Proteomic Analysis of Src Family Kinases Signaling Complexes in

Proteomic Analysis of Src Family Kinases Signaling Complexes in Golgi/Endosomal Fractions Using a Site-Selective Anti-Phosphotyrosine Antibody: Identification of LRP1-Insulin Receptor Complexes ´ ric Winstall,§ Nicolas Bilodeau,# Annie Fiset,# Marie-Chloe´ Boulanger,† Sanjeev Bhardwaj,‡ E † ,# Joseé N. Lavoie, and Robert L. Faure* Department of Pediatrics, CHUL-CRCHUQ, Centre de Recherche en Cance´rologie, HDQ-CRCHUQ, and Plate-forme Proteómique, CHUL-CRCHUQ, Universite´ Laval, Que´bec, G1V 4G2, Canada, and Applied Biosystems, Framingham, Massachusetts 01701 Received May 30, 2009

A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulinmediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (RP42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling proteins. One of these proteins, low density lipoprotein-related protein 1 (LRP1), which is a known SFK substrate, was found to associate with the internalized insulin receptor (IR), suggesting the presence of a co-internalization process. The identification of these proteomes should, thus, contribute to a better understanding of the molecular mechanisms that regulate trafficking events and insulin clearance. Keywords: Liver • fractions • Golgi • endosomes • Src signaling • phosphoproteins • proteomics • insulin receptor • low density lipoprotein receptor-related protein 1 (LRP1) • insulin clearance

Introduction The nonreceptor Src family kinases (SFKs) are involved in almost every aspect of a cell’s life, including mitogenesis, proliferation, survival, cell adhesion and migration.1 The localization of SFKs to the plasma membrane, cytoskeleton, endosomes, Golgi complex, nucleus and a number of other potentially important and unexplored targets within intracellular membranes illustrates the complexity of SFK signaling.2 Recent evidence indicates that SFKs and v-Src reside in endosomes, and upon activation, they are trafficked to the plasma membrane through a Rab11 and actin pathway.3 This suggests that SFKs exploit endosomal traffic to regulate their * To whom correspondence should be addressed: Dr. Robert L. Faure, Cell Biology Laboratory, CHUL/CHUQ Medical Research Center, 2705 Laurier Blvd., Room RC-9800, Que´bec, G1V 4G2, Canada. Tel.: +1 418-656-4141 ext. 48263. Fax: +1 418-654-2753. E-mail: [email protected]. # Department of Pediatrics, Universite´ Laval. † Centre de Recherche en Cance´rologie, Universite´ Laval. ‡ Applied Biosystems. § Plate-forme Proteómique, Universite´ Laval.

708 Journal of Proteome Research 2010, 9, 708–717 Published on Web 11/30/2009

own transport and may also regulate the transport of some effectors. The main argument supporting the role of SFKs in the regulation of the endocytic compartment is that the trafficking of receptor tyrosine kinases requires SFKs and uses the same endosomal and actin-dependent pathway.4 However, it is still unclear where, when and how SFKs act within this pathway. A systematic characterization of the protein content of endosomes and Golgi fractions was recently conducted by mass spectrometry protein identification coupled with organelle purification.5 Despite the accuracy of current mass spectrometers and the high level of purity achieved, it is difficult to identify the signaling elements masked by the presence of abundant proteins under stimulated and unstimulated conditions.6-8 Several experimental approaches were previously described for a comprehensive analysis of a given phosphoproteome.9 In the current work, we describe the analysis of a SFK-targets subproteome of hepatic Golgi/endosomal (G/E) membranes. These studies were based on enrichment of SFK 10.1021/pr900481b

 2010 American Chemical Society

Proteomic Analysis of Src Family Kinases Signaling Complexes substrates and putative partners using a phosphotyrosine antibody, the P42-2 antibody directed toward a tyrosine motif typical of SFK substrates. The P42-2 antibody was raised against the major phosphosite of an adenovirus protein target of SFKs, E4orf4,10 which acts as a substrate and a regulator of SFK signaling at recycling endosomes.11 Because the P42-2 antibody was found to cross-react with up-regulated SFK substrates in response to E4orf4, we reasoned that it could recognize a SFK consensus motif, as shown previously for other phosphospecific antibodies broadly reactive to subclasses of kinase substrates.12 Our data identifies a series of known and putative SFK substrates or associated partners that could control trafficking events in vivo by mechanisms that are yet to be determined. We also provide evidence that the internalized insulin receptor (IR) is associated with the Src substrate LRP1.

