PI(3,4,5)P3 Interactome - American Chemical Society

PI(3,4,5)P3 Interactome Bruno Catimel,*,† Meng-Xin Yin,‡,§ Christine Schieber,‡,§ Melanie Condron,† Heather Patsiouras,† Jenny Catimel,† Diane E. J. E. Robinson,§ Leon S.-M. Wong,§ Edouard C. Nice,† Andrew B. Holmes,§ and Antony W. Burgess† Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Parkville, Victoria, 3052, Australia, and School of Chemistry, Bio21 Institute, University of Melbourne, Parkville, Victoria 3010, Australia Received April 7, 2009

Immobilizing chemically synthesized analogues of PI(3,4,5)P3 onto Affi-10 beads and incorporating them into liposomes allowed their use as affinity absorbents in the comprehensive analysis of the phosphoinositide interactome using cytosolic cell extracts of the LIM1215 colon cancer cell line. This led to the identification of 282 proteins that either interact with PI(3,4,5)P3 or are indirectly captured as part of a complex containing a PI(3,4,5)P3 binding partner. Identification of the proteins was achieved using affinity/LC-MS/MS experiments. Keywords: phosphoinositides • proteomics • interactome • microaffinity purification

Introduction Phosphatidylinositol phosphates, also know as phosphoinositide phosphates (PI(x,y,z)Pn), are known to play an important regulatory role in cell physiology, especially as part of signaling networks that are modulated by proteins, kinases, phosphatases, and phospholipases.1 PIPs act either as phospholipase substrates for the generation of second messengers such as inositol polyphosphates and diacylglycerol (DAG)2 or interact directly with intracellular proteins, affecting their location and/or activity and therefore directing the spatiotemporal organization of key intracellular signal transduction pathways.3,4 Direct signaling is achieved through the binding of the inositol headgroup to a number of defined protein domains.5-7 The functional and structural basis for phosphoinositide binding and specificity has been reported previously.8-15 While PH (pleckstrin homology), FYVE (for Fab1, YOTB, Vac1 and EEA1) and PX (Phagocyte oxidase homology, Phox) domains interact with phosphoinositides in a highly selective manner, cytosolic proteins with these domains exhibit a low affinity and specificity.7,16 Furthermore, these binding sites do not appear to act alone as localization signals17,18 and instead have protein binding partners, forming multidomain complexes with high affinity membrane-protein and/or protein-protein interactions.1,7,15,19,20 Among the signaling networks regulated by phosphoinositides, the phosphatidylinositol 3-kinase (PI3K) pathway has emerged as an important pathway during the regulation of proliferation, growth, apoptosis, cell migration and morphology.21-23 PI3K catalyzes the phosphorylation of the 3-position of the inositol ring, resulting in the formation of PI(3)P, * To whom correspondence should be addressed. Dr Bruno Catimel; Tel, 61 3 9341 3134; Fax, 61 3 9341 3104; e-mail, [email protected]. † Ludwig Institute for Cancer Research. ‡ These authors made equivalent contributions. § University of Melbourne.

3712 Journal of Proteome Research 2009, 8, 3712–3726 Published on Web 05/22/2009

PI(3,4)P2 and PI(3,4,5)P3.21,24 Class IA PI3Ks are activated by a wide variety of cell surface receptors (e.g., receptor tyrosine kinases, growth factors, hormones, neurotransmitters) while Class IB are activated by cell surface receptors utilizing heteromeric G-proteins as their proximal transduction partners.22-24 PI(3,4,5)P3 serves to localize proteins containing a pleckstrin homology (PH) domain (a feature present in many classes of signaling proteins, for example, serine/threonine kinases, tyrosine kinases, GTPase activating proteins, cytoskeletal proteins) to the plasma membrane.19,25 Once recruited to the plasma membrane by PI(3,4,5)P3, PH domain-containing proteins are colocalized with other membrane-associated signaling proteins where they can influence signaling pathways. One PI(3,4,5)P3 target is the protein kinase B (PKB or AKT), a serine/threonine kinase with a wide range of substrates.24 AKT is an important molecule in mammalian cellular signaling that has a profound effect on a number of important functions such as proliferation, apoptosis and growth, cytoskeletal organization, membrane trafficking, membrane ruffling, differentiation, chemotaxis, and glucose homeostasis.24,26-28 AKT/PKB possesses a PH domain that mediates its recruitment to the membrane by 3′-phosphorylated phosphoinositides, where phosphorylation of the regulatory sites of AKT/PKB by PDK1 (and putative PDK2) occurs.23,26,29-31 PDK1 is also recruited to the membrane by interacting with PH-PI(3,4,5)P3.23,26 It is known that genetic defects leading to deficiencies in phosphoinositide-metabolizing enzymes are implicated in a number of diseases including cancer, diabetes, cardiovascular disease, Alzheimer’s disease, bacterial invasion, allergy and autoimmune disorders.32,33 It has also been reported that alterations in the PI3K-AKT signaling pathway are frequent in human cancers.27,34-38 Increased levels of PI(3,4,5)P3 appear to suppress apoptosis and thus contribute to cancer progression, in part through constitutive activation of PKB/AKT.39,40 Furthermore, it has been shown that defects of the phos10.1021/pr900320a CCC: $40.75

 2009 American Chemical Society

PI(3,4,5)P3 Interactome phatases PTEN and SHIP can lead to increased PI(3,4,5)P3 levels.32,35,41,42 However, other PI(3,4,5)P3 regulated proteins may play an important role in inducing the full range of PI3K action.23 While the pleckstrin homology (PH) domain is most commonly associated with PIP-binding proteins, there is no reason to assume that this is a prerequisite for recruiting downstream effector proteins for signal transduction. Therefore, a systematic probe of PIP-binding proteins has the potential to shed light on these aspects and to identify possible downstream targets of PI(3,4,5)P3. While there have been a number of studies that have investigated phosphoinositide binding proteins using a variety of techniques,43 few have focused on the identification of novel binding proteins.44,45 In one study, 16 PI(3,4,5)P3 interacting proteins were identified from pig leucocyte extracts using peptide mass fingerprinting and/or MS/MS-based sequencing.44 The other study on mammalian PIP-binding proteins was performed using macrophages isolated from mouse bone marrow using cleavable phosphoinositide affinity baits and MS/ MS analysis.45 This study resulted in the purification of 10 known and 11 novel potential phosphatidylinositol 3-kinase phosphatidylinositol signaling proteins. We recently reported a novel approach that makes no presumptions about the nature of phospholipid protein binding interactions, but rather relies on identifying proteins which show preferential, specific affinity for the solid-phase or liposome phosphatidylinositol lipid supports.43 This approach combined affinity-based assays using either phosphoinositides immobilized on beads or incorporated into liposomes, followed by nanoRP-HPLC ESI MS/MS analysis43,46,47 to identify the PI(3,5)P2 and PI(4,5)P2 interactomes in a cytosolic extract from the LIM1215 colorectal carcinoma cell line.48 We now report the use of this proteomic approach to characterize the PI(3,4,5)P3 interactome of the LIM1215 colonic carcinoma cell line. Accordingly, an analogue of PI(3,4,5)P3 was synthesized which contained the natural (D) inositol ring configuration and an amino terminal functionality in the saturated fatty acid chain of the diacyl glycerol to enable attachment to an affinity matrix (e.g., Affi-Gel 10).49 A dipalmitoyl PI(3,4,5)P3 analogue was also synthesized and incorporated in liposomes to provide a background of membrane phospholipids in a more physiological context.43

Materials and Methods Synthesis and Characterization of PI(3,4,5)P3 Phosphatidylinositol Phosphate. Sodium salts of dipalmitoyl analogues of phosphatidylinositol-(3,4,5)-triphosphate were synthesized and characterized as described previously.43,50,51 Conjugation of PIPs to Affi-10 Beads. The conjugation and characterization of NH2-PI(3,4,5)P3 to Affi-10 beads (Bio-Rad, Hercules, CA, USA) was performed as previously described for NH2-PI(3,5)P2 and NH2-PI)4,5)P2.43 Production and Purification of Recombinant GST-Tagged PH Domains of Phospholipase C Delta 1 and General Receptor Protein 1. Transformations were carried out using BL21 competent cells with PLCδPH and GRP1PH in pGEXT2TK plasmids (donated by Professor M. Lemmon, University of Pennsylvania, Philadelphia, PA). Protein expression, purification and characterization were carried out as previously described.43 LIM1215 Cytosolic Extracts. A digitonin extraction process was used to produce cytosolic extracts of LIM1215 colonic carcinoma cell lines.48 Briefly, LIM1215 cells (2 × 108 cells) were extracted for 30 min at 4 °C using 10 mLof 10 mM Pipes, 100

