JBC Papers in Press. Published on May 25, 2004 as Manuscript M403044200

Comprehensive proteomic analysis of interphase and mitotic 14-3-3 binding proteins

Sarah E. M. Meek1, William S. Lane2 and Helen Piwnica-Worms 1,3,4*

1Howard Hughes Medical Institute, 2Microchemistry and Proteomics Analysis Facility, Harvard University, 16 Divinity Ave. Cambridge, MA, 3Department of Cell Biology and Physiology, 4Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue St. Louis, MO 63110-1093, USA.

Running title: Interphase and Mitotic 14-3-3 Proteome

* To whom correspondence should be addressed.

Abbreviations SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), DTT (dithiothreitol), FACS (fluorescence activated cell sorting), CDK (cyclin-dependent protein kinase), EDTA (ethylenediamine-tetraacetic acid), TSC2 (tuberous sclerosis protein 2), HDAC (histone deacetylase), HPLC (high pressure liquid chromatography), MS (mass spectrometry), GST (glutathione-S-transferase), PMSF (phenyl methane sulfonyl fluoride).

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Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Summary

14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement following activation of the DNA replication- and DNA damage-checkpoints. Here, we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3z affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary HPLC tandem mass spectrometry analysis. Several known and novel 14-3-3 interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport, as well as several other cellular processes. In some cases, 14-3-3 binding was cell cycle dependent, whereas in other cases the binding was shown to be cell cycle independent. This study adds to the growing list of human 14-3-3 binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes.

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Introduction 14-3-3 proteins belong to a highly conserved, multigene family of small acidic proteins (15).

In mammals there are seven 14-3-3 isoforms, designated with Greek letters

( b, e, g, h, s, t, z). In most cases, 14-3-3s bind to target proteins when the targets are phosphorylated; two canonical 14-3-3-binding motifs have been identified as RSXpSXP and RXY/FXpSXP, where pS represents phosphoserine or phosphothreonine (6,7). 14-3-3 binding can alter the enzymatic activity, subcellular localization, protein-protein interactions, dephosphorylation, and proteolysis of individual target proteins (4,8,9). 14-3-3 proteins bind to and regulate several key cell cycle regulators, including Wee1, Cdc25A, Cdc25B and Cdc25C. In each case, 14-3-3s bind the regulators during interphase but not during mitosis. Entry into mitosis requires activation of the Cdk1 protein kinase, and 14-3-3s control Cdk1 activity indirectly, by regulating the activity of both the kinases and phosphatases that control Cdk1. First, 14-3-3 binds to the C-terminus of Wee1 during interphase to stabilize its kinase activity (10). Second, 14-3-3 binds to Cdc25C in the cytosol during interphase to prevent Cdc25C from accumulating in the cell nucleus (11-14). During late G2, loss of 14-3-3 binding allows Cdc25C to accumulate in the nucleus. 14-3-3 binding to Cdc25B also appears to contribute to the nuclear exclusion of Cdc25B (15). Lastly, 14-3-3s bind to Cdc25A during interphase to prevent Cdc25A from activating Cdk1/cyclin B1 complexes (16). 14-3-3 proteins also play a pivotal role in blocking cell cycle advancement following checkpoint activation. Checkpoints are signaling pathways that monitor the integrity and replication status of the genetic material before cells commit to either replicate (in S-phase) or segregate (in mitosis) their DNA (17). Once activated, checkpoints ultimately interface with cyclin/Cdk complexes to block cell cycle advancement, or induce cell death. 14-3-3 proteins are 3

essential for both the DNA replication and DNA damage checkpoints, in human, Xenopus laevis, and fission yeast cells (13,18-22).

In addition to regulating Cdc25C as described above, the

fission yeast 14-3-3 protein Rad 24 also binds to the Chk1 protein kinase, an essential component of the DNA damage checkpoint (23,24). The binding of 14-3-3 proteins to Chk1 is induced by DNA damage, but the functional consequences of this interaction are not known (25). A recent report indicates that human Chk1 may also bind 14-3-3 proteins (26). Lastly, the p53 tumor suppressor protein is an essential component of the G1 DNA damage checkpoint (27) and also plays a role in maintaining the G2 DNA damage checkpoint (19,28-30). In epithelial cells, 14-33s is induced in a p53-dependent manner in response to DNA damage. Cells lacking 14-3-3s can still arrest in G2, but are unable to maintain this cell cycle arrest, and ultimately die. In these 14-3-3s-deficient cells, Cdk1/cyclin B1 complexes, which normally undergo continual nuclearcytoplasmic shuttling, are now allowed to accumulate in the nucleus, ultimately leading to bypass of the G2 DNA damage checkpoint. The tumour suppressor protein p53 also binds to 143-3 proteins. In this case, dephosphorylation of p53 at serine 376 generates a functional binding motif at phospho-serine 378 and an intact 14-3-3-binding site is required for ATM-dependent p53 activation and effective cell cycle arrest (31). In order to identify novel cell cycle regulatory functions of 14-3-3, we carried out a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-interacting proteins were purified from extracts of both interphase and mitotic HeLa cells, using specific peptide elution from 14-3-3z affinity columns. The eluted proteins were identified by microcapillary HPLC tandem mass spectrometry analysis (LC-MS/MS). Proteins that were identified are involved in multiple biological processes, and include both cell cycle-dependent and cell cycle-independent interactors. 4

Experimental Procedures

Materials The R18/11 peptide, RDLSWLDLEAN, was synthesized by Dr. M. Berne (Tufts University), and HPLC-purified by Dr. J. Gorka (Biomolecules Midwest). Precast SDS-PAGE gels used for MS analysis were from Biorad, and the colloidal Coomassie protein stain was from Invitrogen. Vivaspin and Centricon centrifugal concentrators were from Vivascience and Amersham, respectively. All other reagents were from Sigma or Fisher Scientific, and were reagent grade.

Antibodies, Western blotting and immunoprecipitation Antibodies against 14-3-3 (K19), glutathione S-transferase (GST), Cdc25B, Cdc25C, PCTAIRE 2, PCTAIRE 3, TSC2 (tuberin), nucleolin (C23), nucleophosmin, Chk1, HDAC4 and Wee1 were purchased from Santa Cruz. Antibodies against human Cdc25A were from Neomarkers, and antibodies against EMK/Par1B have been described (32). Cdc25C was detected with ascites generated from a monoclonal Cdc25C antibody (13). C-TAK1 was detected with ascites generated using a monoclonal antibody produced with bacterially purified His-tagged human CTAK1 (hPar-1a). For Western blotting, antibodies were dissolved in 5 % milk in TBST (50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 0.02 % Tween-20), and membranes were washed 3 times in TBST after application of both primary and secondary antibody. Bound primary antibodies were detected with horseradish peroxidase-conjugated goat anti-mouse (Jackson), goat anti-rabbit (Zymed), or donkey anti-goat (Santa Cruz) secondary antibodies, and visualized using the ECL reagent (Amersham). Proteins were immunoprecipitated from 1 mg of total cell lysate, using

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either anti-FLAG (M2) agarose (Sigma), anti-myc (9E10) agarose (Santa Cruz), or anti-TSC2 antibody bound to Protein A beads (Pierce).

14-3-3 Far Western analysis For 14-3-3 Far Western analysis, samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes as for a Western blot. Proteins bound to the membrane were denatured by incubating for at least 1 hour in denaturation buffer (50 mM Tris-HCl pH 8.0, 6 M guanidine-HCl, 6.25 mM EDTA, 1 mM DTT, 10 % glycerol, 0.05 % Tween-20), then renatured by incubating for at least 1 hour in renaturation buffer (denaturation buffer without guanidineHCl or DTT). Membranes were blocked for 1 hour in 5 % milk in TBST, rinsed in TBST, then incubated for 2 hours at room temperature or overnight at 4°C in TBST containing 0.1 mg/ml GST-14-3-3z and s, and 1 mg/ml bovine serum albumin. Where the R18/11 peptide was used to check binding specificity, the GST-14-3-3 protein mixture was pre-incubated with 0.1 mM peptide for 1 hour prior to use. Membranes were washed 3 times in TBST, then incubated for 1 hour with anti-GST primary antibody in 5 % milk / TBST. Bound primary antibody was detected with horseradish peroxidase-conjugated secondary antibody, and visualized using ECL reagent, as for Western blotting.