Experimental Section Reagents and Antibodies. Porcine insulin was obtained from Sigma (St. Louis, MO). The antibody directed against the β-subunit of the IR (rabbit polyclonal, 188430) was obtained from BD Transduction Laboratories (Lexington, KY). The monoclonal anti-phosphotyrosine (PY) antibody (PY20) was obtained from Sigma Chemicals (St. Louis, MO). The antibody against the Transferrin Receptor (TfR) was obtained from Zymed Laboratories, Inc. (South San Francisco, CA). Antibodies against Lyn (sc-7274, H6) and the low density lipoprotein receptor-related protein 1 (LRP1) (A-18, sc-16168; H-80, sc25469) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-phospho-E4orf4 (Y42) 42-2 antibody was produced as reported.13 This antibody was raised against a Tyr motif phosphorylated by SFKs on E4orf4, a viral protein that hijacks SFK signaling at recycling endosomes and dysregulates endosomal traffic by upregulating a selective subset of SFK targets that cofractionate with Rab11a-endosomes.11,14 The serum was adsorbed on immobilized phosphorylated peptide HEGVY[PO3H2]IEPEARGRLC using a SulfoLink Kit (BioLynx, Brockville, ON) and blocked against an excess of nonphosphorylated peptide during immune detection. The specificity of the purified 42-2 antibody was tested by Western blot analysis of E4orf4 immune complexes and total cell lysates from cells transfected with wild-type Flag-E4orf4. The blots were compared to those of cells transfected with mutant FlagE4orf4 (Y42F) alone or together with c-Src or v-Src to induce maximum tyrosine phosphorylation of Ad2 E4orf4 and Src substrates. For immunoblot studies, we used the enhanced chemiluminescence kit, Western Plus (Perkin-Elmer Life Sciences Inc., Boston, MA), and Immobilon-P transfer membranes (Millipore, Bedford, MA). Reagents for SDS-PAGE were obtained from Bio-Rad (Mississauga, ON). The protein tyrosine phosphatase (PTP) inhibitor, bis peroxovanadium 1,10-phenanthroline (bpV(phen)), was synthesized as described.15 All other chemicals were of analytical grade and were purchased from either Fisher Scientific (Sainte-Foy, QC) or Roche Laboratories (Laval, QC). Subcellular Fractionation. Female C57BL/6 mice (12-15 week old, 25-30 g body weight), supplied by Charles River Laboratories, Inc. (Saint-Constant, QC), were maintained under standard laboratory conditions with food and water available ad libitum, except that food was removed 18 h prior to liver collection. All animal procedures were approved by the CPACHUQ (certificate 2008055-2). When mentioned, bpV(phen) was injected (0.3 mg/100 g body weight, intraperitoneal) 16 h

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and 30 min before insulin injection (1.5 µg/100 g body weight). Livers were excised rapidly at the noted times and minced in ice-cold homogenization buffer (250 mM sucrose, 50 mM HEPES (pH 7.4), 40 mM sodium fluoride, 1 mM MgCl2, 1 mM benzamidine, 1 mM PMSF). The G/E fraction was prepared immediately, as described previously.16 The liver weight was 0.85 ( 0.15 g (n ) 103). The yield of the G/E fraction was 0.48 ( 0.09 mg of protein/g of liver weight (n ) 42). The G/E fraction was permeabilized (0.1% Triton X-100) as described.13 The protein recovery following the permeabilization procedure was 66.3 ( 4.2% (n ) 10). Protein content was measured using a modification of the Bradford method with bovine serum albumin (BSA) as the standard. When proteins were separated by 1-D electrophoresis (1-DE), the gel lane containing the permeabilized G/E fraction proteins was cut into 44 gel slices that were used for in-gel tryptic digestion. Affinity Chromatography. Freshly isolated and permeabilized G/E fractions (8.03 ( 0.65 mg proteins, n ) 3) were solubilized (4 °C, 60 min) in the presence of 0.3% Empigen BB and centrifuged at 240 000g for 30 min. Supernatants (7.63 ( 0.04 mg of protein, n ) 3) were loaded on a wheat germ lectin (WGL) affinity column, and the retained proteins were eluted as described previously.17 The WGL eluates (0.29 ( 0.02 mg of protein, n ) 3) were applied to the RP42-2 affinity column (Carbolink Column, Pierce, Rockford, IL), and proteins were eluted with 8 mL of 100 mM glycine, pH 2.4. Eight fractions (1 mL) were obtained and immediately neutralized with 100 µL of neutralizing buffer (Tris 1 M, pH 7.5). Fractions with strong RP42-2 signals were concentrated at 4 °C to a final volume of 80 µL with a single microcon (YM-3, Millipore, Billerica, MA). Purified proteins were then submitted to SDS-PAGE, and the major bands were stained with SYPRO Ruby. Stained bands were excised, placed in 96-well plates and then washed with water. Tryptic digestion was performed with a MassPrep liquid handling robot (Waters, Milford, MA) according to the manufacturer’s specifications and to the protocol of Shevchenko et al.18 with the modifications suggested by Havlis et al.19 Briefly, proteins were reduced with 10 mM dithiothreitol (DTT) and alkylated with 55 mM iodoacetamide. Trypsin digestion was performed with 105 mM modified porcine trypsin (Sequencing grade, Promega, Madison, WI) at 58 °C for 1 h. Digestion products were extracted using 1% formic acid/2% acetonitrile followed by 1% formic acid/50% acetonitrile. The recovered extracts were pooled, vacuum centrifuge dried and then resuspended into 8 µL of 0.1% formic acid. Four microliters were analyzed by mass spectrometry. Mass Spectrometry. Peptides were separated by online reversed-phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ES MS/MS). The experiments were performed with a Thermo Surveyor MS pump connected to a LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, CA). Peptide separation took place on a PicoFrit column BioBasic C18, 10 cm × 0.075 mm internal diameter (New Objective, Woburn, MA), with a linear gradient from 2 to 50% solvent B (acetonitrile with 0.1% formic acid) over a duration of 30 min, at 200 nL/min (obtained by flow-splitting). Mass spectra were acquired using a data-dependent acquisition mode using Xcalibur software version 2.0. Each full scan mass spectrum (400-2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion (30 s Journal of Proteome Research • Vol. 9, No. 2, 2010 709