research articles mM NaCl, 120 mM sucrose 2.5 mM MgCl2, 2 mM EGTA pH 6.8 containing 0.015% w/v digitonin. Cell permeation was assessed under a light microscope using 1% eosin and the cytosolic fraction obtained by sequential centrifugation at 480g for 20 min and 436 000g (Beckman rotor TLA100.2, 100 000 rpm) for 15 min at 4 °C prior to the affinity experiments.43 Affinity Capture Techniques. Affinity capture experiments were performed using two alternative strategies. (1) Affinity Supports: The amino phosphoinositide analogue (ω-amino glycerol derivative) of PI(3,4,5)P3 was conjugated to Affi-gel 10 and incubated with crude LIM1215 cytosolic extracts. Briefly, incubation was performed initially with ethanolamine derivatized Affi-10 blank beads (200 µL beads for 500 µL sample) for 2 h at 4 °C to remove proteins which bind nonspecifically. After removal of the blank derivatized beads by centrifugation (5 min at 480g), the precleared fraction was incubated overnight at 4 °C with PI(3,4,5)P3-Affi-10 or Affi-10 blank derivatized beads (100 µL beads for 500 µL of extract). The beads were then washed 5 times with 1.5 mL PBS containing 0.005% (v/v) Tween 20 before desorbing the bound proteins using 100 µL of SDS-PAGE buffer (LDS NuPAGE sample buffer, Invitrogen) at 95 °C for 5 min and detected using SDS-PAGE and sensitive Coomassie staining.43 (2) Liposomal Capture: Affinity capture was also performed by incubating 100 µL PC/PE/PI(3,4,5)P3 or control PC/PE liposomes overnight at 4 °C with 500 µL of the LIM1215 cytosolic extracts. Liposomes and liposomal bound proteins were separated from unbound proteins by size exclusion chromatography as previously described. Purified liposomal fractions were then analyzed using SDS-PAGE.43 Preparation of Samples for MS Analysis. Reduction, alkylation, tryptic digestion and peptide extraction from protein bands was performed using a Mass-PREP robotic protein handling system (Waters, Rydalmere NSW, Australia). Generated tryptic peptides concentrated to ∼10 µL by centrifugal lyophilization (Savant Instruments, Farmingdale, NY) for nanoRP-HPLC-ESI-IT/MS/MS (LCQ-Deca, Finnigan, San Jose, USA).52,53 LC-MS/MS Analysis. Protein digests (∼10 µL in 0.1% (v/v) formic acid) were transferred into 100 µL glass autosampler vials and fractionated by capillary reversed-phase-HPLC (Model 1100, Agilent, Germany) using a C18, 100 µm × 0.15 mm I.D. RP-capillary column (Acquity UPLC BEH C18, 1.7 µm, Waters, Milford, MA) as described previously. The capillary HPLC was coupled online to a LCQ-Deca ESI-IT mass spectrometer for automated MS/MS analysis of individually isolated peptide ions.43,52,53 Acquired MS/MS spectra were searched against the LudwigNR database (http://www.ludwig.edu.au/archive/ludwigNR/ludwigNR.pdf) using the MASCOT search algorithm (v2.1.04, Matrix Science, U.K.).53 Bioinformatic Analysis. Proteins were classified according to their molecular functions and biological processes using the IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown.edu). Protein domains were determined using the Pfam protein families database (http://pfam.sanger.ac.uk/) and the Database of protein domains, families and functional sites (PROSITE, http:// au.expasy.org/prosite/). The Lipid Metabolites And Pathway Strategies (LIPIDMAPS) database (http://www.lipidmaps.org/) and SwissProt database (http://beta.uniprot.org/uniprot/) were interrogated by protein keywords and lipid class association to identify lipid interacting proteins and lipid-associated protein sequences. Network analyses were performed using the Search Journal of Proteome Research • Vol. 8, No. 7, 2009 3713

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Figure 1. Synthesis and characterization of PI(3,4,5)P3 phosphatidylinositol phosphates. The chemical syntheses of PI(3,4,5)P3 analogues from myo-inositol is based on the following steps: (1) regioselective protection of the hydroxyl groups of myo-inositol orthoformate; regioselective DIBAL-H mediated cleavage of the myo-inositol orthoformate and allyl protection of O-5 followed by removal of the ketal group to form a racemic triol; resolution with camphor acetal; (2) removal of the camphor group and regioselective protection-deprotection sequence; (3) introduction of phosphate groups at the required positions and removal of the protection group at C-2. (4 and 5) coupling to the lipophilic side-chain; (5 and 6) global deprotection.

Tool for the Retrieval of Interacting Genes/Proteins database (String http://string.embl.de/) as previously described.43 Binding of GST-PKN2 to Immobilized Phosphoinositides. Amino derivatives of PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 were covalently immobilized onto anthraquinone photoprobe derivatized plates (Nunc immobilizer amino white) (100 µL at 1 µg/mL) overnight at room temperature according to the manufacturers instructions. After blocking for 1 h with PBS containing 0.5% BSA, 0.05% Tween 20, proteins (GST-GRP1PH, GST-PLCδPH, GST and GST-PKN2 (Invitrogen)) were incubated with the plates at 2 µg/mL in PBS, 0.5% BSA, 0.05% Tween 20 overnight at 4 °C. After washing, plates were incubated with anti-GST IgG at 1 g/mL for 2 h at 37 °C, followed by washing and incubation for 1 h at 37 °C with HRP conjugated goat anti mouse IgG (Biorad, 1/3000 dilution). Plates were developed using chemoluminescence (Supersignal ELISA Fempto Maximum Sensitivity Substrate, Pierce) with a Fluorostar Optima Apparatus (BMG Labtech) with a gain adjustment of 1800.

Results Synthesis and Characterization of PI(3,4,5)P3 Analogues. The protocol used for the syntheses of the PI(3,4,5)P3 analogues is summarized in Figure 1. Analysis of the final products using 1 H NMR showed good agreement with published literature data.50,51 Characterization of Liposomes. The liposomes incorporating the dipalmitoyl analogue of PI(3,4,5)P3 were characterized by their ability to bind specifically to GST-GRP1PH that had been immobilized on a Biacore surface (Supporting Information Figure 1). PI(4,5)P2 containing liposomes and GSTPLCδ1PH were used to confirm specificity. Varying concentrations of GST-PLCδ1PH ((3.5 µM, 1.75 µM, 875 nM, 437 nM, 218 nM, 109 nM)) and of GST-GRP1PH (3 µM, 1.5 µM, 750 nM, 375 nM, 188 nM, 94 nM) were injected over immobilized PC/PE, PC/PE/PI(4,5)P2 and PC/PE/PI(3,4,5)P3 liposomes. As expected, 3714

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GST-PLCδ1PH was found to bind specifically to immobilized PC/PE/PI(4,5)P2 (Supporting Information Figure 1A) over PC/ PE/PI(3,4,5)P3 liposomes (Supporting Information Figure 1B). By contrast, GST-GRP1PH was found to bind specifically to immobilizedPC/PE/PI(3,4,5)P3(SupportingInformationFigure1D) over PC/PE/PI(4,5)P2 liposomes (Supporting Information Figure 1C). Proteomics Analysis. Binding Proteins Identified Using PI(3,4,5)P3 Incorporated into Liposomes. Liposomes containing PI(3,4,5)P3 were incubated with the cytosolic extract of LIM1215 carcinoma cells. Size exclusion chromatography (SEC) was used to separate the liposome-bound proteins from the residual cytosolic proteins (Figure 2). Representative chromatograms obtained with PC/PE and PC/PE/PI(3,4,5)P3 liposomes are shown in Figure 2B and C. The retention time of the liposome bound proteins was between 10 and 12.5 min (Figure 2B and 2C); this peak was not observed in the SEC of the cytosolic extract alone (Figure 2A). SDS-PAGE (Figure 2, gel insets, Figure 3A); analysis of these chromatographic fractions and the corresponding region from the control experiments (blank beads, PC/PE liposomes) allowed the identification of specific PI(3,4,5)P3 proteins (Table 1 of the Supporting Information). Binding Proteins Detected Using NH2-PI(3,4,5)P3 Conjugated to Affi-10 Beads. The level of PI(3,4,5)P3 conjugation to Affi-10 beads was shown to be 0.45 µMole of NH2-PI(3,4,5)P3 per mL of beads (data not shown) by Biacore analysis using a neomycin calibration curve.43 Affinity experiments using the PI(3,4,5)P3 beads and blank beads (as a control) with cytosolic extracts were performed as described in Materials and Methods. A representative SDS-PAGE analysis is shown in Figure 3B and the PI(3,4,5)P3 binding proteins identified using LC-MS/MS analyses are listed in Table 1 of the Supporting Information. Both these PI(3,4,5)P3 binding experiments led to the identification of 282 nonredundant proteins/protein complexes.

PI(3,4,5)P3 Interactome

research articles (PTB/PID) domain (PH like domain) (TBC1 domain family member 4), PH like RanBP1 (Ran-specific GTPase-activating protein) and Phosphatidylinositol transfer protein domains (Phosphatidylinositol transfer protein beta isoform) (Table 1). Additionally, a number of protein domains that are known to interact with phosphoinositides as well as charged phospholipids (e.g., phosphatidylserine) were also present: C1 (PDZ domain-containing protein 8), C2 (1-phosphatidylinositol-4,5bisphosphate phosphodiesterase beta-3, Extended-synaptotagmin-1, Serine/threonine-protein kinase N2), MARCKS (MARCKS related protein), Calponin (R-Actinin-4, Filamin-A, B and C, Transgelin-2 Plectin-1 Microtubule-associated protein RP/EB family member, Ras GTPase-activating-like protein IQGAP1, Calponin-2), Septin (e.g., Septin 2,7,9 and 11) Annexin (Annexin A21 and A2) and Heat domains (Importin subunit beta-1, Sister chromatid cohesion protein PDS5 homologue A, Proteasomeassociated protein ECM29, Cullin-associated NEDD8-dissociated protein 1,Translational activator GCN1) (Table 1).