14-3-3 affinity chromatography All purification steps were carried out at 4°C. Mitotic and interphase HeLa cells were lysed by incubation on ice for 15 min in a hypotonic lysis buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM microcystin-LR, 1 mM DTT, plus Sigma protease inhibitor cocktail), followed by Dounce homogenization. The lysate (lysate 6

containing 150 mg total protein was used for each affinity column) was centrifuged at 12,000 x g for 20 min, and a 0 – 80 % ammonium sulphate fraction was prepared. Precipitated proteins were resuspended in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM microcystinLR, plus protease inhibitors, then dialysed for 2 hours against 2 x 2 liters of 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 1mM benzamidine, and 4 mM PMSF. The sample was clarified by centrifugation at 12,000 x g for 15 min, and incubated end-over-end with a 5 ml GST column for 1 hour, to remove any GST-binding proteins. The precleared sample was then incubated endover-end with either 5 ml GST-14-3-3 beads, or 5 ml GST control beads, for 2 hours. The beads were transferred to a disposable column holder (Biorad) and the flowthrough was collected. The column was washed with 10 column volumes low salt wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT), then with 10 volumes high salt wash buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM DTT), until no further protein was eluted. 14-3-3-binding proteins were specifically eluted from the column with 25 ml of 0.5 mM R18/11 peptide dissolved in high salt buffer, followed by 5 ml high salt buffer (The peptide elution buffer was left on the column for 15 min prior to elution). The first 20 x 2 ml fractions from each column were pooled, concentrated, and dialyzed using Vivaspin then Centricon 10,000 concentrators. Concentrated fractions (35 ml out of a total of 50 ml for each column) were subjected to SDS-PAGE, and proteins were visualized using Colloidal Coomassie stain. One ml of each preparation was used for Far Western analysis.

Peptide Identification by Mass Spectrometry and Bioinformatics Analysis Bands were excised from the SDS-PAGE gel, and pooled into 10 samples from each gel lane according to staining intensity and apparent molecular mass. The protein and Far Western 7

patterns shown are representative of 3 separate purifications, with MS/MS analysis carried out once. After in-gel trypsinization, peptide sequences were determined by microcapillary reversed-phase chromatography coupled to a Finnigan LCQ DECA XP+ quadrupole ion trap mass spectrometer as in (33). To improve data analysis, the resulting MS/MS spectra were preprocessed with inhouse custom software that recombined identical spectra (CombIon), determined precursor charge state without a high resolution scan (ZSA), and increased the accuracy of the precursor assignment (CorrectIon). Interpretation of the resulting MS/MS spectra of the peptides was facilitated by database correlation with the algorithm SEQUEST followed by manual examination of the MS/MS spectra. This large scale evaluation was aided by creating a working superset of peptide sequences with two programs written in-house. The first, ScoreFinal, uses a neural network to synthesize a single normalized score (Sf 0.0–1.0) from the five SEQUEST scores (Sp, RSp, Ions, XCorr, DeltaCn) as well as peptide length, precursor charge state and database size. The second, SigCalc, independently calculates the probability (P) that MS/MS product ions observed would match the peptide’s predicted fragment ions as a random event. The working set were those sequences that met the criteria Sf . >= 0.5, P >= 10-6; or the sum of Sf >= 0.85 for two or more spectra identifying the same protein. From this set, final identifications were always confirmed by rigorous manual validation of the MS/MS spectra.

Expression of recombinant GST and GST-14-3-3, and preparation of GST-14-3-3 and control GST affinity columns BL21 cells were transformed with plasmids encoding GST or GST-tagged 14-3-3z. Cultures were grown in a Bioflo 5000 fermenter (New Brunswick Scientific) at 37°C with TB medium 8

containing 0.2 % glucose, and under 30 % O2, to an A 600 of 0.6. Isopropyl-1-thio-ß-Dgalactopyranoside (IPTG) was added to a final concentration of 1 mM. After growing for an additional 4 h, cells were pelleted by continuous centrifugation. Cell pellets were resuspended in bacterial cell lysis buffer (50 mM Hepes-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA), containing 1.25% N-lauryl sarcosine supplemented with 1 mM DTT, 1 mg/ml lysozyme, and protease inhibitors (4 mM PMSF, 20 µg/ml aprotinin, 40 µM leupeptin, 1 mM benzamidine). After rocking at 4°C for 20 min, cells were lysed by sonication. Lysates were clarified by centrifugation (12,000 x g for 15 min), and Triton X-100 was added to a final concentration of 1%. Proteins were precipitated with glutathione-agarose beads and washed once with bacterial lysis buffer, twice with 10 volumes 50 mM Hepes-HCl pH 7.5 containing 0.5 M LiCl, and 3 times with 10 volumes 50 mM Hepes-HCl pH 7.5, 0.1 M NaCl, 1 mM EDTA, 1 mM DTT. GST fusion proteins were eluted with 20 mM glutathione in the same buffer, and protein-containing fractions were pooled and dialysed overnight against buffer without glutathione, plus 1 mM benzamidine and 4 mM PMSF.

The levels of GST fusion proteins were estimated by

comparison to a BSA standard after SDS-PAGE and Coomassie blue staining. Purified GST or GST-14-3-3 was covalently coupled to activated CH sepharose (Sigma) according to the manufacturer’s instructions. 2.5 mg protein was bound per 1 ml swollen beads. GST and 14-3-3 columns were washed extensively and equilibrated in low salt buffer immediately before use.

GST-14-3-3 pull-down experiments HeLa cells were lysed in 50 mM Tris-HCl pH 8.0, 0.1M NaCl, 5 mM EDTA, 0.5 % NP-40, 1 mM DTT, 1 mM microcystin-LR, plus protease inhibitors.

Lysates were clarified by

centrifugation at 16,000 x g. The lysate was pre-incubated with glutathione agarose without any 9

GST fusion proteins for 1 hour at 4 °C. Meanwhile, bacterial cell lysate expressing GST–14-33z was incubated with glutathione agarose for 1 h at 4 °C. The beads were washed with 0.5 M LiCl in 50 mM Tris-HCl pH 8.0, followed by HeLa cell lysis buffer, then incubated with 1 - 2 mg precleared HeLa cell extract for 1 h at 4 °C. Beads were washed four times in HeLa cell lysis buffer, and bound proteins were eluted by boiling in SDS–PAGE sample buffer.

Cell culture and cell cycle synchronization HeLa cells were routinely maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and streptomycin. To prepare non-nocodazole-treated mitotic cells, asynchronously growing cells were treated with 2 mM thymidine for 16 h, then released from the block by switching to complete growth medium containing 24 µM each of thymidine and deoxycytidine. After 8 h, thymidine was added to the medium to a final concentration of 2 mM, and cells were cultured for an additional 16 h, to synchronize them at the G1-S border. Cells were then rinsed twice with PBS and cultured in complete growth medium for a further 7 – 10 hours, when mitotic cells were collected by shake-off.

