a modified tandem affinity purification technique identifies that 14-3-3 proteins interact with...

A Modified Tandem Affinity Purification Technique Identifies That

14-3-3 Proteins Interact with Tiam1, an Interaction Which Controls

Tiam1 Stability

Simon A. Woodcock,*,§ Richard C. Jones,|,† Ricky D. Edmondson,|,‡ and Angeliki Malliri*,§

Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, University of Manchester,Manchester, M20 4BX, United Kingdom, and National Center for Toxicological Research (NCTR), Food and

Drug Administration (FDA), Jefferson, Arkansas 72079

Received August 11, 2009

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiplecellular processes including proliferation, apoptosis and adherens junction maintenance. Here wedescribe a modified tandem affinity purification (TAP) technique that we have applied to specificallyenrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunctionwith LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seenwith the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors.We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitationexperiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interactionis largely dependent on the N-terminal region of Tiam1; within this region, there are four putativephospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231)are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at threeof these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins tothis region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity;significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is requiredfor the degradation of Tiam1. Thus, we have established and proven methodology that allows theidentification of additional protein-protein interactions in mammalian cells, resulting in the discoveryof a novel mechanism of regulating Tiam1 stability.

Keywords: Tiam1 • 14-3-3 • Rac • stability • phosphorylation • TAP • cross-linking

Introduction

Rho-like GTPases are molecular binary switches in signalingpathways that control cell morphology, adhesion, motility andsurvival, as well as cell-cycle progression.1 Similarly to Ras, Rhoproteins such as Rac, Rho and Cdc42 are guanine nucleotidebinding proteins that cycle between an inactive GDP-boundstate and an active GTP-bound state. In the active state, Rhoproteins undergo a conformational change that exposes theireffector loop; this can now bind various effector molecules,resulting in their activation and downstream signaling events.Guanine nucleotide exchange factors (GEFs) activate smallGTPases by promoting the exchange of GDP for the moreabundant GTP, whereas GTPase-activating proteins (GAPs)promote the intrinsic activity of small GTPases resulting in the

hydrolysis of GTP, returning them back to an inactive GDP-bound state.

Tiam1 (for T-lymphoma invasion and metastasis protein) isa Rac-specific GEF that is known to regulate several cellularprocesses including proliferation, migration, apoptosis andcell-cell adhesions. Like other GEFs for Rho proteins, Tiam1contains a Dbl homology (DH) domain followed by a Pleckstrinhomology (PH) domain, both of which are critical for itscatalytic activity toward Rac. Tiam1 also contains an additionalPH domain nearer the N-terminus, which together with anadjacent extended coil-coil region, is required for the properlocalization of Tiam1 to the membrane.2 In epithelial cells,Tiam1 localizes to intercellular adhesions where it promotesthe maintenance of cadherin-based adhesions through theactivation of Rac.3 Tiam1 interacts with a number of otherproteins, such as Ras (GTP-bound),4 Par3,5,6 JIP27 and theArp2/3 complex.8 We have recently shown that Src, an onco-genic tyrosine kinase, phosphorylates Tiam1 on Y384 creatinga binding site for the small adapter protein Grb2.9 We alsodemonstrated that Tiam1 interacts with extracellular signal-regulated kinase (ERK). These interactions were critical for themechanism that ultimately leads to the degradation of Tiam1,

* To whom correspondence should be addressed. E-mail: (A.M.) [email protected], (S.A.W.) [email protected]. Telephone: 0161 4463122. Fax: 0161 446 3109.

§ University of Manchester.| Food and Drug Administration (FDA).† Current address: NextGen Sciences, Ann Arbor, MI 48108, USA.‡ Current address: Myeloma Institute for Research and Therapy, University

of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.

10.1021/pr900716e CCC: $40.75 2009 American Chemical Society Journal of Proteome Research 2009, 8, 5629–5641 5629Published on Web 11/09/2009

showing the importance of interacting proteins in the regula-tion of Tiam1 function.9

To further understand the regulation of Tiam1, we soughtto identify additional interacting partners from mammaliancells that may modulate its cellular functions. To this end, wehave developed a modified version of the tandem affinitypurification (TAP) technique, which was originally establishedin yeast.10,11 We have optimized this protocol for use withmammalian cells, and have incorporated additional steps toallow the efficient in vivo cross-linking of protein complexeswith formaldehyde. This modified technique allowed themaintenance of weaker/transient interactions throughout thepurification protocol and resulted in the detection of a numberof additional Tiam1-interacting proteins that were not detectedusing the standard protocol. With this technique, we identifiedmultiple 14-3-3 family members as Tiam1-interacting proteins.We determined that there are many phospho-dependent 14-3-3 binding sites on Tiam1, with the majority residing in theN-terminal region. Moreover, we have identified these sites asSer60, Ser172 and Ser231, all of which lie within 14-3-3 bindingmotifs; their mutation to alanine dramatically reduced theinteraction between Tiam1 and 14-3-3 proteins. Significantly,we show that phosphorylation of the 14-3-3 binding motifcontaining Ser60 destabilizes Tiam1 at the protein level; thisindicates that the binding of 14-3-3 proteins to the N-terminusof Tiam1 regulates its stability.

Experimental Procedures

Constructs. Full-length (FL), and N-terminally truncated(C1199 and C580), mouse Tiam1 carrying a C-terminal HA tagin pcDNA3 were provided by J. Collard. The full-lengthconstruct was used as a template to mutate Ser43, Ser60,Ser172, and Ser231 to alanine using the QuikChange site-directed mutagenesis kit (Stratagene) according to manufac-turer’s directions. For the generation of TAP-tagged Tiam1, full-length Tiam1, including the C-terminal HA tag, was cloned inframe from pcDNA3 into a plasmid containing the C-terminalTAP tag in pcDNA4-(Zeor) (provided by H. Clevers). Myc-tagged14-3-3� was provided in pcDNA3.1 by F. Barr. For the genera-tion of GST-tagged 14-3-3� for bacterial expression, a Bam-HI(blunt)-XbaI fragment containing 14-3-3� from pcDNA3.1was cloned into the SmaI-XbaI sites of pGex-KG.