Figure 2. Size exclusion chromatography of PI(3,4,5)P3 liposomes after incubation with LIM1215 cytosolic extracts. Size exclusion chromatography was performed on a Sephacryl S500 HR10/30 column as described in Materials and Methods. (A) Chromatographic profile of LIM1215 cytosolic extract. (B) Chromatographic profile of LIM1215 cytosolic extract incubated with PC/PE control liposomes. (C) Chromatographic profile of LIM1215 cytosolic extract incubated with PI(3,4,5)P3/PC/PE liposomes. Liposomes were found to elute between 10 and 12.5 min (A, B and C). SDSPAGE analyses of proteins bound to liposomes are shown (inset).

Of these, 82 were purified using liposomes, 175 using PIP beads and 25 proteins were common to both data sets (Supporting Information Table 1). Thirty-six of the 282 proteins had been identified in our previous experiments using PI(3,5)P2 and another 41 using PI(4,5)P2. A further 64 proteins bind to all phosphoinositide phosphates.43 Therefore, 141 of the 282 proteins displayed specificity for PI(3,4,5)P3 (Figure 4, Supporting Information Table 1). Previous experiments had identified 264 proteins which bind nonspecifically to either control liposomes or derivatized blank beads under the same conditions used in these studies.43 Identification of Proteins with Known Phosphoinositide Binding Domains. Bioinformatic analysis (see Materials and Methods) identified a number of the proteins binding specifically to PI(3,4,5)P3 by phosphoinositide recognition domains such as PH (e.g., Cytohesin1, 2 and 3, AKT1, Centaurin alpha 1, pleckstrin 2, Dynamin 2, Spectrin beta chain), PX (Sortin nexin 5), FERM (e.g., Erzin, Moesin, Radixin, Talin1, Band 4.1 like protein 1 and 2), PHD (transcription intermediary factor 1 beta), Clathrin adaptor beta (AP-2 and AP-3 adaptor complexes, Phosphatidylinositol 3- and 4-kinase (DNA-dependent protein kinase), Phosphotyrosine-binding/phosphotyrosine-interaction

A number of known lipid binding domains were also identified: Cral-Trio (Rho GTPase-activating protein 1, Rho GTPase-activating protein 8, Tyrosine-protein phosphatase nonreceptor type 9), START (PCTP-like protein) SCP-2 sterol transfer family (Nonspecific lipid-transfer protein, Peroxisomal multifunctional enzyme type 2) and SPFH domain (Prohibitin).54 In addition, 28 proteins were identified as potential lipid associated proteins using the LIPIDMAPS (http://www. lipidmaps.org/) or Swiss-Prot (http://beta.uniprot.org/uniprot/) databases (Supporting Information Table 2). The LIPIDMAPS database identifies proteins using a list of lipid related Gene Ontology (http://www.geneontology.org) and KEEG (http:// www.genome.ad.jp/kegg) terms such as lipid-related enzymatic activity, metabolic processes, lipid and pathways. Classification of Proteins by Molecular Function. We have classified the proteins identified in the PI(3,4,5)P3 interactome by molecular function (Figure 5 and Supporting Information Table 1). The largest cluster of the PI(3,4,5)P3 binding proteins involved transport and trafficking: (61 proteins, 21%, Figure 5, Table 2, Supporting Information Table 1). Among these proteins, 33 proteins that were identified using the PI(3,4,5)P3 beads were not previously pulled down using either of the PIP2 beads (Table 2). A number of solute carriers (SLC)55 were detected: SLC1 high affinity glutamate and neutral amino acid transporter family (solute carrier family 1 member 5, Q59ES3), SLC2 facilitative Glucose transporter family (GTR1 or GLUT1, GTR3 or GLUT3 and GTR14 or GLUT14), SLC7 cationic amino acid transporter family (CTR1) and SLC25 mitochondrial carrier family proteins (Tricarboxylate transport protein, Calciumbinding mitochondrial carrier protein SCaMC-1, Phosphate carrier protein MPCP and Mitochondrial 2-oxoglutarate/malate carrier protein). Interestingly, ATPase Na+/K+ transporting alpha 1 and beta 3 polypeptides, Arsenical pump-driving ATPase and Chloride intracellular channel 1 were also specifically isolated using PI(3,4,5)P3 probes (Table 2). Snare complex proteins (Vesicle-trafficking protein SEC22b, Snare domain), SNARE-associated protein Snapin, Syntaxinbinding protein 5 (Snare binding), Protein NipSnap homologue 1 and 2 (Snap25 domain/Snare binding) and Vesicle-fusing ATPase also showed specificity for PI(3,4,5)P3. Other PI(3,4,5)P3 specific proteins included lysosomal associated protein membrane 1, lethal giant larvae homologue 2, synaptogyrin 2, plasmolipin, and the mitochondrial proteins Sideroflexin-1, TOM34 and TOM70 (Table 2). Journal of Proteome Research • Vol. 8, No. 7, 2009 3715

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Figure 3. SDS-PAGE analysis of proteins purified using PI(3,4,5)P3. (A) Representative SDS-PAGE analysis of proteins purified using PI(3,4,5)P3 liposomes. Specific binding proteins were identified by comparison with control liposomes under the same experimental conditions. (B) Representative SDS-PAGE analysis of proteins purified using PI(3,4,5)P3 Affi-10 beads. Specific binding proteins were identified by comparison with blank derivatized beads used under the same experimental conditions.

Figure 4. Specific PI(3,4,5)P3 interacting proteins. The pie diagram details proteins specifically purified using PI(3,4,5)P3 as well as the number of PI(3,4,5)P3 interacting proteins previously shown to interact with PI(3,5)P2 and PI(4,5)P2.43

A number of proteins (e.g., components of Clathrin complexes, Coatomer subunits, Coat proteins and Nonspecific lipid transporter proteins) were previously identified using PI(3,5)P2 and PI(4,5)P2 (Table 2). Small GTPases and GTPases modulators known to play an important role in the regulation of transport and trafficking (e.g., Rab, Arf (Clathrin coat recruitment) see GTPases as discussed below, Table 3) could also be included in this cluster. A large group of small GTPases or GTPase regulators and activators (17%, 48 proteins) bound to PI(3,4,5)P3 (Figure 5, Table 3, Supporting Information Table 1). Among the GTPases and GTPase regulators, 19 were specifically purified using PI(3,4,5)P3 probes and were not previously detected using PI(3,5)P2 and PI(4,5)P2 affinity probes (in particular HRAS, KRAS and Rheb, Table 3). Members of the Ras family (RRAS, KRAS, Rheb, Ral-A), RHO family (RHO B), ARF family (Arf1, Arf 3, Arf4, Arf5, Sar1a and Arl1 (Arf-like protein 1), Rab family (Rab6A, Rab12) were detected using PI(3,4,5)P3 but not PIP2 beads (Table 3). Similarly, a number of PH domain containing Arf regulatory proteins56,57 were detected only with PI(3,4,5)P3 probes: Cy3716

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tohesin 1, Cytohesin 2/ARNO, Cytohesin 3/GRP1/ARNO 3 and Centaurin alpha 1). Cytohesin 1 and 3 display GEF activity for Arf1 and 3, Cytohesin 2 for Arf 1 and Arf6 while Centaurin alpha 1 is a GTPase activating protein that stimulates Arf-GTP hydrolysis and leads to its inactivation.56,57 Other GTPase regulators specifically purified using PI(3,4,5)P3 probes included TBCD4 (Rab GTPase-activating protein), Rho GTPases regulatory proteins (Rho GTPase-activating protein 8, Rho guanine nucleotide exchange factor 10-like), Ran-specific GTPase activating protein, Ras GTPase activating -like protein 1, Ras suppressor protein 1, RalBP1-associated EPS domain containing protein 1 and EH domain containing proteins (Table 3). A large number of actin binding proteins and cytoskeletal proteins were found: (47 proteins, 17%, Figure 5, Table 4, and Supporting Information Table 1). Proteins specifically purified using PI(3,4,5)P3 probe include Protein diaphanous homologue 1 (DIAPH1), Microtubule-associated protein RP/ EB family member 1 (MARE1), Pleckstrin homology-like domain family B (PH domain) member 1 and Alpha and delta Catenin (Table 4). MARE1 possesses a calponin homology domain while DIAPH1 possesses a nested ERM domain (2e07, Pfam database). Both proteins interact with the tumor suppressor Adenomatous Polyposis Coli (APC) protein, with DIAPH1 acting as a scaffold for MARE1 and APC to stabilize microtubules and promote cell migration. Alpha and delta Catenin were probably purified as part of the Cadherin adhesion complex (see receptor, cell adhesion cluster). A number of actin binding proteins known to interact with PI(4,5)P2 (e.g calponin domain containing proteins (Table 1) and previously identified using PI(4,5)P2 affinity probe were also purified in this study (Table 4). Proteins that bound to PI(3,4,5)P3 also included many kinases and phosphatases (18 proteins, 6.5%) (Figure 5, Table 5, Supporting Information Table 1). RAC-alpha serine/threo-

