The PI(3,5)P2 and PI(4,5)P2 Interactomes - American Chemical Society

The PI(3,5)P2 and PI(4,5)P2 Interactomes Bruno Catimel,*,†,# Christine Schieber,‡,# Melanie Condron,† Heather Patsiouras,† Lisa Connolly,† Jenny Catimel,† Edouard C. Nice,† Antony W. Burgess,† and Andrew B. Holmes‡ 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 July 17, 2008

A comprehensive analysis of the phosphoinositide interactome has been performed using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids which were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts from colonic carcinoma cell lines. Affinity/LC/MS/MS experiments allowed identification of 388 proteins/protein complexes that appeared to interact specifically with the phosphoinositide targets: a number of novel potential phosphoinositide interacting proteins have been identified. Keywords: Phosphoinositides • proteomics • interactome • microaffinity purification

Introduction Phosphoinositides, phosphorylated derivatives of the minor membrane lipid phosphatidylinositol, play an important role in cell biology, either as precursors of second messengers or by directly interacting with proteins to manage the spatiotemporal organization of key intracellular signal transduction pathways.1-4 Over 35 distinct phosphoinositides (PIPs) and soluble inositol phosphates (IPs) have been identified in eukaryotic cells and participate in the transduction of a vast array of extracellular stimuli.5 The phosphoinositide signaling pathways play a central role in vesicle trafficking, exocytosis and secretion, membrane trafficking, actin skeletal reorganization, adhesion, migration, cell proliferation, cell death and cell metabolism.6-11 Direct signaling effects are achieved through the binding of the inositol headgroup to cytosolic proteins or cytosolic domains of membrane proteins, regulating the function of integral membrane proteins or recruiting signaling compounds to the membrane.12-14 Binding of proteins to phosphoinositides involves electrostatic interactions with the negative charge of the phosphate(s) on the inositol ring8,10 Adjacent hydrophobic amino acids can strengthen the interactions through partial penetration into the membrane.8,10 Protein surfaces that interact with phosphoinositides can also consist of stretches of clustered basic and hydrophobic residues (e.g., actin regulatory proteins such as Profilin, Villin, Gelsolin, Cofilin),10 MARCKS (Myristoylated alanine-rich C kinase substrate),15 AKAP79 (A-kinase anchoring protein),16 or folded modules such as PH (Pleckstrin homology),17 FYVE (for Fab1, YOTB, Vac1 and EEA1),18 PX (Phagocyte oxidase homology, Phox),19 ENTH/ ANTH (Epsin or AP180 N-terminal homology),20 PHD (Plant * To whom correspondence should be addressed. Dr. Bruno Catimel, tel, 61 3 9341 3134; fax, 61 3 9341 3104; e-mail, [email protected]. # These authors made equivalent contributions. † Royal Melbourne Hospital. ‡ University of Melbourne. 10.1021/pr800540h CCC: $40.75

 2008 American Chemical Society

homeodomain) zinc finger,21 PDZ (Postsynaptic density protein, Disc large Zona occludens),21 FERM (Band Four-pointone, Erzin, Radixin, Moesin)22 Phosphotyrosine binding (PTB),23 C2 (Protein kinase C homology 2) domain,24 and Gram (Glucosyltransferase, Rab like GTPase activator and Myotubularin)25 domains. Additional phosphoinositide binding domains have also been described such as the Clathrin adaptor AP1 and AP2,26 Beta arrestin -1,10 Tubby proteins27 and SH2 domains.28 Other proteins such as Septins, Annexins (calcium regulated phospholipids binding proteins)29 and Septins30 also have also been shown to display phosphoinositide binding. The functional and structural basis for phosphoinositides binding and specificity have been reported.8,10,28,31-33 Generally, the interaction between phosphoinositides and cytosolic proteins is of low affinity and many of the domains do not possess high enough affinity to act alone as localization signals. For example, some PH domains display high affinity and specificity toward specific phosphatidylinositol phosphates, while others show low affinity and cross reactivity.14 The interaction between PH domains and their binding phospholipid partners range from low micromolar to low nanomolar.34,35 However, most of these binding sites also have protein binding partners, and their protein- and lipid-binding activities influence each other in order to form high affinity membraneprotein and protein/protein interactions as well as promoting signaling complex assembly.11,14,36,37 Identifying phosphoinositide binding protein complexes may provide insights about the function, and regulation of signaling pathways involving these interacting proteins. Approximately 2% of the proteins encoded by the human genome contain canonical phosphoinositide binding domains (e.g., PH, PX).38 However, as phosphoinositide binding domains lack primary sequence similarities, the identification of novel noncanonical phosphoinositides domains using bioinformatics tools is not feasible. Therefore, a number of studies have engaged in the identification of phosphoinositide binding proteins using afJournal of Proteome Research 2008, 7, 5295–5313 5295 Published on Web 11/04/2008

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finity matrices with phosphoinositides targets, liposome incorporation49-52 in vivo assays in yeast,53,54 expressed sequence tag databases search54 and protein chips,55 while binding specificity has been characterized using a variety of techniques ranging from lipid overlay assays, titration calorimetry, vesicle sedimentation, surface plasmon resonance,56 immunoelectron microscopy,57 FRET and nuclear magnetic resonance.33,58 The studies using phosphoinositide affinity matrices have been used successfully to identify PI(3,4,5)P3,39-43,47 PI(3,4),39,40 P(3,5)P2,39,40 PI(3)P,39 Ins(1,3,4,5)P441,44,59 and Ins(1,4,5)P345 binding proteins. Only two large scale studies aimed at the identification of novel phosphoinositide proteins have been performed using PI(3,4,5)P3, PI(3,4)P2 PI(3,5)P2, PI3P and PI(4,5)P2, PI(3,4)P2 PI(3,4,5)P3 phosphoinositide beads in mammalian cells, resulting, respectively, in the identification of 32 phosphoinositide interacting proteins including ARAP3, and 21 known or potential phosphatidylinositol binding proteins.39,40 Phophoinositides incorporated in liposomes have been mainly used to characterize binding affinities of certain set of proteins toward PI(5)P, PI(4,5)P2, PI(3,4,5)P3 phosphoinositides50-52,60,61 using in particular surface plasmon resonance technology.50-52,56 Phosphoinositide liposomes have been used in a genome wide study of yeast PH domains62 as well as for probing a yeast proteome microarray.55 These studies resulted, respectively, in the characterization of 33 PH domains encoded by the Saccharomyces cerevisiae genome and analysis of the phosphoinositide binding specificity for another 150 proteins. PIP liposomes also have the potential to be used as affinity matrices.49 We have therefore carried out a systematic study to identify proteins/protein complexes in cytosolic extract from colorectal carcinoma cells using phosphinositides immobilized on beads or incorporated into liposomes. Our approach was to use a proteomics-based assay63-65 to detect proteins that bind chemically synthesized saturated analogues of PtdInsPn in which all proteins capable of specific binding to particular PtdInsP supports will be considered as potential candidates for cell signaling regulators. These affinity studies involve both the synthesis of saturated phosphatidylinositol polyphosphates having the natural (D) inositol ring configurations66 and the preparation of analogues of the natural phosphatidyl phospholipids carrying a amino terminal functionality in the fatty acid chain of the diacyl glycerol66 so that the phospholipids can be attached covalently to an affinity matrix (e.g., Affigel 10)66 Affinity purified proteins were characterized using 1DSDS-PAGE, tryptic digestion of excised gel strips and nanoRPHPLC ESI MS/MS analysis.65

Materials and Methods Synthesis and Characterization of Phosphatidylinositol Phosphates (PIPs). Sodium salts of the dipalmitoyl forms of various phosphoinositides were synthesized from myo-inositol following the protocol described previously.66,67 Key transformations included a regioselective DIBAL-mediated cleavage of the myo-inositol orthoformate and a resolution-protection step using camphor acetal. Final reductive debenzylation was afforded using palladium black in the presence of sodium hydrogen carbonate. Phosphoinositide analogues were prepared by attaching a terminal fatty acid amino function at the sn-1 position of the glycerol moiety.66 Phosphoinositide analysis was performed using proton NMR. 1H NMR spectra were recorded on Varian Inova 400 (400 MHz) or Varian Inova 500 (500 MHz) instruments at room temperature, using CDCl3 (or 5296

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other indicated solvents) as internal reference deuterium lock. The chemical shift data for each signal are given as δ in units of parts per million (ppm). The multiplicity of each signal is indicated by s (singlet), br s (broad singlet), d (doublet), t (triplet), and m (multiplet). The number of protons (n) for a given resonance is indicated by n H. Coupling constants (J) are quoted in Hz and are recorded to the nearest 0.1 Hz. Conjugation of PIPs to Affi-10 Beads. NH2-PI(3,5)P2 and NH2-PI(4,5)P2 were conjugated to Affi-10 beads (Bio-Rad). Briefly, Affi-10 beads were washed in H2O and incubated with NH2-PI(3,5)P2 or NH2-PI(4,5)P2 overnight at 4 °C with 0.5 mg PIP2/mL of beads in H2O. The beads were the blocked for 2 h at room temperature using 5 mL of 1 M ethanolamine (pH 8.0). Blank beads were prepared under the same conditions by omitting the phosphatidylinositol phosphate. Biosensor Analysis of PIP Conjugation. The efficiency of PIP conjugation to Affi beads was determined by Biacore analysis (Biacore 3000) using neomycin68 immobilized onto a CM5 carboxymethylated biosensensor surface (Biacore) by amine coupling chemistry.69 The level of conjugated PI(3,5)P2 and PI(4,5)P2 was assessed by comparison with a calibration curve generated by injecting various concentration of PIP2 (3.1 µg to 200 µg/mL) over immobilized neomycin. Production and Purification of Recombinant GST-Tagged PH Domains of Phospholipase C Delta 1 and Dynamin 1 Domain. Transformations were carried out using BL21 competent cells with PLCδPH in pGEXT2TK or Dyn1PH in pGSTag plasmid (donated by Professor M. Lemmon, University of Pennsylvania, Philadelphia, PA). Cells were plated out on LB agar containing ampicillin (50 µg/mL) and incubated for 12-16 h at 37 °C. A single colony was inoculated in 50 mL of LB/ ampicillin as a starter culture for overnight growth at 37 °C. A total of 10 mL of starter culture was used to inoculate 500 mL of LB/ampicillin and the bacterial culture was grown to an OD600 of 0.5. The culture was induced with 0.2 mM IPTG for 5 h before harvesting. The resultant bacterial pellet was lysed using CelLytic B cell lysis reagent (Sigma) and the GST fusion proteins were purified using glutathione agarose beads. GSTPLCδ1PH and GST-DYN1PH were further purified using size exclusion chromatography with a Superose 12 column (GE) equilibrated in PBS and connected to an AKTA Purifier System (GE). Protein purity was analyzed using Coomassie stained SDS-PAGE and protein identity was confirmed using LC/MS/ MS analysis.70,71 Preparation and Characterization of Liposomes. Unilammellar vesicles were prepared by an extrusion method with L-RPhosphatidylcholine (PC) (Avanti), L-R-Phosphatidylethanolamine (PE) (Avanti) and the phosphoinositide of interest at a 1:1:0.1 molar ratio, resulting in liposomes with a composition of 2.5 mM L-R-Phosphatidylcholine (PC), 2.5 mM L-R-Phosphatidylethanolamine and 250 µM phosphatidylinositol phosphates (PI(3,5)P2, or PI(4,5)P2.72 Phosphatidylserine was not used in order to reduce background binding to the negatively charged phosphoserine as has been previously observed.56 PC, PE and PIP were dissolved and mixed in chloroform in the required proportions, dried under nitrogen and stored overnight in a desiccator under vacuum. The lipids were then hydrated for 30 min in PBS and subjected to 3 freeze/thaw cycles to increase the efficiency of entrapment of water soluble compounds. Once fully hydrated, the sample was extruded using the Avanti extruder fitted with 100 nm filters.68 Liposomes were characterized using recombinant GSTtagged PH domains of Phospholipase C delta 1 and Dynamin