Cell Lines and Transfection. H293T and Cos7 cells weremaintained in Dulbecco’s Modified Eagle Medium (DMEM;Invitrogen) in the presence of 10% fetal bovine serum (FBS;GIBCO). Tiam1 knockout mouse embryonic fibroblasts12 (MEFs)were maintained in DMEM in the presence of 10% FBS and0.1 mM 2-mercaptoethanol (GIBCO). H293T, Cos7 and MEFcells were transfected using Fugene 6 (Roche), and stabletransfection of MEFs was obtained by selection with Zeocin(250 µg/mL) for at least 10 days. Where indicated, H293T cellswere treated for 25 min with Calyculin-A (20 nM) or Stauro-sporine (400 nM).

Protein Analysis. Lysates of cells were prepared on ice in IPlysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v/v)Triton-X-100, 10% (v/v) glycerol, 2 mM EDTA, 25 mM NaF, and2 mM NaH2PO4) containing a protease inhibitor cocktail(Sigma) and phosphatase inhibitor cocktails 1 and 2 (Sigma)and cleared by centrifugation. Equivalent amounts of proteinwere denatured in 1× SDS-PAGE sample buffer (Nupage;Invitrogen), resolved by SDS-PAGE, and transferred to PVDFmembrane (Immobilon-P; Millipore). Western blotting wasperformed using the following primary antibodies: anti-HA

(Clone 12CA5, CRUK, 1:5000), anti-Rac (BD, 1:1000), anti-14-3-3� (Santa Cruz (C16), 1:500), anti-14-3-3 (Santa Cruz (K19),1:2000), and anti-Tiam1 (Santa Cruz (C16), 1:1000), and sub-sequently horseradish peroxidise-conjugated anti-mouse oranti-rabbit secondary antibodies (GE Healthcare, 1:5000) andvisualized by enhanced chemiluminescence (Perkin-Elmer).Band intensities were quantified using the Genetools software(SynGene).

Affinity Precipitation. For immunoprecipitation (IP) experi-ments, protein lysates were prepared using IP lysis buffer andequivalent amounts of protein were incubated with 2 µg of anti-HA (Clone 12CA5, CRUK), prebound to 20 µL of GammaBindG-Sepharose (GE Healthcare), for 2 h at 4 °C. For GST pulldownexperiments, the pGex-KG constructs were expressed andpurified from Escherichia coli as previously described.13 Mam-malian protein lysates were prepared using IP lysis buffer andequivalent amounts of protein were incubated with immobi-lized GST proteins for 1 h at 4 °C. Following washes in IP lysisbuffer, proteins isolated by IP or GST pulldown were elutedwith 1× SDS-PAGE sample buffer, and processed for Westernblotting as above.

Rac Activity Assay. To measure endogenous Rac GTPaseactivity, cells were lysed on ice in GST-FISH buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1% (v/v) NonidetP-40, 10% (v/v) glycerol, and protease inhibitors (Complete,EDTA-free; Roche)) containing 1 µg of a biotinylated PAK-derived CRIB (Cdc42/Rac interacting binding) peptide perassay. Cleared cell lysates were incubated at 4 °C for 20 min;active Rac/CRIB complexes were precipitated using streptavi-din-conjugated agarose beads (Sigma) for a further 15 min at4 °C. Following washes in GST-FISH buffer, protein sampleswere eluted with 1× SDS-PAGE sample buffer, and processedfor Western blotting with anti-Rac antibody as above.

Immunofluorescence. Cells grown on glass coverslips wereprocessed essentially as described previously14 and were co-stained using anti-HA-Alexa Fluor 594 conjugate (Clone 16B12,Molecular Probes, 1:500) and either anti-Myc-Alexa 488 con-jugate (Clone 4A6, Upstate, 1:1000), or Phalloidin-Alexa 350conjugate (Molecular Probes, 1:100). Cells were mounted inProlong Antifade (Molecular Probes); images were recordedwith a Deltavision microscope system and processed withImaris software.

Protein Stability Assay. To have equivalent levels of expres-sion, 24 h after transfection of the indicated HA-tagged Tiam1constructs (in pcDNA3), Cos7 cells were trypsinized and equallyseeded into 4 wells. The following day, cells were treated with50 µg/mL cycloheximide (CHX, a protein synthesis inhibitor;Sigma) for the indicated times (0, 2, 4, or 6 h), lysed andprocessed for Western blotting with anti-HA as above.

Tandem Affinity Purification. Lysates of cells were preparedon ice in IP lysis buffer and cleared by centrifugation for 20min; this and all subsequent steps were performed at 4 °C.Cleared lysates were incubated with IgG sepharose beads (GEHealthcare) for 2 h before washing four times in TAP washbuffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v)Triton-X-100, 0.5 mM EDTA and 1 mM �-mercaptoethanol),and once in protease cleavage buffer (50 mM Tris-HCl, pH 7.5,150 mM NaCl, 0.1% (v/v) Triton-X-100 and 1 mM DTT). Proteincomplexes were eluted from IgG beads by incubating withTobacco Etch Virus (AcTEV) protease (Invitrogen) in 1 mL ofprotease cleavage buffer for 2 h. The eluate was diluted 1 in 6with calmodulin binding buffer (CBB; 50 mM Tris-HCl, pH 7.5,150 mM NaCl, 0.1% (v/v) Triton-X-100, 1 mM MgCl2, 1 mM

research articles Woodcock et al.

5630 Journal of Proteome Research • Vol. 8, No. 12, 2009

imidazole, 4 mM CaCl2 and 10 mM �-mercaptoethanol) beforeadding to Calmodulin affinity resin (Stratagene), prewashed inCBB, and incubated for 1 h. After incubation, samples werewashed four times with CBB, eluted with 1× SDS-PAGE samplebuffer, and resolved by SDS-PAGE (4-12% Nupage gel, Invit-rogen). Proteins were detected either by Western blotting asabove, or using Coomasie (SimplyBlue SafeStain, Invitrogen)or silver staining (SilverQuest, Invitrogen). For samples run induplicate, 10% (from ∼107 cells) of the sample was stained bysilver staining to visualize proteins, whereas the remaining 90%(from ∼108 cells) of the sample was stained with Coomasie andsubsequently gel lanes were prepared for mass spectrometryas described below.