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PI(3,4,5)P3 Interactome a

Table 1. Summary of PIP/Phospholipid Domains domains

PH

protein ID

DYN2 SPTB2 CYH1 CYH2 CYH3

protein name

domains

protein ID

CENA1 SNX5 EZRI

Dynamin-2 Spectrin beta chain, brain 1 Cytohesin-1 Cytohesin-2 Cytohesin-3 (ARNO3, GRP1) Pleckstrin homology-like domain family B member 1 Pleckstrin-2 RAC-alpha serine/ threonine-protein kinase Centaurin-alpha-1 Sorting nexin-5 Ezrin

MOES

Moesin

IQGA1

RADI Q6NUR7

Radixin Ezrin

CNN2 MARE1

E41L1 E41L2

Band 4.1-like protein 1 Band 4.1-like protein 2

PTB/PID

TLN1 TBCD4

Clathrin adaptor

AP2B1

Talin-1 TBC1 domain family member 4 (Akt substrate of 160 kDa) AP-2 complex subunit beta-1

AP1B1 AP2A2

AP-2 complex subunit beta-1 AP-2 complex subunit alpha-2

AP3S1

AP-3 complex subunit sigma-1

ECM29

Phosphatidylinositol 3- and 4-kinase Phosphatidylinositol transfer protein Inositol monophosphatase family C1

PRKDC

CAND1

C2

PLCB3

DNA-dependent protein kinase catalytic subunit Phosphatidylinositol transfer protein beta isoform 3′(2′),5′-bisphosphate nucleotidase PDZ domain-containing protein 8 1-phosphatidylinositol-4, 5-bisphosphate phosphodiesterase beta-3 Extended-synaptotagmin-1

PHLB1 PLEK2 AKT1

PX FERM

PIPNB BPNT1 PDZD8

ESYT1 PKN2 MARCKS

MRP

Serine/threonine-protein kinase N2 (PK C-related kinase 2) MARCKS-related protein

Septin

SEPT2 SEPT7 SEPT9

Septin-2 (Protein NEDD5) Septin-7 Septin-9

Annexin Calponin

PDZ domain

Septin-11 Annexin A1 Annexin A2 Alpha-actinin-4 Filamin-A

FLNB

Filamin-B

TAGL2 FLNC

Transgelin-2 Filamin-C

PLEC1 PLSI MARE1

Plectin-1 Plastin Microtubule-associated protein RP/EB family member 1 Ras GTPase-activating-like protein IQGAP1 Calponin-2 Microtubule-associated protein RP/EB family member 1 Myosin-XVIIIa PDZ domain-containing protein 8 Syntenin-1 Transcription intermediary factor 1-beta

MY18A PDZD8

PHD

SDCB1 TIF1B

RANBP1 (PH like)

RANG

HEAT

IMB1 PDS5A

SEC14 (CRAL-TRIO)

SPC2

GCN1L

Ran-specific GTPase-activating protein Importin subunit beta-1 Sister chromatid cohesion protein PDS5 homologue A Proteasome-associated protein ECM29 Cullin-associated NEDD8-dissociated protein 1 Translational activator GCN1

RHG01

Rho GTPase-activating protein 1

RHG08

Rho GTPase-activating protein 8

PTN9

Tyrosine-protein phosphatase nonreceptor type 9

HSDL2

Hydroxysteroid dehydrogenase-like protein 2 Nonspecific lipid-transfer protein

NLTP DHB4 START SPFH

protein name

SEP11 ANXA1 ANXA2 ACTN4 FLNA

PCTL PHB

Peroxisomal multifunctional enzyme type 2 PCTP-like protein Prohibitin

a Proteins containing phospholipid-binding domains were identified using the Pfam database (http://pfam.sanger.ac.uk/). Protein accession number, ID and name are as in UniprotKB.

nine-protein kinase (AKT1), PKN2 Serine/threonine-protein kinase N2 (PRK2), Serine /threonine-protein kinase SRPK1, Calcium/calmodulin-dependent protein kinase type II, Uridinecytidine kinase 2 and Low molecular weight phosphotyrosine protein phosphatase were all detected specifically using PI(3,4,5)P3 affinity support.

Figure 5. Molecular function of PI(3,4,5)P3 purified proteins. Proteins are classified according to their molecular functions using the IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown. edu).

A number of the kinases and phosphatases found were previously identified using PI(3,5)P2 and PI(4,5)P2 substrates (e.g., DNA-dependent protein kinase (Phosphatidylinositol 3 and 4-kinase domain), Mitogen activated protein kinase 1, cell division control protein 2, Pyruvate kinase and Protein phosphatase 1) (Table 5).43 Journal of Proteome Research • Vol. 8, No. 7, 2009 3717

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Table 2. Proteins Involved in Transport and Trafficking protein AC protein ID

Q5T8D3

ACBD5

O00170 Q10567 O94973

AIP AP1B1 AP2A2

P63010 Q92572 P04083 P07355 P54709

AP2B1 AP3S1 ANXA1 ANXA2 AT1B3

P05023

AT1A1

O43681 Q6NUK1

ARSA1 SCMC1

O43633 Q9H444 O00299 Q00610 P09496 P53621 P53618 P35606 P48444 O14980 P55060 Q9UIA9 P30825

CHM2A CHM4B CLIC1 CLH1 CLCA COPA COPB COPB2 COPD XPO1 XPO2 XPO7 CTR1

Q6YN16 Q14974 Q6P1M3 P11279 Q15785

HSDL2 IMB1 L2GL2 LAMP1 TOM34

Q02978

M2OM

O94826

TOM70

P22307 P06748 P51659 Q00325 P48739

NLTP NPM DHB4 MPCP PIPNB

Q9Y342 Q9UID3 Q9BPW8 O75323 Q9Y365 P37108

PLLP FFR NIPS1 NIPS2 PCTL SRP14

P61011

SRP54

O76094

SRP72

P49458

SRP09

O95295 Q9Y5X3 Q5T5C0 P46459 P62760 O75396 Q8TDB8 P11169 P11166 P30825 Q59ES3 Q15758 Q9H9B4 O43760 P53007

SNAPN SNX5 STXB5 NSF VISL1 SC22B GTR14 GTR3 GTR1 CTR1 Q59ES3 AAAT SFXN1 SNG2 TXTP

protein name

Acyl-CoA-binding domain-containing protein 5 AH receptor-interacting protein AP-1 complex subunit beta-1 AP-2 complex subunit alpha-2 AP-2 complex subunit beta-1 AP-3 complex subunit sigma-1 Annexin A1 Annexin A2 ATPase, Na+/K+ transporting, beta 3 polypeptide ATPase, Na+/K+ transporting, alpha 1 polypeptide Arsenical pump-driving ATPase Calcium-binding mitochondrial carrier protein SCaMC-1 Charged multivesicular body protein 2a Charged multivesicular body protein 4b Chloride intracellular channel 1 Clathrin heavy chain 1 Clathrin light chain A Coatomer subunit alpha Coatomer subunit beta Coatomer subunit beta Coatomer subunit delta Exportin-1 Exportin-2 Exportin-7 High affinity cationic amino acid transporter 1 Hydroxysteroid dehydrogenase-like protein 2 Importin subunit beta-1 Lethal giant larvae homologue 2 Lysosomal-associated membrane protein 1; Mitochondrial import receptor subunit TOM34 Mitochondrial 2-oxoglutarate/malate carrier protein Mitochondrial import receptor subunit TOM70 Nonspecific lipid-transfer protein Nucleophosmin Peroxisomal multifunctional enzyme type 2 Phosphate carrier protein, Phosphatidylinositol transfer protein beta isoform Plasmolipin; Protein fat-free homologue Protein NipSnap homologue 1 Protein NipSnap homologue 2 PCTP-like protein Signal recognition particle 14 kDa protein Signal recognition particle 54 kDa protein Signal recognition particle 72 kDa protein Signal recognition particle 9 kDa protein SNARE-associated protein Snapin Sorting nexin-5 Syntaxin-binding protein 5 Vesicle-fusing ATPase Visinin-like 1 protein Vesicle-trafficking protein SEC22b Solute carrier family 2, member 14 Solute carrier family 2 member 3 Solute carrier family 2 member 1 Solute carrier family 7 Solute carrier family 1 Solute carrier family 1 member 5 Sideroflexin-1 Synaptogyrin 2 Tricarboxylate transport protein

molecular function

PI(3,5)P2 PI(4,5)P2 PI(3,4,5)P3

Lipid binding Binding; signal transducer activity Membrane traffic protein Transmembrane receptor regulatory/adaptor protein Membrane traffic protein Vesicle coat protein Transfer/carrier protein; Transfer/carrier protein Cation transporter

• • • • • •

Ion channel; Cation transporter;

• • •

• • • • •

•

•

Transporter; Nucleotide phosphatase Mitochondrial carrier protein Molecular function unclassified Transfer/carrier protein Anion channel Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Transfer protein, RNA-binding protein Transporter Transfer/carrier protein Transporter activity Steroid binding Transfer/carrier protein Membrane traffic regulatory protein Membrane traffic regulatory protein Binding

• • •

• • • • •

• • •

• • • • • • • • •

•

•

• • • • • • • • • • • • • • • • • •

Transporter activity

•

Protein binding

•

Steroid, lipid and protein binding Chaperones Steroid binding Transporter activity Tran\?\porter, lipid binding

• •

Transporter activity Transporter activity Membrane traffic protein Membrane traffic protein Transfer/carrier protein RNA-binding protein

•

RNA-binding protein

•

Binding

•

•

• • • • •

• •

• • • • • • •

•

•

Nucleic acid binding

•

Binding Membrane traffic regulatory protein Binding Binding (ion, nucleotide, protein binding) Calmodulin related protein Binding (protein) Carbohydrate transporter Carbohydrate transporter Carbohydrate transporter Anion channel Transporter Transporter Transporter activity Membrane traffic regulatory protein Mitochondrial carrier protein

• • • • • • • • • • • • • • •

•

•

a Phosphoinositide-interacting proteins involved in transport and trafficking were identified using IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown.edu/iproclass/). Small GTPases involved in transport and trafficking are not listed in this Table).

