Proteomic Identification of the Cerebral Cavernous Malformation

Signaling Complex

Thomas L. Hilder,† Michael H. Malone,† Sompop Bencharit,†,‡ John Colicelli,§

Timothy A. Haystead,| Gary L. Johnson,*,† and Christine C. Wu⊥

Department of Pharmacology and the Lineberger Comprehensive Cancer Center, and Department ofProsthodontics, School of Dentistry, University of North Carolina, Chapel Hill, CB #7365,

Chapel Hill, North Carolina 27599-7365, Department of Biological Chemistry, Molecular Biology Institute, andthe Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los

Angeles, Los Angeles, California 90095, Department of Pharmacology and Cancer Biology, Duke UniversityMedical Center, C119 LSRC Research Drive, Durham, North Carolina 27710, and University of Colorado School

of Medicine, Department of Pharmacology/Mail Stop 8303, Fitzsimons RC1 South, 12801 East 17th Avenue,L18-6117, P.O. Box 6511, Aurora, Colorado 80045

Received July 11, 2007

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the centralnervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mappedautosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All threeproteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them.Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and theMAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein,we show that the three CCM proteins are members of a larger signaling complex. To define this complex,epitope-tagged wild type OSM or OSM harboring the mutation of F217 f A, which renders the OSMphosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purifiedby immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-basedmethods combined with electrospray ionization-MS or by multidimensional protein identificationtechnology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3,Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we showfor the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominentproteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disruptedwith the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- andtriphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targetingof the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.

Keywords: cerebral cavernous malformation • KRIT1 • OSM • CCM3 • PDCD10 • MudPIT

IntroductionCerebral cavernous malformations (CCM) are sporadic or

inherited vascular lesions in the brain characterized by endot-helial-lined sinusoids and a sub-endothelial layer of connectivetissue distinct from the neural parenchyma (reviewed in refs 1and 2). The lesions are devoid of mature vessel wall elementslike smooth muscle and elastic tissue. The blood-brain barrieris severely compromised, as gaps at endothelial cell tight

junctions are observed and no astrocytic foot processes borderthe vessels.3 Hemorrhaging of the vessels in affected individualscan occur, leading to chronic headaches, seizures, focal neu-rological deficits, and/or stroke.4,5

The familial form of CCM is autosomal dominant and hasbeen mapped to three loci (ccm1, ccm2, and ccm3),6-9 encodingthe proteins KRIT1, OSM/malcavernin, and PDCD10/CCM3,respectively.10-14 KRIT1 contains ankyrin repeat domains anda FERM domain, both of which coordinate protein-proteininteractions;15,16 however, proteins that interact with KRIT1remain to be defined. KRIT1 also contains multiple NPxY motifsthat are bound by the phosphotyrosine binding (PTB) domainof OSM and of the integrin binding protein ICAP-1.17,18 Ad-ditionally, KRIT1 contains a functional nuclear localizationsignal that enables it to undergo nuclear-cytoplasmic shut-

* To whom correspondence should be addressed. Gary L. Johnson: Phone,(919) 843-3107; Fax, (919) 966-5640; E-mail, glj@med.unc.edu.

† Department of Pharmacology and the Lineberger Comprehensive CancerCenter.

‡ Department of Prosthodontics.§ University of California Los Angeles.| Duke University Medical Center.⊥ University of Colorado School of Medicine.

10.1021/pr0704276 CCC: $37.00 xxxx American Chemical Society Journal of Proteome Research XXXX, X, XXXX-XXXX APAGE EST: 12.2 Published on Web 09/27/2007

tling.17 OSM, in addition to binding KRIT1, is a scaffold for Racand MEKK3 at membrane ruffles for the activation of p38 MAPkinase in response to sorbitol.19 A fraction of OSM colocalizeswith actin and OSM binds F-actin in in vitro binding assays,19

suggesting that OSM organizes a complex capable of couplingRac-dependent actin reorganization to p38 activity. Finally,PDCD10 is a relatively small protein with no discernibleprotein-protein interaction domains or enzymatic activity.PDCD10 was initially characterized as a gene whose expressionwas upregulated upon the induction of apoptosis in humanmyeloid cell lines.14 Mutations in ccm1, ccm2, or ccm3 genesare nonsense, frameshift, or splice site mutations, resulting inloss-of-function alleles.12-14,20 One missense mutation (L198 f

R) has been described in the ccm2 gene,13 which lies withinthe PTB domain of OSM and significantly disrupts the bindingof OSM to KRIT1.17

We hypothesized that OSM, KRIT1, and PDCD10 are scaf-fold- or adaptor-like proteins to organize and localize amacromolecular complex, and mutations in any of the threeCCM proteins could disrupt the function of the CCM complex.To test this hypothesis, we stably expressed a FLAG-taggedversion of OSM in RAW264.7 macrophages and identified theproteins that specifically co-immunoprecipitated with OSMusing nanoelectrospray mass spectrometry and multidimen-sional protein identification technology (MudPIT). Our resultsindicate that OSM, KRIT1, and PDCD10 form a CCM proteincomplex. Previously identified binding partners for OSM andKRIT1 (Rac, MEKK3, and ICAP-1) were identified in ourproteomic analyses. An engineered point mutation within thePTB domain of OSM (F217 f A), which like the L198R patientmutation disrupts OSM binding to KRIT1,17 resulted in the lossof numerous proteins associated with the CCM complex. Theresults provide novel insight into the proteins organized by theOSM-KRIT1 complex and demonstrate that functional disrup-tion of the OSM PTB domain has profound effects on theprotein network assembled by the CCM complex.

Experimental Procedures

Plasmids and Production of Recombinant Proteins. FLAG-tagged KRIT1, OSM, OSMF217A in pRK5, FLAG-LAD in pcDNA3.1,and pEYFPC1-Krit and pECFPN1-OSM were previouslydescribed.17-19,21 For the generation of retroviral constructs,FLAG-OSM and FLAG-OSMF217A were subcloned into thepMSCVpuro vector (Clontech) using standard PCR cloningmethods. Full length cDNA for EF1A1 (I.M.A.G.E. ConsortiumCloneID 3948601)22 was purchased from ATCC and subclonedin-frame into the pcDNA4/myc-His B vector (Invitrogen).

Recombinant 6×His-tagged OSM was described previously.19

Full-length murine PDCD10 was PCR amplified from a mousefibroblast cDNA library and cloned into pMCSG7-His. This full-length 6×His-PDCD10 was expressed in BL21 cells and purifiedby nickel affinity chromatography. The purified recombinant6×His-PDCD10 protein was then coupled to CNBr-Sepharosebeads (GE Biosciences) according to the manufacturer’s pro-tocol.

Cell Culture and Generation of Stable Cell Lines. Phoenixcells and COS7 cells were maintained in Dulbecco’s ModifiedEagle Medium (Invitrogen) supplemented with 10% fetal bovineserum, 100 U/mL penicillin, and 100 µg/mL streptomycin.RAW264.7 mouse macrophages and 1321N1 human astrocy-toma cells were maintained in the same medium, but heatinactivated serum was used and all components were filteredthrough a sterile 0.22 µm vacuum flask (Denville Scientific).