Cross-Linking of Proteins in Vivo. Optimal cross-linkingconditions in MEFs were obtained by adding a range offormaldehyde concentrations (0.0625-1%) in PBS for varioustimes (15-45 min) at 25 °C. The reaction was quenched bythe addition of glycine to a final concentration of 0.125 M for15 min at 25 °C. Cells were washed twice in ice-cold PBS andlysed on ice in IP lysis buffer as above. It was determined thatthe optimal conditions for TAP-tagged Tiam1 were 0.25%formaldehyde for 30 min at 25 °C; these conditions were usedfor subsequent experiments. To reverse the formaldehyde-induced cross-links, samples were heated in 1× SDS-PAGEsample buffer at 95 °C for 20 min.

Systematic Identification of Tandem Affinity PurifiedProteins by LC-MS/MS Analysis and Database Searches. Gellanes from 1D gels (Nupage 4-12%) were manually cut into40 × 1.5 mm bands using a razor blade. Each band was placedin a 96-well plate (PRO10003, Genomic Solutions), and gelbands were processed robotically using a ProGest instrument(Genomic Solutions, Ann Arbor, MI); briefly, bands werereduced with dithiothreitol, alkylated with iodoacetamide anddigested with trypsin (Promega, Madison, WI). The 50 µL digestsupernatant was used directly following acidification. Peptidesolutions were analyzed using nano LC-MS/MS on a LCQ DecaXP Plus ion trap mass spectrometer (Thermo, San Jose, CA). Atotal of 42 µL of sample was loaded using an Enduranceautosampler (Micro-Tech Scientific, Vista, CA) onto an Inte-graFrit (New Objective, Woburn, MA) vented column(v-column) (75 µm × 3 cm) packed with 5 mm Jupiter C12material (Phenomenex, Torrance, CA) at 14 µL/min and elutedwith a 50 min gradient (0.1-30% B in 35 min, 30-50% B in 10min and 50-80% B in 5 min where A ) 99.9% H2O, 0.1%acetonitrile incorporating 0.1% formic acid and B ) 80%acetonitrile, 20% H2O incorporating 0.1% formic acid) at 200nL/min (generated with a split tee from 85 µL/min pump flowrate) using an UltraPlus II capillary HPLC pump (Micro-TechScientific) over a 75 µm × 15 cm IntegraFrit analytical columnpacked also with Jupiter C12 material. The column was coupledto a 30 µm i.d. × 3 cm stainless steel emitter (Proxeon, Odense,Denmark) using a microtee (Upchurch Scientific, Oak Harbor,WA) that also served as a voltage tee via a platinum wire(Goodfellow, Devon, PA). All experiments were performed withfollowing instrument settings: capillary voltage 1.8 kV, heatedcapillary temperature 160 °C. MS/MS was performed on thetop four ions in each MS scan using the data-dependentacquisition mode. Normalized collision energy was set at 35%and 3 microscans were summed following AGC implementation(target values for MS and MS/MS were 5 × 108 and 6 × 107

counts, respectively). Dynamic exclusion and repeat settingsensured each ion was selected only once and excluded for 30 sthereafter.

Product ion data were searched against the combinedforward and reverse mouse protein database (IPI Mouse v3.19)using a locally stored copy of the Mascot search engine v2.0.04(Matrix Science, London, U.K.) via Mascot Daemon v2.0.0.Mascot LCQ_DTA executable generates both doubly and triplycharged versions of each ion selected in the DDA experiment,unless no ions are observed above the parent m/z, in whichcase it is assigned as singly charged. Search parameters wereprecursor mass tolerance 2.5 Da, product ion mass tolerance0.6 Da, 2 missed cleavages allowed, fully tryptic peptides only,fixed modification of carbamidomethyl cysteine, variable modi-fications of oxidized methionine, N-terminal acetylation andpyro-glutamic acid on N-terminal glutamine. Mascot searchresult flat files (.DAT) were parsed to an Oracle database usingin-house software called ProteinTrack;15 all peptides with aMascot score of 15 or greater were saved. The criteria foraccepting a protein identification were determined by calculat-ing the False Discovery Rates (FDR) from the concatenatedforward/reverse database. This resulted in the following cutoffvalues: if there was more than one peptide for a particularprotein, then the total score had to be at least 57 and eachpeptide had to score 23 or greater; if only one peptide wasidentified, it had to score 45 or greater. Peptides had to have amass of at least 600 Da. These criteria resulted in FDR of 1.65%at the protein level for this data set. Of these identified proteins,only those that were detected in Tiam1-TAP samples and notcontrol TAP samples were classified as being Tiam1-specificinteractors.

Identification of Phosphorylation Sites on Tiam1 byLC-MS/MS Analysis and Database Searches. H293T cells weretransfected with wild-type Tiam1-HA; Tiam1 was immunopre-cipitated with anti-HA as above, and after SDS-PAGE, the gelwas stained using Coomasie (SimplyBlue SafeStain, Invitrogen).The putative gel band containing Tiam1 was excised, destainedand digested in-gel with trypsin (Sigma) overnight at 37 °C.The resulting tryptic peptides were acidified to pH 2.0 withformic acid, dried and resuspended in 2% (v/v) acetonitrile/0.1% (v/v) formic acid. Peptide solution was loaded onto a 15cm, 75 µm i.d. reversed phase C18 PepMap analytical column(Dionex). Using an Ultimate pump (LC Packings), peptideseparation was achieved over a 15 min gradient of 2.0-72%(v/v) acetonitrile/0.1%(v/v) formic acid at a flow rate of 200nL/min online to a 4000 QTRAP mass spectrometer (AppliedBiosystems). Spectra were acquired using an informationdependent acquisition method where in a 1 s MS scan of m/z450-1500 the two most intense multiply charged ions (2+, 3+)above a threshold of 100 000 counts were selected for MS/MS.Each selected precursor was dynamically excluded for 2 minafter 2 occurrences. Acquired data were searched againstdatabase NCBInr 2004-09-23 using MASCOT version 2.0(Matrix Science). Searches were restricted to the mouse tax-onomy allowing phosphorylated serine, threonine and tyrosine;oxidized methionine; asparagine and glutamine deamidationas potential variable modifications. Precursor mass tolerancewas 1.0 Da and MS/MS tolerance 0.9 Da; one missed cleavagewas allowed. Manual validation of database search results wasdone by visual inspection of MS/MS spectra.