Cell Adhesion and Receptor Cluster. Twenty-three cell receptors and adhesion molecules were identified as PI(3,4,5)P3 3718

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binding proteins (8%, Figure 5, Table 6, and Supporting Information Table 1). Proteins such as Integrins, E-Cadherin,

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Table 3. GTPases and GTPase Regulators protein AC

protein ID

P61204 P18085 P84085 P62330 P40616 P60953

ARF3 ARF4 ARF5 ARF6 ARL1 CDC42

O75689 Q15438 Q99418 O43739

CENA1 CYH1 CYH2 CYH3

Q9Y295

DRG1

P50570 P62826 Q15382 Q9NR31 P20591

DYN2 RAN RHEB SAR1A MX1

Q3YEC7 P63000

PARF RAC1

P15153

RAC2

P62491 P61106 P59190 P62820 P51148 P20340 P51149 Q6IQ22 P11233 P11234 P10301

RB11A RAB14 RAB15 RAB1A RAB5C RAB6A RAB7A RAB12 RALA RALB RRAS

P01116

RASK

P46940

IQGA1

Q13283

G3BP1

Q15404 P62745

RSU1 RHOB

Q07960 P85298 Q9HCE6

RHG01 RHG08 ARHG EF10L

P43487 Q96D71

RANG REPS1

Q15019 Q16181 Q9UHD8 Q9NVA2 Q9H4M9 Q9H223 O60343 Q7L576

SEPT2 SEPT7 SEPT9 SEP11 EHD1 EHD4 TBCD4 CYFP1

protein name

molecular function

ADP-ribosylation factor 3 ADP-ribosylation factor 4 ADP-ribosylation factor 5 ADP-ribosylation factor 6 ADP-ribosylation factor-like protein 1 Cell division control protein 42 homologue precursor Centaurin-alpha-1 Cytohesin-1 Cytohesin-2 ARNO Cytohesin-3; ARNO3, GRP1

Small Small Small Small Small Small

Developmentally regulated GTP-binding protein 1 Dynamin-2 GTP-binding nuclear protein Ran GTP-binding protein Rheb precursor GTP-binding protein SAR1a Interferon-induced GTP-binding protein Mx1 Putative GTP-binding protein Parf Ras-related C3 botulinum toxin substrate 1 precursor Ras-related C3 botulinum toxin substrate 2 precursor Ras-related protein Rab-11A Ras-related protein Rab-14 Ras-related protein Rab-15 Ras-related protein Rab-1A Ras-related protein Rab-5C Ras-related protein Rab-6A Ras-related protein Rab-7a Ras-related protein Rab-12 Ras-related protein Ral-A Ras-related protein Ral-B precursor Related RAS viral (r-ras) oncogene homologue v-Ki-ras2 Kirsten sarcoma viral oncogene homologue Ras GTPase-activating-like protein IQGAP1 Ras GTPase-activating protein-binding protein 1 Ras suppressor protein 1 Rho-related GTP-binding protein RhoB Rho GTPase-activating protein 1 Rho GTPase-activating protein 8 Rho guanine nucleotide exchange factor (GEF) 10-like Ran-specific GTPase-activating protein RalBP1-associated Eps domain-containing protein 1 Septin-2 Septin-7 Septin-9 Septin-11 EH domain-containing protein 1 EH domain-containing protein 4 TBC1 domain family member 4 Cytoplasmic FMR1-interacting protein 1 (Specifically Rac1-associated protein 1)

GTPase GTPase GTPase GTPase GTPase GTPase

PI(3,5)P2

PI(4,5)P2

• • •

GTPases modulator GTPase regulator Enzyme regulator activity; Enzyme regulator activity; lipid binding Small GTPase

•

PI(3,4,5)P3

• • • • • • • • • •

•

•

• •

• •

•

•

• • • • •

Small GTPases Small GTPase

• •

•

• •

Small GTPase

•

•

•

Small Small Small Small Small Small Small Small Small Small Small

• • • • •

• •

•

•

•

•

• • • • • • • • • • •

Small Small Small Small Small

GTPase GTPase GTPase GTPase GTPase

GTPase GTPase GTPase GTPase GTPase GTPase GTPase GTPase GTPase GTPase GTPase

• •

Small GTPase

•

GTPase regulator

•

•

•

Signaling molecule

•

•

•

Kinase modulator Small GTPase

•

GTPase regulator GTPase regulator Guanine nucleotide exchange factor GTPase regulator Binding (protein, ion) Small GTPase Small GTPase Small GTPase Small GTPase GTPase regulator GTPase regulator GTPase regulator GTPase-modulator

• • •

• • • • •

• • • • • •

• • • • •

•

•

• • • • • • • •

a Phosphoinositide-interacting proteins classified as GTPases and GTPase regulators were identified using IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown.edu). Protein accession number, ID and name are as in UniprotKB.

beta-Catenin, CD44, CD9, CD63, the A33 antigen, MCAM, Galectins, Tetraspanin 8 and HLA class I histocompatibility antigens were included in this cluster. Other Clusters Identified. Proteins were also classified in three other broader groups: enzymes (e.g., ligases, lyases, synthases, hydrolases, oxidoreductases (40 proteins, 14%) binding (17 proteins, 6%) and transcription, translation factors (13 proteins, 4.5%) (Supporting Information Table 1). Seventeen of the PI(3,4,5)P3-binding proteins within the enzyme cluster are involved in cellular lipid metabolic process. Fifteen proteins had unclassified or miscellaneous molecular function (Supporting Information Table 1).

Classification of Proteins Based on Biological Processes. Organizing the PI(3,4,5)P3-interacting proteins by the biological processes in which they are involved reveals that PI(3,4,5)P3 is involved in the regulation of a broad range of processes (see Supporting Information, Table 3). Some of these have been previously identified through the PIP2 binding assays43 (for example molecular transport, protein trafficking, cell-to-cell signaling and interaction, cancer, cell cycle and cellular movement, cellular assembly and organization), but others have also been identified: these include vesicle mediated transport, cell death, cell morphology, cellular development, cellular growth and proliferation, Journal of Proteome Research • Vol. 8, No. 7, 2009 3719

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Table 4. Proteins Associated with the Regulation of the Actin Cytoskeleton System protein AC protein ID

P60709 O43707 P52907 P47756 Q9NZR1 Q9Y490 Q9NYL9 Q1ZYL5 Q5VU58 P15311 P26038 Q15149 P35241 Q9UBC5 Q12965 P19105 P60660 P14649 Q92614 P35579 Q7Z406 P52907 O60610 P35611 Q9UEY8 O43491 Q9H4G0 Q99439 P23528 P60981 P21333 O75369 Q14315 Q13813 Q01082 P37802 P35221 O60716 Q14651 Q13409 O43237 Q14204 P33176 Q86UU1 Q9NYT0 Q15691 Q9UHD9

ACTB ACTN4 CAZA1 CAPZB TMOD2 TLN1 TMOD3 Q1ZYL5 Q5VU58 EZRI MOES PLEC1 RADI MYO1A MYO1E MLRM MYL6 MYL6B MY18A MYH9 MYH14 FCHO2 DIAP1 ADDA ADDG E41L2 E41L1 CNN2 COF1 DEST FLNA FLNB FLNC SPTA2 SPTB2 TAGL2 CTNA1 CTND1 PLSI DC1I2 DC1L2 DYHC KINH PHLB1 PLEK2 MARE1 UBQL2

protein name

molecular function

Actin, cytoplasmic 1 (Beta-actin). Actinin, alpha 4 F-actin capping protein subunit alpha-1 F-actin capping protein subunit beta Tropomodulin-2 Talin-1 Tropomodulin-3 Tropomyosin 1 alpha variant 6 Tropomyosin 3 Ezrin Moesin Plectin-1 Radixin Myosin IA Myosin-Ie Myosin regulatory light chain 2 Myosin light polypeptide 6 Myosin light polypeptide 6B Myosin-XVIIIa Myosin-9 Myosin 14 FCH domain only 2 Protein diaphanous homologue 1 Adducin 1 (alpha) Gamma-adducin Band 4, 1-like protein 2 Erythrocyte membrane protein band 4.1-like 1 Calponin-2 Cofilin-1 (p18) Destrin Filamin A Filamin B Filamin C Spectrin alpha chain, brain Spectrin beta chain, brain 1 Transgelin-2 Catenin (cadherin-associated protein) Catenin delta-1 (p120 catenin) Plastin-1 Dynein 1 intermediate chain 2; cytosolic Dynein 1 light intermediate chain 2; cytosolic Dynein heavy chain, cytosolic (DYHC) Kinesin heavy chain Pleckstrin homology-like domain family B member 1 Pleckstrin-2 Microtubule-associated protein RP/EB family member 1 Ubiquilin-2

