Identification and Validation of Novel Spinophilin-associated Proteins in Rodent Striatum Using an Enhanced ex Vivo Shotgun Proteomics Approach
2010; Elsevier BV; Volume: 9; Issue: 6 Linguagem: Inglês
10.1074/mcp.m900387-mcp200
ISSN1535-9484
AutoresAnthony J. Baucum, Nidhi Jalan‐Sakrikar, Yuxia Jiao, Richard M. Gustin, Leigh Carmody, David L. Tabb, Amy‐Joan L. Ham, Roger Colbran,
Tópico(s)Cellular transport and secretion
ResumoSpinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a χ2 analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, α-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that α-actinin and densin directly interact with residues 151–300 and 446–817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum. Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a χ2 analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, α-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that α-actinin and densin directly interact with residues 151–300 and 446–817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum. Genomic sequencing has revealed the full repertoire of ∼20,000 proteins that can be expressed in most mammals. Innate biochemical or enzymatic activities of many proteins are critical to their function, but these activities are often modified by interactions with other proteins. Moreover, many proteins have no known catalytic activity and are thought to serve structural roles in assembling protein complexes, greatly increasing the efficiency and fidelity of intracellular processes. Thus, systematic definition of protein interactomes promises tremendous insight into biochemical mechanisms underlying the functions of many proteins. A prime example of the importance of protein-protein interactions for modifying biological function is the postsynaptic density (PSD), 1The abbreviations used are:PSDpostsynaptic densityCaMKIIcalcium/calmodulin-dependent protein kinase IICMVcytomegalovirusDAdopamineDMEMDulbecco's modified Eagle's mediumGFPgreen fluorescent proteinHAhemagglutininICQintensity correlation quotientKOknock-outPDParkinson diseasePDZPSD-95/disks large/zonula occludensPP1protein phosphatase 1SpspinophilinSpAPspinophilin-associated proteinTAOthousand and one amino acid kinaseWTwild type6-OHDA6-hydroxydopamineIPIInternational Protein IndexGAPDHglyceraldehyde-3-phosphate dehydrogenaseNFneurofilamentHEKhuman embryonic kidney. an actin-rich organelle localized to neuronal dendritic spines that contains receptors, kinases, phosphatases, and scaffolding proteins (1Kim E. Sheng M. PDZ domain proteins of synapses.Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1243) Google Scholar, 2Kennedy M.B. Beale H.C. 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Selective targeting of the gamma1 isoform of protein phosphatase 1 to F-actin in intact cells requires multiple domains in spinophilin and neurabin.FASEB J. 2008; 22: 1660-1671Crossref PubMed Scopus (28) Google Scholar, 12Stephens D.J. Banting G. In vivo dynamics of the F-actin-binding protein neurabin-II.Biochem. J. 2000; 345: 185-194Crossref PubMed Scopus (23) Google Scholar, 13Terry-Lorenzo R.T. Elliot E. Weiser D.C. Prickett T.D. Brautigan D.L. Shenolikar S. Neurabins recruit protein phosphatase-1 and inhibitor-2 to the actin cytoskeleton.J. Biol. Chem. 2002; 277: 46535-46543Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Allen P.B. Zachariou V. Svenningsson P. Lepore A.C. Centonze D. Costa C. Rossi S. Bender G. Chen G. Feng J. Snyder G.L. Bernardi G. Nestler E.J. Yan Z. Calabresi P. Greengard P. Distinct roles for spinophilin and neurabin in dopamine-mediated plasticity.Neuroscience. 2006; 140: 897-911Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Spinophilin associates with its homolog neurabin, which is also a PP1- and F-actin-binding protein that regulates synaptic plasticity and dendrite morphology (14Allen P.B. Zachariou V. Svenningsson P. Lepore A.C. Centonze D. Costa C. Rossi S. Bender G. Chen G. Feng J. Snyder G.L. Bernardi G. Nestler E.J. Yan Z. Calabresi P. Greengard P. Distinct roles for spinophilin and neurabin in dopamine-mediated plasticity.Neuroscience. 2006; 140: 897-911Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. Neurabin: a novel neural tissue-specific actin filament-binding protein involved in neurite formation.J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (164) Google Scholar, 16Terry-Lorenzo R.T. 