research articles exclusion duration) function was enabled, and the relative collisional fragmentation energy was set to 35%. Isobaric N-Terminal Labeling (iTRAQ). One hundred micrograms of permeabilized G/E proteins was acetone precipitated. Each protein pellet was then solubilized in 0.5 M triethylammonium bicarbonate (TEAB) (pH 8.5) supplemented with 0.1% (w/v) SDS. Cysteine residues were reduced with 5 mM Tris (2-carboxyethyl) phosphine (TCEP) for 1 h at 60 °C and then blocked with 8 mM methyl methanethiosulfonate (MMTS) for 10 min at room temperature. Protein samples were digested with 5 µg of modified porcine trypsin (Promega) in the presence of 10 mM CaCl2 for 18 h at 37 °C. The final SDS concentration at this point in the procedure was 0.05% (w/v). The efficiency of protein digestion was assessed by SDSpolyacrylamide gel electrophoresis using undigested and digested aliquots of proteins. Tryptic peptides from independent experiments were labeled with the iTRAQ 114, 115, 116, and 117 reagents according to the manufacturer’s protocol (Applied Biosystems, Carlsbad, CA). Labeled samples were then combined and dried in a vacuum concentrator. Tryptic peptides were loaded on a 0.1 × 150 mm (5 µm, 100 Å) C18 column and eluted over a 90-min period with a linear gradient of acetonitrile in water and 0.1% TFA. The eluate was mixed online with R-cyano-4-hydroxycinnamic acid (5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid) and Glu-fibrinopeptide (25 fM final concentration) and spotted on a 24 × 24 format MALDI plate. Samples were analyzed on a MALDI-TOF-TOF instrument (4700 Proteomics Analyzer) for protein identification and quantitation. Samples were analyzed in positive ion mode in both reflector and MS/MS acquisitions with laser repetition rate at 200 Hz. In MS/MS mode, 1 kV collision energy without collision gas was used. MS spectra were acquired in the m/z range of 800-4000. MS/MS spectra were acquired on top 10 ions for each spot with a signal-to-noise threshold of 50. Isoelectric Focusing (IEF) of Labeled Peptides. Peptides were resuspended in 325 µL of Milli-Q water containing 0.25% (v/v) ampholytes (Bio-Lyte 3/10 Ampholyte, Bio-Rad). The resulting solution was used to rehydrate an 18-cm IPG gel strip (pH 5-8) for 10 h. Conditions for the isoelectric focusing of peptides were as follows: 250 V for 15 min, 10 000 V for 3 h, 10 000 V for 60 000 V/h and then a hold at 50 V until peptides were extracted. Any excess of overlaying oil was gently blotted away from the IPG strip, and the strip was cut into 36 pieces of 5 mm each. IPG strip pieces were transferred into a 96-well plate, and peptides were eluted from the gel pieces by two successive extractions (15 min each with shaking) that were subsequently pooled. The first extraction was done in 100 µL of a 1% formic acid, 2% acetonitrile solution, and the second extraction was done in 100 µL of 1% formic acid, 50% acetonitrile solution. Extracted peptides were then dried using a vacuum concentrator and resuspended in 25 µL of 0.1% formic acid. Database Searching. All MS/MS samples were analyzed using Mascot (Matrix Science, London, U.K.; version 2.2.0). Mascot was set up to search the uniref100.10.5.10090.Mus_ musculus database (80 682 entries; 1-DE), the uniref100.12.2. 10090.Mus_musculus database (80 481 entries; iTRAQ) and the uniref100.12.7.10090.Mus_musculus database (69 282 entries; IEF Strip) assuming the digestion enzyme, trypsin. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 2.0 Da. Iodoacetamide derivatives of cysteine were specified as a fixed modification, and oxidation of methionine was specified as a variable modification. Two 710