PI(3,5)P2 and PI(4,5)P2 Interactomes 1 domain. PC/PE, PC/PE/PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes were immobilized onto a L1 Chip (Biacore) as described previously73 and various concentration of GST-PLCδ1PH (3.5 µM to 110 nM) and GST-DYN1PH (4 µM to 125 nM) were injected over the immobilized liposomes.73 Experiments were all performed at 25 °C in 10 mM HEPES, pH 7.5, containing 150 mM NaCl. A 30 µL injection of 20 mM NaOH was used to regenerate the lipid surface after each experimental injection. LIM1215 Cytosolic Extracts. Cytosolic extracts of LIM1215 colonic carcinoma cell lines74 were prepared using a digitonin extraction process.75 Briefly, LIM1215 cells were grown to confluence in 10 dishes (10 cm diameter). After removing the culture media and rinsing with cold PBS, cells (2 × 108 cells) were scraped and incubated for 30 min at 4 °C in 10 mL of 10 mM Pipes, 100 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. The cells were removed by centrifugation at 480g for 20 min and the supernatant fluid (cytosolic fraction) was collected and cleared of debris by centrifugation at 436 000g (Beckman rotor TLA100.2, 100 000 rpm) for 15 min at 4 °C prior to the affinity experiments. Affinity Capture Techniques. Affinity capture experiments were performed using two alternative strategies. The amino phosphoinositide analogues (amino function at the sn-1 position of the glycerol moiety) of PI(3,5)P2 or PI(4,5)P2 were conjugated to Affi-gel 10 and mixed either with the crude LIM1215 cytosolic extracts or with fractionated cytosolic extracts obtained using anion exchange chromatography (see below). Briefly, cytosolic extracts were first incubated with Affi-10 blank beads (200 µL of beads for 500 µL of cytosolic extracted fraction) for 2 h at 4 °C to remove proteins which bind nonspecifically. The blank beads were removed by centrifugation (5 min at 480g) and the precleared cytosolic fraction was incubated overnight at 4 °C with PI(3,5)P2-Affi10, PI(4,5)P2 Affi-10 beads or Affi-10 blank beads (100 µL of beads for 500 µL of cytosolic extracts or column fractions). The beads were then washed five times with 1.5 mL of PBS + 0.005% 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 analyzed on a 4-12% SDS-PAGE under nonreducing conditions. Proteins were detected using sensitive Coomassie staining. A similar protocol was used for each of the anion-exchange chromatographic fractions. Affinity capture was also performed using phosphatidylinositol phosphate containing liposomes. Cytosolic extracts of colonic cells (500 µL) were incubated with 100 µL (PC/PE/ PI(3,5)P2, PC/PE/PI(4,5)P2 or control PC/PE) of liposomes overnight at 4 °C. Liposomes and liposomal bound proteins were separated from unbound proteins by size exclusion chromatography using Sephacryl S500 (GE Healthcare) packed in a HR10/30 column (see below). Cytosolic extracts without liposomes were also chromatographed under identical conditions on the size exclusion column. Size Exclusion Chromatography. LIM1215 cytosolic extracts containing liposomes (500 µL) were injected onto a Sephacryl S500 high resolution size exclusion matrix (GE) packed in a HR10/30 column and connected to an AKTA Purifier System (GE). The column was eluted with 40 mL of ammonium bicarbonate buffer (1% (w/v) at a flow rate of 1 mL/min at 25 °C. Fractions (1 mL) were collected. Proteins were detected at 280 nm. Fractions corresponding to liposomes (eluting between 10 and 12.5 mL elution volume) were concentrated to 100 µL

research articles using a Speed Vac concentrator (Christ RVC 2-25), mixed with 25 µL of SDS-PAGE buffer (2× concentrate) and analyzed using 4-12% SDS-PAGE using sensitive Coomassie detection. Anion-Exchange Chromatography. LIM1215 cytosolic extracts (10 mL) were injected using a superloop onto an anionexchange column (MonoQ HR10/10) equilibrated in 10 mM Tris-HCl (pH 7.4) containing 0.005% (w/v) Tween 20. The proteins were eluted at 25 °C from the column using a linear 0-1 M NaCl gradient generated over 25 min at a flow rate of 1 mL/min. Fractions (1 min) were collected automatically (FRAC 100, Pharmacia Biotech). Proteins were detected by absorbance at 280 nm. Three 1-min fractions (3 mL) eluting between 2 and 26 mL were pooled (Fractions 1-8) and incubated with PI(4,5)P2, PI(3,5)P2 or control beads and processed as described above. LC/MS-MS Analysis. The affinity eluates were analyzed on SDS-PAGE gels stained with a sensitive Coomassie dye. Excised gel sections were individually digested with trypsin (0.05 µg) using a robotic digestion station (MassPREP, Micromass, Altrincham, U.K.) and generated peptides were concentrated to ∼10 µL by centrifugal lyophilization (Savant) for electrosprayIon Trap (ESI-IT) tandem mass spectrometry (MS/MS) (LCQDeca, Finnigan, San Jose, CA). Protein digests (∼10 µL in 1% (v/v) formic acid) were transferred into 100 µL glass autosampler vials for fractionation 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). A linear 60-min gradient from 0-100% B with a flow rate of 0.8 µL/min at 45 °C was used. Solvent A was 0.1% (v/v) aqueous formic acid and Solvent B was 0.1% aqueous formic acid/60% (v/v) acetonitrile. The capillary HPLC was coupled online with the ESI-IT mass spectrometer for automated MS/MS analysis of individually isolated peptide ions.70,71 The mass spectrometer was operated in data-dependent mode (triple-play) to automatically switch between MS, Zoom MS (automated charge state recognition), and MS/MS acquisition, selecting the most intense precursor ion for fragmentation using CID. Where four consecutive precursor ions of the same mass were observed, dynamic exclusion was invoked for a period of 240 s. Acquired MS/MS spectra were searched against the LudwigNR database (comprising approximately 5 000 000) using the MASCOT search algorithm (v2.1.04, Matrix Science, U.K.).71 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) and Pfam (http://pfam.sanger.ac.uk/) databases. In-batch searches were also performed against the KEGG Pathway database to display all pathways which are involved and to graphically highlight the input genes within the pathway maps (http://www.genome.jp/kegg/tool/search_pathway.html). The LIPID Metabolites And Pathways Strategy database (http://www.lipidmaps.org/) and Swiss-Prot database (http:// beta.uniprot.org/uniprot/) were also interrogated by protein keywords and lipid class association to identify lipid interacting proteins and lipid-associated protein sequences. Network analyses were performed with the Search Tool for the Retrieval of Interacting Genes/Proteins database (String http://string.embl.de/).76 The network of physical and functional associations was derived from active prediction methods Journal of Proteome Research • Vol. 7, No. 12, 2008 5297

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Figure 1. Schematic of experimental protocols: PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts and chromatographic fractions from colonic carcinoma cell lines. Affinity purified proteins were characterized using 1D-SDS-PAGE, tryptic digestion of excised gel strips and nanoRPHPLC ESI MS/MS analysis.

interrogating experiments, database, and text mining using a high confidence of 0.75 with a network depth of 1.

Results The schematic of the reaction scheme for phosphoinositide synthesis is summarized in Supporting Information Figure 1A. Phosphoinositide analysis using proton NMR showed good agreement with published literature values (Supporting Information Figure 1B).66,77 Affinity experiments were performed using either PI(3,5)P2 or PI(4,5)P2 derivatives incorporated into liposomes or conjugated to Affi-10 beads. Nonspecific binding was determined using blank Affi-10 derivatized beads or control liposomes (PC/ PE). The experimental protocols are summarized in Figure 1. Liposomes Characterization. PI(3,5)P2 and PI(4,5)P2 containing liposomes were characterized by their ability to specifically bind to GST-DYN1 PH and GST-PLCδ1PH (Supporting Information Figure 2). GST-PLCδ1PH was found to bind specifically to immobilized PC/PE/PI(4,5)P2 liposomes as compared to PC/PE and PC/PE/PI(3,5)P2 liposomes.8 GSTDYN1 PH was found to bind both immobilized PC/PE/PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes as compared to PC/PE liposomes.78 Affinity Interaction Experiments Using Incorporation of Phosphatidylinositol Phosphates into Liposomes. Following incubation with LIM1215 cytosolic extracts, liposomes were injected on size exclusion chromatography in order to separate liposomes and liposome-bound proteins from residual cytosolic 5298

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proteins (Figure 2). The chromatograms obtained with PC/PE, PC/PE/PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes are shown in Figure 2, panels. B, C, and D. Liposomes eluted between 10 and 12.5 min retention time (Figure 2B-D). No peak was detected in the cytosolic extract without liposome (Figure 2A). The chromatographic fractions containing liposomes were concentrated using a Speed Vac concentrator and analyzed by SDS-PAGE in order to visualize liposome-bound proteins (Figure 2, gel insets). Representative SDS-PAGE analyses of proteins purified using PI(3,5)P2 with liposomes are shown in Figure 3A. Specific binding proteins identified in the different SDS-PAGE protein bands are listed in Table 1 of the Supporting Information. For comparison, the nonspecific binding proteins identified in the liposomes and control experiments (PC/PS liposomes and blank beads) are listed in Table 2 of the Supporting Information. Affinity Experiments Using Phosphatidylinositol Phosphates Conjugated to Affi-10 Beads. Conjugation of NH2PI(3,5)P2 and NH2-PI(4,5)P2 to Affi-10 Beads: Phosphoinositide conjugation levels between 75% and 95% were achieved for Affi-10 beads as assessed using Biacore analysis with a neomycin calibration curve (Supporting Information Figure 3). This resulted in a yield between 0.35 µmol of PIP/mL and 0.45 µmol of PIP/mL of beads. Affinity experiments were performed using both crude cytosolic extracts and fractions partially purified by anion exchange chromatography as described in Materials and Methods. Nonspecific binding was assessed by incubation with blank derivatized Affi-10 beads.