Amphotropic Phoenix cells were used as packaging cells forthe retroviral gene transfer system.23,24 FLAG-OSM or FLAG-OSMF217A in pMSCVpuro were transfected into Phoenix cellsusing Lipofectamine and PLUS reagents (Invitrogen). Two daysafter transfection, the supernatants were collected, diluted withan equal volume of fresh medium, and Polybrene was addedto a final concentration of 8 µg/mL. The infection mixtures wereadded to RAW264.7 or 1321N1 cells. The next day, the cellswere reinfected using the same method. Twenty-four hoursafter the second infection, selection was started with 4 µg/mLpuromycin in complete DMEM. Stable cells were fully selectedfollowing 1-2 weeks exposure to puromycin.

Cell Lysis and Immunoprecipitation. FLAG pulldowns wereperformed essentially as described.25 Briefly, RAW264.7 or1321N1 cells stably expressing pMSCVpuro empty vector,FLAG-OSM, or FLAG-OSMF217A were lysed in pulldown buffer(50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% NP-40, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate,100 mM sodium fluoride, and 20 mM â-glycerophosphate) andsupplemented with a complete protease inhibitor cocktail tablet(Roche) and 1 mM sodium vanadate. Lysates were clarified bycentrifugation, and 30 mg of protein, diluted to 2 mg/mL inpulldown buffer, was used per pulldown. Protein G-sepharoseresin (Invitrogen) was used to preclear the lysates, and FLAG-tagged proteins were immunoprecipitated with anti-FLAG M2-agarose (Sigma). Following extensive washing in pulldownbuffer, FLAG-OSM was competitively eluted with FLAG peptide(Sigma). For protein identification by nanoelectrospray massspectrometry, the eluates were resolved by SDS-PAGE andvisualized by Sypro Ruby protein staining (Invitrogen) accord-ing to manufacturer’s directions or by silver staining. For theMudPIT analysis, proteins in the eluates were precipitated usingMeOH/CHCl3 as previously described.26

Immunoprecipitation of EF1A1-myc was performed in es-sentially the same manner. COS7 cells transfected with FLAG-tagged constructs and EF1A1-myc/His were lysed in pulldownbuffer, and EF1A1 was immunoprecipitated with the mono-clonal anti-c-myc 9E10 antibody (Santa Cruz Biotechnology).After washing, EF1A1 was eluted from the beads in 2× SDS-loading buffer and immunoblots were performed as describedbelow.

To determine the binding of PDCD10 to OSM and KRIT1,PDCD10-Sepharose or uncoupled Sepharose was tumbledovernight with lysates from RAW264.7 cells stably expressingempty vector or FLAG-OSM, or with lysates from COS7 cellstransiently expressing FLAG-OSM, FLAG-OSMF217A, or FLAG-KRIT1. The beads were then washed four times in pulldownbuffer, proteins were eluted into 2× SDS-loading buffer, andimmunoblots were performed as described below. The amountof PDCD10 present in the pulldowns was measured by stainingthe nitrocellulose membrane with Memcode protein stain(Pierce).

Nanoelectrospray Ionization Mass Spectrometry. Bandsexcised from SDS-PAGE gels were diced into 1 mm2 pieces.Silver-stained bands first destained for 10 min in a 1:1 mixtureof 100 mM sodium thiosulfate:30 mM potassium ferricyanide,then were washed three times in water. The silver-stained orSypro Ruby-stained gel slices were then incubated in a 1:1solution of acetonitrile:100 mM ammonium bicarbonate, anddried in a speed-vac. The gel pieces were re-swelled on ice in30 µL 25 mM ammonium bicarbonate containing 0.5 µgsequencing grade modified trypsin (Promega) for 45 min, then30 µL 25 mM ammonium bicarbonate was added incubated

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overnight at 37 °C. Peptides were recovered from the gel slicesby two successive extractions in 60% acetonitrile with 5%formic acid. The acetonitrile was removed, and the peptideswere concentrated to ∼5 µL by speed-vac. In preparation fornanoelectrospray analysis, the peptides were bound to PorosR2 resin (Applied Biosystems), washed twice in 5% methanolwith 5% formic acid, and eluted into an electrospray needle(Proxeon) with 60% methanol with 5% formic acid.

Identification of the bands was performed by nanosprayelectrospray ionization mass spectrometry (nESI-MS) on anApplied Biosystems QSTAR Pulsar mass spectrometer with anion source voltage of 8 kV. Peaklists were generated (m/z 400-1600 for precursor ions, m/z 130 to twice the mass of the doublycharged precursor ion for fragment ions) and database searcheswere performed with Analyst QS software, version 1 (build 7051;Applied Biosystems), with oMALDI source support, service pack8, and Bioanalyst extensions. For database searches, thefollowing parameters were used: (i) enzyme specificity oftrypsin; (ii) no missed cleavages were allowed; (iii) no fixed orvariable modifications were allowed; and (iv) mass tolerancesof precursor and fragment ions were set at 1.1 and 0.2 Da,respectively. For de novo peptide sequencing, the y-ion withthe highest detectable m/z was selected, and Analyst QS wasused to select the next 3-5 y-ions in the series to generate asequence tag. This sequence tag, along with the masses of they- and b-ions, with or without NH3, were searched against theNational Center for Biotechnology Information nonredundant(NCBInr) database (Apr. 27, 2006 download; 1 596 370 entries).No species restriction was applied, but for all identifications amouse or human homologue was identified (from RAW264.7or 1321N1 cells, respectively). Based on these parameters,greater than 50% of these y- and b-ions fell within the masstolerance specified above. The thresholds applied to thesesequence tags resulted in a single identification with an AnalystQS score between 585 and 696 (below this cutoff, peptideseither had a residue that did not match the tandem MSspectrum or were not tryptic). At least two peptides meetingthese parameters were required for a positive identification.

Immunoblotting. Following anti-FLAG M2-agarose pull-downs or immunoprecipitations, immunoblots were performedfor members of the complex following transfer of the proteinsto nitrocellulose. Primary antibodies were used at manufac-turer’s recommended dilutions and included the following:c-myc 9E10, 6×His, CCT3, CCT6, and KRIT1 (Santa CruzBiotechnology), R- and â-tubulin, actin, and rabbit anti-FLAG(Sigma Aldrich), HSP70 and HSP90 (Cell Signaling Technology),and EF1A1 (Upstate). Rabbit anti-RIN2 was raised against a GSTfusion protein comprising residues 425-895 of human RIN2.Donkey anti-rabbit, donkey anti-goat, and sheep anti-mousesecondary antibodies coupled to HRP were purchased fromJackson ImmunoResearch, Santa Cruz Biotechnology, andAmersham Biosciences, respectively. Detection of HRP wasperformed using SuperSignal West Pico ChemiluminescentSubstrate from Pierce.

Lipid Arrays. PIP arrays (Echelon Biosciences, Inc.) wereblocked in 0.1% ovalbumin in Tris buffered saline with 0.05%Tween-20 (TBS-T) for 1 h and then were incubated with 0.5-1µg/mL of recombinant 6×His-PDCD10 or OSM for 2 h. Afterwashing unbound CCM proteins using TBS-T, the boundproteins were detected by immunoblotting with an anti-6×Hisantibody.