For interphase samples,

asynchronously growing cells were harvested at 60 – 90 % confluence. To obtain nocodazolesynchronised mitotic cells, 100 ng/ml nocodazole was added to the growth medium of asynchronously-growing cells, and cells were harvested by shake-off after 10 – 12 hours. Cell cycle phase was determined by flow cytometry by using a Becton-Dickinson FACScan. Cells were stained with 30!µg/ml propidium iodide for DNA content, and for phospho-histone H3 content to identify mitotic cells as described (34). FACS data were analyzed using CELLQUEST software. Transient transfection of HeLa cells was carried out using Fugene 6 (Roche), according to the manufacturer’s instructions. 10

Dephosphorylation of 14-3-3-binding proteins in vitro Purified 14-3-3-binding proteins (0.1 ml out of a total of 50 ml of each column eluate) were incubated for 30 min at 37°C with 50 U calf intestinal phosphatase (NEB), in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM DTT. Reactions were terminated by the addition of SDS-PAGE sample buffer, and phosphorylation and 14-3-3 binding status were analyzed by Far Western.

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Results Distinct spectrum of 14-3-3-interacting proteins in interphase and mitosis – Far Western analysis was employed to visualize the profile of 14-3-3 binding proteins in mitotic and interphase cells. FACS analysis demonstrated that asynchronously growing populations of HeLa cells consist of ~3% mitotic cells and 97% G1- S- or G2- phase cells (data not shown). Therefore, asynchronous populations of HeLa cells served as the source of interphase cells. Mitotic cells were obtained in two ways. Cells were synchronized in S-phase, released from the S-phase block and then collected when they entered mitosis. Alternatively, HeLa cells were incubated in nocodazole to enrich for mitotic cells. Profiles of 14-3-3 binding proteins obtained using both methods were compared. As seen in Fig. 1, the overall profiles of 14-3-3 binding proteins were similar between the two populations of mitotic cells. However, interphase and mitotic cells exhibited distinct 143-3 binding profiles. The interactions between 14-3-3s and their target proteins were shown to be specific because 14-3-3 binding was specifically competed with an 11 amino acid peptide derived from the R18 peptide (Fig. 1, panel C). R18 is a hydrophobic peptide that binds to the amphipathic target-binding groove of the 14-3-3 dimer (35,36). Although it is not itself phosphorylated, R18 can displace phosphorylated target proteins from the 14-3-3 binding groove. Further evidence for specificity comes from the observation that 14-3-3 binding to target proteins was abolished by protein phosphatase treatment (see Fig. 2B, lanes 11,14). These results demonstrated that there are cell cycle-specific differences in 14-3-3 binding partners that can be detected by Far Western analysis, and that nocodazole can be used to synchronize mitotic HeLa cells for large scale purification of 14-3-3 binding partners.

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Purification of 14-3-3-binding proteins from interphase and mitotic cells - Next, a purification scheme was developed to isolate sufficient quantities of 14-3-3 binding proteins for identification by mass spectrometry (Figure 2A). For large-scale protein purification, HeLa cells were harvested from asynchronously growing cell cultures or from cells that had been synchronized in mitosis with nocodazole. FACS analysis of propidium iodide- and phospho-histone H3-stained cells (34) was used to confirm cell cycle phases: the asynchronous population used for purification contained 97% interphase cells, while the nocodazole-treated population contained at least 90% mitotic cells (data not shown). Cells were snap-frozen, and then thawed in lysis buffer for protein purification. Proteins were precipitated from the cell lysate with 80% ammonium sulphate, resuspended in buffer, and passed over a GST column to remove GST-binding proteins. Samples were then applied to either a GST control column, or a GST-14-3-3z column. The 143-3-binding activity passed through the GST control column (Figure 2B, lanes 5, 6, 15, 16), but a large portion was retained on the GST-14-3-3z affinity column (Fig. 2B, lanes 3, 4, 9, 12). The presence of endogenous 14-3-3s in the HeLa cell lysate or dephosphorylation of target proteins may have prevented some 14-3-3-binding proteins binding to the 14-3-3 column (lanes 3, 4). The 14-3-3-binding proteins remained bound to the 14-3-3 column during the high salt wash (lanes 7, 8) and were eluted by the 14-3-3-binding R18/11 peptide (lanes 9, 12). In contrast, few proteins were eluted from the GST control column by the peptide (lanes 15, 16). Binding of proteins to GST-14-3-3 was phosphorylation-dependent, because phosphatase treatment eliminated binding (lanes 11, 14). Protein yields at various stages of the purification are shown in Figure 2A.

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Proteins eluted from each of the columns were concentrated, subjected to SDS-PAGE, and visualized with colloidal Coomassie stain (Figure 2C). Differences were readily apparent between the mitotic (lane 1) and interphase (lane 2) column eluates. The eluted protein pools should contain proteins that bind to 14-3-3s directly, but may also include proteins that bind to the column due to their association with 14-3-3 binding proteins. Proteins were excised from the gel and pooled according to the apparent molecular mass and abundance of each band. The apparent molecular mass of many, though not all, of the excised bands corresponded to bands that were observed in 14-3-3 Far Westerns (not shown). Proteins were extracted from the gel, trypsinized, and peptides were identified by LC-MS/MS analysis.

Identification of interphase and mitotic 14-3-3-binding proteins – In total, 209 proteins were identified in the mitotic 14-3-3-binding pool, and 184 from the interphase pool (Table 1, and Supplementary Table 1). Of these, 89 were identified in both the interphase and mitotic samples. Identified 14-3-3 interacting proteins included proteins involved in cell signaling, metabolic pathways, protein synthesis, antioxidant and stress responses, cytoskeletal dynamics, RNA binding, DNA binding and chromatin structure, vesicle trafficking, protein folding, ubiquitination and proteolysis, nucleolar function, nuclear transport, transcription, and cell cycle regulation. Many peptides obtained corresponded to proteins that are present in the databases only as EST sequences, with unknown function. Uncharacterized proteins have been listed by provisional name and accession number only. Such proteins were assigned a functional group in Table 1 if a function could be inferred from sequence homology with characterized proteins, from homology with functional protein domains, or from the circumstances under which EST sequences were isolated. 14

Several prominent 14-3-3-interacting proteins seen in Far Westerns (Figure 1, 2B) and by colloidal Coomassie staining (Figure 2C) are dramatically different between interphase and mitotic samples. For instance, much more 14-3-3 binding activity was present at around 120 kDa in mitotic samples, and many more proteins of around that size were purified from mitotic samples. This region of the gel was excised and found to contain at least 18 different proteins; the relative abundance and contribution of each to 14-3-3 binding is not known at this time.

Confirmation of 14-3-3 interaction with identified proteins in interphase and mitosis - It is important to note that although a given protein may have been identified under only one of the two experimental conditions tested (interphase or mitosis), it may in fact have bound to the 14-33 column equally well under both conditions, but only have been detected in one set of samples. Thus, the interaction of 14-3-3 with a subset of identified target proteins was tested in GST-14-33 pull-down experiments (Figure 3). These experiments were performed with interphase and mitotic lysates to confirm 14-3-3 associations and to explore differences between the ability of a given protein to bind to 14-3-3s during interphase and mitosis. As seen in Figure 3, several known 14-3-3 targets bound to 14-3-3 in this assay, including the cell cycle regulator Wee1; the Par-1a (C-TAK1) and Par-1b (EMK) kinases which have been implicated in regulating cell polarity, microtubule dynamics, and the cell division cycle (32,37,38); Histone deacetylase 4, whose nucleo-cytoplasmic shuttling is regulated by 14-3-3 binding (39); and TSC2, a tumor suppressor protein that negatively regulates TOR (40-42). As expected, the known 14-3-3binding proteins Cdc25A and Cdc25C, although not detected in our MS analysis, specifically bound to 14-3-3 in the pull-downs during interphase but not mitosis. Novel 14-3-3 binding partners identified here include the PCTAIRE 2 and 3 protein kinases, and a fragment of 15

