Molecular Cell, Vol. 9, 95–108, January, 2002, Copyright 2002 by Cell Press

Identification of ARAP3, a Novel PI3K EffectorRegulating Both Arf and Rho GTPases, by SelectiveCapture on Phosphoinositide Affinity Matrices

increase in the number of known phosphoinositides toeight (PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2,PtdIns(4,5)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3), makingthe analysis of their functions ever more complex.

Selective interaction between particular phosphoino-

S. Krugmann,1,7 K.E. Anderson,1,7 S.H. Ridley,1,7

N. Risso,1 A. McGregor,1 J. Coadwell,1

K. Davidson,1 A. Eguinoa,1 C.D. Ellson,1

P. Lipp,2 M. Manifava,1 N. Ktistakis,1

G. Painter,4 J.W. Thuring,4 M.A. Cooper,4

sitides and a discriminatory phosphoinositide bindingZ.-Y. Lim,4 A.B. Holmes,4 S.K. Dove,5

domain(s) in involved proteins is central to most phos-R.H. Michell,5 A. Grewal,3 A. Nazarian,3

phoinositide-regulated events. Perhaps the best studiedH. Erdjument-Bromage,3 P. Tempst,3

examples are the products of the class I PI3K signalingL.R. Stephens,1 and P.T. Hawkins1,6

pathway, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, binding to1 Inositide Laboratory andspecific PH domains in their target proteins and causing2 Laboratory of Molecular Signallingtheir translocation to the plasma membrane (Rameh andThe Babraham InstituteCantley, 1999; Lemmon and Ferguson, 2000). Now thereBabraham, Cambridge CB2 4ATis also clear evidence that many FYVE domains, PXUnited Kingdomdomains, and ENTH domains selectively mediate the3 Memorial Sloan-Kettering Cancer Centereffects of PtdIns3P and PtdIns(4,5)P2 on intracellularNew York, New York 10021trafficking events (Simonsen et al., 2001; Simonsen and4 Dept of ChemistryStenmark, 2001).University of Cambridge

Once the phosphoinositide binding properties of par-Lensfield Roadticular domain types were recognized, additional phos-Cambridge CB2 1EWphoinositide binding proteins were speedily identified5 School of Biosciencesby cloning novel molecules found in genome databasesUniversity of Birmingham(e.g., Isakoff et al., 1998; Dowler et al., 2000). However, aBirmingham B15 2TTmajor limitation of this approach is that it cannot identifyUnited Kingdomphosphoinositide binding sites that do not belong toone of these recognized domain families. There are nowmany proteins that have been established to bind phos-Summaryphoinositides, mainly by various ad hoc strategies, butwithin which primary sequence determinants have notWe show that matrices carrying the tethered homologsyet been defined, e.g., several cytoskeletal/focal adhe-of natural phosphoinositides can be used to capturesion proteins (Cockcroft, 2000), signaling proteins (e.g.,and display multiple phosphoinositide binding pro-the MARCKS-protein; Wang et al., 2000), or proteinsteins in cell and tissue extracts. We present the massinvolved in vesicle trafficking (e.g., the AP2 adaptor; Gai-spectrometric identification of over 20 proteins iso-darov and Keen, 1999). The problem of identifying proteinlated by this method, mostly from leukocyte extracts:targets is most severe when studying recently discov-they include known and novel proteins with establishedered phosphoinositides (e.g., PtdIns5P, PtdIns(3,5)P2)phosphoinositide binding domains and also known pro-for which there is no paradigmatic information.teins with surprising and unusual phosphoinositide

An ideal way to get an overview of the constellationbinding properties. One of the novel PtdIns(3,4,5)P3 bind-of proteins that interact specifically with one or moreing proteins, ARAP3, has an unusual domain structure,of the phosphoinositides would be to screen proteinincluding five predicted PH domains. We show thatmixtures from cells or tissues with a minimal assumption

it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulatedproteomic method. This type of methodology would be

Arf6 GAP both in vitro and in vivo, and both its Arf GAP an invaluable tool, not only for discovering novel phos-and Rho GAP domains cooperate in mediating PI3K- phoinositide binding proteins but also for expressiondependent rearrangements in the cell cytoskeleton profiling key phosphoinositide effectors in critical intra-and cell shape. cellular signaling pathways, such as those controlling

cell growth, differentiation, and survival. “Proteomics”Introduction is often taken to mean mapping of the expression of all

proteins (e.g., by 2D gels followed by high throughputPhosphoinositides (PtdIns and its phosphorylated deriv- mass spectrometry) in a manner akin to microarray anal-atives) are membrane phospholipids that dictate the local- ysis of a cell’s entire mRNA complement—but this can-ization and function of many intracellular target proteins. not yield information on protein interactions with otherThese target proteins influence many critical processes proteins, with nucleic acids, or with small molecules. Toin eukaryotic cells, including signaling by cell-surface learn about these, targeted analyses of macromolecularreceptors, vesicle trafficking, cytoskeletal assembly and interactions must be used to define function-critical sub-disassembly. Novel phosphoinositide functions continue sets of the proteome.to emerge (Cockcroft, 2000). Recent years have seen an Affinity matrices derivatized with synthetic phospho-

inositides have the potential to offer one such approach;previous studies using matrices carrying Ins(1,3,4,5)P4-6 Correspondence: phillip.hawkins@bbsrc.ac.uk

7 These authors contributed equally to this work. like structures (isosteric with the PtdIns(3,4,5)P3 head-

Molecular Cell96

group; Hammonds-Odie et al., 1996; Stricker et al., 1997; fractionated NP40 extracts (see Supplemental FigureS2 at http://www.molecule.org/cgi/content/full/9/1/95/Shirai et al., 1998; Tanaka et al., 1997) or linked to biotin-

ylated diC8-PtdIns(3,4,5)P3 (Rao et al., 1999) have been DCI).successful in identifying a small number of PtdIns(3,4,5)P3

binding proteins. We have recently created new affinity Proteins Isolated on PtdIns(3,4)P2, PtdIns(3,5)P2,matrices derivatized with the dipalmitoyl versions of the and PtdIns3P Matricesfull phosphoinositide structures (Painter et al., 2001), We also analyzed cytosol from pig platelet, sheep brain,which we anticipate will more accurately mimic the natu- sheep liver, and rat liver for proteins that bound toral lipids. We now describe the use of these matrices in beads derivatized with PtdIns(3,4)P2, PtdIns(3,5)P2, orsimple competition assays with free phosphoinositides PtdIns3P, focusing on proteins that were not readilyto capture and display multiple phosphoinositide bind- displaced by PtdIns(3,4,5)P3 and so might selectivelying proteins from complex tissue extracts. We describe bind other phosphoinositide(s). While PtdIns(3,4)P2 andthe identification of a substantial number of these pro- PtdIns(3,5)P2 beads showed a limited degree of non-teins from several tissues, particularly neutrophils. Not phosphoinositide-dependent protein binding, there wasonly did we isolate many proteins with established phos- a high background of nonspecific protein adsorption tophoinositide binding domains, including novel proteins PtdIns3P beads, making it hard to characterize genuinewith PH domains and FYVE domains, but we also identi- PtdIns3P-dependent binding (not shown). Several pro-fied members of previously untargeted protein families. teins were isolated from the fractionated cytosols (SR1-We have further characterized the phosphoinositide SR7 and SD1; data not shown). PtdIns(4,5)P2 beads werebinding specificities of some of these proteins and show also created and used successfully to isolate multiplethat one of these, a novel 170 kDa PtdIns(3,4,5)P3 bind- phosphoinositide binding proteins (N.K., unpublisheding protein, is an effector of the PI3K signaling pathway data).regulating growth factor-induced cell shape/cytoskele-tal changes. Identification of the Phosphoinositide

Binding ProteinsSome of the isolated proteins were digested with trypsinResultsand identified by mass fingerprinting and sequencing(Figure 2A gives their tissue and bead origin and FigureThe Use of PtdIns(3,4,5)P3-Derivatized Beads

to Isolate PtdIns(3,4,5)P3 Binding Proteins 2B their domain structures). For PtdIns(3,4,5)P3 bindingproteins, these identified porcine orthologs of four char-from Leukocytes