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Hood S.H. van Maldegem B. Weenink A. Arbuthnott G.W. Morphological changes in the rat neostriatum after unilateral 6-hydroxydopamine injections into the nigrostriatal pathway.Exp. Brain Res. 1993; 93: 17-27Crossref PubMed Scopus (122) Google Scholar). Spine density is regulated by dynamic changes in the F-actin cytoskeleton, and spinophilin regulates dendritic spine density during development (21Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Spinophilin regulates the formation and function of dendritic spines.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9287-9292Crossref PubMed Scopus (325) Google Scholar). Indeed, candidate protein or generic protein-protein interaction screens have identified many additional spinophilin-associated proteins (SpAPs) that modulate F-actin dynamics and/or cell morphology (22Bielas S.L. Serneo F.F. Chechlacz M. Deerinck T.J. Perkins G.A. Allen P.B. Ellisman M.H. Gleeson J.G. 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Although the spinophilin interactome appears to dictate the biological roles of spinophilin, the composition of these complexes in the mature brain is poorly understood. Co-immunoprecipitation is commonly used to confirm the biological relevance of specific bivalent protein-protein interactions in native tissues that were initially identified using generic molecular approaches, such as yeast two-hybrid screening. Prior studies combined this approach with mass spectrometry-based proteomics methods to more broadly characterize the composition of mammalian signaling complexes and the PSD interactome, such as the signalosome associated with synaptic N-methyl-d-aspartate receptors (29Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1029) Google Scholar) and complexes associated with other PSD-enriched proteins (30Collins M.O. Husi H. Yu L. Brandon J.M. 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Moreover, computational approaches are being developed to identify potential protein-protein interactions (32Goñi J. Esteban F.J. de Mendizábal N.V. Sepulcre J. Ardanza-Trevijano S. Agirrezabal I. Villoslada P. A computational analysis of protein-protein interaction networks in neurodegenerative diseases.BMC Syst. Biol. 2008; 2: 52Crossref PubMed Scopus (91) Google Scholar). However, validation of specific interactions among the very large data sets of candidates typically identified using these approaches can be daunting. In addition, most proteomics analyses have relied on a single antibody to the target protein of interest with, at best, an unrelated non-immune IgG as a negative control, necessitating the use of very high quality antibodies. We developed a systematic shotgun proteomics approach to define protein interactomes in a native tissue context. We used this approach to characterize the composition of spinophilin complexes isolated from rodent striatum and confirmed the association of multiple, novel SpAPs. Furthermore, we extensively characterized the interaction of two additional SpAPs, α-actinin and densin, using biochemical and imaging techniques. Our studies directly illustrate the importance of appropriate subcellular fractionation conditions, using multiple antibodies to the protein of interest, and the underappreciated, critical role of analyzing parallel samples prepared from knock-out (KO) animals. Thus, our findings demonstrate a methodological framework with key controls that can be broadly applied to characterizing protein interactomes, in addition to providing novel insights into the role of spinophilin in controlling synaptic signaling. The following antibodies were used as indicated: spinophilin: mouse monoclonal antibody (BD Biosciences 612166; epitope mapping between rat spinophilin residues 238–348), rabbit polyclonal spinophilin antibody (Millipore 06-852; epitope mapping between rat spinophilin residues 286–390), and goat polyclonal antibody (Santa Cruz Biotechnology SC-14774; epitope mapping to residues ∼50–100 of rat spinophilin); CaMKII: goat polyclonal antibody (33McNeill R.B. Colbran R.J. Interaction of autophosphorylated Ca2+/calmodulin-dependent protein kinase II with neuronal cytoskeletal proteins. Characterization of binding to a 190-kDa postsynaptic density protein.J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), mouse monoclonal CaMKIIα antibody (Affinity Bioreagents/Thermo Fisher Scientific MA1-048), and mouse monoclonal CaMKIIβ antibody (Zymed Laboratories Inc./Invitrogen 13-9800); pan-α-actinin, rabbit polyclonal antibody (Santa Cruz Biotechnology SC-15335); densin: goat polyclonal antibodies 450 and 650 (34Jiao Y. Robison A.J. Bass M.A. Colbran R.J. Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization.J. Neurochem. 2008; 105: 1746-1760Crossref PubMed Scopus (20) Google Scholar) and rabbit polyclonal antibody BΔN (34Jiao Y. Robison A.J. Bass M.A. Colbran R.J. Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization.J. Neurochem. 2008; 105: 1746-1760Crossref PubMed Scopus (20) Google Scholar); thousand and one amino acid kinase 1 (TAO1): rabbit polyclonal antibody (Bethyl Laboratories A300-524A); TAO3: rabbit polyclonal antibody (Bethyl Laboratories A300-536A); Tiam1: rabbit polyclonal antibody (Santa Cruz Biotechnology SC-872); P70S6 kinase: rabbit polyclonal antibody (Santa Cruz Biotechnology SC-230); doublecortin: rabbit polyclonal antibody (Cell Signaling Technology 4604); RasGrf1: rabbit polyclonal antibody (Santa Cruz Biotechnology SC-224); PP1γ1: sheep polyclonal antibody (35Colbran R.J. Carmody L.C. Bauman P.A. Wadzinski B.E. Bass M.A. Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits.Methods Enzymol. 2003; 366: 156-175Crossref PubMed Scopus (20) Google Scholar); PSD-95 (NeuroMab 75-028); tyrosine hydroxylase (ImmunoStar 22941); neurabin: mouse monoclonal antibody (BD Transduction Laboratories 611088); myosin Va: rabbit polyclonal antibody (Sigma M4812); neurofilament light polypeptide: rabbit (Cell Signaling Technology 2837) and mouse (Santa Cruz Biotechnology SC-58559) antibodies; Myc: mouse monoclonal antibody (Vanderbilt Monoclonal Antibody Core); HA: mouse monoclonal antibody (Vanderbilt Monoclonal Antibody Core) and rabbit polyclonal antibody (Santa Cruz Biotechnology SC-805); and fluorescent secondary antibodies: donkey anti-mouse Alexa Fluor 546, donkey anti-rabbit Alexa Fluor 488, and donkey anti-goat Alexa Fluor 633 (Molecular Probes). Surgery was performed as described previously (18Brown A.M. Deutch A.Y. Colbran R.J. Dopamine depletion alters phosphorylation of striatal proteins in a model of Parkinsonism.Eur. J. Neurosci. 2005; 22: 247-256Crossref PubMed Scopus (77) Google Scholar). Mice were decapitated without anesthesia, brains were removed, and neostriata (referred to as striata) were rapidly dissected, frozen on dry ice, and stored at −80 °C. Two whole frozen mouse striata (one from each hemisphere; ∼20 mg of total tissue) were pooled and homogenized in 2 ml of an isotonic (150 mm KCl, 50 mm Tris-HCl, 1 mm DTT, 0.2 mm PMSF, 1 mm benzamidine, 10 µg/ml leupeptin, 10 µm pepstatin, and 1 µm microcystin) or low ionic strength (2 mm Tris-HCl, pH 7.4, 2 mm EDTA, 2 mm EGTA, 1 mm DTT, 0.2 mm PMSF, 1 mm benzamidine, 10 µg/ml leupeptin, 10 µm pepstatin, and 1 µm microcystin) buffer with no detergent in a Teflon-glass Wheaton tissue grinder with motorized plunger and incubated at 4 °C for 30–60 min. Samples were adjusted to 0.4–1 mg/ml total protein and then centrifuged at 9,000 × g at 4 °C for 10 min. Supernatants (S1) were saved for immunoprecipitation. The pellet (P1) was resuspended in 1 ml of isotonic or low ionic strength buffer containing 0.5% Triton X-100 (v/v; Sigma) in a microcentrifuge tube, and samples were then adjusted to a final volume of 2 ml. Samples were incubated at 4 °C for 30–60 min and then centrifuged at 9,000 × g for 10 min. Supernatants (S2) were saved for immunoprecipitation, and the P2 pellets were resuspended in 2 ml of isotonic or low ionic strength buffer containing 1% Triton X-100 and 1% sodium deoxycholate (w/v; MP Biomedicals) and sonicated. Following incubation at 4 °C for 30 min, samples were then centrifuged at 9,000 × g for 10 min, and the supernatants (S3) were saved for immunoprecipitation. The final pellet (P3) was resuspended in 2 ml of 2× SDS sample buffer. For co-immunoprecipitations from non-fractionated samples, fresh or frozen brain tissue from adult (3–6-month old) male or female mice (WT or spinophilin KO C57/Bl6) or adult male rats were homogenized in low ionic strength buffer containing 0.5–1% Triton X-100 or isotonic strength buffer containing 1% Triton X-100 and 1% sodium deoxycholate. Solubilized extracts from 1) mouse striata prepared for fractionation studies (S1, S2, or S3) in isotonic or low ionic strength buffer, 2) mouse whole brain homogenized in isotonic (1% Triton X-100 and 1% sodium deoxycholate) or low ionic strength (1% Triton X-100) buffer, or 3) rat or mouse striata or transfected HEK293 cells (see below) prepared directly in low ionic strength buffer containing 0.5–1.0% Triton X-100 were immunoprecipitated essentially as described (17Brown A.M. Baucum A.J. Bass M.A. Colbran R.J. Association of protein phosphatase 1 gamma 1 with spinophilin suppresses phosphatase activity in a Parkinson disease model.J. Biol. Chem. 2008; 283: 14286-14294Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Antibody concentrations used were as follows: goat spinophilin, 4–8 µg; rabbit spinophilin, 7.5–30 µg; mouse spinophilin, 2.5–15 µg; rabbit α-actinin, 12 µg; and appropriate non-immune IgG control of a similar concentration. Western blotting was done as described previously (18Brown A.M. Deutch A.Y. Colbran R.J. Dopamine depletion alters phosphorylation of striatal proteins in a model of Parkinsonism.Eur. J. Neurosci. 