C23/nucleolin, a nucleolar phosphoprotein of unknown function. In addition to these GST-14-33 pull-down experiments, 14-3-3 Far Westerns were carried out where possible, to determine whether 14-3-3 binding to the identified interacting proteins was direct (Figure 4). In some cases, interactions between 14-3-3s and identified target proteins could not be confirmed by GST-14-3-3 pull-down, 14-3-3 Far Western analysis or by co-immunoprecipitation experiments. This was the case for Hus 1, purified histones and nucleophosmin (Table 2 and data not shown). Nucleophosmin binds to the 14-3-3 binding protein nucleolin (43), and may have been isolated on the 14-3-3 column because of its association with nucleolin. Finally, many of the identified proteins could not be confirmed as 14-3-3 targets due to lack of available reagents. The interaction of 14-3-3s with Cdc25 family members and with Wee1 showed the expected cell cycle dependency (Figure 3). Other previously unpublished cell cycle-dependent 14-3-3 interactions were observed in this screen. For example, TSC2 bound to 14-3-3 more in interphase than in mitosis (Figures 3 and 4B). Secondly, although there was more PCTAIRE 2 in the interphase and mitotic input samples, higher levels of mitotic PCTAIRE 2 were pulled down with GST-14-3-3. This suggests that the PCTAIRE 2 / 14-3-3 interaction is cell cycle regulated. In contrast, other interactors such as C-TAK1 (Par-1a) bound 14-3-3s equally well in interphase and mitosis. Interestingly, levels of C-TAK1 in mitotic cells were higher than those in interphase cells.

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Discussion In this study, a global proteomics analysis was undertaken to identify proteins that bind to 14-3-3s during interphase and during mitosis. Many different proteins bound to and specifically eluted from 14-3-3 affinity columns. Some of the identified proteins are known 14-3-3 interactors, including Par-1b and histone deacetylase 4. However, several of the identified proteins have not been previously shown to bind 14-3-3s, such as nucleolin (C23), and PCTAIRE 2 and 3. The 14-3-3-binding proteins identified are involved in many different aspects of metabolism, translation, antioxidant and stress responses, cytoskeletal organization, RNA binding, DNA binding and chromatin structure, vesicle trafficking, protein folding, ubiquitination and proteolysis, nucleolar function, nuclear transport, transcription, cell signaling, and cell cycle regulation. In addition, the identified interactors are localized in multiple places in the cell, including the cytosol, nucleus, mitochondrion, and ER. This is consistent with the observation of 14-3-3 proteins throughout the cell (4,44,45). These results illustrate that 14-3-3s are involved in a very wide range of cellular processes throughout interphase and mitosis. The specificity of 14-3-3 binding to individual proteins identified in this screen remains to be confirmed. Whether each interaction is direct, and whether each varies in a cell cycledependent manner, remains to be examined for many of the identified interactors. However, the fact that several known 14-3-3 targets were identified in this screen suggests that many of the identified proteins are likely to form complexes with 14-3-3s, either directly or indirectly. For instance, sepiapterin reductase is a good candidate 14-3-3 interactor, because two other enzymes in the same biosynthetic pathway, tyrosine and tryptophan hydroxylase, are regulated by 14-3-3 binding (46,47). In addition, the fact that more than half of the identified binding partners were isolated only from mitotic or interphase cells, but not from both, suggests that at least some of 17

these proteins may prove to be either novel regulators of the cell cycle, or to be novel targets of known cell cycle-specific regulatory processes. Perhaps nutrient sensing pathways are regulated in a cell cycle-specific manner: nutrient stimulation of protein translation by mTOR may be more important for the growth phases of the cycle (interphase) than for cell separation during mitosis. 14-3-3 proteins negatively regulate TSC2 which, in turn, negatively regulates mTOR signaling (40-42,48,49).

Interestingly, higher levels of 14-3-3 binding to TSC2 were observed during

interphase than during mitosis (Figures 3 and 4B), inferring that TSC2 may be less able to inhibit mTOR during interphase (growth phases) than during mitosis. While this manuscript was in preparation, Pozuelo Rubio et. al. (2004) published a list of mammalian 14-3-3 binding proteins that were purified from asynchronously growing cultured cells. Despite differences in the methodologies employed, some of the proteins identified here as interphase 14-3-3 interactors were also identified by Rubio et. al. (50) (Table 1). This suggests that many of the identified proteins are authentic 14-3-3 binding partners. In addition, some of the proteins identified in mitotic but not interphase cells in this study (e.g. calmodulin, phosphodiesterase 3A and NuMA) were identified in the screen performed in asynchronous cells by Pozuelo Rubio et. al., suggesting that calmodulin, PDE3A and NuMA may interact with 14-33s during both interphase and mitosis. Given that many 14-3-3 binding partners have now been identified, in this and other studies (50-52), it is now important to further characterize the interactions between the multiple 14-3-3 isoforms and individual target proteins, and to define the cellular consequences of these interactions. Identification of the signal transduction pathways that control binding of 14-3-3s to their multiple binding partners, in response to changing physiological conditions, also remains an important goal of future work. 18

Acknowledgments We thank Dr. B. Vogelstein and Dr. M Matsuoka for cDNAs encoding 14-3-3s and PCTAIRE 2, respectively; Dr. J. Bann for help with using the fermenter; and R. A. Robinson and Dr. J. M. Ren for their LC-MS/MS expertise. We thank members of the laboratory and Dr. T. Muslin for comments on the manuscript. S. M. is an Associate and H.P.W. is an Investigator of the Howard Hughes Medical Institute. This work was supported in part by an NIH grant and a Siteman Cancer Center Research Development Award to H. P.-W.

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Figure legends Figure 1. Far Western Analysis of interphase and mitotic cell extracts. Hela cells were synchronized in S-phase by a double thymidine block and release protocol and mitotic cells were harvested by mitotic shake-off 7 to 9.5 hours later (Mitosis). Alternatively, cells were arrested in mitosis by incubating cultures in the presence of 100 ng/ml nocodazole for 10 h (Nocodazole). Asynchronously growing cells were harvested for the interphase population (Interphase). Lysates were prepared, resolved by SDS-PAGE and proteins were transferred to nitrocellulose membranes. Membranes were washed and then incubated with a mixture of purified GST-14-33z/s protein in the absence (panels A, B) or presence (panel C) of the R18/11 peptide followed by GST antibody. Membranes were incubated with HRP anti-rabbit IgG and proteins were visualized using ECL reagents. Panel B shows a longer (bottom half) and shorter (top half) exposure of Panel A.

Figure 2. Purification and identification of 14-3-3-interacting proteins from interphase and mitotic HeLa cells. (A), Outline of the large-scale purification procedure. Protein yields at each step are indicated. (B), 14-3-3 Far Western analysis of samples at various steps in the purification procedure. Interphase (I) and mitotic (M) lysates prior to fractionation (lanes 1, 2); flow through fractions from the 14-3-3-(lanes 3, 4) and GST- (lanes 5, 6) columns; salt wash of the 14-3-3 column (lanes 7, 8); eluate from the 14-3-3 column (lanes 9,12); eluate from the 14-3-3 column incubated for 10 min at 37°C without (lanes 10, 13) or with (lanes 11, 14) calf intestinal phosphatase. Eluate from the GST column (lanes 15, 16).

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(C), Colloidal Coomassie-stained SDS-PAGE gel of mitotic- (lanes 1, 3) and interphase (lanes 2, 4) eluates from the 14-3-3 affinity column (lanes 1, 2) and GST control column (lanes 3, 4)

Figure 3. Confirmation of interactions between 14-3-3 and a subset of proteins identified in this study. Mitotic and interphase HeLa cell lysates were incubated with GST-14-3-3z coupled to glutathione beads or GST control beads. Bound proteins were resolved by SDS-PAGE and analyzed by Western blotting with the indicated antibodies.