Initial experiments used both tissue extracts and recom- acterized PtdIns(3,4,5)P3 binding proteins (rasGAPIP4BP,BTK, ETK, and centaurin-�), five proteins that were novelbinant PKB (an established PtdIns(3,4,5)P3 target) to

define conditions for identifying proteins that bound spe- at the time of isolation (C, E, F, G/H, and X), and porcineorthologs of seven proteins that were not known to bindcifically to the PtdIns(3,4,5)P3 moiety on PtdIns(3,4,5)P3-

derivatized beads (see Supplemental Figure S1 at http:// PtdIns(3,4,5)P3 (Cdc42-GAP, myosin 1F, megakaryocyteprotein-tyrosine phosphatase [MEG2], Type II inositolwww.molecule.org/cgi/content/full/9/1/95/DCI and data

not shown). In the adopted assay, tissue samples were polyphosphate 5-phosphatase, and/or ezrin [both pres-ent in band D], and the � and � subunits of mitochondrialpreincubated with or without a competing phosphoino-

sitide (usually PtdIns(3,4,5)P3) at near-physiological salt fatty acid oxidase). We cloned the human orthologs ofPtdIns(3,4,5)P3 binding proteins C, E, F, G/H, and X.concentration (�0.1 M NaCl) with a nonionic detergent

(�0.1% NP40) and with reagents likely to inhibit Near-identical ORFs have since been independentlydescribed for F (cytohesin-4; Ogasawara et al., 2000),PtdIns(3,4,5)P3 hydrolysis (�-glycerophosphate, F�, or-

thovanadate, divalent cations chelated). They were then X (DAPP1, Dowler et al., 1999; also termed PHISH, Raoet al., 1999; or Bam32, Marshall et al., 2000), and C (PLC-incubated with PtdIns(3,4,5)P3 beads, and proteins that

were retained by the beads were identified by SDS-PAGE. L2, Otsuki et al., 1999). The sequences of two proteinswith very similar overall domain structure and approxi-Pig leukocyte cytosol was used as an abundant source

of potential PtdIns(3,4,5)P3 binding proteins. The binding mately 50% amino acid similarity to PIP3-G/H have re-cently been submitted to the databases. A more detailedof several cytosolic proteins to the PtdIns(3,4,5)P3 beads

was inhibited by free PtdIns(3,4,5)P3 (D- or L-isomer or alignment of these three sequences (see Figure 2C)reveals further homology in a region of ankyrin-like re-both), but many other proteins interacted with the beads

in a PtdIns(3,4,5)P3-independent way (see Supplemental peats often associated with Arf GAP activity. In agree-ment with P. Randazzo and colleagues, we have namedFigure S1C at http://www.molecule.org/cgi/content/full/

9/1/95/DCI), making it difficult to recover proteins of this family the ARAPs (Arf GAP and Rho GAP with an-kyrin repeat and PH domains) and assigned ARAP1,interest in sufficient purity for unambiguous identifica-

tion on 1D gels. We reduced the protein complexity of ARAP2, and ARAP3 to the sequences TR:AAL12169,TR:AAL12170, and EM:AJ310567, respectively (Miura etthe samples applied to the beads by first fractionating

them by ion-exchange chromatography. Using this ap- al., 2002 [this issue of Molecular Cell]).Type C 6-phosphofructokinase (PFK-C) was isolatedproach, we were able to isolate several proteins in suffi-

cient yield and purity to attempt their identification on PtdIns3P beads, vinculin on PtdIns(3,4)P2 beads, and�-tocopherol transfer protein (ATTP) on PtdIns(3,5)P2(bands A–L and V–Y; Figure 1). We also screened various

detergent and high salt (1 M NaCl) extracts of leukocyte and PtdIns(3,4)P2 beads. The PtdIns3P and PtdIns(3,4)P2

beads also yielded two proteins that were novel at themembranes for PtdIns(3,4,5)P3 binding proteins, andproteins M–U were isolated from chromatographically time of isolation (SR1 and SR3). The human orthologs

Isolation of Phosphoinositide Binding Proteins97

Figure 1. Isolation of Pig Leukocyte Cytosol Proteins that Bind PtdIns(3,4,5)P3

Pig leukocyte cytosol was separated by anion exchange ([A and B] approximately 3 g protein; Q-sepharose HR) or cation exchange ([C andD] approximately 2.4 g protein; S-sepharose HP) chromatography. (A and C) Conductivity and absorbance traces. (B and D) Silver-stainedSDS-PAGE gels showing proteins recovered on PtdIns(3,4,5)P3 beads in the presence or absence of free PtdIns(3,4,5)P3 (50 �M in [B], 25 �Min [D]). PtdIns(3,4,5)P3 inhibited the binding of bands A–L and V–Y, which were isolated from a scaled-up preparation. Figure 2 identifies someof these proteins.

Isolation of Phosphoinositide Binding Proteins99

of SR1 and SR3 were cloned (Figure 2B), and very re- them most effectively (we suspect that during multipleexposures to tissue lysates significant amounts of thecently a near-identical ORF has been described for SR3

(DFCP-1; Derubeis et al., 2000). PtdIns(3,4)P2 tethered to the beads were hydrolyzed toPtdIns3P). The three proteins containing Sec14 domainshad different and unusual binding specificities. ATTP wasThe Phosphoinositide Binding Specificities

of Recombinant Proteins displaced most effectively from PtdIns(3,4)P2 beads byPtdIns(3,4)P2, PtdIns4P, or PtdIns5P. MEG2 was dis-Several of the proteins were made in recombinant form

with N- or C-terminal tags in Cos-7 cells, E. coli, or placed most effectively from PtdIns(3,4,5)P3 beads byPtdIns(3,4,5)P3 or PtdIns(4,5)P2. Cdc42GAP boundbaculovirus-infected Sƒ9 cells. We purified CT-EE-

tagged ARAP3, NT-EE-SR1, NT-EE-SR3, and NT-EE- weakly to PtdIns(3,4,5)P3 or PtdIns(4,5)P2 beads (datanot shown) and was poorly displaced by the phospho-myosin IF from Sƒ9 cells; and GST-Cdc42GAP, GST-

cytohesin-4, GST-MEG2, GST-DAPP1, and HIS-ATTP inositides tested (PtdIns(4,5)P2 and PtdIns(3,4,5)P3 werethe most effective). Myosin 1F was displaced most ef-from E. coli. We could not purify full-length PIP3-E but

obtained a GST fusion of an N-terminal truncation con- fectively from PtdIns(3,4,5)P3 beads by PtdIns(4,5)P2 andPtdIns(3,4,5)P3.taining its PH domain (residues 40-437).

We investigated the relative abilities of various freephosphoinositides to compete with PtdIns(3,4,5)P3 or Surface Plasmon Resonance Analysis

of Phosphoinositide BindingPtdIns(3,4)P2 beads for binding to purified, recombinantproteins and to heterologously expressed N-terminally We used a surface plasmon resonance (SPR) biosensor

to analyze the binding of some of the proteins to lipidmyc- or GFP-tagged proteins in Cos-7 cell lysates (seeFigure 3A). With lysates, assays were run under condi- surfaces of defined composition (Figure 3C). DAPP1 and

cytohesin-4 bound weakly to self-assembled PtdEtn/tions similar to those used to isolate the proteins—withmicellar NP40, physiological salt, EDTA, and other re- PtdSer/PtdCho monolayers on HPA sensorchips, and

binding was promoted by small proportions of phospho-agents to minimize phosphoinositide metabolism. Assayson purified proteins used micellar NP40 in PBS with 1 inositides. In accord with the specificities from the bead

displacement assays, cytohesin-4 bound best to sur-mM MgCl2 to approximate to the physiological cationenvironment and minimize the stripping of Zn2� from faces containing PtdIns(3,4,5)P3 and DAPP1 to surfaces

containing PtdIns(3,4,5)P3 or PtdIns(3,4)P2. PIP3-G/H,FYVE domains. The competing phosphoinositides wereall at one concentration, chosen to achieve just maximal showed high background binding to lipid surfaces as-

sembled on the HPA sensorchip, but this was reducedinhibition by the most effectively competing lipid. Sincethe surface lipid concentrations on the derivatized by using PtdEtn/PtdSer/PtdCho liposomes captured on

the alkane-modified dextran surface of an L1 chip (databeads are unknown and may vary between batches andduring multiple rounds of reuse, the results indicate only not shown). Under these conditions we found that incor-

poration of 6% PtdIns(3,4,5)P3 or PtdIns(3,4)P2, but notthe relative affinities of the relevant binding sites on theproteins for various phosphoinositides. Several of the other phosphoinositides, could significantly promote

binding of PIP3-G/H to this surface (Figure 3C). The beadrelative affinity screens were replicated with pure pro-teins and Cos-7 cell lysates, so the results are likely to displacement assays suggested ARAP3 bound best to