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Figure 2. Size exclusion chromatography (Sephacryl S500 HR10/30) of PIP liposomes after incubation with cytosolic extracts. (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(3,5)P2 liposomes; (D) chromatographic profile of LIM1215 cytosolic extract incubated with PI(4,5)P2 liposomes. Liposomes were shown to elute between 10 and 12.5 min (B-D). SDS-PAGE analyses of proteins bound to liposomes are shown (inset).

Figure 3. SDS-PAGE analysis of purified proteins using PI(3,5)P2. (A) Representative SDS-PAGE analysis of proteins purified using PI(3,5)P2 liposomes. Specific binding proteins were identified by comparison with control liposomes used under the same experimental conditions. (B) Representative SDS-PAGE analysis of proteins purified using PI(3,5)P2 Affi-10 beads. Specific binding proteins were identified by comparison with blank derivatized beads used under the same experimental conditions.

A representative SDS-PAGE analysis corresponding to a crude cytosolic extract pull down using PI(3,5)P2 beads is shown in Figure 3B and a representative affinity experiment using the Mono Q anion-exchange cytosol fractions with PI(4,5)P2 beads is shown in Figure 4. The anion exchange HPLC profile and the corresponding SDS-PAGE analysis of the

proteins captured using PI(4,5)P2 beads are shown, respectively, in Figure 4, panels A and B. Specific binding proteins identified using LC/MS/MS analyses are shown in Figure 4C and Supporting Information Table 1. Affinity experiments led to the identification of 388 proteins/ protein complexes that appear to interact specifically with Journal of Proteome Research • Vol. 7, No. 12, 2008 5299

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Figure 5. Venn diagram analysis of purified phosphoinositide interacting proteins: (A) Venn diagram analysis showing overlapping and nonoverlapping proteins pulled down by PI(3,5)P2 and PI(4,5)P2. (B) Venn diagram analysis showing overlapping and nonoverlapping proteins pulled down using PI(3,5)P2 beads and PI(3,5)P2 liposomes. (C) Venn diagram analysis showing overlap and non overlapping proteins pulled down using PI(4,5)P2 beads and PI(4,5)P2 liposomes.

Figure 4. Interaction of PI(4,5)P2 with cytosolic fractions following anion exchange chromatography on Mono Q. (A) Chromatographic profile of LIM1215 cytosolic extracts on Mono Q (see Materials and Methods for chromatographic conditions). (B) SDSPAGE analysis of proteins purified using PI(4,5)P2 beads. Fractions eluting between 2 and 26 mL were pooled into 3 mL of volume (Fractions 1-8) and incubated with PI(4,5)P2 beads. After incubation and extensive washing, retained proteins were subjected to SDS-PAGE and excised bands (indicated by numbers) were taken for MS-MS analysis. (C) Specific PI(4,5)P2 interacting proteins (B) identified by MS-MS analysis. Specific binding proteins were identified by comparison with blank derivatized beads used under the same experimental conditions. No specific binding proteins were found in bands 27, 37 and 39.

either PI(3,5)P2 or PI(4,5)P2 targets (Figure 5 and Supporting Information Tables 1 and 3). A total of 105 proteins were only found to interact with the PI(3, 5)P2 target, 187 proteins interacted specifically with PI(4, 5), while 96 proteins were found in both the PI(3,5)P2 and PI(4, 5)P2 affinity eluates (Figure 5 and Supporting Information Tables 1 and 3). Among the PI(3,5)P2 purified proteins, 64 were purified using liposomes, 81 using PIP beads and 56 using both beads and liposomes (Figure 5 and Supporting Information Tables 1 and 5300

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3). Within the PI(3,5)P2 purified proteins, 47 bound to liposomes, 177 bound to PIP beads and 59 interacted with both beads and liposomes (Figure 5 and Supporting Information Table 3). An additional 263 proteins bind nonspecifically to either control liposomes and derivatized blank beads (Supporting Information Table 2). Eighty of the nonspecific proteins bound to both control liposomes and beads. PIP2/Phospholipid Domains Containing Proteins. A number of PIP2 affinity purified proteins contained previously reported phosphoinosiditide recognition domains such as PH, PX, FERM, PHD, Clathrin adaptor beta Arrestin, C2, SH2, Phosphatidylinositol-4-phosphate 5-Kinase, Phosphatidylinositol 3- and 4-kinase and Phosphatidylinositol transfer protein domains (Table 1). Additionally a number of protein domains which are known to interact with PIP2 as well as charged phospholipids (e.g., phosphatidylserine) were also present: MARCKS, Calponin, Septin Annexin and Bar domains (Table 1). Fourteen proteins were found to contain HEAT repeats (Table 1). HEAT is a tandemly repeated, 37-47 amino acid long module related to armadillo/beta-catenin-like repeats that occurs in a number of cytoplasmic proteins, including some that are involved in intracellular transport processes.79 Interestingly, the HEAT domains of Importin beta and Serine/threonine-protein phosphatase 2A have been found to be structurally related to the ENTH phosphoinositide binding domain of Epsin,80 suggesting a role in PIP2 binding. The HEAT domain has also been shown to associate with acidic phospholipids at the plasma membrane.81 Additionally, a number of other proteins containing Armadillo like helical domains were also purified including Importin-7, Exportin-7, Exportin-1 (Importin-beta N-terminal domain), Adapter proteins AP-1, AP-2, AP3, and Coatomer subunits (Adaptin N terminal domain), Exportin-T and Exportin-5 (Exportin 1-like domain), Vacuolar proton pump subunit H (V-ATPase subunit H domain) and

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Table 1. Summary of PIP/Phospholipid Domains domains

protein ID

PH

PLCD3 DYN2 OSBP1 PLEK2 ARHG7 MRCKB_ SPTB2 PX SNX1 SNX2 ENTH EPN4 FERM E41L2 EZRI 41 MOES RADI Q6NUR7 TLN1 PHD TIF1B Clathrin adaptor AP1B1 AP2B1 AP2A2 AP3D1 COPB COPG2 beta arrestin ARRB1 Phosphatidylinositol 3-and 4-kinase PRKDC SMG1 Phosphatidylinositol-4-Phosphatidylinositol-5-phosphate 4-kinase PI42C Phosphatidylinositol transfer protein PIPNB SH2 STAT1 STAT3 CSK C2 PLCB3 PLCD3 PA24A NEDD4 CPNE1 ESYT1 KPCA MARCKS MARCS MRP Septin SEPT2 SEPT7 SEPT9 SEP11 Annexin ANX11 ANXA6 ANXA2 ANXA1 ANXA4 ANXA7 Calponin ACTN1 ACTN4 CNN2 FLNA FLNB FLNC PLSI PLEC1 IQGA1 IQGA3 ARHG7 TAGL2 VISL1 Gelsolin CAPG SEC23A Snare STX3 STX7 HEAT CND3 CAND1 PRKDC HEAT2 IMB1 IMB3 IPO4 Q96AG6 ECM29 VAC14 Q8NDA8 SMG1 2AAA Bar

TNPO1 SH3G2

protein name

1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta-3 Dynamin-2 Oxysterol-binding protein 1 Pleckstrin-2 Rho guanine nucleotide exchange factor 7 Serine/threonine-protein kinase MRCK beta Spectrin beta chain, brain 1 Sorting nexin-1 Sorting nexin-2 Clathrin interactor 1 (Epsin-4) Band 4.1-like protein 2 Ezrin Protein 4.1 Moesin Radixin Ezrin Talin-1 Transcription intermediary factor 1-beta AP-1 complex subunit beta-1 AP-2 complex subunit beta-1 AP-2 complex subunit alpha-2 AP-3 complex subunit delta-1 Coatomer subunit beta Coatomer subunit gamma-2 Beta-arrestin-1 (Arrestin beta 1) DNA-dependent protein kinase catalytic subunit Serine/threonine-protein kinase SMG1 phosphate 5-Kinase type-2 gamma Phosphatidylinositol transfer protein beta isoform Signal transducer and activator of transcription 1-alpha/beta Signal transducer and activator of transcription 3 Tyrosine-protein kinase CSK 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-3 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta-3 Cytosolic phospholipase A2 (cPLA2) E3 ubiquitin-protein ligase NEDD4 Copine-1 Synaptotagmin-1 Protein kinase C alpha type Myristoylated alanine-rich C-kinase substrate MARCKS-related protein Septin-2 Septin-7 Septin-9 Septin-11 Annexin A11 Annexin A6 Annexin A2 Annexin A1 Annexin A4 Annexin A7 Alpha-actinin-1 Alpha-actinin-4 Calponin-2 Filamin-A Filamin-B Filamin-C Plastin-1 Plectin-1 Ras GTPase-activating-like protein IQGAP1 Ras GTPase-activating-like protein IQGAP3 Rho guanine nucleotide exchange factor 7 Transgelin-2 Visinin-like protein 1 Macrophage-capping protein Protein transport protein Sec23A Syntaxin-3 Syntaxin-7 Condensin complex subunit 3 Cullin-associated NEDD8-dissociated protein 1 DNA-dependent protein kinase catalytic subunit HEAT repeat-containing protein 2 Importin subunit beta-1 Importin subunit beta-3 Importin-4 KIAA1833 protein Proteasome-associated protein ECM29 homologue Protein VAC14 homologue Putative uncharacterized protein DKFZp434I0113 Serine/threonine-protein kinase SMG1 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform Transportin-1 Endophilin-A1

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

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Figure 6. Molecular function of purified proteins. Proteins were classified according to their molecular functions using the IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown.edu). (A) Proteins purified using PI(3,5)P2 affinity target. (B) Proteins purified using PI(4,5)P2 affinity target.

