines are used for processes for which it has to be determined whether 14-3-3{varsigma} association has an inhibitory or activating effect.

 


   Acknowledgments—
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 Acknowledgments—
REFERENCES
 
We thank Bert Vogelstein for providing cell lines and the members of the Molecular Oncology Group for discussions.


   FOOTNOTES
 
Received, January 18, 2005, and in revised form, March 18, 2005.

Published, MCP Papers in Press, March 18, 2005, DOI 10.1074/mcp.M500021-MCP200

1 The abbreviations used are: HEK, human embryonic kidney; MudPIT, multidimensional protein identification technology; TAP, tandem affinity purification; SIP, 14-3-3{varsigma}-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. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material. Back

Supported by National Institutes of Health Grant RR11823-08. Back

|| Supported by the Max-Planck-Society and the Deutsche Krebshilfe/Dr. Mildred-Scheel-Stiftung (Grant 1945). To whom correspondence should be addressed. Tel.: 49-89-8578-2875; Fax: 49-89-8578-2540; E-mail: herme{at}biochem.mpg.de


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 Acknowledgments—
 REFERENCES
 

  1. Hermeking, H. (2003) The 14-3-3 cancer connection. Nat. Rev. Cancer 3, 931 –943[CrossRef][Medline]

  2. Hermeking, H., Lengauer, C., Polyak, K., He, T. C., Zhang, L., Thiagalingam, S., Kinzler, K. W., and Vogelstein, B. (1997) 14-3-3{varsigma} is a p53-regulated inhibitor of G2/M progression. Mol. Cell 1, 3 –11[CrossRef][Medline]

  3. Ferguson, 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., and Sukumar, S. (2000) High frequency of hypermethylation at the 14-3-3{varsigma} locus leads to gene silencing in breast cancer. Proc. Natl. Acad. Sci. U. S. A. 97, 6049 –6054[Abstract/Free Full Text]

  4. Mhawech, P., Benz, A., Cerato, C., Greloz, V., Assaly, M., Desmond, J. C., Koeffler, H. P., Lodygin, D., Hermeking, H., Herrmann, F., and Schwaller, J. (2005) Downregulation of 14-3-3{varsigma} in ovary, prostate and endometrial carcinomas is associated with CpG island methylation. Mod. Pathol. 18, 340 –348[CrossRef][Medline]

  5. Lodygin, D., Diebold, J., and Hermeking, H. (2004) Prostate cancer is characterized by epigenetic silencing of 14-3-3{varsigma} expression. Oncogene 23, 9034 –9041[CrossRef][Medline]

  6. Lodygin, D., Yazdi, A. S., Sander, C. A., Herzinger, T., and Hermeking, H. (2003) Analysis of 14-3-3{varsigma} expression in hyper-proliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma. Oncogene 22, 5519 –5524[CrossRef][Medline]

  7. Osada, H., Tatematsu, Y., Yatabe, Y., Nakagawa, T., Konishi, H., Harano, T., Tezel, E., Takada, M., and Takahashi, T. (2002) Frequent and histological type-specific inactivation of 14-3-3{varsigma} in human lung cancers. Oncogene 21, 2418 –2424[CrossRef][Medline]

  8. Iwata, 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., and Imai, K. (2000) Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3{varsigma} gene in human hepatocellular carcinoma. Oncogene 19, 5298 –5302[CrossRef][Medline]

  9. Chan, T. A., Hermeking, H., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1999) 14-3-3{varsigma} is required to prevent mitotic catastrophe after DNA damage. Nature 401, 616 –620[CrossRef][Medline]

  10. Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., and Cantley, L. C. (1997) The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961 –971[CrossRef][Medline]

  11. Yaffe, M. B. (2002) How do 14-3-3 proteins work?—gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett. 513, 53 –57[CrossRef][Medline]

  12. Samuel, T., Weber, H. O., Rauch, P., Verdoodt, B., Eppel, J. T., McShea, A., Hermeking, H., and Funk, J. O. (2001) The G2/M regulator 14-3-3{varsigma} prevents apoptosis through sequestration of Bax. J. Biol. Chem. 276, 45201 –45206[Abstract/Free Full Text]

  13. Yang, H. Y., Wen, Y. Y., Chen, C. H., Lozano, G., and Lee, M. H. (2003) 14-3-3{varsigma} positively regulates p53 and suppresses tumor growth. Mol. Cell. Biol. 23, 7096 –7107[Abstract/Free Full Text]

  14. Kino, T., Souvatzoglou, E., De Martino, M. U., Tsopanomihalu, M., Wan, Y., and Chrousos, G. P. (2003) Protein 14-3-3{varsigma} interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway. J. Biol. Chem. 278, 25651 –25656[Abstract/Free Full Text]