Live Cell Imaging. COS7 cells were plated on 25 mm glasscoverslips, and then the cells were transfected with the

indicated constructs fused to mCherry, EYFP, or ECFP. Thecoverslips were placed in an imaging chamber (MolecularProbes) with medium and were imaged using a Zeiss Axiovert200M inverted microscope with a 125-W xenon arc lamp (SutterInstrument Company, Novato, CA), digital charge-coupleddevice camera (CoolSNAP HQ; Roper Scientific, Tucson, AZ),and Slidebook 4.0.10 software (Intelligent Imaging Innovations,Denver, CO). An objective lens (63× oil 1.25-numerical aper-ture, Plan-Neofluar [Zeiss]) was coupled with immersion oilto the bottom face of glass coverslips. Images from three planeswere taken for each of the three channels (CFP [a band-passexcitation filter of 436/20 nm, a 455DCLP band beamsplitter,and a band-pass emission filter of 480/40 nm], YFP [a band-pass excitation filter of 500/20 nm, a 515DCLP band beam-splitter, and a band-pass emission filter of 535/30 nm], andCy5 [a band-pass excitation filter of 620/60 nm, a 660DCLPband beamsplitter, and a band-pass emission filter of 700/75nm]; Chroma). The three planes were deconvolved using thenearest neighbors algorithm.

MudPIT Analysis. The precipitated FLAG peptide eluatesdescribed above were sonicated and resuspended in 0.1%Rapigest (Waters Corp, Milford, MA), reduced with 5 mMdithiothreitol at 60 °C for 15 min and alkylated with 15 mMiodoacetamide at room temperature for 30 min in the dark.The samples were then digested with sequence grade modifiedtrypsin (Promega, Madison, WI) at a 1:50 enzyme/proteinconcentration at 37 °C overnight with gentle shaking in athermomixer (Eppendorf, Westbury, NY). The Rapigest washydrolyzed by the addition of concentrated HCl to a finalconcentration of 200 mM and incubation at 37 °C for 45 min.Insoluble particulates were removed by centrifugation at 20 000g for 10 min. The peptide supernatant was loaded onto adesalting column (1 cm C18-reverse phase) and then connectedto a two-phase, 100 µm inner diameter microcapillary column(10 cm C18-reverse phase, 3 cm strong cation exchange) witha 5 µm tip using an in-line filter assembly (Upchurch). A six-step MudPIT was run using a linear MeCN gradient (5-60%)containing 0.1% formic acid over 120 min with salt pulses (0,50 µL 300 mM, 50 µL 500 mM, 50 µL 700 mM, 50 µL 1 M, 100µL 5M NH4OAc) injected at the beginning of each step. Thismultidimensional separation was performed in a mannersimilar to those described previously.27 However, instead ofusing a quaternary pump to deliver the salt pulses, an au-tosampler was used to deliver a plug of ammonium acetatedirectly in the path of the HPLC flow. Briefly, an Agilent 1100binary pump was run at 200 µL/min and the flow was splitimmediately distal to the multiphasic capillary column usinga microtee as previously described.28 A 50 µm ID capillary wasadded to the waste of the microtee and the length of the splitcapillary was adjusted to produce a flow rate through thecapillary column of ∼250 nL/min. The autosampler was placedin-line between the HPLC and column and a flow splitter isused to add 50 µL ammonium acetate pulses (of variableconcentrations) into the HPLC flow. By running the pump at200 µL/min, the salt pulse reaches the column almost instantlyand only a fraction of the total salt injected (∼0.1%) makes itonto the column. Final salt concentrations required per injec-tion for optimal distribution of peptides between the individualsteps were derived empirically from prior experiments onsimilar mixtures. Fractions were eluted directly onto a Ther-moElectron LTQ mass spectrometer. A spray voltage of 2.4 kVwas applied and mass spectra were acquired in a data-dependent mode, whereby a single full mass scan (m/z 400-

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1400) was followed by five tandem MS scans of the most intensepeaks. The instrument raw files were converted to text files inms2 format29 using libraries supplied by the manufacturer(ThermoElectron). Dynamic exclusion was enabled.

The acquired tandem MS were searched against a mouseRefSeq database (downloaded from NCBI on July 11, 2005 andcontaining 26 996 entries and modified to include the pro-grammed cell death 10 (CCM3) and recombinant FLAG-OSMprotein sequences) concatenated with a shuffled decoy data-base as described30 using a normalized version of Sequest27.31,32 Database selection was determined by cell type (mouseRAW264.7 macrophages). Database search parameters were asfollows: no proteolytic enzyme specificity, no variable modi-fications were considered, and a static modification of +57 wasassumed for all cysteines. Mass tolerance was (1.5 m/z forprecursor ions (average masses), and fragment ions (monoiso-tropic masses) were binned to the nearest integer.

The data was reassembled using DTASelect 1.9,33 andthresholds were set up as follows: Xcorr cutoff for singly,doubly, and triply charged peptides was set at 0.3,31 the ∆Cnwas set to 0.1, only loci with unique peptides were allowed,proteins that are subsets of others were removed, peptides musthave six or more residues, and three tryptic peptides wererequired for a positive identification. False discovery rates forprotein identifications were maintained at <0.01% using themethod previously described.30 Redundancy was handled asfollows. If the database searches were unable to conclude whichisoform was identified because a given set of peptides over-lapped multiple isoforms or subunits of a protein, we groupedthese proteins as a single entry. Otherwise, proteins identifiedby MudPIT had at least one peptide unique to a given protein,isoform, or subunit.

Results

Identification of OSM Binding Proteins. The CCM1 (KRIT1),CCM2 (OSM), and CCM3 (PDCD10) proteins have no discern-ible catalytic domains that can be identified by domainarchitecture, suggesting they function as scaffold and/or adap-tor-like proteins to organize macromolecular protein com-plexes. Therefore, we sought to determine the proteins asso-ciatedwithKRIT1-OSMcomplexes.Toaccomplishthisidentification,we took advantage of a point mutation within the PTB domainof OSM (F217 f A) that disrupts the interaction of KRIT1 andOSM,17 with the prediction that disruption of this interactionwould have profound effects on the composition of thecomplex. Empty vector, wild type FLAG-OSM, or FLAG-OSMF217A were stably expressed in RAW264.7 mouse macroph-ages and human 1321N1 astrocytoma cells. Total cell lysateswere tumbled with anti-FLAG antibody coupled to agarosebeads and FLAG peptide was used to disrupt the FLAG epitope-antibody complex. The eluates were then resolved by SDS-PAGE. Figure 1A and C show representative silver-stained gelswith several proteins specifically co-immunoprecipitated withwild type FLAG-OSM as compared to immunoprecipitates fromcell lysates of empty vector-expressing RAW264.7 mouse mac-rophages or human 1321N1 astrocytoma cells. FLAG-OSMF217A-does not stably bind KRIT1 (Figure 1A and B),17,34 and disrup-tion of the OSM-KRIT1 interaction with the OSMF217A mutantprotein resulted in the loss or significant decrease in abundanceof several proteins in the anti-FLAG immunoprecipitates(Figure 1A and C). The similar profiles of proteins found inempty vector, wild type FLAG-OSM and FLAG-OSMF217A im-

munoprecipitates between the RAW264.7 and 1321N1 cellsdemonstrates that the OSM-associated protein complexes, andthose interactions requiring a functional OSM PTB domain, areconserved in two different cell lines of mouse and humanorigin.

Proteins selectively immunoprecipitated with wild typeFLAG-OSM were excised from the gel and identified by nESI-MS (Table 1; sequenced peptides in Supplemental Table 1, seeSupporting Information). The two most abundant proteins co-immunoprecipitated with wild type FLAG-OSM were KRIT1,an established OSM interacting protein, and EF1A1, a eukary-otic translation elongation factor.35 PDCD10 (CCM3) is alsofound in the complex, demonstrating for the first time that thethree CCM proteins, KRIT1, OSM, and PDCD10, are found ina complex. KRIT1, EF1A1, and â-tubulin were all associatedwith the FLAG-OSM complex requiring a functional OSM PTBdomain, as the co-immunoprecipitation of these proteins wassignificantly reduced in FLAG immunoprecipitations from celllysates expressing OSMF217A.