PtdIns(3,4,5)P3 (Figure 3A) with significant but weakerreflect the intrinsic phosphoinositide binding propertiesof the proteins. All PH domain-containing proteins bound binding to PtdIns(3,4)P2 (data not shown), suggesting

the different forms of lipid presentation in the bead dis-to PtdIns(3,4,5)P3 beads. They were displaced most ef-fectively by PtdIns(3,4,5)P3 (cytohesin-4, ARAP3, PIP3-E) placement and SPR assays can yield differences in the

relative binding affinities of these two lipids. An SPRor equally well by PtdIns(3,4,5)P3 and PtdIns(3,4)P2

(DAPP1). ARAP3 possesses five predicted PH domains, analysis of SR1 and SR3 binding to lipid surfaces con-taining PtdIns3P is published elsewhere (Ridley et al.,any one of which in principle could be responsible for

binding PtdIns(3,4,5)P3. Only the most N-terminal PH 2001).domain, however, fits clearly the current sequence con-sensus for PtdIns(3,4,5)P3 binding (Lemmon and Fergu- ARAP3 Is a Protein with the Potential to Couple PI3K

to the Regulation of Rho and Arf Family GTPasesson, 2000); mutations in this domain predicted to disruptlipid binding (R307/8A) totally abolished ARAP3 binding ARAP3 showed clear PtdIns(3,4,5)P3/PtdIns(3,4)P2 bind-

ing specificity and an unusual domain structure with fiveto the PtdIns(3,4,5)P3 beads (Figure 3B). The FYVEdomain-containing proteins (SR1 and SR3) were har- PH domains as well as domains with predicted Arf and

Rho GAP activities. Given the established importancevested with PtdIns(3,4)P2 beads, but PtdIns3P displaced

Figure 2. Identities of Phosphoinositide Binding Proteins

(A) Proteins isolated on phosphoinositide-derivatized beads. Proteins were excised on nitrocellulose and digested with trypsin, and peptideswere identified by mass fingerprinting and amino acid sequencing. Proteins that were unknown at the time of identification are described asNOVEL. Independent ORFs encoding some of these proteins have since been described, and their names are given in square brackets. EMBLaccession numbers are given for our ORFs where they remain undefined in the databases (PIP3-E, ARAP3, SR1); they define new speciesorthologs (MYO1F) or add significantly to existing entries (SR3). Protein O was tentatively identified on the basis of its size and cochromatographywith the � subunit of the mitochondrial fatty acid oxidase. n.d. designates phosphoinositide binding proteins that have not yet been identified.SW, Swissprot; TR, Trembl; and EM, Embl.(B) Domain structures of proteins isolated on phosphoinositide beads. The SMART program was used to identify domains.(C) An alignment of ARAP1 (TR:AAL12169), ARAP2 (TR:AAL12170), and ARAP3 (EM:AJ310567). A, Ankyrin-like repeat.

Molecular Cell100

Isolation of Phosphoinositide Binding Proteins101

Figure 4. PI3K-Dependent Translocation ofARAP3 to the Cell Periphery

Images shown represent stills taken from live,confocal fluorescent imaging of PC12 cellsor PAE cells transiently expressing GFP-ARAP3 or GFP-R307/8A-ARAP3 (full-lengthmovies are shown in the supplemental mate-rial at http://www.molecule.org/cgi/content/full/9/1/95/DCI). The confocal sections in (A)and (B) were through the center of PC12 cellsand chosen to best visualize translocationsto the plasma membrane and not ARAP3-dependent neurite outgrowths; the GFP fluo-rescence at points on the membrane andcytosol were quantified and expressed as aratio to avoid problems due to fluorescencebleaching. EGF was added at 180 s. In (C),wortmannin (100 nM final) was added 14 minafter PDGF stimulation, and the image wastaken after a further 10 min.

yet lack of clarity in the mechanism by which PI3K signal- ARAP3 was largely cytosolic with a small presence atthe plasma membrane (Figure 4 and data not shown).ing pathways control the cell cytoskeleton, we decided

to further characterize this protein with respect to its Addition of EGF to PC12 cells caused a rapid transloca-tion of some of the GFP-ARAP3 to the plasma mem-function and regulation by type I PI3K.

To examine ARAP3 tissue distribution, pig tissues and brane, and addition of PDGF to PAE cells caused a smalltranslocation of GFP-ARAP3 to the ruffling edges ofseveral cell lines were subjected to Western blotting

with an anti-ARAP3 antiserum. This showed a wide but lamellipodia (Figure 4; these translocations are mosteasily visualized by following the live image recordingsuneven distribution of ARAP3 with strongest expression

in leukocytes and spleen (see Supplemental Figure S3 in the supplemental videos at http://www.molecule.org/cgi/content/full/9/1/95/DCI). Although clearly signifi-at http://www.molecule.org/cgi/content/full/9/1/95/DCI).

We examined the subcellular localization of heterolo- cant, these agonist-dependent translocations weremuch smaller than we have characteristically seen withgously expressed ARAP3 in both PAE cells and PC12

cells, which contain relatively high and low endogenous other PtdIns(3,4,5)P3 binding proteins such as DAPP1(Anderson et al., 2000) or cytohesin-4 (data not shown).levels of the protein, respectively. In both cell types

under serum-starved conditions, GFP- or myc-tagged These translocations were prevented by incubation with

Figure 3. Protein Binding to Derivatized Beads: Inhibition by Free Phosphoinositides

(A) Recombinant proteins (in Cos lysates or purified) were bound to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 beads in the presence of various freephosphoinositides. For PIP3-E, a full-length construct was expressed in Cos cells while a truncated version (residues 40-437) was purifiedfrom E. coli. In the examples shown, all lipids were presented to a particular protein at the same concentration—this was chosen as aconcentration at which the most effective lipid showed just-maximal competition. Unless otherwise indicated, the first lane was loaded witha sample equivalent to 1% (Cos lysates) or 10% (purified proteins) of the protein sample that was incubated with beads. Tagged proteins inCos lysates were detected with antibodies (anti-Myc except ATTP, which was anti-GFP) and purified proteins were detected with antibodies(anti-EE for SR1 and SR3 and anti-His for ATTP) or by silver-staining. For each protein, the data shown are representative of informationcollected in �4 independent experiments.(B) Binding of GFP-ARAP3 and GFP-R307/8A ARAP3 expressed in Cos cell lysates to PtdIns(3,4,5)P3 beads. Proteins were detected with ananti-GFP Western blot.(C) Binding of recombinant DAPP1 and GST-cytohesin 4 to PtdEtn/PtdCho/PtdSer surfaces containing 3 mole % phosphoinositides on anHPA sensorchip or recombinant C-terminally EE-tagged PIP3-G/H to PtdEtn/PtdCho/PtdSer liposomes containing 6 mole % phosphoinositideson an L1 sensorchip. The data represent means � SEM (n � 3–6 independent determinations) of the mass of protein binding at equilibriumto the chip surface after flowing 100 nM recombinant proteins over the chip.