Structural molecule protein, Nonmotor actin binding protein Actin binding Actin binding Actin binding Actin binding Actin binding Actin binding motor protein Actin binding motor protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding motor protein Actin binding motor protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin family cytoskeletal protein Non motor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Nonmotor actin binding protein Structural molecule activity Structural molecule activity Microtubule binding motor protein Microtubule binding motor protein Microtubule binding motor protein; Microtubule binding motor protein Cytoskeletal protein Cytoskeletal protein Protein Binding Protein binding

PI(3,5)P2 PI(4,5)P2 PI(3,4,5)P3

• • •

• • •

•

•

• • • • •

• • •

• • •

• • • •

• •

• •

• • • • • • • • •

•

• • • • • • • •

•

• •

• • •

•

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

a

Phosphoinositide-interacting proteins which have a molecular function involved in actin cytoskeletal regulation were identified using IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research. Protein accession number, ID and name are as in UniprotKB.

further emphasizing the wide array of molecular processes that can be regulated by phosphoinositides. Network Analysis. The comparative network analysis (see Materials and Methods) of the proteins associated with PI(3,4,5)P3 is shown in Figure 2 of the Supporting Information. This confirms that a number of protein complexes that have been identified in the PI(3,4,5)P3 interactome are associated with specific functions such as transport and trafficking, small GTPases, actin cytoskeletal regulation, kinases, cell receptors. Proteins possessing a phosphoinositide/phospholipid binding domains that are able to interact directly with the PI(3,4,5)P3 probe are indicated in the Figure, distinguishing them from those that were obtained as part of protein/protein complexes. Binding of PKN2 to Immobilized Phosphoinositides. The binding of GST-PKN2 to immobilized PI(3,5)P2, PI(4,5)P2 and 3720

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PI(3,4,5)P3 was studied in an ELISA assay (Figure 6). GST-PKN2 was shown to bind preferentially to PI(3,4,5)P3 (Figure 6A) compared to immobilized PI(3,5)P2 and PI(4,5)P2 (Figure 6B and C). GST-GRP1PH- (specific binding to PI(3,4,5)P3, Figure 6A) and GST-PLCδPH- (specific binding to PI(4,5)P2, Figure 6B) were used as positive controls and GST was used as the negative control.

Discussion The specific binding of proteins to phosphoinositide is a key determinant in the spatiotemporal localization of these proteins to their site of function in various cellular processes such as membrane trafficking, permeability and transport, regulation of cytoskeleton and nuclear events, as well as cell adhesion,

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Table 5. Kinases and Phosphatases protein AC protein ID

P23919 P14618 Q01813 P17858 Q13555

KTHY KPYM K6PP K6PL KCC2G

P06493 P78527 P28482 Q9BSD7 P31749 Q96SB4 Q16513 Q9BZX2 Q9BVJ7 P43378 P24666

CDC2 PRKDC MK01 CA057 AKT1 SRPK1 PKN2 UCK2 DUS23 PTN9 PPAC

O95861 P62136

BPNT1 PP1A

protein name

molecular function

Thymidylate kinase Pyruvate kinase isozymes M1/M2 6-phosphofructokinase type C 6-phosphofructokinase, liver type Calcium/calmodulin-dependent protein kinase type II gamma chain Cell division control protein 2 homologue DNA-dependent protein kinase catalytic subunit Mitogen-activated protein kinase 1 Probable UPF0334 kinase-like protein C1orf57 RAC-alpha serine/threonine-protein kinase Serine/threonine-protein kinase SRPK1 Serine/threonine-protein kinase N2 Uridine-cytidine kinase 2 Dual specificity protein phosphatase 23 Tyrosine-protein phosphatase nonreceptor type 9 Low molecular weight phosphotyrosine protein phosphatase 3′(2′),5′-bisphosphate nucleotidase 1 Serine/threonine-protein phosphatase PP1-alpha catalytic subunit

Kinase Carbohydrate kinase Carbohydrate kinase Carbohydrate kinase Serine/threonine protein kinase Serine/threonine Serine/threonine Serine/threonine Kinase Serine/threonine Serine/threonine Serine/threonine Kinase Phosphatase Phosphatase Phosphatase

protein kinase protein kinase protein kinase

PI(3,5)P2 PI(4,5)P2 PI(3,4,5)P3

•

• • •

• • • •

• •

protein kinase protein kinase protein kinase

Phosphatase, Nuclease Phosphatase

• •

• • • • • • • • • • • • • • • • • •

a Phosphoinositide-interacting proteins classified as kinases and phosphatases were identified using IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research. Protein accession number, ID and name are as in UniprotKB.

Table 6. Receptors, Cell Adhesion protein AC

protein ID

P12830 P35222 P16070 P08962 P21926 Q99795 P17931 P56470

CADH1 CTNB1 CD44 CD63 CD9 GPA33 LEG3 LEG4

Cadherin precursor Catenin betaCD44 antigen precursor CD63 molecule CD9 Cell surface A33 antigen Galectin-3 Galectin-4

protein name

molecular function

P17301 P26006 P23229 P16144 P06756 Q8WUM6 P18084 P43121 P19075 P02786 P10319 P04439 P05534 P30466

ITA2 ITA3 ITA6 ITB4 ITAV Q8WUM6 ITB5 MUC18 TSN8 TFR1 1B58 1A03 1A24 1B18

P01889

1B07

Integrin alpha-2 precursor Integrin alpha-3 precursor Integrin alpha-6 precursor Integrin beta-4 precursor integrin, alpha V Integrin, beta 1 Integrin, beta 5 Melanoma cell adhesion molecule; MCAM Tetraspanin-8 Transferin receptor protein 1 HLA class I histocompatibility antigen HLA class I histocompatibility antigen HLA class I histocompatibility antigen HLA class I histocompatibility antigen, B-18 alpha chain HLA class I histocompatibility antigen, B-7 Major histocompatibility complex antigen

Binding signal transducer activity Signal transducer activity, binding Signaling molecule Cell adhesion molecule Cell adhesion molecule Binding; protein, carbohydrate Cell adhesion molecule, signaling molecule Cell adhesion molecule Cell adhesion molecule Cell adhesion molecule Protein binding, signal transduceur activity Cell adhesion molecule Cell adhesion molecule, receptor; Cell adhesion molecule Cell adhesion molecule Signal transducer activity Receptor Major histocompatibility complex antigen Major histocompatibility complex antigen Major histocompatibility complex antigen Major histocompatibility complex antigen

migration, proliferation, metabolism and death. The cellular levels of phosphoinositide are controlled by specific phosphoinositide kinases and phosphatases and the importance of this regulation is reflected by their association with many human diseases including cancer.32,34-37 A number of cancers have been shown to be associated with dysregulation of phosphoinositide-mediated signaling pathways via mutations in phosphoinositide kinases such as phosphatidylinositol 3-kinase (PI3K). The production of PI(3,4,5)P3 is a unique feature of the class I PI3 kinase family members and information about PI(3,4,5)P3 interacting proteins may provide evidence concerning their involvement in functional cellular processes regulated by PI3 kinase.