Proteins that immunoprecipitated nonspecifically, throughtheir association with the anti-FLAG-agarose beads, are alsosummarized in Table 1 (denoted by a letter corresponding tothe labeled gel band in Figure 1A). Of note, actin was the mostprominent protein nonspecifically immunoprecipitated fromempty vector, wild type FLAG-OSM and FLAG-OSMF217A cell

Figure 1. OSM immunoprecipitates a macromolecular proteincomplex. FLAG-OSM (O), FLAG-OSMF217A (F), or pMSCV emptyvector (EV) was stably expressed in RAW264.7 mouse macroph-ages (A) or 1321N1 human astrocytoma cells (C). FollowingFLAG immunoprecipitation and FLAG peptide elution, theeluates were separated by SDS-PAGE and silver stained.Bands were excised and identified by mass spectrometry (seeTable 1). In (A), unique interactions between EV and OSM arenumbered on the right side of the gel, whereas nonspecificinteracting proteins, present in all three samples at equal levels,are lettered on the left. (B) Following FLAG immunoprecipitation(IP) and FLAG peptide elution, the eluates were immunoblotted(IB) for FLAG (top) or endogenous KRIT1 (middle). Whole celllysates (WCL) were blotted for KRIT1 to show equal loading(bottom).

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lysates. Both OSM and EF1A1 are actin-binding proteins,19,36

but this interaction could not be confirmed because of thetrapping of actin on the anti-FLAG agarose beads.

PDCD10 Interacts with OSM. The PDCD10 gene encodes aprotein of unknown function that when mutated results inCCM.14 PDCD10 encodes no known catalytic domains, butthree-dimensional threading analysis suggests it may fold as asix helical bundle characteristic of many adaptor-like proteins(our unpublished observations). PDCD10 was identified (Table1) as a protein that co-immunoprecipitated with FLAG-OSMindicating it is part of a CCM complex that also includes OSMand KRIT1. Figure 2A shows TOF-MS spectrum for PDCD10from which six doubly charged peptides were sequenced, givingsequence coverage of 32%. We validated the interaction bytumbling cell lysates with recombinant PDCD10 covalentlycoupled to Sepharose beads. Figure 2B shows that recombinantPDCD10-Sepharose bound both FLAG-OSM as well as endog-enous OSM from lysates of RAW264.7 cells. Furthermore,PDCD10-Sepharose was capable of capturing FLAG-OSM whenexpressed transiently in COS7 cells (Figure 2C). PDCD10 didnot appear to bind KRIT1 directly, but the binding of PDCD10to OSM was not disrupted by KRIT1, as all three proteins couldbe isolated together as a complex. The interaction betweenOSM and PDCD10 was not dependent upon the PTB domainof OSM, since PDCD10-Sepharose retained the ability tobind OSMF217A, in contrast to the OSM PTB mutation disruptingOSM binding of KRIT1. Thus, PDCD10, OSM, and KRIT1 arefound as a protein complex. The findings are the first todemonstrate that the three CCM proteins are in a complexsuggesting they function coordinately to control vascularintegrity in the CNS.

PDCD10 and OSM Bind Phosphatidylinositides. In additionto binding distinct NPxY motifs within proteins, PTB domainsalso may bind headgroups of phosphatidylinositides.34 This,combined with the evidence that a fraction of OSM and KRIT1translocates to the membrane in a stimulus-dependent man-ner,17,19 led us to hypothesize that the CCM proteins may bindlipids. Therefore, purified His-tagged OSM or PDCD10 wereoverlaid onto phosphotidylinositol phosphate arrays and blot-ted for bound protein using an anti-His antibody. Figure 3shows that OSM preferentially bound monophosphorylatedphosphatidylinositols including PtdIns(3)P, PtdIns(4)P, and

PtdIns(5)P. PDCD10 selectively bound the diphosphorylatedphosphatidylinositols PtdIns(3,4)P2 and PtdIns(4,5)P2 as wellas the triphosphorylated PtdIns(3,4,5)P3. The domain archi-tecture responsible for PDCD10 binding to specific PIP2 andPIP3 phosphatidylinositols is unknown at this time and iscurrently being investigated. The fact that OSM binds PIPs andPDCD10 binds both PIP2 and PIP3 phosphatidylinositols dem-onstrates the CCM protein complex is able to bind to mem-brane lipids and suggests that phosphatidylinositol 3-kinasemay in part regulate localization of the CCM protein complexin cells.

Validation that OSM Binds EF1A1. EF1A1 is a multi-functional translation elongation factor that has been shownto charge tRNA for translation,37 bind F-actin protein andâ-actin mRNA, and regulate actin cytoskeletal dynamics.36,38

FLAG-OSM immunoprecipitation using anti-FLAG antibodiesresults in co-immunoprecipitation of EF1A1 (Figure 1, Table1). EF1A1 is 92% identical at the protein level to EF1A2, butwe identified a unique doubly charged peptide from EF1A1 ofm/z ) 702.8077. MS/MS de novo sequencing of this peptiderevealed a valine as the y10 ion (Figure 4A and B), whereasEF1A2 has an isoleucine at this position in the analogouspeptide, thereby accounting for the difference in the massesof the parent ions. Following FLAG-OSM immunoprecipitationfrom RAW264.7 cell lysates, we were able to immunoblotendogenous EF1A1 (Figure 4C), validating the interaction andconfirming the dependence of the interaction on the PTBdomain of OSM. Furthermore, cotransfection of FLAG-KRIT1,FLAG-OSM, and EF1A1-myc/6×His in COS7 cells showed thatall three proteins could be co-immunoprecipitated in a com-plex (Figure 4D). Cumulatively, the findings suggest that thebinding of EF1A1 maybe via KRIT1 binding to the OSM PTBdomain.

It was important to show that the interaction of EF1A1 withthe OSM-KRIT1 complex was specific, since EF1A1 is a highabundance protein that is often found nonspecifically inimmunoprecipitation analyses.25,39 We used FLAG-tagged LAD,a scaffold protein that we have shown binds MEKK2,21,40 aMAPK kinase kinase closely related to MEKK3 (which bindsOSM).19 FLAG-LAD and EF1A1 were coexpressed and EF1A1-myc was immunoprecipitated. Figure 4E shows that EF1A1 didnot co-immunoprecipitate with FLAG-LAD, but effectively

Table 1. Proteins Identified from SDS-PAGE Gel and nESI-MS

band protein name accession number

peptides

sequenced

sequence

coverage

Proteins that selectively interact with FLAG-OSM1 HSP90 (Heat shock protein 1 beta) gi|40556608|ref|NP_032328.2| 5 8.2%2 KRIT1 (cerebral cavernous malformation 1) gi|13994219|ref|NP_109600.1| 8 11.7%3 HSP70 (heat shock protein 8) gi|31981690|ref|NP_112442.2| 5 9.0%4 Tubulin, alpha 4 gi|6678467|ref|NP_033473.1| 5a 16.5%