Figure 4. Direct binding of 14-3-3 to a subset of target proteins. (A), HeLa cells were mock-transfected (control), or transfected with constructs encoding FLAGEMK, FLAG-C-TAK, myc-Cdc25A, myc-Wee1, or a truncated form of Flag-EMK that does not bind to 14-3-3. GST-14-3-3 Far Western blots of anti-FLAG or anti-myc immunoprecipitates are shown. Although levels of ectopically-expressed FLAG-C-TAK were low (lane 10), the protein was visible in anti-C-TAK Western blots (lane 13), and interaction of FLAG-C-TAK with 14-3-3 was visualized by Far Western (lane 4). (B), TSC2 was immunoprecipitated from mitotic or interphase cell lysates. GST-14-3-3 Far Western blots and anti-TSC2 Western blots of the immunoprecipitates are shown.

Table 1 (abbreviated version) and Supplementary Table 1 (full version). Identification of proteins that interact with the 14-3-3 affinity column. Peptides were identified by LC-MS/MS analysis. Each protein identification was manually confirmed to ensure that no other human proteins matched the peptide sequences obtained. For proteins with multiple isoforms, an individual isoform is listed only if at least one peptide was 21

observed that is unique to that isoform. Proteins identified by a single peptide are listed only if the sequence was derived from a unique human accession number.

For instance, the Wee 1

protein kinase was identified from only one peptide, but its interaction with 14-3-3s has been confirmed in this study and elsewhere (see text). The protein name given in the table is from the database entry where the protein was most functionally informative. In several cases, multiple gi accession numbers match the identified protein; only one is listed.

Many peptides

corresponding to 14-3-3, keratins (including the 14-3-3 interactors keratin 8 and 18) and several corresponding to GST were obtained; these are not listed. All proteins listed are human proteins. The number of peptides of each protein that were seen in each sample are listed separated by commas. It is of note that several proteins that were identified by a large number of peptides are also functionally uncharacterized. Caution must be used in interpreting relative protein abundance from the number of peptides observed, because the two do not necessarily correlate. The identification of a protein in multiple gel bands may indicate protein degradation or smearing on the gel; in some cases the repeated identification of a given protein increases the likelihood that it has been correctly identified as present in the screen. * represents a known 14-3-3 interactor. + represents a 14-3-3 interactor also published by Pozuelo Rubio et. al. (2004).

Table 2. Confirmation status of identified 14-3-3 interactors. GST-14-3-3 pull-downs and GST-14-3-3 Far Western blots were carried out as described for Figures 3 and 4.

22

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27

Nocodazole

Mitosis

C

Interphase

Nocodazole

Mitosis

B

Interphase

Nocodazole

Mitosis

A

Interphase

Fig 1

190

115

85 66 59 59

37

Probe membrane with:

GST-14-3-3

GST-14-3-3 (alternative exposures)

R18/11 peptide + GST-14-3-3

Fig 2A Interphase cells (150 mg protein)

Mitotic cells (150 mg protein)

Interphase cells (150 mg protein)

Mitotic cells (150 mg protein)

Lyse cells hypotonically in presence of protease and phosphatase inhibitors 0 - 80 % NH4SO4 precipitation Resuspend pellet in buffer containing 150 mM NaCl Preclear with GST column GST control column

GST-14-3-3 column

Flowthrough 69 mg

Flowthrough 66 mg

Flowthrough 69 mg

Flowthrough 60 mg

Low salt buffer wash (150 mM NaCl) High salt buffer wash (500 mM NaCl)

Fig 2C

Elute with 0.25 mM R18/11 peptide in high salt buffer

GST14-3-3 M I

GST M I

Concentrate and dialyse 20 µg

43 µg

0 µg

7 µg

120 100 90 80 70 60

SDS-PAGE

compare

Far Western

220 160

Colloidal Coomassie stain

50

Excise bands of interest

40

LC-MS/MS

30 25 20

Fig 2B

Total lysate

14-3-3 GST column column FT FT

I

M

I

M

I

M

1

2

3

4

5

6

I

GST column eluates

14-3-3 column eluates

Wash

Interphase

M

Mitosis

I

M

190kDa

115 kDa

85 kDa 66kDa

59 kDa

37 kDa

7

8

9

10

11

12

13

14

15

16

1

2

3

4

63 kDa

mitotic form interphase form

86 kDa 60 kDa

50 kDa

250 kDa

GST-14-3-3ζ

I

GST

I

M GST-14-3-3ζ

M

GST-14-3-3ζ

input

GST

beads only

Pull-downs

GST

Fig 3

α-Cdc25A

α-Cdc25C

α-Wee1 full-length

α-PCTAIRE 2

α-TSC 2

150 kDa

86 kDa

α-C-TAK/Par-1a background band

86 kDa 173 kDa

α-EMK/Par-1b α-HDAC4

113 kDa 62.5 kDa 53 kDa

70 kDa

α-PCTAIRE 3

α-C23 (nucleolin)

Cell cycledependent 14-3-3 binding

1

Probe with:

Fig 4B 250kDa

250kDa

150kDa

2 3

Interphase 4 5 6 7

GST-14-3-3ζ

Mitosis α-TSC2

150kDa

GST-14-3-3 Far Western 8 9 10 11

α-FLAG/myc

12

FLAG-C-TAK/Par-1a

Myc-Wee1

Myc-Cdc25A

FLAG-C-TAK/Par-1a

FLAG--∆EMK

FLAG-EMK/Par-1b

No transfection

Myc-Wee1

Myc-Cdc25A

FLAG-C-TAK/Par-1a

FLAG-∆EMK

FLAG-EMK/Par-1b

No transfection

Fig 4A

100kDa

75kDa

50kDa

IgG

13

α-C-TAK

Table 1: Selected 14-3-3 Interactors Function Cell division

Interphase

Mitosis NuMA

+

+

MCM3

Pericentriolar material 1 (PCM1) NUDE (nuclear distribution gene E homolog)

NUDE (nuclear distribution gene E homolog) Apoptosis stimulating of p53 protein 2 (ASPP2) / p53binding protein 2 (53BP2)

Replication factor C (RFC) 37 kDa subunit Rec 14 Wee1* Signaling - Kinases PCTAIRE-3

PI3 kinase C2 beta

PCTAIRE-2

FRAP2 (MTOR2)

EMK / Par1B*

C-TAK / Par1A*

C-TAK / Par1A*

B-raf *

B-raf *

PCTAIRE 3 DNA-PK, catalytic subunit PAK 4 (p21-activated kinase 4)

Signaling – Small GTPase related

Rho-interacting protein 3 (RIP3) RhoGAP10

+

Rho-interacting protein 3 (RIP3) RhoGAP10

IQGAP1

+

IQGAP1 +

+

ARHGEF 16

ARHGEF 16

G protein coupled receptor 69A Signaling - Misc.

Liprin beta 1

Liprin beta 1

LL5 beta

Tuberin / Tuberous sclerosis complex 2 (TSC2) *

Insulin receptor substrate 2 (IRS2)*

+

Insulin receptor substrate 2 (IRS2)*

+

Phosphodiesterase (PDE) 3A * Epithelial protein lost in neoplasia B (EPLINb) / Sterol regulatory element binding protein 3 (SREBP3) Calmodulin*

+

+

Proline-rich AKT substrate * Protein tyrosine phosphatase PTPH1* Yes-associated protein (YAP)-like protein, 65 kDa

PAR3 / ASIP*

+

A kinase anchoring protein (AKAP)

A kinase anchoring protein (AKAP) APC (adenomatous polyposis coli) Nucleolar proteins

NOLC1 (nucleolar phosphoprotein p130) nucleolin/C23

Proteolysis/ ubiquitination

+

NOLC1 (nucleolar phosphoprotein p130) Nucleolin / C23

+

Nucleophosmin / B23

Nucleophosmin / B23

Ki Antigen / proteasome activator subunit

Ki Antigen / proteasome activator subunit

COP 9 subunit 4

NEDD4-like ubiquitin ligase Deubiquitinating enzyme 8 (USP8)