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Bilodeau et al. miss-cleavage sites were allowed. For iTRAQ experiments, oxidation of methionine and Applied Biosystems iTRAQ multiplexed quantitation chemistry of lysine, tyrosine and the N-terminus were specified as variable modifications. Peak areas for each of the four reporter ions (m/z: 114, 115, 116 and 117) were corrected to account for isotopic overlap according to the manufacturer’s instructions. Protein quantification results were calculated and viewed using ProteinPilot version 2.0. Protein iTRAQ ratios were corrected for experimental bias using the median average protein ratio as the correction factor. Quantification data obtained from very low intensity spectra were removed from the protein ratio calculation. Protein identification data is presented in Supplementary data Mouse liver P-G/E ITRAQ Reports in Supporting Information. Criteria for Protein Identification. Scaffold (Proteome Software, Inc., Portland, OR; version 2.1.03) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm.20 Protein probabilities were assigned by the Protein Prophet algorithm.21 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Immunofluorescence Microscopy. MDCK ts v-Src cells cultured at 40.5 °C (restrictive temperature) or 35 °C (permissive temperature) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2. MDCK ts v-Src cells were generated from MDCK cells infected with the retroviral vector pBabe/puro/ts72-v-Src driving the expression of a thermosensitive v-Src mutant isolated from RSV tsNY72, as described.22 The clonal cell line, Pi34, was isolated by limiting dilutions of a pool of infected cells after a 15 day selection period at the restrictive temperature in the presence of puromycin (2 µg/mL). This cell line displays a normal cytoskeletal organization composed of actin stress fibers at the restrictive temperature. For immunostaining, cells were fixed in 3.7% formaldehyde in Luftig buffer (0.2 M sucrose, 35 mM PIPES (pH 7.4), 5 mM EGTA, 5 mM MgSO4) for 20 min at 37 °C. Fixation-induced fluorescence was quenched with 50 mM NH4Cl for 15 min at room temperature. All immune incubations were performed in PBS with 4% BSA and 0.4% saponin as described.23 Equal amounts of each total cell lysate were loaded on SDS-PAGE for Western blot analysis. Electron Microscopy. The G/E fractions were immediately fixed with 2.5% glutaraldehyde and 100 mM sodium cacodylate, pH 7.4. Samples were rinsed and postfixed in 1% ferrocyanide osmium tetroxide, dehydrated in a graded series of ethanol and then processed for embedding in EPON. Ultrathin sections of each block were cut and placed on copper grids and then stained with uranyl acetate and lead citrate. Sections were examined with a Philips EM 301 electron microscope.13

Results and Discussion Analysis of Permeabilized G/E Fractions by Mass Spectrometry. The characterization of the mouse hepatic G/E fraction in terms of electron microscopy, 1-DE electrophoresis, ligand-mediated endocytosis and protein content is shown in Figure 1. Typical lipoprotein-filled tubulovesicular structures consisting of 70 nm vesicles and 200-400 nm vesicles were observed (Figure 1A, left panel). The luminal depletion procedure resulted in empty membrane vesicular structures13 with

Proteomic Analysis of Src Family Kinases Signaling Complexes

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Figure 1. Characterization of mouse liver G/E fractions. (A) Electron microscopy of the mouse G/E fractions, typical tubulovesicular structures as well as lipoprotein-filled vesicles of 70, 200-400 nm are observed (left panel). Empty vesicular structures are observed following depletion of luminal content (right panel). (B) SDS-PAGE analysis (20 µg of protein) of intact (C) or permeabilized (P) G/E membranes. Some major proteins depleted in the permeabilized fraction are indicated by arrows including the 66 kDa band. (C) Immunodetection of the IR in the permeabilized G/E fraction following insulin injection (20 µg of protein; 1.5 µg insulin/100 g body weight) using anti-IRβ and anti-PY antibodies (PY20). (D) Tryptic peptides from depleted G/E fractions prepared independently were labeled with the iTRAQ 115 or 117 isobaric markers. Labeled samples were then combined before MS/MS analysis. On average, 8438 mass spectra samples have been collected, and 1331 nonredundant peptides and 241 unique proteins with an average 115:117 iTRAQ ratio of 1.0 (n ) 3) have been identified.

an average 34% loss of total proteins (Figure 1A, right panel). The disappearance of luminal proteins, including the abundant 66 kDa albumin protein band, was assessed by 1-DE (Figure 1B). Following the injection of a single dose of insulin (1.5 µg/ 100 g body weight), the IR accumulated in endosomes with a peak occurring at 2 min postinjection, as previously measured in rat liver G/E fractions24 (Figure 1C). The reproducibility of the depletion procedure was verified here using a quantitative N-terminal peptide labeling procedure (iTRAQ). We identified 241 proteins with a 115:117 iTRAQ ratio of 1.0 for the majority (80%) of the proteins, indicating that fractions were homogeneous from one experiment to another (Figure 1D and Supplementary data Mouse liver P-G/E iTRAQ Reports in Supporting Information). From this protein survey, it is already clear that the protein composition of the G/E membranes is influenced by contributions from Golgi, ER and endosomes components. In particular, 15% of identified proteins fell into the vesicular transport category. Of interest, 21% have unknown functions (Figure 2). The number of identified proteins markedly increased when tryptic peptides were separated by IEF prior to LC/MS/MS