  15. Rothblum-Oviatt, C. J., Ryan, C. E., and Piwnica-Worms, H. (2001) 14-3-3 binding regulates catalytic activity of human Wee1 kinase. Cell Growth Differ. 12, 581 –589[Abstract/Free Full Text]

  16. Urano, T., Saito, T., Tsukui, T., Fujita, M., Hosoi, T., Muramatsu, M., Ouchi, Y., and Inoue, S. (2002) Efp targets 14-3-3{varsigma} for proteolysis and promotes breast tumour growth. Nature 417, 871 –875[CrossRef][Medline]

  17. Laronga, C., Yang, H. Y., Neal, C., and Lee, M. H. (2000) Association of the cyclin-dependent kinases and 14-3-3{varsigma} negatively regulates cell cycle progression. J. Biol. Chem. 275, 23106 –23112[Abstract/Free Full Text]

  18. Subramanian, R. R., Masters, S. C., Zhang, H., and Fu, H. (2001) Functional conservation of 14-3-3 isoforms in inhibiting bad-induced apoptosis. Exp. Cell Res. 271, 142 –151[CrossRef][Medline]

  19. Liu, M. Y., Cai, S., Espejo, A., Bedford, M. T., and Walker, C. L. (2002) 14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s). Cancer Res. 62, 6475 –6480[Abstract/Free Full Text]

  20. Stavridi, E. S., Chehab, N. H., Malikzay, A., and Halazonetis, T. D. (2001) 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. 61, 7030 –7033[Abstract/Free Full Text]

  21. Sekimoto, T., Fukumoto, M., and Yoneda, Y. (2004) 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1). EMBO J. 23, 1934 –1942[CrossRef][Medline]

  22. Mils, V., Baldin, V., Goubin, F., Pinta, I., Papin, C., Waye, M., Eychene, A., and Ducommun, B. (2000) Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase. Oncogene 19, 1257 –1265[CrossRef][Medline]

  23. Uchida, S., Kuma, A., Ohtsubo, M., Shimura, M., Hirata, M., Nakagama, H., Matsunaga, T., Ishizaka, Y., and Yamashita, K. (2004) Binding of 14-3-3ß but not 14-3-3{varsigma} controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B. J. Cell Sci. 117, 3011 –3020[Abstract/Free Full Text]

  24. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Seraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218 –229[CrossRef][Medline]

  25. Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M., and Yates, J. R., III (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676 –682[CrossRef][Medline]

  26. Tabb, D. L., McDonald, W. H., and Yates, J. R., III (2002) DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome. Res. 1, 21 –26[Medline]

  27. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030 –1032[CrossRef][Medline]

  28. Yu, J., Zhang, L., Hwang, P. M., Rago, C., Kinzler, K. W., and Vogelstein, B. (1999) Identification and classification of p53-regulated genes. Proc. Natl. Acad. Sci. U. S. A. 96, 14517 –14522[Abstract/Free Full Text]

  29. Washburn, M. P., Wolters, D., and Yates, J. R., III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242 –247[CrossRef][Medline]

  30. Obenauer, J. C., Cantley, L. C., and Yaffe, M. B. (2003) Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic. Acids. Res. 31, 3635 –3641[Abstract/Free Full Text]

  31. Aitken, A., Baxter, H., Dubois, T., Clokie, S., Mackie, S., Mitchell, K., Peden, A., and Zemlickova, E. (2002) Specificity of 14-3-3 isoform dimer interactions and phosphorylation. Biochem. Soc. Trans. 30, 351 –360[CrossRef][Medline]

  32. Chaudhri, M., Scarabel, M., and Aitken, A. (2003) Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo. Biochem. Biophys. Res. Commun. 300, 679 –685[CrossRef][Medline]

  33. Ridley, A. J., Schwartz, M. A., Burridge, K., Firtel, R. A., Ginsberg, M. H., Borisy, G., Parsons, J. T., and Horwitz, A. R. (2003) Cell migration: integrating signals from front to back. Science 302, 1704 –1709[Abstract/Free Full Text]

  34. Meek, S. E., Lane, W. S., and Piwnica-Worms, H. (2004) Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins. J. Biol. Chem. 279, 32046 –32054[Abstract/Free Full Text]

  35. Rubio, M. P., Geraghty, K. M., Wong, B. H., Wood, N. T., Campbell, D. G., Morrice, N., and Mackintosh, C. (2004) 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. Biochem. J. 379, 395 –408[CrossRef][Medline]