Results

A Modified Tandem Affinity Purification (TAP) StrategyIdentifies Additional Tiam1-Interacting Proteins. Tiam1 is alarge, multidomain protein that has the potential to interactwith many targets (Figure 1a). To identify proteins that interact

TAP Technique Identifies 14-3-3 Proteins Interacting with Tiam1 research articles

Journal of Proteome Research • Vol. 8, No. 12, 2009 5631

with Tiam1 in mammalian cells, we made use of the tandemaffinity purification (TAP) technique. The chosen TAP tagincorporates two Protein-A domains and a Calmodulin bindingpeptide (CBP) separated by a consensus sequence for the site-specific protease TEV (Figure 1b). This technique allows thesequential purification and elution of tagged proteins undernear-physiological conditions, resulting in a significant enrich-ment of associated protein complexes.10,11,15 We initiallyconfirmed that the addition of the TAP tag to the C-terminusof Tiam1 did not affect activity toward its substrate Rac; Cos7cells expressing HA-tagged or TAP-tagged Tiam1 showed asimilar increase in active Rac over control cells (Figure 1c).These data indicate that the addition of the TAP tag does notcompromise Tiam1 function in cells.

To remove any competition from endogenous Tiam1 duringthe purification procedure, we made use of Tiam1 knockout (KO)mouse embryonic fibroblasts (MEFs). Hence, Tiam1 KO MEFsexpressing TAP-tagged Tiam1 (Figure 1d) or a control TAP tagwere generated by stable transfection, and used in subsequentpurifications. Using the standard TAP technique described above,TAP-tagged Tiam1 or a control TAP tag was purified from proteinlysate of these stable lines and the resulting samples were resolvedby SDS-PAGE. Following silver staining (Figure 1e), and alsoWestern blotting and mass spectrometry (data not shown), it was

evident that Tiam1, still tagged with the CBP, was successfullyenriched throughout the strategy. We noted, however, that otherthan Tiam1-CBP, we were unable to clearly visualize by silverstaining any discrete bands corresponding to copurifying proteins(Figure 1e). Similar gels were stained with Coomasie Blue, andgel lanes systematically laddered; these samples were subse-quently destained and trypsinized, and the resulting peptidemixture was subjected to liquid chromatography-coupled tandemmass spectrometry (LC-MS/MS). Following database searches, thisled to the identification of over 40 proteins that were specificallypurified in complex with Tiam1; these proteins will be describedelsewhere (Reeves, Woodcock, Jones and Malliri; data not shown).

We hypothesized that one reason that discrete bands ofcopurifying proteins were not visualized could be that ad-ditional protein interactions were extremely transient or lowaffinity and, therefore, were lost during the TAP technique. Thiswould also be compounded by the increased duration of atandem, compared to a single, affinity purification technique.To test this hypothesis, we explored the use of protein cross-linking agents within mammalian cells. We found that treat-ment with increasing concentrations of the cross-linker form-aldehyde resulted in an increasing amount of Tiam1 appearingas a smear of Tiam1-containing complexes that migrated athigher molecular weights than non-cross-linked Tiam1 (Figure

Figure 1. Tandem Affinity Purification (TAP) strategy for the purification of Tiam1-interacting proteins. (a) Schematic representation offull-length Tiam1 showing tandem PEST sequences (P), N-terminal Pleckstrin homology (PHn) domain with adjoining coiled-coil region(CC), Ras-binding domain (RBD), PDZ domain, Dbl (DH) and C-terminal PH (PHc) domains. The location of C1199 and C580 truncationsis shown. (b) Schematic of C-terminal TAP tag and outline of TAP strategy used to purify TAP-tagged Tiam1. (c) Rac activity wasanalyzed from lysates of Cos7 cells transfected with HA-tagged Tiam1, TAP-tagged Tiam1, or a control empty vector. Total levels ofRac are also shown. (d) Levels of Tiam1 were detected by immunoblotting lysates prepared from wild-type (WT) mouse embryonicfibroblasts (MEFs), Tiam1 knockout (KO) MEFs, or Tiam1 KO MEFs stably expressing TAP-tagged Tiam1. A loading control (LC) is alsoshown. (e) TAP-tagged Tiam1 or a control TAP-tag was purified by the strategy described in panel b from Tiam1 knockout (KO) mouseembryonic fibroblasts (MEFs). Tiam1 and copurifying proteins were resolved by SDS-PAGE and detected by silver staining. The bandcorresponding to Tiam1-CBP, as detected by mass spectrometry, is indicated.



2a). Concomitantly, there was a resultant decrease in therelative levels of non-cross-linked Tiam1. Reversal of formal-dehyde-induced cross-links, using high temperature, resultedin the loss of the Tiam1-containing protein smear, and a gainin the amount of non-cross-linked Tiam1 (Figure 2b). Thesedata indicate that, within mammalian cells, Tiam1 interactswith a number of proteins that can be covalently linked toTiam1 by formaldehyde cross-linking.

We next determined whether the use of this formaldehydecross-linking procedure would allow the maintenance of ad-ditional interactions throughout the TAP technique and, there-fore, permit the identification of proteins that we were unableto detect using the standard TAP technique. After purificationof TAP-tagged Tiam1 or a control TAP tag, the cross-links werereversed and samples were resolved by SDS-PAGE. Followingsilver staining, other than the presence of a band the expectedmolecular weight of Tiam1-CBP, we also now visualized anumber of additional protein bands (Figure 2c) that we didnot detect using the standard technique (Figure 1e). Duplicategels were stained with Coomasie Blue, and gel lanes processedas described above for analysis with LC-MS/MS. Followingdatabase searches, we now identified over 30 proteins that werespecifically purified in complex with Tiam1, half of whichoverlapped with those identified using the standard TAPdescribed above; selected proteins are summarized in Table 1.These included multiple 14-3-3 family members, which couldreadily be visualized by silver staining in the Tiam1-TAP

sample, following the modified TAP strategy, as two prominentbands at approximately 30 kDa (Figure 2c).