analysis. Hence, 514 proteins were identified with 11% of them in the vesicular transport category and 24% unknown (Figure 2, Supplementary data Mouse liver P-G/E IEF Reports in Supporting Information). By comparison, separation of proteins by 1-DE before slicing into 40 gel bands and the LC/MS/MS analysis as described previously25 resulted in the identification of 124 proteins (Figure 2, Supplementary data Mouse liver P-G/E 1-DE Reports in Supporting Information). Thus, the depletion procedure was reproducible, and we identified 666 proteins in the permeabilized G/E fraction (Table 1). A majority of the proteins fell into the vesicular transport category, including a number of small GTPases of the Rab subfamily, clathrin heavy chains and AP-1/2 adapters, proteins of the SEC series, SNAREs and vacuolar ATPase subunits. We also included proteins involved in the ubiquitin pathway, such as ubiquitin, UBIE1 and noticeably the ubiquitin-like protein UFM1, in this category. A majority of the other proteins identified were arbitrarily distributed in the metabolism/redox/chaperone category (50%), and the remainder had unknown functions (26%). In the signaling category (6%), we identified the tyrosine Journal of Proteome Research • Vol. 9, No. 2, 2010 711

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Figure 2. Classification of identified proteins. Absolute numbers and percentages are indicated for each methodology. Table 1. Proteins Identified for Each Method method

number of proteins

unique proteins

% of unique proteins

iTRAQ IEF Strip 1-DE Total

241 514 124 666

115 357 31 503

23 71 6 100

kinase EGF receptor (EGFR) and we also identified the SFK Lyn, a less characterized tyrosine kinase in liver. Analysis of rP42-2 Signals in G/E Membranes. To further analyze SFK signaling in the context of G/E membranes, we first confirmed the presence of Lyn using a monospecific antibody (Figure 3A). We then used the RP42-2 antibody to detect putative SFK substrates and their associated proteins in the permeabilized G/E fraction under basal conditions and also following insulin stimulation in the presence or absence of bpV(phen), which causes accumulation of PY phosphosites. Under these circumstances, we detected a series of insulindependent signals with maximal accumulation reached at 15 min postinsulin injection (Figure 3B). The results confirmed that Lyn is present in the G/E fraction and suggested that the RP42-2 antibody can be a useful tool to purify SFK substrates and associated partners. Subcellular Localization of rP42-2 Signals in MDCK Cells Overexpressing v-Src. We further verified the subcellular localization of RP42-2 signals by an alternative approach. As a model, we used normal epithelial cells expressing a thermosensitive mutant of v-Src, (MDCK ts v-Src). When MDCK cells were switched to the permissive temperature (35 °C), they rapidly underwent dramatic changes in cell morphology that reflected v-Src activation. Specifically, the MDCK cell morphology converted from a well-spread morphology at 40.5 °C to a more refractile and rounded cell shape at 35 °C. This change in morphology was associated with the loss of stress fibers, as visualized by F-actin staining, and the formation of invasive actin-rich structures called podosomes with concomitant marked increase in cellular PY that is consistent with v-Src activation (Figure 4). Tyrosine phosphorylation signals were detected with 712


Figure 3. Identification of Src phosphosites in the permeabilized G/E fraction. G/E fractions were prepared and processed at the indicated times postinjection of insulin (1.5 µg insulin/100 g body weight) in the presence or absence of the PTP inhibitor, bpV(phen). Proteins were separated by SDS-PAGE (80 µg, 7.5% resolving). (A) The internalized IR was detected using the anti-IR β-subunit antibody (IR 95 kDa β-subunit) or the RPY20 antibody (95 kDa RPY). The SFK Lyn was immunodetected on the same membranes. (B) Tyrosine-phosphorylated protein bands were detected by immunoblotting using the anti-phospho-E4orf4 (Y42) 42-2 antibody (RP42-2).

both the RP42-2 antibody and the PY20 anti-PY antibodies. Yet the RP42-2 displayed strong signals for a series of v-Srcregulated phosphoproteins (Figure 4A). Moreover, the RP42-2 antibody revealed a distinctive localization pattern of PY proteins (Figure 4B). The PY proteins labeled by the RP42-2 were largely localized to tubulovesicular membranes. To visualize the endocytic compartment, recycling endosomes were labeled with the transferrin receptor (TfR), which is internalized in coated pits and is recycled to the cell surface after transit in the juxtanuclear endocytic recycling compartment, where other endosomes carrying nonclathrin-dependent cargo proteins also transit.26-28 The results showed an overlapping distribution with endosomes carrying the TfR at the perinuclear endocytic recycling compartment (Figure 4B, arrowheads). A distinctive