Molecular Cell102

Figure 5. Characterization of ARAP3 GAP Activities

(A) ARAP3, Rho GAP activity. 3.0 �M GST-Rac1, RhoA, or Cdc42/G25K was incubated for 15 min with [32P]-GTP and 0.5 �M ARAP3,Cdc42GAP, or its vehicle (see Experimental Procedures). After termination of the reaction, [32P]Pi was extracted and quantified by scintillationcounting. Data represent means � SD (n � 3). Where indicated, PtdIns(3,4,5)P3 was added to 10 �M prior to addition of the [32P]GTP.(B and C) ARAP3, Arf GAP acitivity. HA-Arfs were immunoprecipitated from transiently transfected Cos-7 cells and loaded with [�32P]-GTP inthe presence of lipid vesicles composed of 200 �M PtdEtn. For each assay point, similar amounts of GTP-loaded Arf were incubated for 15min with 0.05 �M ARAP3 or its vehicle and lipid vesicles which did or did not contain PtdIns(3,4,5)P3 (20 �M unless otherwise indicated). Afterthe assay was stopped, the beads were washed extensively, and bound nucleotides were extracted, freeze dried, and resolved by thin-layerchromatography. (B) Comparison of the ability of ARAP3 to stimulate the GTPase activity of Arf1, 5, and 6; the insert in the left panel showsGTP loading of immunoprecipitated Arf1 compared to an immunoprecipitation from mock-transfected Cos-7 cells. The GDP spot obtainedfrom Arfs incubated with PtdEtn vesicles without ARAP3 was arbitrarily taken as “1” and did not change from the start to the end of theassay; this represents [32P]-GDP contaminating the [32P]-GTP preparation or produced by GTPases associated with the immunoprecipitationduring the preincubation with [32P]-GTP. This confirms much data indicating Arf proteins possess very low intrinsic GTPase activity. Eachgraph shows the values obtained in one representative experiment. (C) Further characterization of ARAP3 Arf6 GAP activity: (left panel) ARAP3Arf6 GAP activity in the presence of increasing concentrations of PtdIns(3,4,5)P3; (right panel), ARAP3 Arf6 GAP activity in the presence ofvesicles composed of (1) 200 �M PtdEtn or 200 �M PtdEtn and 15 �M (2) DD dipalmitoyl-PtdIns(3,4,5)P3, (3) LL dipalmitoyl-PtdIns(3,4,5)P3,(4) DD stearoyl-, arachidonyl-PtdIns(3,4,5)P3, (5) LL stearoyl-, arachidonyl-PtdIns(3,4,5)P3, (6) DD dipalmitoyl-PtdIns(3,4)P2, (7) LL dipalmitoyl-PtdIns(3,4)P2, or (8) DD dipalmitoyl PtdIns(4,5)P2. Each graph represents the mean from four experiments. GDP spots in the Arf6 control (PEvesicles, no ARAP3) were subtracted from the other values as blanks.

the PI3K inhibitor wortmannin and by mutating residues GTPase and the ARAP3 potentiated GTPase activity ofthe Rho family proteins by the same proportions; FigureR307/8A in the N-terminal PH domain of ARAP3 (Figure

4 and data not shown), suggesting that they were driven 5A and data not shown). The in vitro Arf GAP activity ofARAP3 against Cos-derived Arf1, Arf5, and Arf6 wasby direct binding to PtdIns(3,4,5)P3 generated at the

plasma membrane by agonist-stimulated type I PI3Ks. insignificant in the absence of phosphoinositides. How-ever, in the presence of PtdIns(3,4,5)P3 (and to a lesserextent of PtdIns(3,4)P2), ARAP3 acted as a specific GAPARAP3 Is a PI3K-Dependent, GTPase-Activating

Protein In Vitro and In Vivo for Arf6 (Figures 5B and 5C). The assays were con-structed such that they were linear with respect to time,To address whether ARAP3 can act as a Rho and/or

Arf GAP in vitro and whether such an activity might be to the concentration of added recombinant ARAP3, andto the concentration of added PtdIns(3,4,5)P3, and careregulated by PI3K, we conducted GAP assays with repre-

sentative members of the Rho and Arf family GTPases in was taken to ensure similar concentrations of [32P]-GTP-loaded Arfs were introduced into the assays. Thus, thesethe presence and absence of PtdIns(3,4,5)P3. Recombi-

nant ARAP3 possessed significant GAP activity toward results indicate that ARAP3 is a highly PtdIns(3,4,5)P3-dependent, highly selective Arf6 GAP in vitro.bacterially derived Rac-1, RhoA, and Cdc42 in vitro. A

comparison with the GAP activity of Cdc42GAP is shown To see whether this clear specificity for Arf6 was ex-hibited in vivo, GFP-tagged ARAP3 and HA-tagged Arfin Figure 5A. Inclusion of 10 �M PtdIns(3,4,5)P3 in these

assays, either included in liposomes containing PtdSer/ proteins were expressed in porcine aortic endothelial(PAE) cells. We assessed the ability of ARAP3 to affectPtdCho or alone, had no measurable effect on this activ-

ity (PtdIns(3,4,5)P3 routinely reduced both the intrinsic the cellular localization and phenotype caused by the

Isolation of Phosphoinositide Binding Proteins103

Figure 6. ARAP3 Acts as an Arf6 GAP In Vivo

PAE cells were cotransfected with vectors encoding HA-tagged Arf6 and either GFP or various GFP-ARAP3 constructs. Seven hr aftertransfection, cells were starved for 12 hr, fixed in 4% paraformaldehyde, and stained for either the HA-tag (using 12CA5 coupled to TRITC-conjugated anti-mouse) or for filamentous actin (alexafluor568-phalloidin). Arrows point to Arf6-dependent membrane ruffles.

heterologously expressed Arfs. We found that coexpres- of GFP-ARAP3-expressing cells (Figure 7). Upon addi-tion of PDGF, cells overexpressing GFP-ARAP3 exhib-sion of GFP-ARAP3 with Arf6 reversed Arf6-dependentited dramatic alterations in their normal lamellipodia andmembrane ruffling and caused Arf6 redistribution frommembrane-ruffling response. These cells developedthe cell periphery into an intracellular, particulate com-very irregular and convoluted cell edges combined withpartment (Figure 6). These results are consistent withan increased loss of filamentous actin (Figure 7). Real-previous observations that Arf6 can regulate membranetime confocal imaging suggests that this PDGF-stimu-ruffling (Radhakrishna et al., 1996) and undergoes cy-lated symptom was defined by edge contractions, oftencling from the plasma membrane to an intracellular com-resulting in an increased incidence of “doughnut-like”partment on conversion from a GTP- to GDP-boundholes in thin, flattened areas of the cell (see Supplemen-form (Peters et al., 1995). Coexpression of C504A Arftal Videos S7 and S8 at http://www.molecule.org/cgi/GAP or R307/8A PH domain point mutants of GFP-content/full/9/1/95/DCI for two examples of differing se-ARAP3 were unable to affect Arf6 distribution or activityverity). This effect of ARAP3 was abolished by the R307/(Figure 6), indicating that this effect of ARAP3 is likely8A PH domain mutation (Figure 7). It was also abolishedto be mediated by its PtdIns(3,4,5)P3-regulated Arf GAPby dual mutations in both the Rho GAP and Arf GAPactivity. We also found that coexpression of GFP-ARAP3domains (Figure 7) and greatly reduced by individualcould reverse the lamellipodia formation and extensivemutations in each of these two domains (Figure 7), sug-membrane-ruffling phenotype of heterologously ex-gesting that both domains cooperate in this phenome-pressed HA-Arf5 (data not shown). However, given ournon downstream of a PI3K input. PC12 cells overex-observations that ARAP3 overexpression in these cellspressing GFP-ARAP3 characteristically possessed one

has broad effects on the cell cytoskeleton (see below),or more neurite outgrowths (approximately 67% of over-

it is possible that these effects were indirect. We found expressing cells compared to 5% of nonexpressingno evidence that coexpression of GFP-ARAP3 affected neighbors; see Supplemental Figure S4 at http://the perinuclear distribution of HA-Arf1 or endogenous www.molecule.org/cgi/content/full/9/1/95/DCI), and in�-COP, a marker for Golgi-derived structures affected a small number of cells these were quite long andby Arf1 activity (Aoe et al., 1997 and data not shown). branched (data not shown). Addition of EGF to PC12

cells caused extensive PI3K-dependent membrane ruf-ARAP3 Causes PI3K-Dependent Changes fling around the cell periphery, but in cells overexpress-in Cell Morphology ing ARAP3 this ruffling was reduced and confined to theBoth Arf and Rho GTPases are involved in the modula- ends and edges of the projections (see Supplementaltion of the actin cytoskeleton. To determine whether Figure S4 at http://www.molecule.org/cgi/content/full/ARAP3 might participate in the regulation of dynamic 9/1/95/DCI). These properties of ARAP3 were abolishedactin rearrangements, we analyzed the filamentous actin by the R307/8A mutation and reduced by either theof PAE and of PC12 cells that had been transiently trans- C508A or R987A mutations (see Supplemental Figurefected with GFP-ARAP3 constructs. GFP-ARAP3 in se- S4 at http://www.molecule.org/cgi/content/full/9/1/95/rum-starved PAE cells caused a reduction in internal, DCI and data not shown), again suggesting the activityphalloidin-stainable filamentous actin, with some build- of both domains can cooperate downstream of a direct

PI3K signal.up of cortical actin and small membrane ruffles in 65%

Molecular Cell104

Figure 7. Heterologous Expression of ARAP3 Causes PI3K-Dependent Cytoskeletal Changes

PAE cells were transiently transfected with vectors encoding GFP or various GFP-ARAP3 constructs. Seven hours after transfection, cellswere starved for 12 hr, fixed, and processed for direct visualization of GFP fluoresence and filamentous actin. PDGF stimulation was for 5min with 10 ng/ml PDGF.