Confirmation That Tiam1 Interacts with 14-3-3 Proteins.14-3-3 proteins are a highly conserved family of abundantdimeric proteins involved in many vital cellular processes.16,17

In mammals, there are seven family members that act asphospho-serine/phospho-threonine binding proteins and in-teract with a wide array of partners. These partners arerestricted by nature of additional binding specificity of 14-3-3proteins. To validate the interaction of Tiam1 with 14-3-3proteins, we performed a TAP technique on a smaller scale,and confirmed by Western blotting that 14-3-3� was detectedin samples purified from cells expressing TAP-tagged Tiam1,but was absent from control samples (Figure 3a). We furtherassessed whether Tiam1 could be affinity precipitated frommammalian cell lysate using immobilized 14-3-3�. Indeedaround 18% of the total endogenous or exogenously expressedTiam1 in H293T cells could be efficiently precipitated by GST-14-3-3� (Figure 3, panels b and c, respectively), whereas noTiam1 could be precipitated by a GST only control. It is knownthat a majority of interactions with 14-3-3 proteins require thetarget protein to be phosphorylated on specific serine orthreonine residues.16,17 We therefore tested whether the inter-action with Tiam1 could be modulated by treating H293T cellswith either a general protein Ser/Thr-phosphatase inhibitor(Calyculin-A) to increase protein phosphorylation, or a generalprotein kinase inhibitor (Staurosporine) to decrease protein

Figure 2. Combined cross-linking and TAP identifies 14-3-3 proteins as Tiam1 interactors. (a and b) Tiam1 KO MEFs stably expressing TAP-tagged Tiam1 were either left untreated or protein complexes were cross-linked in vivo using (a) various concentrations of formaldehyde(from 1% down to 0.0625%) for 30 min at 25 °C, or (b) 0.25% formaldehyde for 30 min at 25 °C. Cells were subsequently lysed, and whereindicated, formaldehyde cross-links were reversed by heating at 95 °C for 20 min. TAP-tagged Tiam1 and cross-linked Tiam1-containingcomplexes were detected in cell lysates by immunoblotting using anti-HA. (c) TAP-tagged Tiam1 or a control TAP-tag was purified by thestrategy described in Figure 1b, from Tiam1 KO MEFs that had been cross-linked in vivo using 0.25% formaldehyde for 30 min at 25 °C. Thecross-linked samples were reversed as in panel b after the final elution from calmodulin beads. Tiam1 and copurifying proteins were resolvedby SDS-PAGE and detected by silver staining. The bands corresponding to Tiam1-CBP and 14-3-3 proteins, as identified by mass spectrometry,are indicated.



phosphorylation. Treatment with Calyculin-A increased theproportion of endogenous Tiam1 that was precipitated by GST-14-3-3�; conversely, treatment with Staurosporine dramaticallydecreased the proportion precipitated (Figure 3b). Similarresults were found using exogenous Tiam1 expressed in H293Tcells (Figure 3c). Finally, we also investigated where Tiam1 and14-3-3 proteins localize in cells. When coexpressed in Cos7cells, we found that Tiam1 and 14-3-3 proteins showedextensive colocalization (Figure 3d); this was particularlyevident in actin-rich membrane ruffles (arrowheads in Figure3d and data not shown). Thus, these data indicate that Tiam1and 14-3-3 proteins interact in a phosphorylation-dependentmanner, and that they colocalize in cells.

14-3-3 Proteins Predominantly Bind the N-TerminalRegion of Tiam1. Next, we began to address the mechanismbehind the Tiam1/14-3-3 interaction. As Tiam1 is a relativelylarge, multidomain protein, we initially determined whether aparticular region of Tiam1 is required for the interaction with14-3-3 proteins. Truncated versions of Tiam1 (Figure 1a) wereexpressed in H293T cells and their ability to interact withimmobilized 14-3-3� was tested. We found that the removal ofthe N-terminal 392aa of Tiam1 was sufficient to dramaticallyreduce the proportion of Tiam1 that was precipitated by 14-3-3� (from ∼18% of full-length Tiam1 to ∼2% of C1199-Tiam1)(Figure 4a,b). Additional truncation of Tiam1 failed to reducethe proportion that was precipitated by 14-3-3� any further(∼2% of C580-Tiam1) (Figure 4a,b), indicating the residualbinding is likely to be due to this C-terminal region (C580-Tiam1) and not the intervening region of Tiam1. We nextaddressed whether this result could also be verified by coim-munoprecipitation (Co-IP) experiments. Initially, it was con-firmed that the interaction between exogenous Tiam1 andendogenous 14-3-3 proteins in H293T cells could be detectedby Co-IP (Figure 4c). Consistent with data above, the removalof the N-terminal region of Tiam1 (C1199-Tiam1) dramaticallyreduced the Co-IP of endogenous 14-3-3 proteins (Figure 4c,d);also, the additional truncation of Tiam1 (C580-Tiam1) did notappear to further reduce the relative proportion of endogenous14-3-3 that was coimmunoprecipitated (Figure 4d). Together,these data indicate that 14-3-3 proteins preferentially interactwith the N-terminal region of Tiam1.