Tubulin, alpha 6 gi|6678469|ref|NP_033474.1|5 Tubulin, beta 5 gi|7106439|ref|NP_035785.1 6 15.3%6 Eukaryotic elongation factor 1 alpha 1 gi|51873060|ref|NP_034236.1| 6 11.9%7 Capping protein (actin filament) muscle Z line, alpha 1 gi|33468887|ref|NP_033927.1| 3 14.3%8 Ribosomal protein S3 gi|6755372|ref|NP_036182.1| 7 31.3%9 Programmed cell death 10 (CCM3) gi|31560391|ref|NP_062719.2| 6 32.1%10 Solute carrier family 25, mitochondria carrier,

adenine nucleotide translocator 1gi|51764087|ref|XP_134169.2| 2 8.4%

Proteins that nonspecifically interact with anti-FLAG antibody-conjugated beadsa Flightless I homologue gi|11528490|ref|NP_071292.1| 8 7.5%b Serine/threonine kinase 38 gi|19527344|ref|NP_598876.1| 5 12.3%c Actin, gamma, cytoplasmic 1 gi|6752954|ref|NP_033739| 7 24.8%d Tropomodulin 3 gi|8394460|ref|NP_058659.1| 4 12.5%

a Sequenced peptides overlapped both isoforms

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bound the OSM complex. Thus, EF1A1 binds the OSM-KRIT1complex and the OSMF217A mutation disrupts this interaction.The co-immunoprecipitation of OSM-KRIT1 complexes withEF1A1 is specific and other FLAG-tagged scaffold proteins donot show EF1A1 binding.

Colocalization of EF1A1, KRIT1 and OSM in Living Cells.To ensure that the CCM proteins colocalize with EF1A1, COS7cells were transiently transfected with YFP-KRIT1, CFP-OSMor CFP-OSMF217A, and either mCherry alone or mCherry-EF1A1to analyze their localization in live cells. Expression of mCherry-EF1A1, YFP-KRIT1, and CFP-OSM in COS7 cells resulted in astrong colocalization of the three proteins in the cytoplasm andat the cell periphery (Figure 4G). In contrast, mCherry alone isprimarily nuclear (Figure 4F). Expression of CFP-OSMF217A ledto the translocation of KRIT1 to the nucleus (Figure 4H and aspreviously shown),17 whereas EF1A1 remained cytosolic. Al-though EF1A1 and OSMF217A remain cytosolic in this assay, theydo not co-immunoprecipitate (Figure 4C, D).

MudPIT Identification of Proteins Associated with OSM-KRIT1 Complexes. Although our gel-based approaches pro-vided a number of new binding partners for the CCM scaffoldcomplex, we were unable to identify lower abundance, knowninteracting proteins such as Rac, MEKK3, and ICAP-1 using thismethod. Therefore, to determine the complex organized by thethree CCM protein scaffolds, we analyzed the immunoprecipi-tated proteins by MudPIT. Following FLAG-agarose immuno-precipitation and FLAG peptide elution of the complex, theproteins were digested in-solution and identified by LC-MS/MS. MudPIT runs were performed for immunoprecipitates fromeach RAW264.7 cell lysate (empty vector, and stably expressedFLAG-OSM or FLAG-OSMF217A). The sequenced peptides, spec-tral counts, and sequence coverages for every protein identifiedin the MudPIT analysis are listed in Supplemental Table 2. Eachprotein in Supplemental Table 2 has been identified bysequencing of at least three unique peptides; the one exception,PDCD10, was found at low levels in the MudPIT analysis (only

Figure 2. Identification and validation of PDCD10 as an OSM-KRIT1 complex member. (A) TOFMS of peptides from band 9. *)Doublycharged peptides subjected to MS/MS that identified PDCD10. (B) Lysates from RAW264.7 macrophages stably expressing emptyvector or FLAG-OSM were tumbled with either Tris-Sepharose or Sepharose coupled to recombinant PDCD10. OSM was identifiedfollowing PDCD10-sepharose pulldown by immunoblotting (IB; top), and CCM3 loading was demonstrated by staining the membranewith Memcode protein stain. (C) COS7 cells were transfected with FLAG-tagged KRIT1, OSM, or OSMF217A, and lysates were tumbledwith Tris- or PDCD10-sepharose. FLAG immunoblots were performed following the pulldown (top, left). Whole cell lysates (WCL) wereblotted for expression of FLAG-tagged proteins (right).

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one peptide was identified in the FLAG-OSM and FLAG-OSMF217A immunoprecipitates). It was included in the data set,however, since the gel-based nESI-MS and biochemical ap-proaches (Figure 1 and 2, Table 1) substantiate PDCD10 as acomplex member.

A total of 339 unique proteins were identified in the MudPITanalysis. This includes 238, 209, and 174 proteins in theimmunoprecipitates from empty vector, FLAG-OSM, and FLAG-OSMF217A stably expressing cells, respectively. Of the proteinsthat were identified in the empty vector immunoprecipitates,99 were found in the empty vector sample only, 24 were foundin all three samples at roughly equal levels, and 57 were foundin all three samples but at significantly higher levels in theempty vector sample compared to OSM and OSMF217A. Theseproteins were considered nonspecific interactions and wereexcluded as potential complex members, leaving 159 as possiblecandidates. Of the remaining 159 proteins, 45 were distributedamong those that co-immunoprecipitated with OSMF217A only,those that were found in both empty vector and OSMF217A

immunoprecipitates but not in wild type OSM samples, andthose that were only found in empty vector and OSM immu-noprecipitates but not OSMF217A. These proteins were alsoconsidered nonspecific. This left a total of 114 proteins thatwere likely immunoprecipitated by the presence of wild typeOSM and were regarded as complex members. Of these,53 were found only in the OSM immunoprecipitates, 11associated with OSM and OSMF217A complexes at similar levels,21 associated with OSM in a PTB domain-dependent manner(i.e., protein abundance was much lower in the OSMF217A

sample compared to wild type OSM), nine were identifiedin all three samples but were found at much higher levels inthe OSM and OSMF217A immunoprecipitates than in emptyvector samples, and 20 were present in all three samples butco-immunoprecipitated with OSM at much higher levels thanthe empty vector sample and displayed a PTB domain-dependence.

We have summarized those proteins that selectively co-immunoprecipitated with FLAG-OSM in Table 2. Since OSMlocalizes to the cytoplasm,17,19 we have excluded those proteinsthat were identified from other organelles, as these are mostlikely artifacts of cell lysis and/or contaminants of the antibody-bead pulldowns. We have also included the spectral counts foreach protein, as there is a linear correlation between spectral

counts (the total number of MS/MS spectra matched to aparticular protein) and relative protein abundance.41 For theidentification of some complex members, such as KRIT1, ICAP-1, and RIN2, the spectral counts are absent from the emptyvector immunoprecipitates, suggesting that the interaction ofthese proteins with the OSM complex is clearly specific. Otheridentifications are made slightly more complicated by thepresence of peptides in the empty vector immunoprecipitates,but the spectral counts still provide a level of specificity. Forexample, EF1A1 is known to bind actin,36,42-45 and actinnonspecifically bound anti-FLAG antibody beads from lysatesof all three stable RAW264.7 cell lines (see Figure 1 and Table1). Therefore, there was a strong chance of recovering EF1A1in the immunoprecipitates from empty vector lysates. Visuallyin silver-stained gels, EF1A1 appears to be absent in thepulldown from the cells expressing empty vector (band 6,Figure 1A). However, the spectral counts for EF1A1 fromimmunoprecipitates from cells expressing empty vector, FLAG-OSM, or FLAG-OSMF217A were 33, 402, and 186, respectively.So although a small amount of EF1A1 immunoprecipitatedfrom the empty vector cell lysates, a significantly greateramount was pulled down in the presence of FLAG-OSM andthe interaction was markedly disrupted with the FLAG-OSMF217A

mutant. This result is in accordance with the immunoprecipi-tation experiments shown for EF1A1 in Figure 4. Therefore,proteins with such spectral count relationships are also con-sidered members of the OSM protein complex.