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Figure 4. Subcellular localization of RP42-2 signals in MDCK cells overexpressing v-Src. (A) Immunodetection with the indicated antibodies of equal amounts of cell extracts (25 µg of proteins) isolated from MDCK ts v-Src cells maintained at 40.5 °C, or switched to 35 °C for an overnight period (O/N). (B) Single plane confocal images of MDCK ts v-Src cells stained with anti-TfR and RP42-2 antibodies. Arrows point to cells with robust actin structures resulting from v-Src transformation. Arrowheads designate the vesicular staining of RP42-2 that partially overlaps with TfR (a recycling receptor) at the perinuclear recycling compartment, as emphasized in enlarged views of the boxed regions. Bar, 10 µm.

staining pattern of PY proteins within tubular and vesicular elements was also observed at the perinuclear region of MCF7 cells overexpressing activated c-Src using the RP42-2 antibody, in addition to the more typical phosphotyrosine staining at podosome-like structures (data not shown). On the basis of these results, we were prompted to use the RP42-2 antibody to affinity purify SFK-regulated PY proteins in the G/E fraction. Enrichment and Identification of G/E rP42-2 Targets. The general scheme of the method is shown in Figure 5A. We used permeabilized G/E membranes that were isolated at the peak time of protein tyrosine phosphorylation (15 min postinsulin injection, in the presence of bpV(phen)) (Figure 3B). Proteins were solubilized (0.3% Empigen BB) before subjecting them to WGL affinity chromatography. This latter step was selected here as it was previously shown to concentrate a series of tyrosine-phosphorylated proteins in rat hepatic endosomes stimulated by insulin.17 Retained proteins were eluted with N-acetylglucosamine, after which they were loaded on the P42-2 affinity column (Figure 5A, left panel). Most of the tyrosine-phosphorylated protein bands detected in G/E membranes were recovered in the WGL eluates, particularly above apparent MW 50 kDa (Figure 5A, right panel). A subset of proteins was consistently recovered after P42-2 affinity chromatography with immunoblot signals detected above an apparent MW range of 66-120 kDa (Figure 5B). The eluted proteins were further concentrated and separated by 1-DE, and proteins were stained with SYPRO Ruby (Figure 5C, right panel). While there were clear signals on the P42-2 Western blot that presumably corresponded to some of the protein bands, the signal was barely detectable for others. This suggested that some proteins could have been recovered by virtue of their association with SFK substrates. Ten bands were excised and submitted to trypsinolysis and LC-MS/MS identification procedures. Sixteen proteins were identified and were classified in five functional categories: (1) glucose/energy metabolism, (2) recycling shuttles, (3) actin network, (4) vesicular transport and (5) signaling. In each category, known SFK substrates or SFK partners were identified (Table 2, Supplementary data P42-2 affinity chromatography Reports in Supporting Informa-

Figure 5. Enrichment of phosphoproteins from permeabilized (G/ E) membranes. (A) Flow diagram representing the enrichment of RP42-2 signals for each fraction generated. G/E, Golgi/ endosomes; WGL, wheat germ lectin (left panel). Immunoblots of the numbered fractions (30 µL aliquot), using the RP42-2 antibody (right panel). (B) Detection of RP42-2 signals in fractions collected from the RP42-2 column. (C) Fractions 3 and 4 eluted from the RP42-2 column were concentrated and proteins were separated by SDS-PAGE (7.5% resolving) and stained with SYPRO Ruby. Proteins bands (arrows) were excised and submitted to mass spectrometry analysis for identification. Journal of Proteome Research • Vol. 9, No. 2, 2010 713

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Table 2. RPY42-2-Affinity Chromatography protein molecular Weight (kDa)

number of peptides

protein identification probability

SFK signaling

protein category

protein name

accession numbers

Metabolism/Proteases

Dipeptidyl peptidase 4 (EC 3.4.14.5) (DPP IV) similar to glyceraldehyde3-phosphate dehydrogenase similar to pyruvate kinase 3 (LOC315231), mRNA Insulin receptor precursor (EC 2.7.10.1) Leukemia inhibitory factor receptor (LIF receptor) similar to solute carrier family 26, member 10 isoform 2 similar to low density lipoprotein receptor-related protein 1 (LRP1) similar to centromere protein F (350/400kD) similar to SIRP beta 1 cell surface protein Gamma-catenin PDZ domain-containing protein 1 Annexin A1 Protein transport protein Sec23A similar to ATPase, H+ transporting, V1 subunit A, isoform 1 Sorting nexin-27 Sept9 protein (Fragment)