Stress or checkpoint + + Thioredoxin , peroxiredoxin 1 responses

Apoptosis

+

Thioredoxin , peroxiredoxin 1

+

KARP-1-binding protein (KAB) 1, 2

DNA damage-binding protein 1

PCD6 (programmed cell death 6) / alg2

Hus1 B-cell receptor-associated protein 31 (BAP31) / CDM protein

+

BH3-only domain protein 3 Nucleotide metabolism

CTP synthase Poly(p)/ATP NAD kinase Nucleoside diphosphate kinase +

CTP synthase NAD kinase +

Chromatin structure, HDAC4 , 7a * DNA binding mitochondrial ssDNA-binding protein Histone acetyltransferase B subunit 2

+

HDAC4 , 7a * mitochondrial ssDNA-binding protein DNA primase large subunit

Histone H4, 2B, 1 DNA methyltransferase 1 CGI-46 / DNA helicase RNA binding

+

+

+

hnRNP F, H 2, K , X, C, A1

hnRNP X, I, U +

Translation

Dead-box RNA binding protein 1 , 3 (DDX1 , 3) Angiogenin 2 (RNase) Ribosomal protein S3 / UV endonuclease Ribosomal protein P0, L19, L5, P2, S7 Glu, Gly-tRNA synthetase Poly(A) binding protein 2, 4 (PABP2, 4) HsGCN1 eIF 4G I, 3 epsilon +

+

eEF 2 , 1A1 , 1B Cytoskeleton

Dead-box RNA binding protein (DDX) 9 Decapping protein Dcp1a Ribosomal protein S3 / UV endonuclease Ribosomal protein L10a, P0 Val, Ile, Leu, Ala-tRNA synthetase HsGCN1 eIF 4E, 4A, 3 theta

+

+

+

Kinesins * mitotic kinesin-like protein 1 Dynein heavy chain Tubulin, alpha, beta Vimentin* Lamin A/C, B1

Kinesins *

+

+

+

Phosphofructokinase isoforms *

+

+

+

Fatty acid synthase

+

Long-chain fatty acid coenzyme A ligase 4 (LACS4) Enoly CoA hydratase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) * creatine kinase Lactate dehydrogenase isoforms Sepiapterin reductase H+-ATP synthase alpha, beta *

+

+

Very long-chain acyl coA synthetase (VLCS) Phosphoglycerate kinase 1 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) + * creatine kinase Lactate dehydrogenase isoforms Arginosuccinate synthase Panthothenate kinase

+

Chaperones

+

Annexin A2, 1 Actin Spectrin alpha, beta Talin 1 Profilin 1, cofilin 1 * Hornerin

Phosphofructokinase isoforms * Fatty acid synthase

+

Dynein heavy chain Tubulin, alpha, beta Vimentin* Hornerin

Annexin A2, 1 Actin Fascin 1 E-cadherin binding protein E7 Profilin 1, cofilin 1 * Metabolism

+

eEF 2 , 1A1 , 1B

+

Pyruvate kinase, M2 * Adenosylhomocysteinase (AdoHcyase) Enolase Pyridoxal kinase Triose phosphate isomerase Chaperonin containing t complex (CCT) subunits Cytosolic, ER and mitochondrial heat shock proteins

Pyruvate kinase M2-type * Adenosylhomocysteinase, AdoHcyase Enolase Triose phosphate isomerase Chaperonin containing t complex (CCT) subunits +

+

Cytosolic, ER and mitochondrial heat shock proteins

+

+

Nuclear transport

Importins Importin alpha re-exporter (Cellular apoptosis susceptibility protein)

Transcription

KRAB-associated protein KAP1 Human capicua Homolog of bovine peptidoglycan recognition protein LMO7 (Lim-domain only 7) KIAA1429 (viriliser-related)

NFAT2 Human capicua Homolog of bovine peptidoglycan recognition protein LMO7 (Lim-domain only 7) KIAA1429 (viriliser-related)

Wilms tumor 1 associating protein 1 (WTAP) Unnamed KIAA0853 Protein associated with myc, PAM

Wilms tumor 1 associating protein 1 (WTAP) Copine 1 KIAA0264 Protein associated with myc, PAM

Sugar-binding Unknown

+

Vesicle trafficking / Clathrin heavy chain* + formation

Importins Importin alpha re-exporter (Cellular apoptosis susceptibility protein)

Clathrin heavy chain * Fer1-like protein 3

+

Table 2. Confirmation status of identified 14-3-3 interactors. Protein PCTAIRE 2 PCTAIRE 3 Nucleolin (C23) Nucleophosmin (B23) Hus 1 HDAC 4 hPar1a hPar1b Wee 1 TSC2

14-3-3 interaction in pull-down Yes Yes Yes No

14-3-3 interaction in Far Western Yes N/D N/D No

No Yes Yes Yes Yes Yes

No N/D Yes Yes Yes Yes

Supplementary Table 1 (A) Mitosis

Function

Matched protein

Cell division

NuMA Pericentriolar material 1 (PCM1) NUDE (nuclear distribution gene E homolog) Replication factor C (RFC) 37 kDa subunit Rec 14 Wee1 * PCTAIRE-3 PCTAIRE-2 EMK / Par1B* C-TAK / Par1A* B-raf *

Signaling - Kinases

Signaling – Small GTPase related

Signaling - Misc.

Nucleolar proteins

Matched unique peptides

GenBank accession number (gi)

9, 2 3, 25 1

35119 5453856 8923110

1

4506491

1 1 2 4 6, 11, 2 15, 4 12 5, 2

13376840 1085293 20178302 266426| 11067437 3089349 213599 21740322

Rho-interacting protein 3 (RIP3) GEF-H1 1 RhoGAP10 15, 10 IQGAP1 3, 1 KIAA1521 14 ARHGEF 16 4 ARL6-interacting protein 2 4 Liprin beta 1 25, 15 LL5 beta 9, 2, 7 Insulin receptor substrate 2 (IRS2) * 3, 2, 37, 23 Phosphodiesterase (PDE) 3A * 3, 10 Yes-associated protein (YAP) 2 Epithelial protein lost in neoplasia B 2 (EPLINb) / Sterol regulatory element binding protein 3 (SREBP3) calmodulin* 4 PAR3 / ASIP* 2 BA416N2.2, FISH-like 2, 3, 1 signal-induced proliferation2 associated 1 like 3 Arg/Abl interacting protein 2 7, 8 A kinase anchoring protein (AKAP) 42 APC (adenomatous polyposis coli) 3 NOLC1 (nucleolar phosphoprotein 54, 64, 5, 27, 20, p130) 14, 32, 23, 11, 6 Nucleolin/C23 2 Nucleophosmin / B23 4

19744759 25535866 1170586 20521930 30353993 11641303 29294627 21955172 14537854 20070127 1363003 7705373

multiple 11275612 10334638 42661622 10947118 21493029 4557319 434765 21750187 10835063 1

Proteolysis/ ubiquitination

Stress or checkpoint responses

Apoptosis Nucleotide metabolism

Chromatin structure, DNA binding

RNA binding

Ki Antigen / proteasome activator subunit 26S proteasome regulatory subunit COP 9 subunit 4 Thioredoxin Peroxiredoxin 1 KARP-1-binding protein (KAB1) KARP-1-binding protein 2 (KAB2) PCD6 (programmed cell death 6) / alg2 CTP synthase Carbamoyl-phosphate synthase 1 (CPS1) Poly(p)/ATP NAD kinase Nucleoside diphosphate kinase Thymidine kinase UMP-CMP kinase HDAC4 * HDAC7A* Y-box binding protein 1 mitochondrial ssDNA-binding protein Histone acetyltransferase B subunit 2 Histone H4 Histone H2 B Histone 1 (histone 2A G) SUPT6H DNA methyltransferase 1 CGI-46 / DNA helicase RuvB-like (ATP helicase) hnRNP F hnRNP H2 hnRNP K hnRNP X hnRNP C hnRNP A1 LRPPRC (LRP130, GP130, Leucine-rich PPR motif containing protein) Dead-box RNA binding protein 1 (DDX1) Dead-box RNA binding protein 3 (DDX3) DKFZP434I116 protein isoform 1 Unnamed