The data set in Table 2 is presented with three differentparameters to categorize specificity of protein interactions withthe CCM complex based on the spectral counts. First, proteinsare listed that were found solely in FLAG immunoprecipitationsfrom FLAG-OSM stable cell lysates. These proteins includechaperonin containing t-complex protein 1 (CCT) subunits 1,3, 6a, and 7, and RIN2, a Ras-interacting Rab5 guaninenucleotide exchange factor.46 Second, proteins are listed thatwere found in anti-FLAG immunoprecipitations from both theFLAG-OSM and FLAG-OSMF217A cell lysates, but which have areduced affinity for the complex with the OSMF217A PTB domainmutant. These proteins include KRIT1 and its binding partnerICAP-1, as well as CCT subunits 2, 4, 5, and 8, and the DnaJ/HSP40 subfamily A member 2. The final parameter is proteinsthat were identified from anti-FLAG pulldowns from lysates ofall three stable cell lines, but higher spectral counts wereobtained with FLAG-OSM, and similar or reduced spectralcounts in immunoprecipitates from FLAG-OSMF217A cell lysates.This group of proteins includes EF1A1, R- and â-tubulins, andHSP90. Each R- and â-tubulin isoform listed in Table 2 had atleast one unique peptide, indicating that the complex binds asubstantial portion of these cytoskeletal proteins.

To validate the association of identified complex memberswith FLAG-OSM, we took the eluates generated after FLAGimmunoprecipitation and FLAG peptide elution and immu-noblotted for a number of the complex members (Figure 5).The immunoblots generally reflect the magnitude differencebetween the pulldowns from the three stable cell lysates. Twoproteins listed in Supplemental Table 2, actin and heat shockprotein 8 (HSP70), were included in the validations. Actin hadroughly equal spectral counts in pulldowns from all threelysates and was therefore deemed nonspecific. HSP70 hadhigher spectral counts in the pulldown from the OSMF217A

mutant as compared to wild type OSM. Again, the relativedifferences between the spectral counts for these two proteinswere reflected in the immunoblot validation in Figure 5.

Figure 3. OSM and PDCD10 bind phosphatidylinsitol phosphates.PIP arrays were overlaid with recombinant 6×His-tagged OSMor PDCD10, and bound protein was detected by anti-6×Hisimmunoblotting. PIP concentrations ranged from 100 to 1.56pmol/spot.

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Therefore, our MudPIT analysis has identified a number of newmembers of the macromolecular complex organized by theCCM proteins, which were validated by the immunoblot data

following the FLAG immunoprecipitation and the fact that allknown interacting proteins identified in previous studies werefound by this proteomic approach.

Figure 4. Identification and validation of EF1A1 as a CCM protein complex member. (A) MS/MS spectrum of the doubly chargedprecursor peptide of m/z ) 702.8077, giving the sequence YYVTIIDAPGHR. (B) Ion series for the spectrum in panel A. Masses in boldfell within the 0.2 Da mass tolerance used for the NCBInr database search. (C) Lysates from RAW264.7 macrophages stably expressingempty vector (EV), FLAG-OSM (O), or FLAG-OSMF217A (F) were used for FLAG immunoprecipitations (IP). Following FLAG peptideelution, the eluates were separated by SDS-PAGE and immunoblotted (IB) for endogenous EF1A1, and whole cell lysates (WCL) wereblotted for total EF1A1. (D) EF1A1-myc/6×His was expressed with FLAG-KRIT1, FLAG-OSM, or FLAG-OSMF217A in COS7 cells. EF1A1was immunoprecipitated with an anti-myc antibody, and the CCM proteins were detected by FLAG immunoblotting. WCL were blottedfor FLAG or 6×His to monitor expression and for HSP70 as a loading control. (E) EF1A1-myc/6×His was expressed with FLAG-OSM orFLAG-LAD. Myc immunoprecipitation and immunoblots were performed as in (D). (F-H) Live cell imaging of COS7 cells transfectedwith: mCherry empty vector (EV), YFP-KRIT1, and CFP-OSM (F); mCherry-EF1A1, YFP-KRIT1, and CFP-OSM (G); or mCherry-EF1A1,YFP-KRIT1, and CFP-OSMF217A (H). Magnification: 63×.

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Table 2. Proteins Identified by MudPIT Analysis That Selectively Interact with FLAG-OSM

spectral counts sequence coverages

accession number description EV OSM F217A EV OSM F217A

Previously identified CCM complex membersgi|33468949|ref|NP_036077.1| MEKK3 (mitogen activated protein kinase kinase kinase 3) 0 4 0 0 6.9 0gi|6679601|ref|NP_033034.1| Rac (RAS-related C3 botulinum substrate 2) 0 3 0 0 14.6 0gi|6680488|ref|NP_032429.1| ICAP-1 (integrin beta 1 binding protein 1) 0 215 19 0 82 24gi|13994219|ref|NP_109600.1| KRIT1 (cerebral cavernous malformations 1) 0 150 38 0 49.7 23.4gi|13994219|ref|NP_109600.1| Programmed cell death 10 (CCM3)a 0 1 2 0 6.1 6.1

Cytoskeletal proteinsgi|33598964|ref|NP_780469.1| myosin heavy chain 10, non-muscle 0 12 0 0 5.5 0gi|27754056|ref|NP_080749.2| tubulin, beta 6 29 56 40 17 38.5 31.8gi|7106439|ref|NP_035785.1| tubulin, beta 5 49 175 125 48.2 57.4 54.1gi|22165384|ref|NP_666228.1| tubulin, beta, 2 46 134 102 39.3 53.9 47.2gi|6680924|ref|NP_031713.1| cofilin 1, non-muscle 11 48 40 33.7 68.1 55.4gi|6678467|ref|NP_033473.1| tubulin, alpha 4 31 81 62 32.8 56.2 39.1gi|6678469|ref|NP_033474.1| tubulin, alpha 6 39 101 88 39.4 55 52.8gi|6755901|ref|NP_035783.1| tubulin, alpha 1 39 94 85 39.2 60.1 49.4

Protein translation and foldinggi|51873060|ref|NP_034236.1| EF1A1 (eukaryotic translation elongation factor 1 alpha 1) 33 402 186 39.8 47 45.2gi|6753320|ref|NP_033966.1| chaperonin subunit 3 (gamma) 0 14 0 0 28.3 0gi|6753324|ref|NP_033968.1| chaperonin subunit 6a (zeta) 0 9 0 0 21.3 0gi|31982472|ref|NP_031664.2| chaperonin subunit 7 (eta) 0 7 0 0 13.1 0gi|7305565|ref|NP_038714.1| chaperonin subunit 1 (t-complex protein 1) 0 5 0 0 9.9 0gi|6754736|ref|NP_035977.1| DnaJ (Hsp40) homolog, subfamily B, member 6 0 3 0 0 15.7 0gi|9789937|ref|NP_062768.1| DnaJ (Hsp40) homolog, subfamily A, member 2 0 25 3 0 39.8 13.3gi|6671700|ref|NP_031662.1| chaperonin subunit 2 (beta) 0 16 4 0 25.2 14.4gi|6671702|ref|NP_031663.1| chaperonin subunit 5 (epsilon) 0 15 6 0 16.1 10.5gi|31560613|ref|NP_033970.2| chaperonin subunit 8 (theta) 0 12 5 0 15.1 9.5gi|6753322|ref|NP_033967.1| chaperonin subunit 4 (delta) 0 6 3 0 9.6 7.2gi|6680297|ref|NP_032324.1| DnaJ (Hsp40) homolog, subfamily A, member 1 3 15 7 15.6 28.5 21.2gi|40556608|ref|NP_032328.2| HSP90 (heat shock protein 1, beta) 10 30 16 14.9 14 15.1