Discussion spect. Our methods are validated by the retrieval ofseveral, now authenticated phosphoinositide bindingproteins with established lipid binding specificities, e.g.,We describe here the use of novel phosphoinositide

affinity matrices to capture and identify a substantial the PtdIns(3,4,5)P3 effectors BTK and DAPP-1 (Li et al.,1997; Anderson et al., 2000; Dowler et al., 1999; Marshallnumber of phosphoinositide binding proteins from tis-

sue extracts. We reisolated 14 previously known pro- et al., 2000) and the PtdIns3P-targeted endosomal pro-tein SR1 (Ridley et al., 2001). Given the clarity with whichteins and identified, cloned, and characterized seven

new phosphoinositide binding gene products, the ORFs our affinity matrices isolated these proteins even fromquite complex mixtures, it should be possible to usefor three of which remain undefined in the databases.

We also used these matrices to establish the phospho- this approach with modern 2D gel/high throughput massspectrometry technologies to rapidly screen for varia-inositide binding selectivities of some of the proteins

we isolated. We provide data on nine proteins that are tions in the expression of phosphoinositide binding pro-teins between tissue samples, for example, comparingeither novel or previously uncharacterized in this re-

Isolation of Phosphoinositide Binding Proteins105

normal or disease states. As proof of principle, we note binding protein with unknown Arf GAP specificity (Ven-kateswarlu et al., 1999); cytohesin 4, a new member ofthat BTK was clearly displayed on our PtdIns(3,4,5)P3

beads (PIP3-B; Figure 1A), and mutations in its PH do- the established ARNO/cytohesin family of PtdIns(3,4,5)P3-regulated Arf-GTP exchangers (Jackson and Casanova,main which destroy PtdIns(3,4,5)P3 binding are known

to cause X-linked immunodeficiency (Vetrie et al., 1993). 2000), and ARAP3, defining a new family of proteins withArf GAP and Rho GAP activities. There is convincingWe identified several phosphoinositide binding proteins

that earlier screens did not detect. Some were novel evidence that the ARNO/cytohesin family of exchangersare PI3K effectors, but the precise role these proteins(PIP3-E, ARAP3, SR1), while others were previously known

proteins whose phosphoinositide affinities were not play in the regulation of Arf proteins is less clear. Theyappear to exhibit variable selectivity in vitro for the differ-previously recognized (e.g., ATTP, MEG2, Cdc42GAP,

mitochondrial fatty acid oxidase, Type C phosphofructo- ent Arf family members but there is strong evidence thatthey may regulate Arf6 at the cell surface in vivo (e.g.,kinase). It was also clear from inspection of the original

gels of fractionated cytosol and membrane extracts that Santy and Casanova, 2001). Arf6 is thought to be in-volved in the recycling of plasma membrane compo-many more phosphoinositide binding proteins were

present than we have identified (we estimate more than nents which is crucial for the formation of actin-richmembrane protrusions (membrane ruffling) in coopera-30 proteins in leukocyte cytosol alone). We note, for

example, that some important PtdIns(3,4,5)P3 targets tion with the small GTPase Rac (Zhang et al., 1999;Radhakrishna et al., 1999). These properties are consis-(e.g., PKB and PDK-1) have not yet emerged from this

screen. tent with widely seen PI3K effects on cell ruffling andmovement (Wennstrom et al., 1994; Dekker and Segal,Our identification of a trio of polyphosphoinositide

binding proteins that contain SEC14-like domains 2000). However, just how Arf6 participates in theseevents and the role of the PI3K signaling system are still(ATTP, MEG2, and Cdc42GAP) was unexpected. Desig-

nation of a SEC14 domain relies on sequence homology obscure.We present evidence that ARAP3 is also a genuinewith the canonical yeast PtdIns transfer protein Sec14p

(Araviond et al., 2001). Eukaryote databases currently effector of the PI3K signaling system. In vitro, ARAP3binds PtdIns(3,4,5)P3 specifically via its N-terminal PHcontain more than one hundred SEC14 domain-con-

taining proteins but with no consensus on any shared domain, and in vivo it undergoes small but convincingPI3K and PH domain-dependent translocations to thecommon function. It has been reported that two SEC14

domain-containing proteins from soybean, Ssh1p, and plasma membrane. Further, ARAP3 is a PtdIns(3,4,5)P3-stimulated Arf6 GAP both in vitro and in vivo. We wereSsh2p, bind PtdIns(4,5)P2 and/or PtdIns(3,5)P2 (Kearns

et al., 1998), but polyphosphoinositide binding by ATTP, unable to measure any discrimination in the Rho GAPactivity of ARAP3 toward Rac, Rho, and Cdc42 or itsMEG2, and Cdc42GAP, which have no obvious links to

phosphoinositide function, was still a surprise. These regulation by PtdIns(3,4,5)P3 in vitro; also, its Rho GAPspecificity was not apparent through simple measure-proteins are thought to have very different functions:

ATTP is involved in intracellular �-tocopherol trafficking ments of cytoskeletal changes in vivo (data not shown).However, the phenotypic effects of ARAP3 overexpres-(Arita et al., 1995), Cdc42GAP stimulates GTP hydrolysis

by the small GTPase Cdc42 (Lancaster et al., 1994; Bar- sion in cells suggest that both domains cooperate in amanner controlled by the N-terminal PH domain. Thus,fod et al., 1993), and MEG2 is a hematopoietic protein-

tyrosine phosphatase (Gu et al., 1992). They show no ARAP3 may provide a mechanism whereby PI3K signal-ing can control not only the GTP loading of Arf (viasequence similarity outside the SEC14 domain, so their

SEC14 domains probably bind polyphosphoinositides, ARNO/cytohesin) but also its GTP hydrolysis (viaARAP3), thereby controlling the complete Arf6 cycle.suggesting that a subset of SEC14 domain-containing

proteins constitute an unrecognized class of proteins Further, ARAP3 provides the potential to link this cyclewith the function of a Rho family member. Other Arfwith a propensity for binding PtdIns and/or phosphory-

lated PtdIns derivatives. This idea is intriguing given GAPs have also been suggested to be specific for Arf6.Thus, the ACAP family would appear to be good candi-that SEC14 domains often occur in proteins involved in

signaling, for example: additional protein-tyrosine phos- dates for regulating Arf6 function at the cell periphery,but they are not PtdIns(3,4,5)P3-regulated in vitro andphatases, GEFs, and GAPs that regulate the guanine

nucleotides status of Rho and Ras (including neurofi- there is no data to link them with PI3K signaling in vivo(Jackson et al., 2000). The Arf GAP activity of the GITbromin-related protein NF-1), and a diacyglycerol ki-

nase-related protein from Drosophila (Araviond et al., family can be specifically activated by high concentra-tions of PtdIns(3,4,5)P3 in vitro, but they are not Arf iso-2001).