UPI0000502C41

84

21

over 95%

Yes

UPI0000508467

34

2

over 95%

Yes

UPI0000503FDF

55

1

80%-95%

Yes

P15208

157

15

over 95%

-

O70535

122

2

over 95%

-

UPI0000DA2E1A

68

1

80%-95%

-

UPI0000504CBE

505

12

over 95%

Yes

UPI0000DA3A27

354

3

over 95%

-

UPI0000194382

44

1

80%-95%

-

P70565 Q9JJ40

82 57

2 6

over 95% over 95%

-

P07150 Q01405

39 86

4 15

over 95% over 95%

Yes -

UPI00001D0792

68

8

over 95%

-

Q3UHD6 B2GVB4

61 62

1 1

80%-95% 80%-95%

-

Recycling Receptors

Signaling

Structure

Vesicular traffic

tion). They included LRP1,29,30 Annexin A1,31,32 the dipeptidyl peptidase DPP IV13 and sPK3 (also named pyruvate kinase M2, single peptide identification).33 Other proteins we identified are either involved in F-actin remodeling (catenin complexes34), have unknown functions or are involved in the control of trafficking events, particularly in the endosomal recycling compartment. This is the case for CENPF (Lek1),35 Sorting nexin 27,36 Septin 937 and solute carrier family 26 (single peptide identification).38 The IR itself is not a known SFK substrate, but because LRP1 Tyr63 was shown to be phosphorylated by multiple tyrosine kinases29 and because it was recently identified as insulinresponsive in adipocytes,39,40 we verified the possible association of IR and LRP1. We did, in fact, detect a pool of activated IR in LRP1 immunocomplexes, which suggests that these two highly recycling receptors in the liver can associate with one another (Figure 6). In the current study, we used a method for the analysis of SFK signaling complexes in a previously characterized G/E fraction. The permeabilization procedure used prior to analysis of the G/E fraction decreased the abundance of major cargos that otherwise masked the identification of less abundant membrane-associated proteins. One disadvantage of this method may be the loss of some permeabilization-sensitive membrane microdomains. Nonetheless, the 666 proteins reported here represent a large proteome of the hepatic G/E membranes. A majority of identified proteins fell into the vesicular transport category. They included major coat proteins (clathrin heavy chains, AP-1/2 adapters and proteins of the COP I and COP II complexes), a series of small GTPases of the Rab subfamily, 714


vacuolar ATPase subunits and SNAREs. We also detected proteins of the ubiquitin pathway, including ubiquitin itself, UBIE1 and UFM1. The latter was recently classified as an ubiquitin-like protein on the basis of its folding and protein attachment properties41,42 and was recently involved in diabetes.43 Three SFKs, Yes, Src and Lyn, were previously identified as major tyrosine kinases in hepatocytes.44 The use of the RP42-2 antibody as an affinity tool resulted in the identification of five classes of proteins (Table 2, tentative identifications with P-values >0.05 are also listed in the Supporting Information table). LRP1 (class 1) is a large shuttling protein with the capacity to bind diverse ligands, including transmembrane receptors, and is a known substrate for Src. LRP1 undergoes constitutive endocytosis and recycling, and can be recruited in caveolar compartments following insulin stimulation.45 Kinetic parameters of IR association and dissociation with LRP1 are not measured with the current experimental approach. However, the fact that kinase-activated IR was present in LRP1 immunocomplexes with no increase in the total amount of associated IR indicates that the pool of LRP1/IR does not occur during the solubilization process. LRP1 is phosphorylated by serine/threonine-specific protein kinases, and its cytoplasmic tail includes two NPXY internalization motifs that are recognized targets for phosphorylation at Tyr63 by tyrosine kinases, including v-Src.29 All of these phosphorylation events affect the efficiency of LRP1 endocytosis.45 The finding that LRP1 associates with the internalized IR suggests a model in which LRPs could be viewed as a shuttle that displace insulin-IR complexes in the hepatic recycling compartments where SFKs are present.


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was recently reported that Cyt/LEK1 suppression alters TfR recycling and that Cyt/LEK1 also forms complexes with SNAP25, Rab11a, VAMP-2 and syntaxin, which are partners in the membrane recycling process.35 Whether CENPF/CytLEK1 is a partner that controls the routing of recycling receptors, such as the TfR (and presumably LRP1, IR and Slc27), remains to be investigated. Similarly, Sorting nexin 27 redirects traffic to early endosomes and was found associated with TfR-containing vesicles.36 Annexin A1 is tyrosine-phosphorylated by kinases, including Src,54,55 and is involved in the confinement of internalized EGFR in multivesicular bodies.56 Slc 27/FATP (single peptide identification, supplementary data P42-2 affinity chromatography Reports in Supporting Information) is an insulin-regulated fatty acid transporter that cycles from endosomes to the plasma membrane.38 Further characterization of these proteins should provide clues for studying crosstalk between receptor recycling, proteins involved in trafficking processes, the actin network and metabolism.

Figure 6. Association of low density lipoprotein receptor-related protein 1 (LRP1) with the internalized insulin receptor (IR). G/E membranes were prepared at the indicated times following insulin injection. (Upper panel) Immunodetection (50 µg of protein) of the internalized IR (IR 95 kDa β-subunit); 95 kDa RPY (antibody: RPY20); and LRP1 (LRP1 β-subunit). (Lower panel) LRP1 was immunoprecipitated (LRP1 R-subunit) from solubilized G/E membranes (0.5 mg of protein). Immunoprecipitated proteins were immunoblotted (10% resolving) using RPY20 (95 kDa RPY), anti-IR β-subunit (IR 95 kDa β-subunit), and anti-LRP1 β-subunit (LRP1 β-subunit) antibodies. Lane C: control without primary antibody.