Signalinggi|37497108|ref|NP_083000.3| RIN2 (Ras and Rab interactor 2) 0 59 0 0 22.9 0gi|22726177|ref|NP_080667.1| protein phosphatase 2A, regulatory subunit B,

delta isoform0 6 0 0 5.5 0

gi|8394024|ref|NP_059070.1| protein phosphatase 2a, catalytic subunit, beta isoform orprotein phosphatase 2a, catalytic subunit, alpha isoformb

0 4 0 0 12.9 0

gi|6680047|ref|NP_032169.1| guanine nucleotide binding protein (G protein), betapolypeptide 2 like 1

0 4 0 0 26.2 0

gi|6680045|ref|NP_032168.1| guanine nucleotide-binding protein, beta-1 subunit 0 3 0 0 11.5 0gi|8394027|ref|NP_058587.1| protein phosphatase 2, alpha isoform of regulatory

subunit A0 3 0 0 6.3 0

gi|31980772|ref|NP_038664.2| protein phosphatase 1, catalytic subunit, gamma isoform 0 10 6 0 29.7 18.6Vesicular transport proteins

gi|11596855|ref|NP_035768.1| transferrin receptor 0 5 0 0 9.6 0gi|15426055|ref|NP_203534.1| coatomer protein complex, subunit beta 1 0 5 0 0 5 0gi|31981828|ref|NP_034068.2| coatomer protein complex subunit alpha 0 5 0 0 3.4 0gi|6680716|ref|NP_031502.1| ADP-ribosylation factor 1, 2, 3, 4, or 5b 0 4 0 0 27.6 0gi|8567338|ref|NP_059505.1| coatomer protein complex, subunit gamma 0 3 0 0 3.2 0

Metabolism and biosynthetic proteinsgi|58037293|ref|NP_082549.1| hypothetical protein LOC72542 0 5 0 0 7 0gi|12963491|ref|NP_075608.1| enolase 1, alpha non-neuron 0 3 0 0 11.5 0gi|51093867|ref|NP_076014.1| carbamoyl-phosphate synthetase 2, aspartate

transcarbamylase, and dihydroorotase0 32 7 0 13.4 2.7

gi|13385434|ref|NP_080215.1| phosphoribosylaminoimidazole carboxylase 0 3 3 0 8.5 9.4Miscellaneous

gi|68448551|ref|NP_081655.2| male sterility domain containing 2 0 12 0 0 16.3 0gi|27370424|ref|NP_766512.1| hypothetical protein LOC244895 0 11 0 0 11.4 0gi|31088858|ref|NP_034510.2| histocompatibility 2, D region locus 1 0 10 0 0 23.8 0gi|21630283|ref|NP_660212.1| 2′-5′ oligoadenylate synthetase 1A or 2′-5′ oligoadenylate

synthetase 1Gb0 6 0 0 15.8 0

gi|6754724|ref|NP_034947.1| proteasome (prosome, macropain) 26S subunit,non-ATPase, 7

0 3 0 0 16.8 0

gi|19923070|ref|NP_598879.1| glycoprotein, synaptic 2 0 6 3 0 18.2 8.1gi|13811697|ref|NP_112735.1| histocompatibility 28 0 11 9 0 29.6 14.7gi|19527086|ref|NP_598632.1| interferon-induced protein 44 0 9 9 0 14.2 11.6

a Identified by manual inspection of peaks. b Sequenced peptides overlapped multiple isoforms; accession number corresponds to the first protein in thelist (see Supplemental Table 2 for complete list of peptides and accession numbers).

Proteomic Identification of the CCM Complex research articles

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DiscussionWe have shown for the first time that the proteins whose

expression is altered in CCM can exist as a complex in cells.We have previously shown that KRIT1 (CCM1) and OSM(CCM2) are in a complex in cells,17 and we now show thatPDCD10 (CCM3) is also a member of this complex. The factthat none of the three CCM proteins have definable catalyticfunctions characterized by domain architecture within proteindatabases strongly suggests that the function of KRIT1, OSM,and PDCD10 is to organize protein complexes in cells. Ourproteomic analysis identified the known binding partners forOSM, namely KRIT1, MEKK3, and Rac. In addition, we foundthe KRIT1 binding partner, ICAP-1, in the complex. The factthat no other kinase was found in the FLAG-OSM immuno-precipitation complex validates the specificity of the analysisand reinforces the prediction that MEKK3 plays an importantrole in the CCM protein complex. This, combined with the factthat targeted deletion of MEKK3 causes severe vascular de-fects,47 is consistent with a role of dysregulated MEKK3 signal-ing in the pathogenesis of CCM. Our work also defined bothOSM and PDCD10 as binding proteins for phosphatidylinositolphospholipids. OSM binds phosphatidylinositol monopho-phospates (PtdIns(3)P, PtdIns(4)P and PtdIns(5)P) whereasPDCD10 binds phosphatidylinositol di-and triphosphates(PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3). It is likely thatOSM binds PIPs via its PTB binding domain, which helpslocalize and stabilize its interaction with membrane-associatedproteins.34 The fact that PDCD10 binds PIP2 and PIP3 suggestsits function will be controlled in part by phosphatidylinositiol3-kinase activation.

The fact that PDCD10 was found at higher levels in the gel-based MS approach compared to the MudPIT analysis (sixpeptides versus one following FLAG-OSM immunoprecipita-tion) may have occurred for a number of reasons. PDCD10 mayhave been poorly resolubilized following the MeOH/CHCl3

precipitation in preparation for the MudPIT analysis. This

step was not performed prior to the gel-based analysis. Asecond explanation is that the complex organized by thesescaffolds is highly dynamic. For example, our previousstudies demonstrated increased fluorescence resonanceenergy transfer between KRIT1 and OSM in response tohyperosmotic stress.17 Since KRIT1 binds ICAP-1, providing alink to integrins, the composition of the complex may bedramatically affected by cell attachment to matrix proteins, cellcycle, or other extracellular stimuli. Regulation of the proteincomplex organized by OSM is an important area of interestfor future studies.

Our studies had an important control for analysis of specific-ity in FLAG-OSM co-immunoprecipitations that many pro-teomic studies do not have: namely, the comparison of wild-type FLAG-OSM to the PTB domain-defective FLAG-OSMF217A

mutant protein. The FLAG-OSMF217A protein was stably ex-pressed in RAW264.7 mouse macrophages and 1321N1 humanastrocytoma cells at similar levels as the wild-type FLAG-OSMprotein but has a defective PTB domain unable to bind NPxYmotifs.17 OSMF217A is unable to bind KRIT1 and would presum-ably be defective in binding other proteins that encode NPxYmotifs capable of interacting with the OSM PTB domain. Atpresent, we have not identified proteins other than KRIT1 thatdirectly bind the OSM PTB domain but it is probable that suchproteins exist. Thus, we are not simply comparing OSM versusnonspecific co-immunoprecipitations from cells expressingempty vector alone, but, in addition, are comparing theproteins that are co-immunoprecipitated with OSM that aredependent on a functional OSM PTB domain.