Determination of the 14-3-3 Binding Motifs in the N-Terminal Region of Tiam1. As mentioned previously, 14-3-3proteins have additional binding specificity for their phosphory-lated targets. This specificity has allowed the identification of twohigh affinity 14-3-3 binding motifs, RXXpSXP (mode 1) andRXXXpSXP (mode 2), with pS representing phospho-serine.18

Furthermore, as dimers, 14-3-3 proteins can bind two motifs

simultaneously, either on a single target protein, or on twoseparate proteins.18 We made use of Scansite analysis19 in orderto locate potential 14-3-3 binding sites in the N-terminal regionof Tiam1. This revealed one high scoring and three mediumscoring sites that corresponded to the 14-3-3 mode 1 bindingmotif RXXpSXP (Figure 5a). Sequence alignment showed thatthese motifs are well-conserved in Tiam1 throughout vertebrates(Figure 5a), whereas they are not found in the related protein Stef/Tiam2 (data not shown). We assessed whether any of these motifsof Tiam1 are phosphorylated on the serine residues in vivo usingmass spectrometry. Ser172 and Ser231 were both identified asbeing phosphorylation sites (Figure 5b,c). However, we did notdetect phosphorylation of the medium/high scoring 14-3-3 bind-ing sites Ser43 and Ser60, although it should be pointed out thatSer60 lies within a very large tryptic peptide that is unlikely to bedetected in this experiment.

To test whether any of these motifs of Tiam1 are importantfor the interaction with 14-3-3 proteins, the critical serine residueswere mutated to nonphosphorylatable alanine by site-directedmutagenesis. Motifs were altered singly, or in a variety ofcombinations, and the ability of the various mutant Tiam1proteins to be affinity precipitated by immobilized GST-14-3-3�was tested. Mutation of Ser43, a medium scoring interaction motif,did not noticeably affect the interaction with 14-3-3� (Figure 5d,e).However, the S60A, S172A and S231A single mutants all showedreduced binding to 14-3-3� compared to the wild-type control(Figure 5d,e). The double S172A, S231A mutant showed a largerreduction in binding than either single mutation alone; further-more, a mutant with mutations in all four consensus motifs (4A)showed the largest reduction in binding to 14-3-3� (Figure 5d,e),and was reduced to a similar level as the N-terminally truncatedTiam1 (C1199). Together, these data indicate that, within theN-terminal region of Tiam1, three motifs containing Ser60, Ser172and Ser231 contribute to its interaction with 14-3-3 proteins, andthat Ser172 and Ser231 of Tiam1 are confirmed as bona fide sitesof phosphorylation in vivo.

Phosphorylation of the N-Terminal 14-3-3 BindingMotifs in Tiam1 Does Not Affect Its Activity but RegulatesIts Protein Stability. To determine the function of the interac-tion between 14-3-3 proteins and the identified phospho-dependent binding motifs in the N-terminus of Tiam1, wemade use of the various Tiam1 nonphosphorylatable alaninemutants. Initially, we assessed the ability of these Tiam1mutants to increase Rac-GTP levels in cells; however, wedetected no differences between these mutants and wild-typeTiam1 (Figure 6a). Furthermore, when expressed in Cos7 cells,all of the 14-3-3 binding mutants were able to induce, andlocalize to, actin-rich membrane ruffles (Figure 6b), which are

Table 1. Summary of Selected Proteins Identified after a Modified Tandem Affinity Purification of Tiam1

accession number protein name Mascot score unique peptides spectral count % coverage

IPI00119006 T-lymphoma invasion and metastasis-inducing protein 1 5262 114 972 54.0IPI00230682 14-3-3 beta 714 17 42 47.6IPI00116498 14-3-3 protein zeta/delta 662 16 36 49.8IPI00118384 14-3-3 protein epsilon 541 13 34 46.3IPI00230707 14-3-3 protein gamma 532 12 32 37.4IPI00408378 14-3-3 protein theta 539 13 30 44.1IPI00227392 14-3-3 protein eta 505 13 25 41.2IPI00420385 Septin-11 343 9 18 19.6IPI00224626 Cell division cycle 10 homologue (Septin-7) 181 5 5 11.0IPI00110588 Moesin 76 2 4 2.8IPI00330476 SHYC 123 3 3 2.7IPI00667471 Protein phosphatase 2A (PP-2A) regulatory subunit B 107 3 3 8.3IPI00353563 Fascin 75 3 3 7.1IPI00330729 Casein kinase I, alpha 1 58 1 1 6.2



known to require the activation of Rac.20 Together, these dataindicate that the binding of 14-3-3 proteins to the identifiedmotifs in the N-terminus of Tiam1 does not significantly affectits activity toward its substrate Rac.

The N-terminal region of Tiam1, where 14-3-3 proteinspredominantly interact, has limited known function; however,it does contain tandem PEST sequences. These sequences oftenserve as a signal for protein degradation,21 although to ourknowledge this has not yet been directly determined in the caseof Tiam1. To assess whether the interaction with 14-3-3

proteins may regulate Tiam1 protein stability, we performedprotein turnover assays on wild-type Tiam1 and the variousnonphosphorylatable alanine mutants. Inhibition of proteinsynthesis, by the addition of cycloheximide (CHX), revealed thatthe steady state levels of wild-type Tiam1 protein are regulatedby degradation (Figure 7a). With the use of protein degradationcurves determined from a number of such assays (Figure 7b),the average protein half-life of wild-type Tiam1 expressed inCos7 cells was calculated to be 6.5 h. However, similar proteinturnover assays of the single S60A mutant Tiam1 indicated a

Figure 3. Confirmation that Tiam1 interacts with 14-3-3 proteins. (a) TAP-tagged Tiam1 or a control TAP-tag was purified by the strategydescribed in Figure 1b from Tiam1 KO MEFs cross-linked in vivo using 0.25% formaldehyde. The cross-linked samples were reversedafter the final elution from Calmodulin beads, and 14-3-3� that copurified was detected by immunoblotting. Levels of 14-3-3� in theinput are also shown. (b and c) H293T cells either (b) untransfected, or (c) transfected with Tiam1-HA for 24 h, were left untreated, orwere treated with Calyculin A or Staurosporine for 25 min. Lysates were incubated with either immobilized GST-14-3-3� or GST only.Affinity precipitated (AP) endogenous Tiam1 was detected with anti-Tiam1 (b), whereas exogenous Tiam1-HA was detected with anti-HA (c), and the relative levels in the lysates are also shown. The intensities of Tiam1 bands were quantified and are shown graphicallyas a percentage of the total Tiam1 that was affinity precipitated by GST-14-3-3�. (d) HA-tagged Tiam1 and Myc-tagged 14-3-3� werecotransfected into Cos7 cells for 24 h and fixed. Fixed cells were costained with anti-HA to detect Tiam1, and anti-Myc to detect 14-3-3�. Arrowheads indicate colocalization with actin-rich membrane ruffles. Scale bars, 20 µm.



significantly reduced rate of degradation (Figure 7a,b), resultingin an average protein half-life of around 15 h. All othernonphosphorylatable alanine mutants tested showed no sig-nificant differences in protein turnover, or protein half-lives,when compared to wild-type Tiam1. Taken together, these dataindicate that the interaction of 14-3-3 proteins with theN-terminus of Tiam1 regulates its protein stability.