A striking feature was how many of the proteins that form-specific, nor is this effect mediated via any recog-nizable PtdIns(3,4,5)P3 binding domain (Vitale et al.,we harvested are involved directly (myosin 1F, ezrin,

vinculin) or indirectly (via the regulation of Rho and Arf 2000); there is also no evidence they are PI3K-regulatedin vivo. Thus, ARAP3 has the clearest credentials of anyfamily GTPases) in control of the cytoskeleton. It is be-

coming ever clearer that phosphoinositides have essen- Arf GAP for operating in a PI3K-regulated Arf6 cycle.Precedent suggests that other Arf GAPs can form pro-tial roles in coordinating the complex spatial and tempo-

ral events underlying cell adhesion and movement. tein contacts which govern their Arf selectivity and thecellular context for their function (e.g., in focal adhesionRelating the phosphoinositide binding properties of indi-

vidual proteins to an understanding of these processes turnover; Curtis, 2001). Given its large size and the pres-ence of established protein binding modules (e.g., theis a major challenge. We isolated three proteins regulat-

ing the function of the Arf family of small GTPases: SAM domain), it would seem that investigating potentialbinding partners for ARAP3 offers a real opportunity tocentaurin-�, a previously established PtdIns(3,4,5)P3

Molecular Cell106

using centrifugal filters (Centricon). They were stored at �80C inprogress in our understanding of how PI3K signaling50% glycerol. Baculovirus-driven expression of N-terminally EE-controls Arf6-regulated events.tagged SR1, SR3, and myo 1F, and C-terminally EE-tagged ARAP3is detailed in the supplemental material at http://www.molecule.org/Experimental Procedurescgi/content/full/9/1/95/DCI.

Isolation of Cells, Preparation of Cell Fractions,Use of Phosphoinositide-Derivatized Beads to Investigateand Protein Fractionationthe Specificities of Recombinant ProteinsDetailed procedures describing the preparation of cytosol and mem-107 Cos-7 cells were transfected by electroporation with 20 �gbrane extracts from various tissues and their fractionation by anion-expression vector (usually Myc- or GFP-tagged) and allowed toand cation-exchange chromatography are available in the supple-recover in DMEM containing 10% FBS in 2 � 5 cm diameter dishesmental material at http://www.molecule.org/cgi/content/full/9/1/95/for 36–48 hr. They were washed in PBS and lysed into 5 ml perDCI.dish of 1.0% NP40, 20 mM HEPES (pH 7.5), 0.12 M NaCl, 5 mM EDTA,5 mM EGTA, 5 mM �-glycerophosphate, 1 mM orthovanadate, andSynthesis of Phosphoinositides and Phosphoinositide-10 mM NaF. Lysates were centrifuged (190,000 g; 30 min).Coupled Beads

Samples of the supernatants were diluted up to 4-fold in lysisSynthesis of the dipalmitoyl forms of the various phosphoinositidesbuffer, depending on expression levels, and mixed with indicated(and their nonbiologial L-stereoisomers) are described elsewhereconcentrations of phosphoinositide in 1 ml for 10 min on ice. They(Painter et al., 1999). Lipid stocks were stored as dry films at �80C;were transferred to 5–20 �l phosphoinositide beads in lysis bufferthe sodium salts of PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(3,5)P2, andand mixed gently for 45 min. Sedimented beads were washed (4�,PtdIns(4,5)P2 were adjusted to pH 7.0 and dried under vacuum;�15 min) in modified lysis buffer (0.1% NP40). Proteins were elutedPtdIns3P, PtdIns4P, and PtdIns5P were converted to the free acidswith SDS sample buffer, separated by SDS-PAGE, and detected bythrough a CHCl3/MeOH/0.1 M HCl (1:1:0.9) phase partition, and theimmunoblotting (anti-myc 9E10 or anti-GFP antiserum; Clontech).lower phases were dried. Stocks (4–10 mM) were prepared by bath-

Assays with purified-recombinant proteins (prepared via expres-sonicating dry lipid in H2O (PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(3,5)P2,sion of EE-, GST-, or His-tagged versions in E. coli or Sƒ9 cells; seeand PtdIns(4,5)P2) or DMSO (PtdIns3P, PtdIns4P, and PtdIns5P); con-above) were as just described, except that 0.2–2 �M proteins werecentrations were determined by organic phosphorus assay.incubated with indicated concentrations of phosphoinositides inCoupling of phosphoinositides to Affigel-10 beads is describedPBS, 1 mM DTT, 1 mM MgCl2, 0.1% NP40, and 0.5 mg ml�1 BSAelsewhere (see Supplemental Figure S1A at http://www.molecule.(omitted when bound protein was assessed by silver-staining). Theorg/cgi/content/full/9/1/95/DCI; Painter et al., 2001). Derivatized andbeads were washed in PBS, 1 mM MgCl2, 0.1% NP40, and 0.1 mgwashed beads were stored at 4C in 0.1 M sodium phosphate bufferml�1 BSA (no BSA for silver-staining), and proteins were eluted(pH 7.0), 0.01% azide.in sample buffer, separated by SDS-PAGE, and detected—eitherdirectly by silver-staining or after transfer to Immobilon by immu-Identification of Phosphoinositide Binding Proteinsnoblotting with anti-EE ascites fluid (Babco), anti-His-monoclonalBefore samples were subjected to binding studies; they were gener-(Clontech), or anti-GST antiserum (Amersham-Pharmacia).ally adjusted to 20–30 mM HEPES/Na or Tris/HCl (pH 7.2–7.5), 5

mM �-glycerophosphate, �0.1 mM EDTA (in excess of the freeBinding of Recombinant Proteins to Phosphoinositide-Mg2�), �0.1 M NaCl, 10 mM NaF, 0.1% NP40, and 1 mM sodiumContaining Biacore Chipsorthovanadate. They were kept on ice for 15 min with or withoutBinding of recombinant proteins to lipid surfaces assembled onfree phosphoinositides (typically 5–50 �M), transferred onto phos-HPA or L1 Biacore chips was performed as described elsewherephoinositide-derivatized beads (typically 1 ml onto 10 �l beads [ana-(Ridley et al., 2001; see Supplemental Methods S9 at http://lytical] or 30–100 ml onto 0.1–0.3 ml beads [preparative]), mixed,www.molecule.org/cgi/content/full/9/1/95/DCI).and returned to ice (45 min). Beads were washed (three times; 8

min) with 20 mM HEPES (pH 7.2), 0.2 M NaCl, 5 mM EDTA, 5 mMImmunofluorescence Staining and Confocal ImagingEGTA, 5 mM �-glycerophosphate, 10 mM NaF, and 0.1% NP40, andCells were cultured and transiently transfected with various con-then once in 5 mM HEPES (pH 7.2). Proteins were eluted with SDS-structs, and fixed and live cells were processed for direct and indi-PAGE sample buffer (95C for 5 min), separated by SDS-PAGE, andrect fluorescence microscopy as described previously (Anderson etsilver-stained (analytical experiments) or transferred to nitrocellu-al., 2000).lose (preparative). Beads, recycled by washing in SDS-PAGE buffer

followed by 0.1 M sodium phosphate buffer (pH 7.0), were used upto five times. Proteins that showed specific binding to phosphoinosi- Arf GAP Assaystide beads were digested with trypsin and processed for mass spec- Transiently transfected COS-7 cells expressing HA-tagged Arf1,trometric fingerprinting (Erdjument-Bromage et al., 1998; see sup- Arf5, or Arf6 were lysed on ice in lysis buffer (150 mM NaCl, 40 mMplemental material at http://www.molecule.org/cgi/content/full/9/1/ HEPES [pH 7.4] at 4C, 1% triton X-100, 1 mM MgCl2, 1 mM EGTA,95/DCI for details). 1 mM PMSF, and 10 �g/ml each of leupeptin, antipain, pepstatin

A, and aprotinin). Insoluble material was pelleted by centrifugation,Cloning of Phosphoinositide Binding Proteins and the soluble material was incubated with 25–40 �l covalentlyDetails of the cloning strategies used to obtain the seven novel open coupled anti-HA sepharose beads at 4C for 4 hr, followed by fivereading frames and also those of several known proteins are given in washes in lysis buffer and three washes in loading buffer (40 mMthe supplemental material at http://www.molecule.org/cgi/content/ HEPES [pH 7.4] at room temperature, 0.1% triton X-100, 1 mM EDTA,full/9/1/95/DCI. 2.5 mM MgCl2, 100 mM NaCl, 25 mM KCl, and 1 mM DTT). Arfs

were loaded with GTP for 40 min at 30C in a total volume of 65 �lin loading buffer supplemented with 3 mM phosphoenol pyruvate,Recombinant Proteins