The proteins identified in our proteomic analysis suggest asignificant interaction of the CCM protein complex with thetubulin cytoskeleton. Both tubulins and several tubulin-associ-ated proteins were identified in both the gel-based and MudPITanalyses that may be associated with the CCM protein complexthrough the FERM domain of KRIT1. Consistent with thisdiscovery using mass spectrometry is the immunohistochemi-cal analysis of KRIT1, suggesting it can localize in part to thetubulin cytoskeleton,48 although KRIT1-GFP does not demon-strate a KRIT1 association with the tubulin cytoskeleton.17 Theidentification of all eight subunits of the TRiC/CCT complexin OSM co-immunoprecipitations reinforces the interaction ofthe OSM protein complex with the tubulin cytoskeletal system.Various subunits of the TRiC/CCT complex have been identifiedin proteomic screens in which â-tubulin has been identifiedas an interacting protein,25,39,49 indicating that this complex maydirectly associate with the cytoskeleton. Furthermore, the TRiC/CCT complex is required for proper folding of tubulin andactin,50,51 as well as other proteins we identified as co-immunoprecipitating proteins with OSM such as proteinphosphatase 2A subunits and G protein â subunits.51 It is likelythat the TRiC/CCT complex and proteins specifically requiringthe chaperonin complex for proper folding are co-immuno-precipitated with OSM complexes. Based on the immunoblotsin Figure 5, the association of tubulin and the TRiC/CCTsubunits with FLAG-OSM but not from control empty vectorlysates reinforces the specificity of these proteins in the OSMprotein complex. The co-immunoprecipitation of tubulin,TRiC/CCT complex, and proteins known to require the chap-eronin for folding is largely dependent on a functional OSMPTB domain. Given the suggested interaction of KRIT1 withtubulin,48 it is likely that the association of OSM with thechaperonin complex is via the interaction of KRIT1 with thetubulin cytoskeleton.

Figure 5. Validation of complex members identified by MudPIT.Lysates from RAW264.7 cells stably expressing empty vector(EV), FLAG-OSM (O), or FLAG-OSMF217A (F) were subjected toanti-FLAG agarose immunoprecipitation (IP). Following FLAGpeptide elution and separation of the eluates by SDS-PAGE,immunoblots (IB) were performed for the indicated complexmembers.

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A second prominent FLAG-OSM co-immunoprecipitatingprotein was EF1A1. This translation factor has a complexcellular regulatory function including control of the actincytoskeleton in addition to translation. EF1A1 is also a highabundance protein that is found in many proteomic studieslooking at protein complexes,25,39 and we were naturallyconcerned about the specificity of our results. Therefore, wespent considerable time characterizing the OSM-EF1A1 inter-action. Endogenous EF1A1 co-immunoprecipitates with FLAG-OSM and the co-immunoprecipitation is lost with the OSMF217A

mutant protein. There are no NPxY or conserved NPxY-likesequences known to bind a PTB domain in EF1A1, so it is likelythat the association of EF1A1 in OSM complexes is dependenton an intermediate protein. The functional role of EF1A1 inthe OSM protein complex is currently not understood. Interest-ingly, in addition to its role in protein translation, EF1A1 hasbeen established as an actin binding protein.42-45 In yeast,EF1A1 has been shown to bundle actin and mediate cell shapechanges,38 and in fibroblasts, EF1A1 binds F-actin and concur-rently anchors â-actin mRNA at cellular protrusions,36 suggest-ing that actin mRNA is locally translated at sites where dynamicactin polymerization is occurring. Furthermore, as mentionedabove, the TRiC/CCT complex is required for the proper foldingof tubulin and actin.50-52 Therefore, the CCM protein complexmay colocalize with EF1A1 and the chaperonin complex at siteswhere actin turnover is rapidly occurring, akin to the OSM-MEKK3 localization to sites of newly formed actin fibers inlammelipodia-like structures that we have previously demon-strated.19

An additional protein of great interest identified in theMudPIT analysis is RIN2. This Ras effector protein binds toactivated Ras53,54 and has GEF activity for Rab5.46 RIN2 has alsobeen shown to regulate E-cadherin internalization,55 whichspeculatively could be involved in regulation of endothelial celltight junction formation. Given the binding of OSM andPDCD10 to phosphatidylinositol phospholipids, there mayindeed be a link of OSM protein complexes with the control ofvesicle trafficking. The RIN2 interaction was dependent on afunctional OSM PTB domain. There are no good consensusNPxY-like motifs in RIN2 suggesting the interaction with theCCM protein complex may not be by directly binding to OSM.Alternatively, it is possible that the binding of many proteinsto the CCM protein complex is in part stabilized by the OSMPTB domain binding to KRIT1 or other NPxY-encoding pro-teins. This could partially explain the strong dependence for afunctional PTB domain for EF1A1 and RIN2 binding to the OSMprotein complex.

In conclusion, our analysis has, for the first time, providedthe beginning of a working model of a protein networkorganized by the CCM protein complex. This network involvesa complex of KRIT1, OSM, and PDCD10 capable of interactingwith the cytoskeleton and membrane phospholipids. Theidentification of the integrin binding protein ICAP-1 in thecomplex is consistent with a role of the complex in integrinsignaling.18,56 Integrin engagement with the extracellular matrixenhances Ras activation and membrane sorting and actin fiberrearrangements consistent with a function for RIN2 and EF1A1,respectively, in this signaling process organized by the CCMcomplex. These new discoveries related to the OSM proteinnetwork will provide new directions that are needed to definethe molecular basis of CCM.

AbbreviationsCCM, cerebral cavernous malformation; CCT, chaperonin

containing TCP-1; EF1A, eukaryotic translation elongationfactor 1A; GEF, guanine nucleotide exchange factor; ICAP-1,integrin cytoplasmic domain-associated protein-1; nESI-MS,nanoelectrospray ionization-mass spectrometry; OSM, osmo-sensing scaffold for MEKK3; PIP, phosphatidylinositol phos-phate; RIN2, Ras and Rab interactor 2; TCP-1, t-complexpolypeptide-1; TRiC, TCP-1 ring complex.

Acknowledgment. This work was supported by theAmerican Heart Association grant 0625466U (to T.L.H.), theNHLBI and NINDS grant 5F32HL084971 (to M.H.M.), and NIHgrants GM30324 and GM62388 (to G.L.J.).

Supporting Information Available: SupplementalTable 1: Proteins identified by SDS-PAGE and nESI-MS, alongwith peptide sequences and Analyst QS peptide scores. Supple-mental Table 2: All proteins identified by MudPIT analysis,including peptide sequences, spectral counts for each peptideand summations for each protein, and percent sequencecoverage for each protein identified. This material is availablefree of charge at http://pubs.acs.org.

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(8) Gunel, M.; Awad, I. A.; Anson, J.; Lifton, R. P. Mapping a genecausing cerebral cavernous malformation to 7q11.2-q21. Proc.Natl. Acad. Sci. U.S.A. 1995, 92 (14), 6620-6624.

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