7

2135531

1 1 2, 2, 1

6174930 7022321 10120659

14 11 4 1

4505591 7662142 5734603 12230420

4 6

14495609 21361331

11, 5 3 1 1 23, 8, 11, 18

8480400 1421609 225602 2497487 5174481

2, 15, 2, 7 5 3

30913097 181468 2624694

2

3334209

7 5 4 6, 9 1 5 1 2 1 4 13, 5 9 4 3

122098 10800138 10800130 1136404 20137608 4929561 28201891 16876910 9624998 585911 5453854 13097279 133254 1730078

3, 4

6919862

1

3023628

2 3

10434568 21750187 2

Translation

Cytoskeleton

Metabolism

La autoantigen 2 Angiogenin 2 (RNase) 1 Ribosomal protein S3 / UV 5 endonuclease Ribosomal protein P0 14 Ribosomal protein L19 2 Ribosomal protein L5 4 Ribosomal protein P2 6 Ribosomal protein S7 4 Glutamyl-tRNA synthetase * 1 Poly(A) binding protein * 2 3, 5 (PABP2) Poly(A) binding protein* 4 5 (PABP4) Glycyl-tRNA synthetase 2 HsGCN1 1 eIF 4G I 3 eIF3-epsilon 2 eEF 2 9 eEF 1 alpha 1 14 eEF 1B 2 Elongation factor Ts 2 Mitochondrial ribosomal protein S27 2 Kinesin heavy chain* 63 Kinesin 2 27, 23 Kinesin light chain 2 8, 2 mitotic kinesin-like protein 1 3 Dynein heavy chain 6 Tubulin, alpha, beta 14, 4 Vimentin* 28, 21 Lamin A/C 56, 14 Lamin B1 7 Annexin A2 16 Annexin I 4 Talin 1 2 Actin 10, 13 Fascin 1 2 E-cadherin binding protein E7 6 Profilin 1 3 Cofilin 1 * 1 Phosphofructokinase, platelet type 5 Phosphofructokinase heart isoform* 17 Fatty acid synthase 8 Long-chain fatty acid coenzyme A 2 ligase 4 (LACS4) Enoly CoA hydratase 2 Glyceraldehyde-3-phosphate 12 dehydrogenase (GAPDH) *

10835067 2500561 12848978 12654583 4506609 1173054 133061 134000 4758294 12229876 4504715 21264523 2282576 21634449 4503519 4503483 4503471 119163 12644268 13129094 4758648 14250822 10433849 20143967 30581065 multiple 2119204 27436946 5031877 18645167 4502101 14916725 multiple 13623415 13376204 30584265 5031635 11321601 11933149 21618359 12669909 12707570 31645 3

Chaperones

Nuclear transport

Transcription Sugar-binding

dehydrogenase (GAPDH) * creatine kinase Lactate dehydrogenase (heart type) Lactate dehydrogenase (muscle type) Sepiapterin reductase H+-ATP synthase alpha subunit* H+-ATP synthase beta subunit Pyruvate kinase, M2 * Adenosylhomocysteinase (AdoHcyase) Transketolase Aldolase Enolase Pyridoxal kinase Triose phosphate isomerase Chaperonin containing t complex (CCT) theta Chaperonin containing t complex (CCT) zeta Chaperonin containing t complex (CCT) eta Chaperonin containing t complex (CCT) beta Chaperonin containing t complex (CCT) gamma Chaperonin containing t complex (CCT) alpha Chaperonin (HSP 60) Heat shock protein 70 kDa (BiP) Heat shock protein 90 kDa alpha Heat shock protein 90 kDa beta heat shock 105kDa protein Heat shock protein 27 kDa Calreticulin Cyclophilin A Mitochondrial heat shock protein 75 (MTHSP75) DnaK-type chaperone Importin 7 Importin beta 1 Importin beta 2 Importin alpha re-exporter (Cellular apoptosis susceptibility protein) KRAB-associated protein KAP1 Human capicua Homolog of bovine peptidoglycan recognition protein

8 11, 4 7

180570 13786847 13786849

2 2 3 23 1

3885362 15030240 114562 125604 9951915

5 6 20 1 3 12

14250367 229674 30593767 13543317 17389815 1136741

10

4502643

8

5453607

4

5453603

3

2136253

1

135538

41, 3 41, 42, 10, 3 5 29, 25 2 10 4 5 27, 21

306890 16507237 123678 20149594 42544159 4504517 30583735 1633054 292059

49 2 11 7 3

2119712 11544639 20981701 1613834 20141403

2

1699027

3, 9 17

16507208 27808640 4

recognition protein Beta galactoside soluble lectin Unknown

Vesicle trafficking / formation

4 42, 22, 36, 54, 68, LMO7 (Lim-domain only 7) 13 KIAA1429 (viriliser-related) 81, 9, 23 Wilms tumor 1 associating protein 1 22, 4, 3 (WTAP) FLJ34154 2 Unnamed 3 Unnamed 26, 23, 48, 6 KIAA1458 9 KIAA0853 20, 3, 6, 3 TRIM28 2 sterol regulatory element binding 2 protein 3 trinucleotide repeat containing 15; 8 Grb10 interacting GYF protein 2 dJ469A13.1 3 KIAA1458 7 FLJ38426 2 Unnamed 2 KIAA0642 4 Unnamed 8 KIAA0731 4 KIAA0456 2 FLJ10211 4 Valosin-containing protein (VCP), 5 transitional ER ATPase FLJ38426 3 CGI-99 4 N8, long isoform 2 Polyposis locus protein 1 – like 1 1 Protein associated with myc, PAM 49 63, 7, 13 Clathrin heavy chain*

227920 17225574 7243239 21361159 21749840 10434568 2747767 14722175 31982941 33873597 7705373 42476299 14717080 14722175 27734703 23468240 20521117 21740322 3882183 25535897 37551910 2984586 22734703 7706322 1488414 14198280 7662380 4758648

5

(B) Interphase Matched unique peptides

GenBank accession number (gi)

Function

Matched protein

Cell division

MCM3 1 6631095 NUDE (nuclear distribution gene E 2 8923110 homolog) Apoptosis stimulating of p53 protein 1 16197705 2 (ASPP2) / p53-binding protein 2 (53BP2) PI3 kinase C2 beta 2, 3, 6 13632400 FRAP2 (MTOR2) 9 3282239 C-TAK / Par1A* 3 3089349 B-raf * 3 213599 PCTAIRE 3 16 16160923 DNA-PK, catalytic subunit 4 13606056 PAK 4 (p21-activated kinase 4) 2 12585288 3 7657261 PDZ-containing GEF1 ARHGEF16 8 30353993 RhoGAP10 4, 3 20977541 IQGAP1 (Ras GTPase-activating- 5 1170586 like protein) Rap1GAP 2 106198 Rho-interacting protein 3 (RIP3) 11, 3, 2 21740322 Unnamed, similar to ADP3 10435296 ribosylation factor-like 6 interacting protein 2) G protein coupled receptor 69A 21 5174445 SRGAP1 2 31753111 AF-6 3, 2 12644018 Lbc (a GEF) 3, 3 15207794 Rab GDP dissociation inhibitor 1 15290266 ARL6-interacting protein 8 14424435 liprin beta 1 14, 4, 3 3309539 Tuberin / Tuberous sclerosis 6 3522922 complex 2 (TSC2) * Insulin receptor substrate 2 (IRS2) * 7, 10, 13, 6, 2, 2, 3, 14537854 7, 6 Proline-rich AKT substrate * 5 14150199 Protein tyrosine phosphatase 1 131530 PTPH1* Yes-associated protein (YAP)-like 8, 12, 12 23398532 protein, 65 kDa A kinase anchoring protein (AKAP) 61 15986729

Signalling - kinases

Signalling – small GTPase related

Signalling - misc.