PI(3,5)P2 PI(4,5)P2 PI(3,4,5)P3

• • •

• •

•

•

• • • • •

•

• • • •

•

• • • • • • • • • • • • • • • • • • • • • • •

However, few proteomic studies have been performed to date to identify proteins which interact with PI(3,4,5)P3 in complex biological samples. Of these, only two PI(3,4,5)P3 affinity-based proteomic studies have involved mammalian cells.44,45 One study using pig leucocyte extracts from 35 L of blood,44 led to 16 PI(3,4,5)P3 interacting proteins being identified using peptide mass fingerprinting and/or MS/MS-based sequencing. Proteins containing PH domains (e.g., Tyrosineprotein kinases BTK and ETK, Centaurin alpha, Dual Adaptor of Phosphotyrosine and 3-Phosphoinositides (DAPP1), Cytohesin 4, ARAP3, Phosphoinositide-binding protein PIP3-E, Phospholipase C-L2, RasGAPIP4BP), FYVE domains (SR1, SR3) were specifically purified in this study. Although only a Journal of Proteome Research • Vol. 8, No. 7, 2009 3721

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Figure 6. ELISA analysis of the interaction of PNK2-GST with immobilized phosphoinositides. N-Terminal amino derivatives of PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3 (100 µL at 1 µg/mL) were immobilized onto anthraquinone photoprobe derivatized plates and incubated with GST-PKN2, GST- GRP1PH, GST-PLCδ1PH and GST (2 µg/mL) as described in Materials and Methods. GST-GRP1PH and GST-PLCδ1PH were used as positive controls for immobilized PI(3,4,5)P3 and PI(4,5)P2 respectively, while GST was used as a negative control. Binding was detected using a polyclonal anti-GST antibody and horseradish peroxidase conjugated anti-GST-IgG. Detection was performed using luminescence with a Fluorostar Optima Luminometer.

relatively small number of phosphoinositide interacting proteins were identified in this study, Krugmann et al. suggested that many more PIP binding proteins were present in the cell lysate and remained to be identified.44 The second study on mammalian PIP binding proteins45 reported the use of a cleavable phosphoinositide affinity probes in experiments with macrophages isolated from mouse bone marrow. MS/MS analysis identified 10 known and 11 novel potential PI3 kinase phosphatidylinositol signaling proteins. In agreement with our results, some of the proteins isolated in that study contained phosphoinositide recognition domains: PH domain containing proteins (e.g., AKT, PDK1, BKT, IIk, TEC, Vav, GAP1) were purified along with cytoskeletal proteins (Gelsolin, Talin) and a number of potential PIP binding proteins (hexokinase, Coronin-like protein p57, Rab5c, Ras GTPase activating 3 GAP1, PRDX1, Vacuolar protein sorting-associated protein 29, Sphingosine1 phosphate receptor Edg8).45 We have recently performed a comprehensive proteomic analysis of the phosphoinositide interactome using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids as affinity reagents with cytosolic extracts from the LIM1215 colonic carcinoma cell line.43 In that study we identified 388 proteins/protein complexes that appeared to interact specifically with either PI(3,5)P2 or PI(4,5)P2.43 We have now used a similar experimental protocol to analyze the PI(3,4,5)P3 interactome, using analogues of PI(3,4,5)P3, either immobilized onto Affi-10 beads or incorporated into liposomes, to pull down binding complexes from cytosolic extracts of LIM1215 colonic carcinoma cells. Digitonin was used to prepare cell cytosolic extracts.43 Digitonin is a detergent that binds and precipitates sterols, forming pores in cellular membranes and allowing extraction of cytosolic cellular contents. Our extraction protocol produced an enriched cytoplasm fraction as shown by the classification of 217 proteins (77% of phosphoinositide purified proteins) as intracellular (Gene Ontology GO0005622) Among them, 190 proteins were classified as cytoplasmic (Gene Ontology GO:0005737). This extract also contained 76 proteins (27%) classified as integral or intrinsic to membrane (Gene Ontology GO:0016021 and GO:0031224) with 31 proteins being integral to plasma membrane (Gene Ontology GO:0005887). The pres3722

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ence of integral membrane proteins may be explained by the disruption of cholesterol rich domains within the plasma membrane (e.g., lipid rafts), resulting in the release of proteins contained within these domains. Preclearing and stringent control experiments were conducted using blank derivatized beads and control liposomes under the same experimental conditions.43 Affinity/SDS-PAGE/LC-MS/MS proteomic experiments using PI(3,4,5)P3 affinity beads led to the identification of 282 protein/protein complexes. Of these proteins 141 displayed specific interaction with PI(3,4,5)P3 and had not been previously identified using either PI(4,5)P2 and PI(3,5)P2 affinity targets (Figure 4). Eighty-two PI(3,4,5)P3 interacting proteins were purified using liposomes, 175 proteins using PIP beads and 25 proteins were common to both data sets (Supporting Information Table 1). These distinct protein pools were also observed in our previous proteomic experiments (177 proteins were purified using PI(4,5)P2 beads, 47 using PI(4,5)P2 liposomes and 59 using both PI(4,5)P2 liposomes and beads, 81 proteins were purified using PI(3,5)P2 beads, 64 using PI(3,5)P2 liposomes and 56 on both PI(3,5)P2 liposomes and beads.43 Proteins purified specifically using liposomes may bind using a mechanism that involves recognition of the phosphatidylinostidol headgroup as well as a lipophilic environment, membrane curvature or insertion of a hydrophobic loop in the lipid layer.15,82 There is also an additional size exclusion chromatographic step in the liposome affinity protocol during which low affinity binders may be dissociated. Lastly the P1(3,4,5)P3 liposomes have a lower loading of phosphoinositide (approximately 27 µg/100 µL liposomes) than the beads (approximately 45 µg/100 µL of beads). Our affinity method is validated by the purification of 64 proteins that possess known phosphoinositide/phospholipid binding domains (e.g PH, PX, FERM, PTB/PID, Phosphatidylinositol 3- and 4-kinase, RANBP1, C1, C2, MARCKS, PDZ, PHD, Septin, Annexin, Clathrin adaptor, Calponin, Phosphatidylinositol transfer protein, HEAT, Inositol monophosphatase family, Cral-Trio, SPC2, START, SPFH). Twenty-two small GTPases of Ras, Rab Arf and Rho families, also known to interact with phosphoinositide phosphates, were also purified. Thus at least 30% of the total number of proteins identified in

PI(3,4,5)P3 Interactome our study are able to interact directly with PI(3,4,5)P3. An additional 28 proteins were identified as potential lipid associated proteins using either LIPIMAPS or the SwissProt database (Supporting Information). These proteins may provide a primary anchor site in phosphoinositide binding while the remaining proteins are presumably purified as part of interactome complexes. Furthermore, phosphoinositide binding proteins are known to engage multiple binding partners by coincidence detection using multidomain cooperation. (e.g., Phospholipid binding domain that cooperate with a SH2 domain in the recruitment of phosphorylated proteins, PH domains binding to both phosphoinositides and small GTPases).1,15,19 The engagement of ligands other than phosphoinositides has been suggested to be a common mechanism employed by a variety of phosphoinositide binding modules to enhance avidity for surfaces and to impose a restriction upon their localization.1,15,19 An interesting feature of our affinity studies is the number of purified proteins that have molecular function and processes corresponding to previously well characterized cellular functions of PI(3,4,5)P31 (Supporting Information Tables 1 and 3). A large number of the purified proteins are involved in transport, trafficking, endocytosis and exocytosis (e.g., ARF and Rab GTPases, Snare proteins, Solute carrier proteins) in cytoskeletal regulation (e.g Arf family GTpases, Rho GTPase, actin binding proteins), and in the regulation of the spatial and temporal events that coordinate cell adhesion and movement, growth and proliferation (e.g Ras, AKT, Integrins, Catenins). In total, 61% (173 proteins) of the purified proteins were classified in 4 major functional clusters: small GTPases, transport and trafficking, actin regulation and cell receptors). Furthermore, a large number of purified proteins are reported to have direct and indirect interactions with the PI3K/ AKT signaling pathway. The solute carriers proteins (e.g., SLC1, SLC2 (GLUT1, GLUT3, GLUT14), SLC7 and SLC25) specifically isolated using PI(3,4,5)P3 (Table 2), are low abundance, hydrophobic, proteins involved in the transport of ions and solutes across membranes along their electrochemical gradients.55 The PI3kinase/AKT pathway has been shown to be involved in the regulation of synthesis, activity and trafficking of SCL2 proteins such as GLUT1 (GTR1), GLUT2, GLUT3, GLUT4 and GLUT5.58,59 Furthermore the expression and trafficking of a wide variety of nutrient transporters is controlled by the PI3 kinase/AKT/mTOR pathway.60 Ion channel proteins were also indentified in our experiments (e.g., ATPase Na+/ K+ transporting polypeptides, Arsenical pump-driving ATPase and Chloride intracellular channel). These active transporters have also been shown to be regulated by membrane phosphoinositides.61 Snare and Snare binding proteins, also specifically purified using PI(3,4,5)P3 probes (Table 2), are involved in vesicular transport.62,63 Regulation of vesicular trafficking is known to be regulated by PI3 kinase which influences a number of trafficking events such as cargo selection, vesicle formation, movement and membrane fusion.22,28 These events are often mediated through stimulation of actin turnover and are regulated by Rho family GTPase effectors. Regulation of Rac1and Cdc42 by PI3kinases links receptor-ligand interaction to the induction of endocytosis as well as to actin mediated cellular functions. A large number of small GTPases of the Ras, Rho, Rab and ARF families as well as GTPase regulators and activators (e.g., Cytohesins, Rho GTPase activating protein 8) were also specif-