Discussion

Tiam1 is a Rac-specific GEF that has functions in multiplebiological processes; identifying proteins that can interact withTiam1 is important in order to increase our understanding ofhow it is regulated. This has recently been shown to be thecase as we demonstrated that both Grb2 and ERK bind toTiam1, and these interactions are critical in its regulation

during Src-induced epithelial-mesenchymal transition (EMT).9

This regulation results in the selective degradation of Tiam1localized at adherens junctions, removing a key moleculeinvolved in maintaining cell-cell adhesions, thereby facilitatingtheir breakdown during Src-induced EMT.

Other binding partners have been identified through yeast two-hybrid screening using the PHn-CC-Ex region of Tiam1. Theseinclude JIP27 and the Arp2/3 complex8 and have been shown todirect signaling events downstream of Rac. In the case of theTiam1-JIP2 complex, JIP2 acts as a scaffold for the p38 mitogen-activated protein kinase (MAPK) signaling cascade, and links theRac-GEF Tiam1 to the Rac-effector MLK3, resulting in increasedp38-dependent gene transcription;7 whereas Tiam1 and theArp2/3 complex appear to be mutually dependent for properlocalization and activity. It is suggested that a positive-feedback

Figure 4. 14-3-3 proteins predominantly bind the N-terminal region of Tiam1. (a) H293T cells were transfected with full-length (FL)Tiam1-HA, or the Tiam1 truncation mutants C1199-HA and C580-HA for 24 h. Lysates were incubated with either immobilized GST-14-3-3� or GST only. Affinity precipitated exogenous Tiam1 was detected with anti-HA antibody, with the relative levels in the lysatesalso shown. (b) The intensities of Tiam1 (FL or truncation mutants) bands from panel a were quantified and the percentage of the totalTiam1 (FL or truncation mutants) that was affinity precipitated by GST-14-3-3� was calculated. (c) H293T cells transfected as in panela were lysed and exogenous Tiam1 was immunoprecipitated with anti-HA antibody; endogenous 14-3-3 that coimmunoprecipitatedwas detected using anti-14-3-3 antibody. Relative levels of 14-3-3 in the lysate are also shown. (d) The intensities of Tiam1 (FL ortruncation mutants) and 14-3-3 bands from panel c were quantified and the relative amount of endogenous 14-3-3 (normalized to the14-3-3 input levels and the amount of Tiam1 (FL or truncation mutants) immunoprecipitated) that coimmunoprecipitated with Tiam1(FL or truncation mutants) was determined.



Figure 5. Identification of the 14-3-3 binding motifs on Tiam1. (a) The amino acid sequences of various vertebrate Tiam1 orthologues werealigned, and a consensus sequence is also shown. The Mode 1 14-3-3 binding motif RXXpS/TXP is highlighted in red along with the Scansiteranking. The potential phosphorylation site for each motif is indicated on the top. (b) Product ion spectrum of a peptide precursor ion of521.7 Da. Sequence confirmation is derived from a number of fragment ion masses labeled. The site of serine phosphorylation is confirmedto be residue 3 (Ser172) by the mass difference between y5 and y6 equivalent to phosphoserine at 167 Da. Elimination of phosphoric acidfrom the phosphoserine residue generating dehydroalanine (y6-H3PO4) is further evidence for phosphorylation of residue 3. (c) Product ionspectrum of a peptide precursor ion of 630.28 Da. Sequence confirmation is derived from a number of fragment ion masses labeled. Thesite of serine phosphorylation is confirmed to be residue 3 (Ser231) by the mass difference between y8 and y9 equivalent to phosphoserineat 167 Da. Elimination of phosphoric acid from the phosphoserine residue generating dehydroalanine (y9-H3PO4) is further evidence forphosphorylation of residue 3. (d) H293T cells were transfected for 24 h with either full-length (FL) Tiam1-HA, the Tiam1 truncation mutantC1199-HA, or full-length Tiam1 with the indicated single, double or quadruple alanine substitution mutants. Lysates were incubated withimmobilized GST-14-3-3�, and affinity precipitated exogenous Tiam1 was detected with anti-HA antibody, with the relative levels in thelysates also shown. (e) The intensities of Tiam1 bands from panel d were quantified and are presented as a percentage of the total Tiam1that was affinity precipitated by GST-14-3-3�.



Figure 6. Tiam1 localization and activity are not regulated by phosphorylation of the N-terminal 14-3-3 binding motifs. (a and b) Cos7 cellswere transfected as indicated with either empty vector (Control), HA-tagged full-length wild-type (WT) Tiam1, or various HA-tagged alaninemutant full-length Tiam1 constructs. (a) Relative levels of active Rac were determined from cell lysates. (b) Cells were fixed and costainedwith anti-HA to detect Tiam1, and Phalloidin to detect filamentous Actin (F-Actin). Arrowheads indicate example localization of Tiam1 toactin-rich membrane ruffles. Scale bars, 30 µm.