6

Nucleolar proteins

Proteolysis/ ubiquitination

Stress or checkpoint responses

Apoptosis

Nucleotide metabolism

Chromatin structure, DNA binding

RNA binding

Translation

BA416N2.2, FISH-like SH3 domain binding protein 4 NOLC1 (nucleolar phosphoprotein p130) Nucleolin / C23 Nucleophosmin / B23 26S proteasome regulatory subunit Ki antigen, proteasome activator subunit 3 Proteasome theta chain NEDD4-like ubiquitin ligase NAP1 Deubiquitinating enzyme 8 (USP8) TIP120 Ubiquitin-activating enzyme E1 DNA damage-binding protein 1

2 1 22, 15, 10, 4

10334638 76757562 434765

16, 7 6 3 4

21750187 10835063 25777600 30410796

2 9 2 3 2 7 3

22538465 21361472 4758754 731046 21361794 23510338 12643730

Hus1 8 Thioredoxin 2, 3, 2, 3, 2, 6, 2 Peroxiredoxin 1 10 B-cell receptor-associated protein 31 2 (BAP31) / CDM protein BH3-only domain protein 3 1 CAD trifunctional protein 3, 14, 30 CTP synthase 2, 11 UMP-CMP kinase 1 NAD kinase 11, 5 Histone deacetylase 4 * 24, 5

4758576 10120659 4505591 1705725

Histone deacetylase 5 * Histone deacetylase 7A * Histone acetyltransferase B subunit 2 Y-box binding protein 1 mitochondrial ssDNA-binding protein DNA primase large subunit LRPPRC (leucine-rich PPR motif containing protein) hnRNP X hnRNP I hnRNP U Decapping protein Dcp1a Regulator of nonsense transcript stability Dead-box RNA binding protein (DDX) 9 Ribosomal protein L10a

2 15 2

30353988 13259522 3334209

9 4

181468 2624694

1 12

510408 31621305

4 2 2 1 2

14141166 14165464 14044052 8923767 1575536

2

13514822

8

2202029

21361979 18105007 14495609 2497487 12804579 5174481

7

Cytoskeleton

Metabolism

Ribosomal protein P0 Splicing factor 3B, subunit 3 Splicing coactivator Srm300 Ribosomal protein S3 / UV endonuclease Poly(A) binding protein 4 (PABP4) Poly(A) binding protein 2 (PABP2) Valyl tRNA synthetase Isoleucyl tRNA synthetase Leucyl tRNA synthetase Alanyl tRNA synthetase Mitochondrial ribosomal protein S27 HsGCN1 eIF 4E type 3 eIF 3 theta eIF 4A eEF2 eEF 1 alpha 1 eEF 1 B Kinesin heavy chain* Kinesin-like protein 5 Kinesin 2 Kinesin light chain 2 Kinesin light chain 3 Tubulin, alpha, beta Dynein heavy chain Hornerin Filamin Talin 1 Vimentin* Annexin A2 Annexin I Cofilin* Profilin Cingulin Desmoyokin Desmoplakin I Ezrin Spectrin alpha II Spectrin beta 2 Actin Vinculin Alpha actinin 4 Phosphofructokinase heart isoform (PFK2) * Phosphoglucose isomerase

9 3 13, 4, 2 15, 6

12654583 11034823 19923466 13097759

8 11, 27, 32 2 2, 5 8 3 2

4504715 12229876 15215421 31873360 22496789 15079238 13129094

4, 3 4 1 7 34 10 2 5, 8, 102 3 29, 16 33 4, 6 6, 8, 2, 11, 14, 4 4 1 2, 7 2 52, 11, 8 13 3 3 3 6 7, 4 3 1 3, 4 2 24, 8, 21 3, 1 5 22, 3, 32

20521848 29839292 4503509 4503529 4503483 4503471

4, 3

14488680

4758648 20143967 14250822 13878553 13878563 multiple 30581065 28557150 1203969 14916725 2119204 16306978 4502101 116850 30584265 16262452 627367 2134996 119717 1805280 30315658 multiple 4507877 12025678 11933149

8

Chaperones

Nuclear transport

Phosphoglycerate mutase 1 5 Phosphoglycerate kinase 1 10 Fatty acid synthase 2, 22 Very long-chain acyl coA synthetase 1 (VLCS) creatine kinase 8, 3 Glyceraldehyde-3-phosphate 16 dehydrogenase (GAPDH) Lactate dehydrogenase (heart type) 16 Lactate dehydrogenase (muscle 8 type) Pyruvate kinase M2-type * 4, 9, 8 Panthothenate kinase 1 Panthothenate kinase 2 1 Adenosylhomocysteinase, 1 AdoHcyase Arginosuccinate synthase 2 Adenylosuccinate synthase 1 Cystathionase 1 Enolase 28 Aldolase 8 Transketolase 12 Triose phosphate isomerase 9 Transferrin receptor 4 Chaperonin containing t complex 19 (CCT) delta Chaperonin containing t complex 15 (CCT) eta Chaperonin containing t complex 3 (CCT) theta Chaperonin / HSP 60 9, 7 Calreticulin 4 cyclophilin A 7 Heat shock protein 70 kDa (BiP) 12 Heat shock 70kDa 9B / mortalin 9, 8 Heat shock 70kDa 8 40 Heat shock protein 90 kDa alpha 6 Heat shock protein 27 6 Protein disulphide isomerase (PDI) 2 FK506 binding protein 4 1 Heat shock protein A4 7 heat shock 105kDa protein 2 ER heat shock protein 90 (gp96) 7 HSP 90 related 30 Importin 7 4, 3 CRM1/exportin Importin alpha 1 9

130348 4505763 21618359 4503653 180570 31645 13786847 13786849 125604 27805667 27805667 9951915 16950633 1172749 345809 30593767 229674 14250367 17389815 4507457 2559008 5453607 1136741 306890 30583735 1633054 16507237 12653415 27805925 123678 19855073 2507460 12804069 20547107 42544159 15010550 11277141 11544639 13543657 9

Importin beta 2 3, 3, 5 Importin alpha re-exporter (Cellular 5 apoptosis susceptibility protein) Transcription Sugar-binding Unknown

Vesicle trafficking / formation

1613834 20141403

NFAT2 1 1082855 Human capicua 4 16507208 Homolog of bovine peptidoglycan 16, 3 27808640 recognition protein LMO7 (Lim-domain only 7) 6, 13, 5, 6 17225574 Wilms tumor 1 associating protein 1 21 21361159 (WTAP) cytoplasmic FMRP (fragile X 13 24307969 mental retardation protein) interacting protein 1 Valosin-containing protein (VCP), 9 2984586 transitional ER ATPase S100A6 / calcyclin binding protein 4 7656952 Copine 1 1 12231878 SET 2, 2 21618461 HSPC117 5 12652799 KIAA0264 2 1665795 Protein associated with myc, PAM 28 7662380 Unnamed 33, 5, 9, 9, 11, 9 2747767 TBC1 (Tre2, Bub2, CDC16) domain 30, 9, 6, 3, 4, 53 7662198 protein 4 (TBC4) KIBRA 4, 4 24307969 Retinoic acid induced 14 2 13470086 Major vault protein (MVP) 1 19913412 Neoplasm-related C140 product 1 546831 Epithelial cell marker protein 1 8 631131 Polyposis locus protein 1 – like 1 1 14198280 53, 30, 3, 2, 24, 53, 3882183 KIAA0731 7 KIAA0456 22, 3 25535897 KIAA0747 7 14149680 KIAA1401 2 7243183 KIAA1720 5 12697985 FLJ34154 2 21749840 FLJ1128 6 10437164 Unnamed 2 27469519 Clathrin heavy chain* 7, 26 1705916 Fer1-like protein 3 4, 7 secretory carrier membrane protein 2 1

10834587 5730031

10