Catenin complexes are responsible for the dynamic anchorage of the actin cytoskeleton to cadherin-based adherens junctions.46 In these complexes, β-catenin can be phosphorylated at Tyr333, Tyr604 or Tyr654 residues.47 A current prevailing viewpoint is that tyrosine phosphorylation of β-catenin promotes its dissociation from R-catenin/E-cadherin complexes.34,48 According to a model proposed by Weis and Nelson, the release of R-catenin homodimers from adherens junctions can antagonize Arp2/3 functions and/or β-catenin can interact with actin by binding to other proteins such as ZO-1 and afadin.49 Therefore, the catenin complexes tyrosine phosphorylation/ dissociation/degradation sequence has the capacity to act on the dynamics of the juxta-membrane cortex, and can, thus, facilitate the formation of internalization foci. In the metabolism category, we identified the spliced form of the pyruvate kinase, PKM2 (also named sPK3, single peptide identification, see supplementary data P42-2 affinity chromatography Reports in Supporting Information), which is expressed during cell differentiation.50 Of interest, PKM2 activity is regulated by the association of putative tyrosine-phosphorylated proteins and was associated with the Warburg effect.33 Also, the identification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is reminiscent of recent findings showing that the phosphorylation of compartmentalized GAPDH at Tyr41 by Src affects the formation of Rab2-mediated retrograde vesicles51 (Table 2). Other proteins of apparent unrelated functions, such as CENPF (Table 2), are also of interest. CENPF belongs to the emerging CENPF/LEK1/mitosin family of proteins52 and undergoes post-translational cleavages that produce an N-terminal peptide (CytLEK1) that distributes in the cytoplasm.53 It

Conclusions The present work supports the idea that the identified signaling molecules, shuttling receptors, regulators of vesicular traffic and actin cytoskeleton regulators build, in a concerted manner, the specialized membrane conditions required for hepatic traffic in vivo. The main site for insulin clearance is the liver, which removes approximately 80% of the total insulin during portal passage.57 This adaptive mechanism is still not completely understood and involves several mechanisms, including binding with the IR at the cell surface, internalization of the resulting complexes into the endosomal apparatus and recycling of the deactivated IR back to the cell surface.58 Confirmation and detailed analysis of each of these players may define the molecular mechanism involved and lead to a better understanding of diabetes.

Acknowledgment. This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (RF: OGPO157551) and by the Canadian Diabetes Association (CDA, RF) and by the Canadian Institutes of Health Research Operating Grant MOP-49450 (to J.N.L.). J.N.L. is a chercheur-boursier (Senior, FRSQ). N.B. and A.F. are supported by the CDA, the Canadian Institutes of Health Research (CIHR) and Stars Foundation scholarships. M.C.B. is supported by a FRSQ fellowship. Suzanne Fortier is greatly acknowledged for expert technical assistance. Supporting Information Available: Mouse liver P-G/E ITRAQ Reports: Excel file comprising three sheets presents the complete listing of all distinct proteins identified from permeabilized G/E membranes resolved by ITRAQ coupled with LC/ TOF/TOF mass spectrometry, plus the entire listing of spectra obtained and the spectra with the fragmentation table associated with all of the single peptide protein identifications. Proteins are listed according to their reported or predicted functions. Mouse liver P-G/E IEF Reports: Excel file comprising three sheets presents the complete listing of all distinct proteins identified from permeabilized G/E membranes. Tryptic peptides were separated by IEF prior to mass spectrometry analysis. The entire listing of spectra obtained and the spectra with the fragmentation table associated with all the single peptide proteins identifications are included. Proteins are listed according to their reported or predicted functions. Mouse liver P-G/E 1-DE Reports: Excel file comprising three sheets presents Journal of Proteome Research • Vol. 9, No. 2, 2010 715

research articles the complete listing of all distinct proteins identified from permeabilized G/E membranes resolved by 1-DE. The entire listing of spectra obtained and the spectra with the fragmentation table associated with all the single peptide proteins identifications are included. Proteins are listed according to their reported or predicted functions. Mouse liver G/E 2-DE Reports: Excel file comprising three sheets presents the complete listing of all distinct proteins identified from permeabilized G/E membranes resolved by 2-DE before spectrometry analysis of the excised spots (200). The entire listing of spectra obtained and the spectra with the fragmentation table associated with all the single peptide proteins identifications are included. P42-2 affinity chromatography Reports: Excel file comprising three sheets presents the complete listing of all the proteins identified following RP42-2 affinity chromatography (see Table 2). The entire listing of spectra obtained and the spectra with the fragmentation table associated with all the single peptide proteins identifications are included. This material is available free of charge via the Internet at http:// pubs.acs.org.

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