ORFs were subcloned into expression vectors by standard restric- 1.25 �/ml pyruvate kinase, 20–40 �Ci �[32P]GTP, and lipid vesicles(200 �M PtdEtn). The beads were washed once in GAP buffer (40tion enzyme cloning and PCR. PCR products were verified by se-

quencing. pGEX4T (Pharmacia) and pQE (Qiagen) vectors were used mM HEPES [pH 7.4], 2.5 mM MgCl2, 100 mM NaCl, 0.5 mM GTP,1 mM DTT, and 0.1% triton X-100), aliquotted into minifuge tubesfor bacterial expression of N-terminally GST- or His-tagged proteins,

respectively. GST proteins were purified on glutathione-Sepharose- containing 5 �l preequilibrated protein G sepharose beads to in-crease bead volume, and aspirated tightly. GTP hydrolysis was al-4B (Pharmacia), and His-tagged proteins on metal affinity resin

(Talon, Clontech). Proteins were recovered from the elution buffers lowed to proceed at 30C for 15 min in a total volume of 40 �l ofGAP buffer supplemented with lipid vesicles (200 �M PtdEtn andby gel filtration on PD10 columns (Pharmacia) in PBS, 1 mM EGTA,

and 0.01% azide. Thrombin cleavage of GST proteins was carried 20 �M PtdIns(3,4,5)P3 or as indicated in individual figure legends)and 0.05 �M ARAP3 or its vehicle. The assay was stopped by theout on the glutathione-Sepharose beads in PBS, 1 mM DTT for 16

hr at 4C. Where needed, proteins were concentrated to �1 mg ml�1 addition of 1 ml ice-cold wash buffer (50 mM HEPES [pH 7.4] at

Isolation of Phosphoinositide Binding Proteins107

4C, 5 mM MgCl2, 0.5 M NaCl, and 0.1% triton X-100) and tubes ing and characterisation of a novel FYVE finger protein from a bonemarrow cDNA library. Gene 255, 195-203.placed onto ice. Beads were washed four times with 1.25 ml wash

buffer, twice with 1.25 ml of 10 mM MgCl2, 10 mM HEPES (pH 7.4) Dowler, S., Currie, R.A., Downes, C.P., and Alessi, D.R. (1999).at 4C and were aspirated tightly. Nucleotides were eluted into 100 DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinosi-�l 2 M formic acid, 50 �M GTP, and 50 �M GDP followed by freeze tides. Biochem. J. 342, 7–12.drying. Pellets were taken up in 4 �l 50 mM NaPO4, and 10 mM

Dowler, S., Currie, R.A., Campbell, D.G., Deak, M., Kular, G., Downes,NaP2O7 (pH 7.0), spotted onto PEI cellulose thin-layer chromatogra-C.P., and Alessi, D.R. (2000). Identification of pleckstrin-homologyphy plates, and resolved in 1 M NaH2PO4. Quantification of GDP anddomain containing proteins with novel phosphoinositide-bindingGTP was by scintillation counting or on a phosphoimager (Molecularspecificities. Biochem. J. 351, 19–31.Dynamics).Erdjument-Bromage, H., Lui, M., Lacomis, L., Grewal, A., Annan,R.S., MacNulty, D.E., Carr, S.A., and Tempst, P. (1998). Micro-tipRho GAP Assaysreversed-phase liquid chromatographic extraction of peptide pools0.5 �M recombinant ARAP3 or Cdc42-GAP and 3.0 �M GST-RhoA,for mass spectrometric analysis. J. Chromatogr. 826, 167–181.Rac1, or Cdc42/G25K were mixed on ice in 20 �l PBS and 1 mM

MgCl2. 1 �l PBS containing 0.02 �Ci [32P]GTP was added, and Gaidarov, I., and Keen, J.H. (1999). Phosphoinositide-AP-2 interac-tubes were placed at 25C. Assays were terminated by the addition tions required for targeting to plasma membrane clathrin-coatedof 150 �l ice-cold 1.5 M perchloric acid and 1 mM Pi. Released pits. J. Cell Biol. 146, 755–764.[32P]Pi was extracted with 1 volume of 2% ammonium molybdate and Gu, M., Warshawsky, I., and Majerus, P.W. (1992). Cloning and ex-4 volumes isobutanol:toluene (1:1), and quantitated by scintillation pression of a cytosolic megakaryocyte protein-tyrosine-phospha-counting. tase with sequence homology to retinaldehyde-binding protein and

yeast SEC14p. Proc. Natl. Acad. Sci. USA 89, 2980–2984.Acknowledgments Hammonds-Odie, L.P., Jackson, T.R., Profit, A.A., Blader, I.J., Turck,

C.W., Prestwich, G.D., and Theibert, A.B. (1996). Identification andWork reported here has been supported by the BBSRC (Babraham cloning of centaurin-�. J. Biol. Chem. 271, 18859–18868.Laboratory and grant 8/B11586), the Issac Newton trust, the MSKCC

Isakoff, S., Cardozo, T., Andreev, J., Li, Z., Ferguson, K., Abagyan,and an NCI Core Grant (Memorial Sloan-Kettering Cancer Center),R., Lemmon, M.A., Aronheim, A., and Skolnik, E.Y. (1998). Identifica-and by the MRC and the Royal Society (R.H.M. and S.K.D.). S.K. istion and analysis of PH domain-containing targets of phosphatidyl-a DFG research fellow, P.T.H. is a BBSRC fellow, and K.E.A. is ainositol 3-kinase using a novel in vivo assay in yeast. EMBO J. 17,Beit Memorial Fellow and acknowledges support of the Australian5374–5387.National Health and Medical Research Council. Z.-Y.L. thanks the

Cambridge Commonwealth Trust, the ORS scheme, New Hall (Cam- Jackson, C.L., and Casanova, J.E. (2000). Turning on ARF: the Sec7bridge), and the Tan Kar Kee Foundation (Singapore). We would like family of guanine nucleotide exchange factors. Trends Cell Biol. 10,to thank C. Petit (Institut Pasteur, Paris), P. Majerus (University of 60–67.Washington, St. Louis), and A. Hall (UCL, London) for kind gifts of Jackson, T.R., Brown, F.D., Nie, Z., Miura, K., Foroni, L., Sun, J.,reagents. We also thank Babraham Technix for DNA sequencing Hsu, V.W., Donaldson, J.G., and Randazzo, P.A. (2000). ACAPs areand the EPSRC Mass Spectrometry Service (Swansea) for mass Arf6 GTPase-activating proteins that function in the cell periphery.spectra. J. Cell Biol. 151, 627–638.

Kearns, M.A., Monks, D.E., Fang, M., Rivas, M.P., Courtney, P.D.,Received February 26, 2001; revised November 2, 2001. Chen, J., Prestwich, G.D., Theibert, A.B., Dewey, R.E., and Bankaitis,

V.A. (1998). Novel developmentally regulated phosphoinositide-References binding proteins from soybean whose expression bypasses the re-

quirement for an essential phosphatidylinositol transfer protein inAnderson, K.E., Lipp, P., Bootman, M., Ridley, S.H., Coadwell, J., yeast. EMBO J. 17, 4004–4017.Ronnstrand, L., Lennartsson, J., Holmes, A.B., Painter, G.F., Thuring, Lancaster, C.A., Taylor-Harris, P.M., Self, A.J., Brill, S., Van Evp,J., et al. (2000). DAPP1 undergoes a PI 3-kinase-dependent cycle H.E., and Hall, A. (1994). Characterization of rho GAP. A GTPase-of plasma membrane recruitment and endocytosis upon cell stimu- activating protein for rho-related small GTPases. J. Biol. Chem. 269,lation. Curr. Biol. 10, 1403–1412. 1137–1142.Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P.J., and Hsu, Lemmon, M.A., and Ferguson, K.M. (2000). Signal-dependent mem-V.W. (1997). The KDEL receptor, ERD2, regulates intracellular traffic brane targeting by pleckstrin homology (PH) domains. Biochem. J.by recruiting a GTPase-activating protein for ARF1. EMBO J. 16, 350, 1–18.7305–7316.