Vacuolar proton pump subunit H (CAS/CSE domain). The relatively high occurrence of the HEAT and Armadillo like domains in our affinity purified proteins suggests a role for phospholipids/phosphoinositide binding in the function of these proteins. Lipid binding domains were also identified: START (PCTPlike protein), SCP-2 sterol transfer family (Nonspecific lipidtransfer protein) and Apolipoprotein-L (Apolipoprotein-L2). In addition, a number of proteins were identified as potential lipid associated proteins using LIPIDMAPS (Lipid Metabolites and Pathways Strategy) (http://www.lipidmaps.org/) or SwissProt (http://beta.uniprot.org/uniprot/) database. The LIPIDMAPS database identified 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 (Supporting Information Table 4). Molecular Function Classification. The proteins identified in our affinity studies can be classified by molecular function (Figure 6 and Supporting Information Table 1). Interestingly, a large number of proteins were classified as small GTPases or GTPase regulators and activators (17% (35 proteins) for PI(3,5)P2 and 13% (39 proteins) for PI(4,5)P2) (Figure 6, Table 2). Phosphoinositides have been shown to regulate the recruitment of GAPs and GEFs to membranes and to also act as coreceptors with small GTPases in the recruitment of cytosolic proteins to specific subcompartments.11 It has been also recently shown that PI(4,5)P2 and PI(3,4,5) P3 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.82 Rho GTPases that have been implicated in the control of cytoskeletal events and the coordination of diverse cellular 5302

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functions (e.g., cell shape, attachment and motility, vesicular trafficking endocytosis and exocytosis) have been identified in our studies along with Rho GTPases regulatory proteins.83-85 RhoG was identified using PI(3,5)P2 and Rho C, Cdc42, Rac1 and Rac2 using both phosphoinositides targets (Table 2). Rho GTPases regulatory proteins (Rho GDP dissociation inhibitor, Rho guanine nucleotide exchange factor 7 (PH domain) and DOCK1/ELMO1 complex were copurified with the GTPases. Rho GDP dissociation inhibitor is also known to interact with Rac1, Rac2 and cdc42,83 while Rho guanine nucleotide exchange factor 7 and DOCK1/EMO1 act as Rac1 guanine nucleotide exchange factor.86 FMR1-interacting protein 1, involved in formation of membrane ruffles and lamellipodia, was also copurified (Supporting Information Table 1), probably via its interaction with the active GTP-bound form of Rac1. ELMO has also been recently shown to interact with ERM proteins (Erzin, Radixin, Moesin, phosphoinositide interacting proteins), playing a possible role in localizing ERM proteins in certain cellular sites.87 ERM proteins have also been shown to displace the GDP-bound form of Rho proteins from Rho GDI.83 Three members of the Arf family of small GTPases (Arf1, Arf4, Arf5, Table 2) that are regulators of protein and membrane intracellular trafficking, and cytoskeletal remodeling were also purified using PI(3,5)P2 (Arf5), PI(4,5)P2 (Arf4) or both phosphoinositides targets (Arf1).83 ADP-ribosylation factor-like protein 2 and Brefeldin A-inhibited guanine nucleotide-exchange protein 2, that promote guanine-nucleotide exchange on Arf1 and Arf5, were also found (Table 2). A number of Rab GTPases, important regulators of vesicular transport and transport in specific intracellular compartments, were identified:83 Rab5a, Rab10, Rab11b and Rab14 were associated with PI(4,5)P2, Rab 15 and Rab21 with PI(3,5)P2,

research articles

PI(3,5)P2 and PI(4,5)P2 Interactomes a

Table 2. GTPases and GTPase Regulators AC

ID

protein name

molecular function

PI(3,5)P2

PI(4,5)P2

P08134 P84095 P11234 P63000 P15153 P60953 P62820 P20339 P61020 P51148 P51149 P61006 P61026 P62491 Q15907 P61106 P59190 Q9UL25 P84077 P36404 P18085 P84085 P62826 Q9Y295 Q9Y6B6 Q15019 Q9UHD8 Q16181 Q9NVA2 O00429 P50570 P20591 Q9NTK5 P46060 Q07960 Q15436 Q7L576 P46940 Q13576 Q86VI3 P52566 P31150 Q9H223 Q9H4M9 Q15404 Q9Y6D5 Q14155 Q14185 Q9H7D0 Q3YEC7

RHOC RHOG RALB RAC1 RAC2 CDC42 RAB1A RAB5A RAB5B RAB5C RAB7A RAB8A RAB10 RAB11A RAB11B RAB14 RAB15 RAB21 ARF1 ARL2 ARF4 ARF5 RAN DRG1 SAR1B SEPT2 SEPT9 SEPT7 SEPT11 DNM1L DYN2 MX1 OLA1 RGP1 RHG01 SC23A CYFP1 IQGA1 IQGA2 IQGA3 GDIS GDIA EHD4 EHD1 RSU1 BIG2 ARHG7 DOCK1 DOCK5 PARF

Rho-related GTP-binding protein RhoC Rho-related GTP-binding protein RhoG Ras-related protein Ral-B Ras-related C3 botulinum toxin substrate 1 Ras-related C3 botulinum toxin substrate 2 Cell division control protein 42 homologue Ras-related protein Rab-1A Ras-related protein Rab-5A Ras-related protein Rab-5B Ras-related protein Rab-5C Ras-related protein Rab-7a Ras-related protein Rab-8A Ras-related protein Rab-10 Ras-related protein Rab-11A Ras-related protein Rab-11B Ras-related protein Rab-14 Ras-related protein Rab-15 Ras-related protein Rab-21 ADP-ribosylation factor 1 ADP-ribosylation factor-like protein 2 ADP-ribosylation factor 4 ADP-ribosylation factor 5 GTP-binding nuclear protein Ran Developmentally regulated GTP-binding protein 1 GTP-binding protein SAR1b Septin-2 Septin-9 Septin-7 Septin-11 Dynamin-1-like protein (Dynamin-like protein) Dynamin-2 (EC 3.6.5.5) Interferon-induced GTP-binding protein Mx1 Obg-like ATPase 1 Ran GTPase-activating protein 1 Rho GTPase-activating protein 1 Protein transport protein Sec23A Cytoplasmic FMR1-interacting protein 1 Ras GTPase-activating-like protein IQGAP1 Ras GTPase-activating-like protein IQGAP2 Ras GTPase-activating-like protein IQGAP3 Rho GDP dissociation inhibitor 2 Rab GDP dissociation inhibitor alpha EH domain-containing protein 4 EH domain-containing protein 1 Ras suppressor protein 1 BrefeldinA-inhibited guanine nucleotide exchange protein 2 Rho guanine nucleotide exchange factor 7 Dedicator of cytokinesis protein 1 Dedicator of cytokinesis protein 5 Putative GTP-binding protein Parf

Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase Small GTPase, Small GTPase, Small GTPase, Small GTPase Small GTPase Small GTPase Small GTPase G-protein G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator G-protein modulator Kinase modulator Enzyme regulator GEF GEF GEF Nucleotide binding

• • • • • • •

•

• • • • • • • • •

• • • • • • • • • • • • • • •

• • • • • • • • •

• • • • • • • • • •

• • • • • • •

• • • • • • • • • • •

• • • •

a Phosphoinositides 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.

while Rab1a, Rab5b, Rab5c, Rab7a, Rab8a and Rab11a were isolated using both phosphoinositides (Table 2). Three additional GTPases belonging to the small GTPase family have been purified: Ran (purified using PI(3,5)P2 and PI(4,5)P2), GTP binding Sar1b, (PI(4,5)P2) and Ras-related protein Ral-B (PI(3,5)P2) (Table 2). A putative GTP-binding protein Parf was also identified using PI(3,5)P2. Ran is involved in nucleocytoplasmic transport and was purified with Ran GTPase activating protein 1 as well as a number a protein also involved in nuclear transport (e.g., Importins, Exportins) (Table 2, Table 3, Table 1 in Supporting Information).83 Sar1b has been reported to be involved in recruitment of membrane lipids into assembling COPII coated transport carriers (Sec23/24, Sec13/ 31, Table 3)83,88 and the transport of chylomicrons from the endoplasmic reticulum to the Golgi apparatus.88 RalB, located primarily in intracellular vesicles, has been involved in a wide range of functions, including protein trafficking, vesicle transport, cytoskeleton dynamics and mitogenic regulation.83,89 Ral

GTPases are regulated by a diverse group of guanyl nucleotide exchange factors that are either Ras-responsive or are mobilized by phosphatidylinositol second messenger through a PH domain.83,90 A number of GTPases that do not belong to the Ras superfamily were also purified: for example, Dynamin 2, Interferon-induced GTP binding protein MX1 (Dynamin family), Dynamin 1 like protein, Developmentally regulated GTP binding protein 1 and Septin 2, 7, 9 and 11 (Table 2). Septins have been shown to play a role in cytokinesis, formation of focal adhesion complexes and cell polarity complexes.30 Dynamins have been shown to be involved in clathrin-mediated endocytosis.91 A second cluster encompasses proteins involved in transport and trafficking: 18% (37 proteins) for PI(3,5)P2 and 16% (44 proteins) for PI(4,5)P2 (Figure 6, Table 3). In particular, components of clathrin complexes (Clathrin, Adaptor proteins AP-1, AP-2 and AP-3), COPI (coatomer subunits) and COPII Journal of Proteome Research • Vol. 7, No. 12, 2008 5303

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Table 3. Proteins Involved in Transport and Trafficking AC

ID

protein name

molecular function

P04083 P50995 P07355 P09525 P08133 P20073 Q10567 O94973 P63010 O14617 Q92572 Q9BQE5 O43681 O43264 O43633 Q9H444 Q00610 P53675 Q14677 P09496 P53618 P53621 P35606 P48444 Q9Y678 Q9UBF2 P61923 Q8WTW3 Q9H9E3 Q9Y2V7 Q99829 Q96A65 O14980 P55060 Q9C0E2 Q9HAV4 Q9UIA9 O43592 O60763 O00410 Q14974 Q8TEX9 O95373 Q14847 O60664 Q13765 P22307 P22059 Q9Y365 P48739 Q15436 O94979 Q13596 O60749 Q86V81 O43617 Q92973 P38606 P21281 P21283 P36543 Q9UI12 Q96QK1 P49407 Q9H4M9 Q13277 O15400

ANXA1 ANX11 ANXA2 ANXA4 ANXA6 ANXA7 AP1B1 AP2A2 AP2B1 AP3D1 AP3S1 APOL2 ARSA1 ZW10 CHM2A CHM4B CLH1 CLH2 EPN4 CLCA COPB COPA COPB2 COPD COPG COPG2 COPZ1 COG1 COG4 COG6 CPNE1 EXOC4 XPO1 XPO2 XPO4 XPO5 XPO7 XPOT USO1 IMB3 IMB1 IPO4 IPO7 LASP1 M6PBP NACA NLTP OSBP1 PCTL PIPNB SC23A SC31A SNX1 SNX2 THOC4 TPPC3 TNPO1 VATA VATB2 VATC1 VATE1 VATH VPS35 ARRB1 EHD1 STX3 STX7