2005; 22: 247-256Crossref PubMed Scopus (77) Google Scholar). Immune complexes were fractionated by SDS-PAGE (10% acrylamide) and stained with colloidal Coomassie Blue G-250 (Invitrogen). Each gel was loaded with only one sample (and molecular weight markers) to limit cross-contamination. Entire gel lanes, with or without prominent IgG bands, were excised in two to three molecular weight ranges, finely chopped, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Tryptic peptides were resolved using a reverse phase packed capillary tip (100 µm × 11 cm) packed with C18 resin (Jupiter C18, 5 µm, 300 Å; Phenomenex, Torrance, CA) and a precolumn (100 µm × 6 cm) with the same resin using a frit generated from liquid silicate Kasil 1 (36Cortes H.J. Pfeiffer C.D. Richter B.E. Stevens T. Pourous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography.J. High Resolut. Chromatogr. Chromatogr. Commun. 1987; 10: 446-448Crossref Scopus (138) Google Scholar) essentially as described previously (37Licklider L.J. Thoreen C.C. Peng J. Gygi S.P. Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column.Anal. Chem. 2002; 74: 3076-3083Crossref PubMed Scopus (185) Google Scholar). Mobile phase A was 0.1% formic acid; mobile phase B was acetonitrile with 0.1% formic acid. The peptides were eluted from the column using a 95-min protocol, including a 15-min wash period (100% A for the first 10 min followed by a gradient to 98% A at 15 min) at 1 µl/min to allow for solid phase extraction and removal of residual salts followed by a 35-min linear gradient from 98% A to 75% A at 0.7 µl/min, then a steeper gradient to 10% A at 65 min, and an isocratic phase at 10% A to 75 min. Fractions were analyzed by tandem mass spectrometry using a ThermoFinnigan LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Thermo Surveyor HPLC pump, Nanospray source, and Xcalibur 2.0 instrument control software. MS/MS spectra of the peptides were obtained using data-dependent scanning in which one full MS spectra was followed by three MS/MS spectra. The msconvert tool from ProteoWizard (38Kessner D. Chambers M. Burke R. Agus D. Mallick P. ProteoWizard: open source software for rapid proteomics tools development.Bioinformatics. 2008; 24: 2534-2536Crossref PubMed Scopus (1259) Google Scholar) exported mass spectra to the mzML v1.1 format. Intensity and m/z values were reported to 32-bit precision, and both MS and MS/MS scans were exported in centroid format. MyriMatch 1.6.0 (39Tabb D.L. Fernando C.G. Chambers M.C. MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis.J. Proteome Res. 2007; 6: 654-661Crossref PubMed Scopus (450) Google Scholar) identified peptides corresponding to the MS/MS scans. Each scan was assessed as a singly charged precursor if 90% of fragment intensity fell below the precursor m/z; otherwise, the scans were identified under both doubly charged and triply charged precursor assumptions. The sequence database for immunoprecipitations in rat tissue included the IPI rat database (v3.66 with 39,677 sequences), the IPI mouse database (v3.66 with 56,791 sequences), and a database of 71 common contaminant sequences, including five proteases, 42 Ig constant regions, 14 human keratins, and 10 proteins from wool, cotton, and saliva. The sequence database for immunoprecipitations in mouse tissue included all but the IPI rat sequences. In both cases, all sequences were present in both normal and reversed orientations to enable estimation of false discovery rates. Each of the following modifications was allowed to be present or absent on the corresponding residues: carbamidomethyl Cys (+57), oxidized Met (+16), and pyro-Glu from N-terminal Gln (−17). Only one end of each peptide was required to match an expected trypsin cleavage site, and missed cleavages were not counted. Precursor ions were required to fall within 1.25 m/z of peptide average mass, whereas fragments were expected w
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