Li, Z., Wahl, M.I., Eguinoa, A., Stephens, L.R., Hawkins, P.T., andAraviond, L., Neuwald, A.F., and Ponting, C.P. (2001). Sec14p-like Whitte, O.N. (1997). Phosphatidylinositol 3-kinase- activates Bru-domains in NF1 and Dbl-like proteins indicate lipid regulation of Ras ton’s tyrosine kinase in concert with src family kinases. Proc. Natl.and Rho signaling. Curr. Biol. 9, R195–197. Acad. Sci. USA 95, 13820–13825.Arita, M., Sato, Y., Miyata, A., Tanabe, T., Takahashi, E., Kayden,

Marshall, A.J., Niiro, H., Lexner, C.G., Yun, T.J., Thomas, S., Dis-H.J., Arai, H., and Inone, K. (1995). Human �-tocopherol transfer

teche, C.M., and Clark, E.A. (2000). A novel B lymphocyte-associatedprotein: cDNA cloning, expression and chromosomal localization.

adaptor protein, Bam32, regulates antigen receptor signalling down-Biochem. J. 306, 437–443.

stream of phosphatidylinositol 3-kinase. J. Exp. Med. 191, 1319–Barfod, E.T., Zhang, Y., Kuang, W.J., Hart, M.J., Evans, T., Cerione, 1331.R.A., and Ashkenazi, A. (1993). Cloning and expression of a human

Miura, K., Jacques, K.M., Stauffer, S., Kubosaki, A., Zhu, K., Hirsch,CDC42 GTPase-activating protein reveals a functional SH3-binding

D.S., Resau, J., Zheng, Y., and Randazzo, P.A. (2002). ARAP1: adomain. J. Biol. Chem. 268, 26059–26062.

point of convergence for Arf and Rho signaling. Mol. Cell 9, thisCockcroft, S. ed. (2000). Biology of Phosphoinositides, Frontiers in issue, 109–119.Molecular Biology 27 (New York: Oxford University Press).

Ogasawara, M., Kim, S.-C., Adamik, R., Togawa, A., Ferrans, V.J.,Curtis, I. (2001). Cell migration: GAPs between membrane traffic Takeda, K., Kirby, M., Moss, J., and Vaughan, M. (2000). Similaritiesand the cytoskeleton. EMBO Rep. 2, 227–281. in function and gene structure of cytohesin-4 and cytohesin-1, gua-

nine nucleotide-exchange proteins for ADP-ribosylation factors. J.Dekker, L.V., and Segal, A.W. (2000). Signals to move cells. ScienceBiol. Chem. 275, 3221–3230.287, 982–985.

Derubeis, A.R., Young, M.F., Jia, L., Robey, P.G., and Fisher, L.W. Otsuki, M., Fukami, K., Kohno, T., Yokota, J., and Takenawa, T.(1999). Identification and characterization of a new phospholipase(2000). Double FYVE-containing protein-1 (DFCP-1): isolation, clon-

Molecular Cell108

C-like protein, PLC-L (2). Biochem. Biophys. Res. Commun. 266, sitol 3,4,5-trisphosphate-stimulated GTPase-activating proteins forARF6. J. Biol. Chem. 275, 13901–13906.97–103.

Wang, J., Arbuzora, A., Hangyas-Mihalyne, G., and McLaughlin, S.Painter, G.F., Grove, S.J.A., Gilbert, I.H., Holmes, A.B., Raithby, P.R.,(2000). The effector domain of myristoylated alanine-rich C kinaseHill, M.L., Hawkins, P.T., and Stephens, L.R. (1999). General synthe-substrate (MARCKS) binds strongly to phosphatidylinositol 4,5-bis-sis of 3-phosphorylated myo-inositol phospholipids and derivatives.phosphate (PIP2). J. Biol. Chem., in press.J. Chem. Soc. Perkin Trans. 1, 923–935.Wennstrom, S., Hawkins, P.T., Cooke, F., Hara, K., Yonezawa, K.,Painter, G.F., Thuring, J.W., Lim, Z.-Y., Holmes, A.B., Hawkins, P.T.Kasuga, M., Jackson, T., Claesson-Welsh, L., and Stephens, L.and Stephens, L.R. (2001). Synthesis and biological evaluation of a(1994). Activation of phosphoinositide 3-kinase is required for PDGF-PtdIns(3,4,5)P3 affinity matrix. Chem. Commun. 2001, 645–646.stimulated membrane ruffling. Curr. Biol. 4, 385–393.

Peters, P.J., Hsu, V.W., Ooi, C.E., Finazzi, D., Teal, S.B., Ooschot,Zhang, Q., Calafat, J., Janssen, H., and Greenberg, S. (1999). ARF6V., Donaldson, J.G., and Klausner, R.D. (1995). Overexpression of wtis required for growth factor- and Rac-mediated membrane rufflingand mutant Arf1 and Arf6: distinct perturbations of nonoverlappingin macrophages at a stage distal to Rac in membrane targeting.membrane compartments. J. Cell Biol. 128, 1003–1017.Mol. Cell. Biol. 19, 8158–8168.

Radhakrishna, H., Klausner, R.D., and Donaldson, J.G. (1996). Alu-minium fluoride stimulates surface protrusions in cells overexpress-

Accession Numbersing the ARF6 GTPase. J. Cell Biol. 134, 935–947.

Radhakrishna, H., Al-Awar, O., Khachikian, Z., and Donaldson, J.G. The sequences TR:AAL12169, TR:AAL12170, and EM:AJ310567 re-(1999). ARF6 requirement for Rac ruffling suggests a role for mem- ported in this paper are designated as ARAP1, ARAP2, and ARAP3,brane trafficking in cortical actin rearrangements. J. Cell Sci. 112, respectively.855–866.

Rameh, L.E., and Cantley, L.C. (1999). The role of phosphoinositide3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347–8350.

Rao, V.R., Corradetti, M.N., Chen, J., Peng, J., Yuan, J., Prestwick,E.D., and Brugge, J.S. (1999). Expression cloning of protein targetsfor 3-phosphorylated phosphoinositides. J. Biol. Chem. 274, 37893–37900.

Ridley, S.H., Ktistakis, N., Davidson, K., Anderson, K.E., Manifava,M., Ellson, C., Lipp, P., Bootman, M., Coadwell, J., Nazarian, A., etal. (2001). Fens-1 and DFCP1 are FYVE-domain containing proteinswith distinct functions in the endosomal and Golgi compartments.J. Cell Sci., 114, 3991–4000.

Santy, L.C., and Casanova, J.E. (2001). Activation of Arf6 by ARNOstimulates epithelial cell migration through downstream activationof both Rac1 and phospholipase D. J. Cell Biol. 154, 599–610.

Shirai, T., Tanaka, K.-I., Terada, Y., Sawada, T., Shirai, R., Hashimoto,Y., Nagata, S., Iwamatsu, A., Okawa, K., Li, S., et al. (1998). Specificdetection of phosphatidylinositol 3,4,5-trisphosphate binding pro-teins by the PIP3 analogue beads: an application for rapid purificationof the PIP3 binding proteins. Biochim. Biophys. Acta 1402, 292–302.

Simonsen, A., and Stenmark, H. (2001). PX domains: attracted byphosphoinositides. Nat. Cell Biol. 3, E179–E182.

Simonsen, A., Wurmser, A.E., Emr, S.D., and Stenmark, H. (2001).The role of phosphoinositides in membrane transport. Curr. Opin.Cell Biol. 13, 485–492.

Stricker, R., Hulser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich,F., and Reiser, G. (1997). cDNA cloning of porcine p42IP4, a mem-brane-associated and cytosolic 42 kDa inositol (1,3,4,5) tetrakis-phosphate receptor from pig brain with similar high affinity forPtdIns(3,4,5)P3. FEBS Lett. 405, 229–236.

Tanaka, K., Imajoh-Ohmi, S., Sawada, T., Shirai, R., Hashimoto, Y.,Iwasaki, S., Karbuchi, K., Kanaho, Y., Shirai, T., Terada, Y., et al.(1997). A target of phosphatidylinositol 3,4,5-trisphosphate with azinc finger motif similar to that of the ADP-ribosylation-factorGTPase-activating protein and two pleckstrin homology domains.Eur. J. Biochem. 245, 512–519.

Venkateswarlu, K., Oatey, P.B., Tavare, J.M., Jackson, T.R., andCullen, P.J. (1999). Identification of centaurin-�1 as a potential invivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that isfunctionally homologous to the yeast ADP-ribosylation factor (ARF)GTPase-activating protein, Gcs1. Biochem. J. 340, 359–363.

Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter,F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., et al.(1993). The gene involved in X-linked agammaglobulinaemia is amember of the src family of protein tyrosine kinases. Nature 361,226–233.

Vitale, N., Patton, W.A., Moss, J., Vaughan, M., Lefkowitz, R.J., andPremont, R.T. (2000). GIT proteins, a novel family of phosphatidylino-