Annexin A1 Annexin A11 Annexin A2 Annexin A4 Annexin A6 Annexin A7 AP-1 complex subunit beta-1 AP-2 complex subunit alpha-2 AP-2 complex subunit beta-1 AP-3 complex subunit delta-1 AP-3 complex subunit sigma-1 Apolipoprotein-L2 Arsenical pump-driving ATPase Centromere/kinetochore protein zw10 homologue Charged multivesicular body protein 2a Charged multivesicular body protein 4b Clathrin heavy chain 1 (CLH-17) Clathrin heavy chain 2 Clathrin interactor 1 (Epsin-4) Clathrin light chain A Coatoamer subunit beta Coatomer subunit alpha Coatomer subunit beta Coatomer subunit delta Coatomer subunit gamma Coatomer subunit gamma-2 Coatomer subunit zeta-1 (Zeta-1 COP) Conserved oligomeric Golgi complex component 1 Conserved oligomeric Golgi complex component 4 Conserved oligomeric Golgi complex component 6 Copine-1 (Copine I) Exocyst complex component 4 Exportin-1 Exportin-2 (Importin-alpha re-exporter) Exportin-4 (Exp4) Exportin-5 (Exp5) (Ran-binding protein 21) Exportin-7 (Exp7) (Ran-binding protein 16) Exportin-T (tRNA exportin) General vesicular transport factor p115 Importin beta-3 (RanBP5) Importin subunit beta-1 Importin-4 Importin-7 (RanBP7) LIM and SH3 domain protein 1 (LASP-1) Mannose-6-phosphate receptor-binding protein 1 Nascent polypeptide-associated complex subunit R Nonspecific lipid-transfer protein Oxysterol-binding protein 1 PCTP-like protein Phosphatidylinositol transfer protein beta isoform Protein transport protein Sec23A Protein transport protein Sec31A Sorting nexin-1 Sorting nexin-2 THO complex subunit 4 Trafficking protein particle complex subunit 3 Transportin-1 (Importin beta-2) Vacuolar ATP synthase catalytic subunit A Vacuolar ATP synthase subunit B Vacuolar ATP synthase subunit C 1 Vacuolar ATP synthase subunit E 1 Vacuolar ATP synthase subunit H Vacuolar protein sorting-associated protein 35 Beta-arrestin-1 EH domain-containing protein 1 Syntaxin-3 Syntaxin 7

Transfer/carrier protein; Transfer/carrier protein; Transfer/carrier protein Transfer/carrier protein; Transfer/carrier protein; Transfer/carrier protein Membrane traffic protein Membrane traffic protein Membrane traffic protein Membrane traffic protein Vesicle coat protein Protein, lipid binding Transporter;Nucleotide phosphatase Microtubule family cytoskeletal protein Molecular function unclassified Transfer/carrier protein Vesicle coat protein Vesicle coat protein Membrane traffic protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Vesicle coat protein Transporter Transporter Transporter Membrane traffic protein Membrane traffic protein Transfer protein, RNA-binding protein Transporter Transfer/carrier protein Transfer protein receptor Transfer/carrier protein Transfer protein, RNA-binding protein Membrane traffic protein Transporter Transfer/carrier protein Transfer/carrier protein; Transporter Nonmotor Actin binding protein Transfer/carrier protein Basic helix-loop-helix transcription factor transferase, lipid binding Transfer/carrier protein Transfer/carrier protein tranporter, lipid binding Vesicle coat protein Vesicle coat protein Membrane traffic regulatory protein Membrane traffic regulatory protein RNA-binding protein Transporter Transfer/carrier protein Hydrolase, Hydrogen transporter; Hydrolase, Hydrogen transporter; Hydrolase, Hydrogen transporter; Hydrolase, Hydrogen transporter; Hydrolase, Hydrogen transporter; Membrane traffic protein Regulatory molecule G-protein modulator; Membrane traffic SNARE protein SNARE protein

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

• • •

• • •

• • •

•

•

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

• • • • • • • • • • • •

• • • • • • • • • • •

• • • • • • • • • • • • • • •

• •

• • • • • • • • •

a

Phosphoinosites interacting proteins which have a molecular function involved in transport and trafficking were identified using IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research. Small GTPases involved in transport and trafficking are not listed in this Table).

(coat protein complex) (Sec 23, Sec31) and the small GTPase, Sar1b, were purified in our studies. While Clathrin mediates endocytic protein transport, and transport from ER to Golgi, coatomer proteins primarily mediate intra-Golgi transport, as well as the reverse Golgi to ER transport.92 CPOII proteins are involved in vesicle budding from the ER to the Golgi.93 Clathrin 5304

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interactor 1, that contains an ENTH PIP binding motif, was copurified with Clathrin. Adaptor proteins have been shown to interact with phosphoinositides,10,94 while Sec 23 has also been reported to display affinity for acidic phospholipids.93 Beta Arrestin 1, a specific regulator of GPCRs, was also purified. It has been shown to bind to phosphoinositide10,94 and to support

PI(3,5)P2 and PI(4,5)P2 Interactomes endocytosis of GPCR via its interaction with Clathrin in a phosphoinositide dependent manner.10,95 Sorting nexin 1 and 2 (PX domain) were also identified and are reported to play a role in vesicular transport.96 Syntaxins (Snare proteins) have been shown to play a important role in membrane fusion and exocytosis.97 Additional proteins involved in protein export and secretory pathways98 (e.g., Signal recognition particle 54, 68, and 72 kDa), protein transport (e.g., Vacuolar protein sorting-associated protein 35, Solute carrier family 1, General vesicular transport factor p115) and lipid transport and transfer (Oxysterol-binding protein 1, Phosphatidylinositol transfer protein, Nonspecific Lipid-transfer protein, Copine 1) were also identified (Table 3). Proteins involved in nuclear import and export (e.g., Importins and exportins (Table 3)) were copurified with Ran, suggesting a role of phosphoinositides in the regulation of nuclear import/ export. Small GTPases, known to play an important role in transport and trafficking (e.g., Rab, ARF and Ran GTPases, see GTPases as discussed above, Table 2) could also be included in this cluster. Proteins kinases and phosphatases were also well represented among the PIP2 affinity purified proteins: 8% (16 proteins) for PI(3,5)P2 and 10% (29 proteins) for PI(4,5)P2) (Figure 6, Table 4). Three kinases were found to belong to the phosphatidylinositol kinase family: DNA-dependent protein kinase and Serine/threonine-protein kinase SMG1 (Phosphatidylinositol 3- and 4-kinase domain) and Phosphatidylinositol5-phosphate 4-kinase type-2 gamma. DNA-dependent protein kinase, purified using PI(3,5)P2 and PI(4,5)P2, is known to play a major role in the repair of DNA strand breaks, DNA replication, and gene transcription.99 This kinase has been suggested to be the missing Phosphoinositide-dependent protein kinase-2 kinase.100 SMG1 kinase is involved in both mRNA surveillance and genotoxic stress response pathways.101 Protein kinase CR (purified using PI(4,5)P2) is regulated by the second messengers calcium and diacylglycerol and plays a critical role in multiple signaling pathways.102 Recent studies have demonstrated that the C2 domain of PKCs interacts specifically with PtdIns(4,5)P(2) through its C2 domain, this interaction being critical for the membrane localization of these enzymes in living cells.103 Serine/Threonine Kinase MRCK (Myotonic dystrophy protein kinase-like beta, isolated using both PI(3,5)P2 and PI(4,5)P2) contains a number of functional domains, among them a Protein kinase C domain, a Phorbol esters/diacylglycerol binding domain, a Cdc42/Rac-binding p21 binding domain, a Pleckstrin homology (PH) domain and a Citron domain. The Citron homology domain is often found after cysteine rich and Pleckstrin homology (PH) domains at the C-terminal end of the proteins.104 This kinase has been shown to interact with the GTP bound form of Cdc42 and Rac, and may act as a downstream effector of Cdc42 in cytoskeletal reorganization.104 Ste20-like kinase was purified using both phosphoinositides. It is known to be part of a microtubule-associated complex that is targeted to adhesion sites105 and implicated in the regulation of cytoskeletal dynamics as well as cell cycle progression.106 GSK3β, a key downstream target of PI3-kinase/AKT survival signaling pathway,102 was purified using PI(3,5)P2. Axin, a known binding partner of GSK3β within the Wnt signaling pathways, was also purified with PI(3,5)P2 beads. A potential phospholipid binding site has been identified in the C-terminal DIX domain of axin that may mediate vesicle interaction.107 Cdc2 (Cell division control protein 2 homologue, CDK1) that

research articles is known to play a key role in the control of the eukaryotic cell cycle was also purified using PI(3,5)P2 substrate.108 The proliferating cell nuclear antigen (PCNA), whose role in the cell cycle control is recognized on the basis of the interaction with cyclins and cyclin-dependent kinases,109 was copurified in our studies. The extracellular signal-regulated kinases Mitogen activated protein kinase 1 (MAPK1 or ERK2) and Mitogen activated protein kinase 3 (ERK1) that regulates cell proliferation and cell differentiation were isolated on PI(3,5)P2 and PI(4,5)P2, respectively, while the stress-activated MAPK8 (c-Jun N-terminal kinase 1) was identified using PI(4,5)P2. Six carbohydrate kinases were purified, among them the splice variant of Pyruvate kinase (PMK2)110 (Table 4). This protein, which was isolated using both PIP substrates, is involved in aerobic glycolysis and has recently been shown to be important for cancer metabolism and cell growth.110 It is negatively regulated by binding to phosphotyrosine peptides, in particular Enolase and Lactate dehydrogenase peptides:110,111 both Enolase and Lactate dehydrogenase were purified in our studies. Other kinases associated with the PIP2 affinity eluate were CSK kinase (C-Src kinase), serine/threonine protein kinases OSR1 and WNK1 which have been, respectively, involved in down regulation of Src family kinases,112 regulation of downstream kinases in response to environmental stress and regulation of electrolyte homeostasis through its effects on synaptotagmin function.113 WNK1 has also been shown to phosphorylate and activate OSR1.114 Three serine/threonine-protein phosphatases were purified (Table 4): Protein phosphatase 1(PP1), Protein phosphatase 2A (PP2A) and Protein phosphatase 5 (PP5). Both PP1 and PP2 are known to be involved in the regulation of a variety of cellular processes and signaling pathways.115,116 PP5 is regulated by mTOR117 and has been shown to interact with DNAPKcs118 and to regulate Raf-1 signaling pathways.119 Protein Set, isoform 2 (SET/I2PP2A), a potent inhibitor of Protein phosphatase 2A, was also purified in our proteomic pull down. SET/I2PP2A was recently shown to associate with Rac1 and act as a signaling amplifier for Rac1-mediated cell migration.120 The inositol phosphatase BPNT1 was also isolated in our studies (Table 4). Actin Cytoskeletal Cluster. A large number of actin binding proteins were identified: 13% (27 proteins) for PI(3,5)P2 and 16% (45 proteins) for PI(4,5)P2) (Figure 6, Table 5). Many of these map to the canonical actin cytoskeletal pathways (KEGGS, Supporting Information Figure 4). Many actin-binding proteins that sequester actin monomers, sever actin filaments and cap the fast growing barbed end of filaments are regulated by specific interactions with phosphoinositides (in particular PI(4,5)P2).121 Increases in the concentration of PIP(4,5)P2 tends to promote actin assembly, while PI(4,5)P2 hydrolysis causes actin disassembly.121 Cytoskeletal and actin interacting proteins have been shown to interact with phosphoinositides using either PIP domains (e.g., FERM, PH), positively charged and/ or hydrophobic residues (e.g., Gelsolin and Calponin domains) or stretches of positively charged residues (MARCKS). Myosin Ic has been shown to possess a putative Pleckstrin homology domain that mediates binding to PIP(4,5)P2.122 This site is conserved within Myosin I isoforms. Phosphoinositides present in actin rich structures may recruit Myosin-I isoforms to function in endocytosis, secretion and membrane retraction.122 Alternatively, the barbed-end directed motor activity of myosin I isoform may orientate the growing ends of the actin Journal of Proteome Research • Vol. 7, No. 12, 2008 5305