Figure 7. Phosphorylation of the N-terminal 14-3-3 binding motifs regulates Tiam1 stability. (a) Cos7 cells were transfected with HA-taggedfull-length (FL) Tiam1 that was either wild-type (WT) or carried a single S60A mutation. Transfected cells were treated with cycloheximide(CHX) for the indicated times, and the turnover of Tiam1 protein was detected by immunoblotting total cell lysates with anti-HA. Levels ofthe stable protein Actin are shown as a loading control. (b) The intensities of Tiam1 bands from a number of protein turnover assays, suchas in panel a, were quantified. The level of Tiam1 at the zero time point (CHX - 0 h) for each experiment was set as 100%, with subsequenttime points calculated relatively. Data are presented as mean ( SEM, *p < 0.02. (c) Potential model for the regulation of Tiam1 stability bythe phosphorylation of key serine residues (Ser60, Ser172, Ser231) in its N-terminus and the resulting interaction of 14-3-3 dimers.



mechanism occurs through Rac signaling via WAVE or PAK1 toactivate the Arp2/3 complex, which then increases Tiam1 activity,resulting in rapid and abundant actin polymerization.8

During the current study, we sought to identify Tiam1-interacting proteins from mammalian cells under near physi-ological conditions. One major advantage of this approach overyeast two-hybrid screening, among others, is the potential toidentify binding partners that are dependent on post-transla-tional modifications, which we have previously shown can beinstrumental in the regulation of Tiam1.9 However, an obstacleto identifying this type of binding partners is that they are morelikely to interact in a transient manner, which can readilybe reversed by removal of the modification. Here we have madeuse of in vivo cross-linking with formaldehyde in order tomaintain such transient interactions throughout a purificationprocedure. A benefit of using formaldehyde is the short bridgingdistance relative to other cross-linkers, resulting in only thosemolecules essentially in contact in the noncovalent state beinglinked together, and hence reducing false-positives. We coupledthis in vivo cross-linking protocol with a tandem affinitypurification strategy, which resulted in a sample highly en-riched for Tiam1-containing complexes.

Two prominent protein bands that could now be detectedcopurifying with Tiam1 were identified as 14-3-3 family mem-bers. It was confirmed by various GST-pulldown, immunopre-cipitation and colocalization experiments that Tiam1 doesinteract with 14-3-3 proteins in cells, and that the interactionis phosphorylation-dependent. To attempt to understand themechanism behind the interaction, we initially identified thatthe N-terminal region of Tiam1 contained the major bindingsites for 14-3-3 proteins. This was further refined to three serineresidues that lie within 14-3-3 binding motifs, namely, Ser60,Ser172 and Ser231. Using nonphosphorylatable alanine mu-tants of these sites, which impaired the interaction with 14-3-3 proteins, we determined the function of the interaction.While no affect was found on Tiam1’s ability to activate Rac,we found that the single S60A mutation resulted in thestabilization of Tiam1 at the protein level. As indicated above,the N-terminus of Tiam1 contains tandem PEST sequences,which commonly serve as signals for the degradation ofproteins. Consistent with this, truncation of the first PESTsequence of Tiam1 dramatically stabilizes the protein (Wood-cock and Malliri; data not shown), as does truncation of theentire N-terminus (C1199). Because of the effect of the S60Amutation on Tiam1 stability, it is therefore likely that theinteraction between 14-3-3 proteins and the N-terminus ofTiam1 directly regulates the influence that the tandem PESTsequences have on Tiam1 stability. One potential model wouldinvolve a 14-3-3 dimer binding simultaneously to motifsflanking the tandem PEST sequences (e.g., Ser60 and Ser172/Ser231), thus, inducing a conformational change in Tiam1 dueto the rigid structure of the 14-3-3 dimer, causing the PESTsequences to become exposed and destabilize Tiam1. Alterna-tively, absence of the Ser60 binding motif (e.g., the S60A singlemutant) may now force a 14-3-3 dimer to simultaneously bindthe Ser172 and Ser231 motifs, causing the inactivation of thePEST sequences, potentially through sterical hindrance or aconformational change that makes the PEST sequences inac-cessible to the degradation machinery, thereby stabilizingTiam1. We favor the latter of these models (see Figure 7c) asthe 4A Tiam1 mutant, which has all the 14-3-3 binding motifsmutated, has a similar protein half-life as wild-type Tiam1,

indicating that, in the absence of the Ser172 and Ser231 bindingmotifs, mutation of the Ser60 motif does not stabilize Tiam1.

It will be important to determine the kinases and phosphatasesthat directly modify Tiam1 on Ser60, Ser172 and Ser231, and theextracellular cues that regulate them. Analysis of the sequencesurrounding these sites suggests that they are potential substratesfor various protein kinases, including Protein kinase A (PKA),Protein kinase B (PKB), Protein kinase G, Calmodulin-dependentkinase II (CaMDKII), Casein kinase and Ribosomal S6 kinase(RSK). Potentially, it will be these enzymes that ultimately controlthe balance of phosphorylation on these sites, thereby directingwhere the 14-3-3 dimer binds and the prospective outcome onTiam1 function or stability. Of note, during the modified TAPdescribed, we found that a subunit of the Ser/Thr-phosphatasePP-2A, and Casein kinase I, were identified as putative Tiam1-interacting proteins, suggesting these enzymes may regulate thephosphorylation events on Tiam1.

In summary, we have devised a modified tandem affinitypurification technique for use with mammalian cells that incor-porates an in vivo formaldehyde cross-linking procedure. Thisallows the maintenance of lower affinity or transient interactionsthroughout the purification, and therefore has the potential toidentify additional interacting proteins that could be missed usingthe standard technique. We would therefore recommend that thismodified TAP technique should be used alongside the standardone in order to maximize the number of interacting proteinsdetected. The fact that this form of screening for interactingproteins occurs in mammalian cells will allow the use of thistechnique in the future to identify Tiam1 interactors that only bindunder certain conditions. This useful strategy has the potentialto tease out the regulatory mechanisms behind Tiam1’s widerange of cellular functions, and could be easily adapted for usewith other proteins in order to maintain lower affinity or transientinteractions throughout purification strategies.

Acknowledgment. We thank J. G. Collard, F. Barr, andH. Clevers for reagents, members of the Cell Signalling Groupfor helpful discussions, A. Gambus and K. Labib for advice onthe TAP strategies, and Y. Connolly and D. Smith for criticalreading of the manuscript and very helpful discussions, adviceand assistance in setting up the modified TAP strategy, andmass spectrometry work at the PICR. We are also grateful toSteve Bagley for his help with microscopy. This work wassupported by Cancer Research UK [CR-UK] grant numberC147/A6058 to A.M.

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