  36. Jin, J., Smith, F. D., Stark, C., Wells, C. D., Fawcett, J. P., Kulkarni, S., Metalnikov, P., O’Donnell, P., Taylor, P., Taylor, L., Zougman, A., Woodgett, J. R., Langeberg, L. K., Scott, J. D., and Pawson, T. (2004) Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr. Biol. 14, 1436 –1450[CrossRef][Medline]

  37. Pinheiro, E. M., and Montell, D. J. (2004) Requirement for Par-6 and Bazooka in Drosophila border cell migration. Development 131, 5243 –5251[Abstract/Free Full Text]

  38. Hurd, T. W., Fan, S., Liu, C. J., Kweon, H. K., Hakansson, K., and Margolis, B. (2003) Phosphorylation-dependent binding of 14-3-3 to the polarity protein Par3 regulates cell polarity in mammalian epithelia. Curr. Biol. 13, 2082 –2090[CrossRef][Medline]

  39. Rodriguez, L. G., and Guan, J. L. (2005) 14-3-3 regulation of cell spreading and migration requires a functional amphipathic groove. J. Cell. Physiol. 202, 285 –294[CrossRef][Medline]

  40. Benzinger, A., Popowicz, G. M., Joy, J. K., Majumdar, S., Holak, T. A., and Hermeking, H. (2005) The crystal structure of the non-liganded 14-3-3 sigma protein: insights into determinants of isoform specific ligand binding and dimerization. Cell Research 15, 219 –227[CrossRef][Medline]

  41. Dent, P., Yacoub, A., Fisher, P. B., Hagan, M. P., and Grant, S. (2003) MAPK pathways in radiation responses. Oncogene 22, 5885 –5896[CrossRef][Medline]

  42. Tzivion, G., and Avruch, J. (2002) 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol. Chem. 277, 3061 –3064[Free Full Text]

  43. Hirota, T., Kunitoku, N., Sasayama, T., Marumoto, T., Zhang, D., Nitta, M., Hatakeyama, K., and Saya, H. (2003) Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114, 585 –598[CrossRef][Medline]

  44. Marie, H., Pratt, S. J., Betson, M., Epple, H., Kittler, J. T., Meek, L., Moss, S. J., Troyanovsky, S., Attwell, D., Longmore, G. D., and Braga, V. M. (2003) The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with {alpha}-catenin. J. Biol. Chem. 278, 1220 –1228[Abstract/Free Full Text]

  45. Lee, J., Kumagai, A., and Dunphy, W. G. (2001) Positive regulation of Wee1 by Chk1 and 14-3-3 proteins. Mol. Biol. Cell 12, 551 –563[Abstract/Free Full Text]

  46. Peng, C. Y., Graves, P. R., Ogg, S., Thoma, R. S., Byrnes, M. J., III, Wu, Z., Stephenson, M. T., and Piwnica-Worms, H. (1998) C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. Cell Growth Differ. 9, 197 –208[Abstract]

  47. Kuriyama, R., Gustus, C., Terada, Y., Uetake, Y., and Matuliene, J. (2002) CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis. J. Cell. Biol. 156, 783 –790[Abstract/Free Full Text]

  48. Santamaria, A., Fernandez, P. L., Farre, X., Benedit, P., Reventos, J., Morote, J., Paciucci, R., and Thomson, T. M. (2003) PTOV-1, a novel protein overexpressed in prostate cancer, shuttles between the cytoplasm and the nucleus and promotes entry into the S phase of the cell division cycle. Am. J. Pathol. 162, 897 –905[Abstract/Free Full Text]

  49. Marchler-Bauer, A., Anderson, J. B., DeWeese-Scott, C., Fedorova, N. D., Geer, L. Y., He, S., Hurwitz, D. I., Jackson, J. D., Jacobs, A. R., Lanczycki, C. J., Liebert, C. A., Liu, C., Madej, T., Marchler, G. H., Mazumder, R., Nikolskaya, A. N., Panchenko, A. R., Rao, B. S., Shoemaker, B. A., Simonyan, V., Song, J. S., Thiessen, P. A., Vasudevan, S., Wang, Y., Yamashita, R. A., Yin, J. J., and Bryant, S. H. (2003) CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res. 31, 383 –387[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. M. Atkins, L. Thomas, R. T. Youker, M. J. Harriff, F. Pissani, H. You, and G. Thomas
HIV-1 Nef Binds PACS-2 to Assemble a Multikinase Cascade That Triggers Major Histocompatibility Complex Class I (MHC-I) Down-regulation: ANALYSIS USING SHORT INTERFERING RNA AND KNOCK-OUT MICE
J. Biol. Chem., April 25, 2008; 283(17): 11772 - 11784.
[Abstract] [Full Text] [PDF]