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Table 4. Kinases and Phosphatases AC

ID

P17252 P27361 P28482 P45983 P49841

KPCA MK03 MK01 MK08 GSK3B

Q9H4A3

WNK1

Q9Y5S2

MRCKB

Q96Q15 P78527

SMG1 PRKDC

O95747 P06493

OXSR1 CDC2

Q9H2G2

SLK

P41240

CSK

O00764 Q8TBX8

PDXK PI42C

Q9BSD7

CA057

Q13057 P14618 P08237 P17858 Q01813 Q8N0W3 Q9Y223

COASY KPYM K6PF K6PL K6PP FUK GLCNE

Q3LXA3 P23919 P11908

DAK DTYMK PRPS2

Q9UNW1

MINP1

O95861 Q92562 P30153

BPNT1 SAC3 2AAA

P53041

PPP5

P62136

PP1A

P63151

2ABA

Q01105

SET

Q5VXV2 Q8IZ21 O43681

Q5VXV2 PHAR4 ARSA1

protein name

Protein kinase C alpha type (PKC-A) Mitogen-activated protein kinase 3 Mitogen-activated protein kinase 1 Mitogen-activated protein kinase 8 Glycogen synthase kinase-3 beta (GSK-3 beta) Serine/threonine-protein kinase WNK1 Serine/threonine-protein kinase MRCK beta Serine/threonine-protein kinase SMG1 DNA-dependent protein kinase catalytic subunit Serine/threonine-protein kinase OSR1 Cell division control protein 2 homologue (Cyclin-dependent kinase 1) (CDK1) STE20-like serine/threonine-protein kinase Tyrosine-protein kinase CSK (C-SRC kinase) Pyridoxal kinase Phosphatidylinositol-5-phosphate 4-kinase type-2 gamma Probable UPF0334 kinase-like protein C1orf57 Bifunctional coenzyme A synthase Pyruvate kinase isozymes M1/M2 6-phosphofructokinase, muscle type 6-phosphofructokinase, liver type 6-phosphofructokinase type C L-fucose kinase Bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase Dihydroxyacetone kinase Thymidylate kinase Ribose-phosphate pyrophosphokinase 2 Multiple inositol polyphosphate phosphatase 1 precursor 3′(2′),5′-bisphosphate nucleotidase 1 SAC domain-containing protein 3 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A Serine/threonine-protein phosphatase 5 (PP5) Serine/threonine-protein phosphatase PP1-alpha catalytic subunit (PP-1A) Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B Protein SET (Phosphatase 2A inhibitor I2PP2A) SET translocation Phosphatase and Actin regulator 4 Arsenical pump-driving ATPase

molecular function

Serine/threonine Serine/threonine Serine/threonine Serine/threonine Serine/threonine

protein protein protein protein protein

PI(3,5)P2

PI(4,5)P2

kinase kinase kinase kinase kinase

•

Serine/threonine protein kinase

•

Serine/threonine protein kinase

•

•

Serine/threonine protein kinase Serine/threonine protein kinase

•

• •

Serine/threonine protein kinase Serine/threonine protein kinase

•

Serine/threonine protein kinase

•

Tyrosine protein kinase

•

• •

• • •

•

•

Kinase Kinase

• •

Kinase

•

Kinase; Carbohydrate Carbohydrate Carbohydrate Carbohydrate Carbohydrate Carbohydrate

kinase kinase kinase kinase kinase kinase

Nucleotide binding, transferase Nucleotide kinase Transferase ligase

•

•

• • • • • • •

• • •

Nucleotide phosphatase

•

Nuclease; phosphatase Phosphatase Phosphatase

•

• • •

Phosphatase

•

Phosphatase

•

Phosphatase

•

Phosphatase inhibitor

•

Phosphatase inhibitor Phosphatase modulator Nucleotide phosphatase

• • • •

a Phosphoinosites 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.

filament toward the membrane and PIP cytoskeletal regulators.122 There is also evidence that PI(4,5)P2 bind to Tubulin without affecting tubulin GTP binding or GTP hydrolysis.123 These observations may explain the presence of Tubulin and microtubule family cytoskeletal protein in our experiments. Pleckstrin 2, that has been shown to interact with PI3K and to regulate actin organization and cell spreading, was also purified using PI(4,5)P2 substrate. Proteins were also classified in 2 other broader groups: binding (receptor, protein, peptide, lipid, nucleic acid binding) 5306

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representing 14% (27 proteins) for PI(3,5)P2 and 14% (42 proteins) for PI(4,5)P2 and enzymes (e.g., ligases, lyases, synthases, hydrolases, oxidoreductases, metalloproteases) corresponding to 9% (18 proteins) for PI(3,5)P2 and 16% (48 proteins) for PI(4,5)P2. Additional minor functional groups that were identified include cell adhesion molecules (6% for PI(3,5)P2 and 3% for PI(4,5)P2), transcription/translation factors (3% for PI(3,5)P2, 4% for PI(4,5)P2), and microtubules proteins (3% for PI(3,5)P2 and 3% for PI(4,5)P2) and unclassified molecular functions (9% for PI(3,5)P2, 3% for PI(4,5)P2).

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PI(3,5)P2 and PI(4,5)P2 Interactomes a

Table 5. Proteins Associated with the Regulation of the Actin Cytoskeleton System AC

ID

P60709 P63261 P62736 P61163 P14649 P15311 P19105 P26038

ACTB ACTG ACTA ACTZ MYL6B EZRI MLRM MOES

P35241 Q15149 Q5T928 Q6NUR7 P60660 P48059

RADI PLEC1 Q5T928 Q6NUR7 MYL6 LIMS1

O00159 O00160 O43795 O94832 P06753 P35579 P35749 Q12965 Q7Z406 Q9UM54 Q13182 O43491 O75369 P11171 P12814 P21333 P23528 P35611 P37802 P40121 P60981 Q01082 Q13813 Q14315 Q14651 Q14847

MYO1C MYO1F MYO1B MYO1D TPM3 MYH9 MYH11 MYO1E MYH14 MYO6 Q13182 E41L2 FLNB 41 ACTN1 FLNA COF1 ADDA TAGL2 CAPG DEST SPTB2 SPTA2 FLNC PLSI LASP1

Q99439 Q9Y6W5

CNN2 WASF2

Q9Y490 O15145

TLN1 ARPC3

P52907

CAZA1

P47755

CAZA2

P47756 P40121 O43491

CAPZB CAPG E41L2

protein name

Actin, cytoplasmic 1 (Beta-actin). Actin, cytoplasmic 2 (Gamma-actin) Actin, aortic smooth muscle Alpha-centractin Myosin light polypeptide 6B Ezrin (p81) (Cytovillin) (Villin-2) Myosin regulatory light chain 2 Moesin (Membrane-organizing extension spike protein) Radixin Plectin-1 Sorbin and SH3 domain containing 1 Villin 2 Myosin light polypeptide 6 LIM and senescent cell antigen-like-containing domain protein 1 Myosin-Ic Myosin-If Myosin-Ib Myosin-Id. Tropomyosin alpha-3 chain Myosin-9 Myosin-11 Myosin-Ie Myosin 14 Myosin-VI Myosin regulatory light chain Band 4.1-like protein 2 Filamin B Protein 4.1 Alpha-actinin-1 Filamin A Cofilin-1 (p18) adducin 1 (alpha) Transgelin-2 Macrophage-capping protein Destrin Spectrin beta chain, brain 1 Spectrin alpha chain, brain Filamin C Plastin-1 LIM and SH3 domain protein 1 (LASP-1) Calponin-2 Wiskott-Aldrich syndrome protein family member 2 Talin-1 Actin-related protein 2/3 complex subunit 3 F-actin capping protein subunit alpha-1 F-actin capping protein subunit alpha-2 F-actin capping protein subunit beta Macrophage-capping protein Band 4.1-like protein 2

molecular function

PI(3,5)P2

PI(4,5)P2

Structural molecule Structural molecule Structural molecule Actin and actin related protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein Actin binding cytoskeletal protein

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• • • • • • • •

Actin Actin Actin Actin Actin Actin

binding binding binding binding binding binding

cytoskeletal cytoskeletal cytoskeletal cytoskeletal cytoskeletal cytoskeletal

protein protein protein protein protein; protein

Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin binding motor protein Actin family cytoskeletal 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 Nonmotor actin binding protein

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• • • • • • •

• • • •

• • • •

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

Nonmotor actin binding protein Nonmotor actin binding 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

•

•

•

• •

a Phosphoinosites 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.

Biological Process Classification. The PIP2 interacting proteins can also be organized according to their molecular processes (Supporting Information, Table 5). These molecular processes included protein trafficking, molecular transport, cell signaling, cellular assembly and organization, cellular function and maintenance, cell-to-cell, signaling and interaction, cell cycle, cellular movement, tissue development and function, cancer, small molecule biochemistry, DNA replication, recombination, and repair, nucleic acid, carbohydrate and lipid metabolism. This classification further reflects the broad array of molecular processes regulated by phosphoinositides that have been implicated by our experiments.

Network Analysis. Network analysis (see Materials and Methods) of the proteins associated with PI(3,5) P2 and PI(4,5)P2 confirms that a number of protein complexes associated with specific functions have been identified from our data (Supporting Information Figures 5 and 6, respectively). These network diagrams also highlight the proteins that can interact directly with PIP2 among the purified protein/protein complexes. The PI(4,5)P2 network encompasses more protein interactions, reflecting the larger number of proteins purified using this phosphoinositide. Some significant differences were observed between the two interacting networks. Within the PI(3,5)P2 network (Supporting Information Figure 5), small Journal of Proteome Research • Vol. 7, No. 12, 2008 5307

research articles GTPases regulation, cell/cell interaction (Integrins, CD81, CD9, CD44, RDX, ERZ, MSN), meiosis and mitosis regulation (PKRDC, Cdc2, MAPK3, PCNA) nuclear transport (Ran, Importins, Exportins), and small GTPases regulation (RHO G, ARHG7) were uniquely identified. By contrast, within the PI(4,5)P2 network (Supporting Information Figure 6), transport and trafficking (Clathrin, Clathrin adaptors, Coat I proteins), actin cytoskeletal modulation, regulation of signal transduction (STAT1, STAT2), signalosome complexes (COP proteins (CSN2, CSN3,CSN6, CSN8)), carbohydrate metabolism (Pyruvate kinsase, Enolase) and small GTPases regulation (DOCK1, ELMO, RHGO1) were more prominent.