research articles ically purified in this study using PI(3,4,5)P3 probes (Table 3). It has been shown that phosphoinositides regulate the recruitment of Guanine Activating Proteins and Guanine Exchange Factors to membranes and also act as coreceptors with small GTPases in the recruitment of cytosolic proteins to specific subcompartments.1 PI(4,5)P2 and PI(3,4,5)P3 have been reported to target members of Ras, Rab Arf and Rho families to the plasma membrane via their polybasic amino acid clusters and palmitoyl, prenyl and myristoyl modifications.64 Members of the Ras and Rho family are particularly important for PI3 kinase function. PI3 kinase is a direct downstream effector of Ras and is activated by GTP-bound Ras binding to PI3 kinase class IA catalytic subunits.1,65 Rheb is involved in mTor activation within the PI3kinase pathway27 while RhoB is required for stability and nuclear trafficking of AKT1/AKT.65 AKT was also reported to mediate Ras down-regulation of RhoB.66 RalA and RalB GTPases are regulated by a diverse group of guanyl nucleotide exchange factors that are either Rasresponsive or are mobilized by phosphatidylinositol second messenger through a PH domain.67 Arf GTPases have been postulated to act together with members of the Rho GTPase family that have been implicated in actin cytoskeletal regulation under the control of the PI3 kinase cascade. ARF regulatory proteins (Cytohesin 1, 2, 3 and Centaurin alpha 1) were also specifically purified in our studies. These proteins are known to interact with PI(3,4,5)P3 via the specific binding property of their PH domains.56,57 A number of Rab GTPases, important regulators of vesicular transport and transport in specific intracellular compartments68 were found along with TBCD4 rab activating protein. TBCD4 was found only using PI(3,4,5)P3 and is known to be phosphorylated by AKT in response to insulin.69 Within the kinase/phosphatase cluster (Table 4), two kinases were of particular interest: RAC-alpha serine/threonine-protein kinase (AKT1) and PKN2 Serine/threonine-protein kinase N2 (PRK2). AKT1 is known to interact specifically with PI(3,4,5)P3 via its PH domain and to signal downstream of phosphatidylinositol 3-kinase. This kinase mediates the effect of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). It is also known to phosphorylate TBCD4, which was also purified in this study. The protein kinase C-like 2 PKN2 is a Rho-dependent and lipid-dependent protein kinase that can also interact and regulate 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) activity.70-73 It has also been shown to bind and inhibit AKT phosphorylation, thus inhibiting AKT downstream signaling.74 PKN kinases are known to play a role in cytoskeletal regulation, vesicle transport, glucose transport, cell adhesion, meiotic maturation and embryonic cell cycle regulation73 PKN2 possesses a C2 phospholipid binding domain and therefore is able to interact with phosphoinositides. PI(3,5)P2 and PI(3,4,5)P3 have both been shown to stimulate PKN kinase activity.73 Using an Elisa-based assay with immobilized phosphoinositides, PKN2 was shown to bind to all immobilized phosphoinositides but demonstrated stronger binding to immobilized PI(3,4,5)P3 as compared to PI(3,5)P2, PI(4,5)P2 (Figure 6). A number of receptors and cell adhesion molecules were also present (Table 5). Interestingly, proteins belonging to the cadherin adhesion complex (E-cadherin, alpha 1 and delta catenin, beta-catenin) were uniquely purified using PI(3,4,5)P3 probes. A key target for E-cadherin signaling is PI3 kinase, which is recruited to cadherin-based adhesive contacts and Journal of Proteome Research • Vol. 8, No. 7, 2009 3723

research articles subsequently activated, as shown by membrane recruitment of AKT and production of PI(3,4,5)P3 at cadherin contacts.75 This recruitment involves the binding of the p85 regulatory subunit of PI3 kinase to beta catenin.76,77 Cell adhesion molecules such as integrins can also act as upstream factors in the regulation of PI3 kinase.78 PI3K inhibitors have also been recently implicated in shifting activated integrins toward a resting state.79 Other purified receptors have also been shown to interact with PI3K: Galectin-3 is involved in tumor metastasis by interacting with integrins and promotes PI3K activation.80 Major histocompatibility complex (MHC) class I molecules, reported to activate the PI3K/Akt cascade on the surface of endothelial cells by antibody ligation81 were also identified. While a large number of the proteins identified in this study showed specificity toward the PI(3,4,5)P3 affinity probe, 50% of the proteins had been previously identified using PIP2 probes (36 using PI(3,5)P2, 41 using PI(4,5)P2 and 64 using both PIP2 supports). For example, many actin binding proteins were identified in our previous study as well as a number of known PI(4,5)P2 interacting proteins (e.g., Annexins, Septins, Clathrin, Adaptor proteins, COPI and COPII proteins).47 The number of PI(4,5)P2 interacting protein identified could reflect promiscuity, as domains that interact with PI(4,5)P2 are more diverse in structure and do not require the same stringency and affinity to those interacting with PI(3,4,5)P3.11 Furthermore, some cross reactivity may be explained by the in vitro context of our proteomic analysis; the phosphoinositide probes interact with their binding partners in the absence of the cellular lipid bilayer membrane and will therefore differ in local concentration and true cellular environment.82 If some PH domains bind with highly stereospecific recognition via the inositol headgroup, a number of phosphoinositide binding domains require additional membrane insertion and/or oligomerization components.11,15 Alternatively, incubation of the PI(3,4,5)P3 probes with cytosol extracts may have resulted in some hydrolysis of PI(3,4,5)P3 to PI(4,5)P2 by 3-phosphatase, despite the use of phosphatase inhibitors in our samples. PI(3,4,5)P3 interacting proteins were also organized using the String database in a network of both physical (direct binding interaction) and functional associations (transcriptional regulation, substrate sharing within a metabolic pathway or participation in larger multiprotein assemblies) (Supporting Information Figure 2). The establishment of such a network from our data suggests that a large number of proteins were purified as part of relatively intact interactome complexes. However, it also underlines the difficulties of discriminating between proteins that binds directly to the phosphoinositide targets and those that are purified via subsequent protein/protein interactions. However, proteins possessing phospholipid and phosphoinositide binding domains should provide a primary anchor site for such protein complexes. More than 30% of the proteins purified in this study have the capacity to interact directly with the phosphoinositide substrate (Table 1, Table 2 and Supporting Information Table 2). Additionally, phosphoinositide binding proteins are known to engage multiple binding partners by coincidence detection using multidomain cooperation. (e.g., PH domains binding to both phosphoinositides and small GTPases).15,19

Conclusion We have carried out a comprehensive proteomic analysis of PI(3,4,5)P3 interacting proteins using synthetic analogues of the PI(3,4,5)P3 phosphatidyl phospholipid. Cytosolic extracts and 3724

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Catimel et al. chromatographic fractions of LIM1215 colonic carcinoma cells were probed using phosphoinositides incorporated into liposomes or covalently linked to beads, resulting in the purification of 282 PI(3,4,5)P3 interacting proteins. Domain analysis of the purified proteins revealed that a large number of proteins possessed phosphoinositide/phospholipid binding domains (e.g., PH, PX, FERM, PTB/PID, Clathrin adaptor domains, C1, C2, Phosphatidylinositol 3- and 4-kinase, MARCKS, Calponin, Septin, PDZ, Heat and Annexin domains). Among the purified proteins, 141 proteins displayed specific interaction with PI(3,4,5)P3 and had not been previously identified using either PI(4,5)P2 and PI(3,5)P2 affinity targets. These specific PI(3,4,5)P3 proteins include solute carrier proteins (SLC1, SLC2, SLC7, SLC25 family), RRAS, KRAS, Rheb, RalA and RhoB small GTPases, ARF regulatory proteins (Cytohesin 1, 2, 3 and Centaurin alpha 1), RAC-alpha serine/threonine-protein kinase (AKT1), PKN2 Serine/threonine-protein kinase N2 (PRK2) and proteins belonging to the cadherin adhesion complex (Ecadherin, alpha 1, beta and delta catenin. Proteins could be classified into a number of molecular functions and molecular processes corresponding to the known biological function of phosphoinositides (e.g., molecular transport, protein trafficking, vesicle mediated transport, actin cytoskeletal regulation and GTPases regulated function, cell-to-cell signaling and interaction, cellular development, movement, assembly organization, growth and proliferation). Database searches and network analyses have suggested that PI(3,4,5)P3 interacting proteins were affinity purified via direct protein interaction with the phosphoinositide target as well as part of intact interactome complexes. The biological relevance of these findings will need to be validated using alternative techniques (e.g., vesicle sedimentation analyses, lipid overlay assays surface plasmon resonance,) as well as with cellular studies (e.g., fluorescent imaging, FRET). This study was performed using the LIM1215 carcinoma colonic cell line that possesses a wild type PI3 kinase protein. Further studies will now be performed using carcinoma cell lines having mutated PI3 kinase (e.g., LIM2550 (p110 chain mutation), LIM2537 p85 chain mutation)83 to assess the downstream effects of these mutations on protein signaling complexes.

Acknowledgment. This work was supported by the Australian Research Council, Discovery Project, Grant DP0770668. General support was provided by CSIRO and VESKI (Victorian Endowment for Science, Knowledge and Innovation). We thank Professor Mark Lemmon, University of Pennsylvania, Philadelphia, PA, for his generous gift of PLCδPH and Dyn1PH in plasmids. Supporting Information Available: Figure SI-1, Figure SI-2, Tables SI-1-SI-3. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Di Paolo, G.; De Camilli, P. Phosphoinositides in cell regulation and membrane dynamics. Nature 2006, 443 (7112), 651–657. (2) Berridge, M. J. Inositol trisphosphate and calcium signalling mechanisms. Biochim. Biophys. Acta 2008. doi:, 10.1016/j.bbamcr. 2008.10.005. (3) Cantley, L. C. The phosphoinositide 3-kinase pathway. Science 2002, 296 (5573), 1655–1657. (4) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso, R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D. Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 2001, 70, 535–602.

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