Discussion The number of cellular processes that have previously been reported to be regulated by phosphoinositides is very broad and includes signal transduction at the cell surface, membrane trafficking, permeability and transport, regulation of cytoskeleton and nuclear events, as well as adhesion, migration, cell proliferation, cell metabolism and cell death.2,4-7,124 Despite the vast array of proteins interacting with phosphoinositides, few proteomic studies have been performed to date to identify proteins which interact with phosphoinositides in complex biological samples. Two affinity-based studies using phosphatidylinositol phosphate derivatives as bait (PI(3,4,5)P3, PI(3,4)P2, PI(3,5)P2 and PI(3)P) were performed using mammalian cells.39,40 In these studies, using pig leucocyte extracts from 35 L of blood,39 over 30 phosphoinositide interacting proteins were identified using peptide mass fingerprinting and/or MS/MSbased sequencing. This work was performed principally using PI(3,4,5)P2 and PI(3,4)P2, making detailed comparison with our studies (done with PI(3,5)P2 and PI(4,5)P2) difficult.39 However, similar to our results, a number of proteins with phosphoinositide binding domains were found (e.g., PH domain,) as well as a number of proteins with similar molecular function, for example, small GTPase regulators (Ras-GAP, CDC42-GAP, ARAP3, R Centaurin, and Cytohesin-4), Phospholipase L2, Myosin, Phosphofructokinase and an Inositol phosphatase. The other study on mammalian PIP binding proteins40 was performed using macrophages isolated from mouse bone marrow using cleavable PI(4,5)P2 PI(3,4)P2 and PI(3,4,5)P3) phosphoinositide affinity baits and MS/MS analysis. This resulted in the purification of 10 known and 11 novel potential phosphatidylinositol signaling proteins. Again, some of the proteins isolated in that study contained phosphoinositide recognition domains: PH, FERM and gelsolin.40 Several of the proteins they identified were also found in our analysis: Talin, Rab5c, PRDX and Guanine nucleotide-binding protein subunit beta 2. Pasquali et al.40 also found Vacuolar protein sortingassociated protein 29, a component of the retromer complex. A different component of this complex, Vacuolar protein sorting-associated protein 35, as well as related proteins (Vacuolar protein sorting-associating protein 4B, Vacuolar protein sorting-associated protein VTA1), were identified in our experiments. A novel assay in yeast,53 involving the transformation of a yeast strain which is temperature sensitive for CDC25 with a construct that expresses activated Ras with the PH domain of interest (AKT, BTK), resulted in the identification of several new PH domain-containing proteins (Gab12, Dos, Myosin X and Sbf1) that bind (PI(3,4)P2 and/or PI(3,4,5)P3. The only largescale study was performed using a yeast proteome microarray representing 5800 different yeast proteins that were screened 5308

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Catimel et al. 55

for their ability to interact with phospholipids. A total of 150 proteins were found to bind to phosphoinositides (PI(3)P, PI(4)P, PI(4,5)P2, PI(3,4)P2, PI(3,4,5)P3) with some specificity as compared to binding to phosphatidylcholine. In this study, 35% of the lipid binding proteins were uncharacterized, while 45% of the known proteins were found to be either membrane associated or to have a membrane spanning region or lipid modification (e.g., prenylation). Identified proteins that we can relate to our studies are cell cycle regulating kinases, Mitogen activated protein kinases, Pyruvate kinase, protein phosphatases PP1 Septins, GTPase-activating protein, Meiosisspecific protein with similarity to Phospholipase B, Sorting proteins involved in vacuolar targeting, Vacuolar ATPases, Nuclear transport factor (karyopherin/importin), components of the Dynactin complex, Clathrin adaptor proteins, myosin motors, Protein disulfide isomerase, Nucleolar DEAD-box protein and Ubiquitin conjugating enzymes.55 The remaining proteins isolated such as ribosomal proteins, histones, elongation factors, DNA polymerase, Aspartyl-tRNA synthetase, ATP synthases, and CTP synthase were classified in our study as nonspecific as they display binding to control beads or liposomes (Supporting Information Table 2). A possible limitation of the yeast proteome microarray study55 is that the GST-yeast recombinant proteins used may not have been correctly folded or have the correct post-translational modifications. Furthermore, proteins arrayed on the chip55 displayed little phosphoinositide binding specificity. Confirming this observation, a genome-wide analysis of membrane targeting by S. cerevisiae Pleckstrin homology domains showed that high specificity and specific phosphoinositide binding is not a common property of yeast PH domains.62 Among all 33 PH domains encoded by this genome, only one PH domain was found to bind with high affinity and specificity, while six bound with moderate affinity but little specificity.62 We have performed a large-scale mammalian proteomic study using phosphoinositdides (PI(3,5)P2 or (PI(4,5)P2) as targets with cytosolic extracts of human colonic carcinoma cells as the starting material. Digitonin was used in our studies to prepare cell cytosolic extracts. Digitonin is a detergent that binds and precipitate sterols, forming pores in cellular membranes and allowing extraction of cyosolic cellular contents.125 Our solubilization procedure (2 × 108 cells/10 mL, 30 min incubation at 4 °C with 0.0125% digitonin) led to an enriched cytoplamic extract (Supporting Information, Table 1) as shown by the cellular distribution of the purified proteins: only 12% of proteins were found to have specific membrane localization, 10% specific cytoskeletal localization and 5% specific nuclear localization and cytoskeletal localization. Affinity studies were performed with phosphoinositides conjugated to beads or incorporated into liposomes in order to provide a background of membrane phospholipids in a more physiological context. The use of either beads or liposomes as bait allowed the purification of different pools of proteins. For example, small GTPases and GTPas regulators (e.g., RhoC, Rho G, Rab, Rho guanine nucleotide exchange factor 7, DOCK1), Septins and cell adhesion molecules (e.g., integrins, A33 antigen, CD44, CD9) were preferentially found using phosphoinositide incorporated in liposomes (Supporting Information Tables 1 and 3,). In contrast, proteins involved in actin cytoskeletal dynamics and transport were mostly purified using phosphoinositide derivatized beads. Other proteins such as kinases and phosphatases were found on both substrates.

research articles

PI(3,5)P2 and PI(4,5)P2 Interactomes Previous studies have shown that prefractionation steps (e.g., chromatography, gradient centrifugation techniques) increase proteome coverage by reducing sample complexity.126 The combination of prefractionation and specific affinity capture techniques allowed deeper mining of proteins present in biological samples at low levels.126 Therefore, an anion-exchange chromatographic step was performed prior to affinity pull down, resulting in the identification of additional proteins such as Mitogen activated kinase 1, 3 and 8, Signal transducer and activation of transcription 1 and 3, Glycogen phosphorylase, Fatty acid synthase, Phospholipase A, Rho activating protein 1, beta Arrestin, Pyridoxal kinase, Protein farnesyltransferase, Multiple inositol polyphosphate phosphatase, Serine/threonine-protein phosphatase 1 and 5, Serine/threonine-protein kinase OSR1, Sorbin and SH3 domain containing 1, Proliferating cell nuclear antigen and Centromere/kinetochore protein zw10. A crucial part of all affinity studies is the ability to discriminate between specific and nonspecific binding. Low-abundance proteins that bind specifically to the phosphoinositide bait can be masked by more abundant specific and nonspecific proteins binding to the chromatographic matrix. Competition experiments using free phosphoinositide were not performed due to limited amount of phosphoinositide derivatives. Therefore, preclearing and control experiments were conducted using blank derivatized beads and control liposomes under the same experimental conditions. In both set of experiments, 31% of nonspecific proteins were found to bind to blank beads or PC/PE liposomes A number of the nonspecific proteins identified in this study are commonly found in proteomic experiments carried out in our laboratory; for example, heat shock proteins (12 proteins), histones and chromosomal proteins (10), cytoskeletal keratins (14), tubulins (9), 14-3-3 proteins (4), translation initiation factors (24) and nuclear ribonucleoprotein (10). A large number of ribosomal proteins were found in the affinity experiments (52) with 33 of them identified in control experiments. These were all classified in the nonspecific pool. Overall, 60% of proteins identified in the nonspecific pool displayed nucleic acid/nucleotide binding properties. Nucleic acids have recently been identified as a source of contamination and false results in affinity experiments by contributing to general background and mediating interactions between bait and ligand.127 Another recent study has also highlighted a number of individual proteins, protein families and functionally related protein groups that are highly represented in all proteomic studies, regardless of the experiment, tissue or species.128 These include keratin proteins, tubulins, glutathione S-transferases, stress proteins (heat-shock proteins), proteasome subunits, heterogeneous ribonucleoprotein particle subunits and elongation factors: all of these were identified as nonspecific in our analysis (Supporting Information Table 2). Annexin, Pyruvate kinase and Enolase were also found to be present in many studies.128 These proteins were classified in our study as specific (Supporting Information Table 1) since they were not found in control experiments using either the blank beads or control lysosomes. The proteomic strategy we have described has enabled us to probe phosphoinositide interacting proteins in complex mammalian samples (2 × 107 cells /mL lysate) and has led to the identification of potential novel phosphoinositide interacting proteins and protein domains: 388 proteins/protein complexes appeared to interact specifically with phosphoinositide targets (105 proteins interacted with PI(3, 5)P2, 187 proteins with PI(4, 5) and 96 with both PI(3,5)P2 and PI(4, 5)P2 (Figure 5 and Supporting Information Tables 1 and 3)).

A larger number of proteins was purified using PI(4,5)P2, reflecting the cellular abundance of this phosphoinositide and the least stringent requirement of PI(4,5)P2 interacting domains as compared to the 3-phosphoinositide binding domains.37 A number of phosphoinositide binding domains (e.g., PH, PX, ENTH, FERM, PHD, Clathrin adaptor, beta Arrestin, Phosphatidylinositol-4-phosphate 5-Kinase domain, Phosphatidylinositol 3- and 4-kinase, C2, SH2, MARCKS, Septin, Annexin, Calponin, Gelsolin, Snare) have been identified among the phospholipids/phosphoinositide recognition. In summary, 85 proteins have been identified with known phosphoinositide binding domains, with a further 22 small GTPases that also have phosphoinositide binding properties. Thus, approximately 27% of the total number of proteins identified in our study appears to be capable of interacting directly with our PIP substrates. However, the presence of lipid binding domains such as START, SCP-2 sterol transfer and the identification of 38 proteins with potential lipid binding properties (Supporting Information Table 4) suggest that binding may also be achieved through the diacylglycerol chain instead of the polyphosphorylated inositols. As an example, axin possesses a potential phospholipid binding site that has been identified in the C-terminal DIX domain of the molecule.107 With the use of biosensor and vesicle sedimentation analyses, purified axin DIX domain has been shown to bind PC, PC/PE, PC/PS and PC/PIP liposomes with a similar affinity (Supporting Information Figure 7). A limitation of our study is the difficulty to discriminate between proteins that binds directly to the phosphoinositide targets and presumably purified as part of interactome complexes. However, proteins possessing phospholipid and phosphoinositide binding domains should provide a primary anchor site in protein complexes binding. Furthermore, proteins can be purified as part of interactome complexes due to the fact that 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).36,129 Further characterization of the interactome complexes might be facilitated by the addition of additional nondenaturing separation steps such as size exclusion chromatography or sucrose gradient in order to further resolve individual protein/ protein complexes prior to anion exchange chromatography and affinity pull down using beads and liposomes. This step will help to identify more precisely which proteins are found interacting in specific complexes. It is also possible to include subcellular fractionation as part of the original sample preparation step to further define the role of the signaling networks identified. The development of such proteomic strategies can be used in the study of the cellular responses mediated by phosphoinositides, to identify signaling pathways that are affected by a change of phosphoinositide cellular concentration (e.g., 3-phosphoinositide signaling cascade, PI3kinase mutations) and as well as a tool in drug discovery and biomarker development.

Conclusion With the use of analogues of the natural PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids, we have carried out a comprehensive proteomic analysis of phosphoinositide interacting proteins. The combination of alternative purification strategies (phosphoinositides incorporated into liposomes or covalently linked to beads or anion-exchange fractionation of cytosolic cell extracts) combined with stringent control experiments allowed the purification of a Journal of Proteome Research • Vol. 7, No. 12, 2008 5309

research articles significantly larger number of proteins compared to previous proteomic studies.39,40 Domain analysis of the purified proteins revealed that a large number of proteins possessed phosphoinositide binding domains (e.g., PH, PX, FERM, clathrin adaptor domains, β-Arrestin, C2, SH2, Phosphatidylinositol-4-phosphate 5-Kinase domain, Phosphatidylinositol 3- and 4-kinase, Marcks, Calponin, Septin and Annexin domains). A potentially novel phosphoinositide binding domain (HEAT domain) was also identified. Proteins could be classified into a number of groups corresponding to the known biological function of phosphoinositides (transport, trafficking, cell cycle, actin cytoskeletal regulation and GTPases regulated function). Database searches and pathways analyses have suggested that protein/proteins complexes were affinity purified via specific proteins interacting directly with the phosphoinositide targets. In addition, a large number of potential PI(3,5)P2 and PI(4,5)P2 binding proteins have also been identified. The biological relevance of these findings will ultimately need to be validated using alternative techniques (e.g., surface plasmon resonance, lipid overlay assays) as well as with cellular studies (e.g., fluorescent imaging, FRET). This study provides an initial detailed assessment of the phosphoinositide interactome and suggests potential phosphoinositide specificity for further biochemical and biological characterization.

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 1: synthesis and characterization of phosphatidylinositol phosphates (PIPs). (A) Summarized reaction scheme of PI(3,5)P2 and PI(4,5)P2 synthesis. (B) Proton NMR data of PI(3,5)P2, PI(4,5)P2 and aminoterminal analogues. Figure 2: binding of recombinant GST-tagged PH domain of Phospholipase C delta 1 (PI(4,5)P2 binding) and Dynamin 2 (PI(3,5)P2 and PI(4,5)P2 binding). PC/PE, PC/PE/ PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes were immobilized onto a L1 chip by injecting 100 µL liposomes at 10 µL/min over the sensor chip surface. After washing with 30 µL of 20 mM NaOH, stable immobilization levels of approximately 6800RU, 7200RU and 6800RU were obtained, respectively, for PC/PE, PC/PE/ PI(3,5)P2 and PC/PE/PI(4,5)P2. Various concentrations of GSTPLCδ1PH and GST-DYN1 were injected over immobilized liposomes. The sensorgrams shown have been subtracted with the corresponding signal obtained when the sample was passed over immobilized PC/PS liposomes. No binding was observed when various concentration of GST (4 µM to 125 nM) was injected over the immobilized liposomes (not shown). (A) Injection of GSTPLCδ1PH (3.5 µM, 1.75 µM, 875 nM, 437 nM, 218 nM, 109 nM) over immobilized PC/PE/PI(3,5)P2. (B) Injection of GST-PLCδ1PH (3.5 µM, 1.75 µM, 875 nM, 437 nM, 218 nM, 109 nM) over immobilized PC/PE/PI(4,5)P2. (C) Injection of GST-DYN1 (4 µM, 2 µM, 1 µM, 500 nM, 250 nM, 125 nM) were injected over immobilized PC/PE/PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes. (D) Injection of GST-DYN1PH (4 µM, 2 µM, 1 µM, 500 nM, 250 nM, 125 nM) were injected over immobilized PC/PE/PI(3,5)P2 and PC/PE/PI(4,5)P2 liposomes. Figure 3: phosphoinositide conjugation monitored using immobilized neomycin on biosensor surface. (A) Immobilisation of neomycin onto a CM5 sensor chip using NHS/EDC chemistry. An immobilization level of 0.3 ng/ mm2 was obtained. (B) Injection (30 µL at 10 µL/min) of various 5310

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Catimel et al. concentration of PI(4,5)P2 (200, 100, 50, 25, 12.5, 6.25, 3.1 µL/ mL)onto immobilized neomycin. (C) Calibration curve of PI(4,5)P2 binding to immobilized neomycin. (D) Injection of PI(4,5)P2 (50 µL/mL) before and after conjugation to Affi-10 beads. Figure 4: mapping PI(3,5)P2 and PI(4,5)P2 purified proteins onto the canonical actin cytoskeletal regulation pathway (KEGG number). In-batch search was performed with the phosphoinositide purified proteins against the KEGG Pathway database to display the canonical actin pathway and to graphically highlight the input proteins than are involved in the pathway map. Figure 5: PI(3,5)P2 protein interaction network. Proteins purified using PI(3,5)P2 targets were organized via direct interactions and molecular function using the Search Tool for the Retrieval of Interacting Genes/Proteins database (String http://string.embl.de/).76 The network of physical and functional associations was derived from active prediction methods interrogating experiments, database, and text mining using a high confidence of 0.75 with a network depth of 1. Figure 6: PI(4,5)P2 protein interaction network; proteins purified using PI(4,5)P2 targets were organized via direct interactions and molecular function the Search Tool for the Retrieval of Interacting Genes/Proteins database (String http:// string.embl.de/). The network of physical and functional associations was derived from active prediction methods interrogating experiments, database, and text mining using a high confidence of 0.75 with a network depth of 1. Figure 7: analysis of axin-DIX domain binding to PC, PC/PE, PC/PE/PI(3,5)P2 and PC/PE/ PI(4,5)P2. (A and B) Biosensor analysis of human axin-DIX (residues 742-762) to immobilized phospholipid liposomes onto a L1 chip. (A) Axin-DIX domain (reduced and alkylkated, monomeric form) was injected (30 µM, 30 µL at 10 µL/min) over (1)PC, (2)PC/PE, (3)PC/PE/PI(3,5)P2 and (4)PC/PE/PI(4,5)P2 (2.5 mM PC, 2.5 mM PE and 250 µM PIP2). Axin-DIX domain was found to bind the different liposomes with a similar affinity. (B) Axin-DIX domain (nonreduced and nonalkylkated, tetrameric form, 30 µM) was injected (30 µL at 10 µL/min) over immobilized (1)PC, (2)PC/ PE, (3)PC/PE/PI(3,5)P2 and (4)PC/PE/PI(4,5)P2. Axin-DIX domain was found to bind the different liposomes with a similar affinity. (C-E) Sedimentation analysis of the binding of axin-DIX domain, GST-PLCδ1PH domain and GST. (C)Axin-Dix domain (reduced and alkylkated, tetrameric form, 7 µM) was incubated for 15 min at 25 °C with 75 µL of sucrose-loaded unilamellar phospholipid composed of 2.5 mM PC, 2.5 mM PE and 250 µM PI(3,5)P2. After centrifugation at 85 000 rpm for 15 min, the proportion of protein in the lipid pellets and supernatants was analyzed using Coomassie-stained SDS-PAGE. Axin-DIX domain was found to bind similarly to PC, PC/PS and PC/PE/PI(3,5) liposomes. (D) Sedimentation analysis was also performed as a positive control with GST-PLCδ1PH domain under the same experimental condition as axin_DIX domain. GST-PLCδ1PH was found do bind specifically to PC/PE/PI(3,5)P2 liposomes. (E) Sedimentation analysis was also performed as a negative control with GST- under the same experimental conditions as axin_DIX domain. GST was found in the supernatant and therefore did not bind to the phospholipid liposomes. Table 1: specific proteins purified using PI(3,5)P2 and PI(4,5)P2 targets. Proteins identified for each phosphoinositide are shown (shaded according to their purification using liposomes, beads or both liposomes and beads substrates). Protein accession number, ID and name are as in UniprotKB. Table 2: Venn diagram data analysis of purified phosphoinositide interacting proteins; Excel sheet1, Proteins isolated using PI(3,5)P2 liposomes; Excel sheet2, Proteins isolated using PI(3,5)P2 beads; Excel sheet3, Proteins isolated using both PI(3,5)P2 liposomes and PI(3,5)P2 beads; Excel sheet4, Proteins isolated using PI(4,5)P2

PI(3,5)P2 and PI(4,5)P2 Interactomes liposomes; Excel sheet5, Proteins isolated using PI(5,5)P2 beads; Excel sheet 6, Proteins isolated using both PI(4,5)P2 liposomes and PI(4,5)P2 beads, Table 3: nonspecific proteins identified using PI(3,5)P2 and PI(4,5)P2 targets; Proteins identified on liposomes or beads are shown (shaded shaded according to their purification using liposomes, beads or both liposomes and beads substrates)). Protein accession number, ID and name are as in UniprotKB. Table 4: potential lipid associated proteins (LIPID Metabolites and Pathways Strategy database (http://www.lipidmaps.org/) and Swiss-Prot database (http://beta.uniprot.org/uniprot/). The databases were interrogated by protein keywords and lipid class association to identify lipid interacting proteins and lipid-associated protein sequences. Table 5: biological processes classification for PI(3,5)P2 and PI(4,5)P2 purified proteins. Proteins were classified according to their biological processes using the IProClass Integrated Protein Informatics Resource for Genomic & Proteomic Research (http://pir.georgetown.edu). This material is available free of charge via the Internet at http://pubs.acs.org.

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