Microtubule-Associated Protein 1B Binds Glyceraldehyde-3

Vaudois et Université de Lausanne, CERY, 1008 Prilly-Lausanne, Suisse. Received February 15, 2007. Microtubule-associated protein 1B, MAP1B, is a maj...
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Microtubule-Associated Protein 1B Binds Glyceraldehyde-3-phosphate Dehydrogenase Nathalie Cueille,† Corinne Tallichet Blanc,† Ire` ne M. Riederer,‡ and Beat M. Riederer*,†,‡ De´partement de Biologie Cellulaire et de Morphologie (DBCM), Universite´ de Lausanne, 9 Rue du Bugnon, 1005 Lausanne, Suisse, and Centre des Neurosciences Psychiatriques (CNP), Centre Hospitlaier Universitaire Vaudois et Universite´ de Lausanne, CERY, 1008 Prilly-Lausanne, Suisse Received February 15, 2007

Microtubule-associated protein 1B, MAP1B, is a major cytoskeletal protein during brain development and one of the largest brain MAPs associated with microtubules and microfilaments. Here, we identified several proteins that bind to MAP1B via immunoprecipitation with a MAP1B-specific antibody, by one and two-dimensional gel electrophoresis and subsequent mass spectrometry identification of precipitated proteins. In addition to tubulin and actin, a variety of proteins were identified. Among these proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 8, dihydropyrimidinase related proteins 2 and 3, protein-L-isoaspartate O-methyltransferase, β-spectrin, and clathrin protein MKIAA0034, linking either directly or indirectly to MAP1B. In particular, GAPDH, a key glycolytic enzyme, was bound in large quantity to the heavy chain of MAP1B in adult brain tissue. In vitro binding studies confirmed a direct binding of GAPDH to MAP1B. In PC12 cells, GAPDH was found in cytoplasm and nuclei and partially co-localized with MAP1B. It disappeared from the cytoplasm under oxidative stress or after a disruption of cytoskeletal elements after colcemid or cytochalasin exposure. GAPDH may be essential in the local energy provision of cytoskeletal structures and MAP1B may help to keep this key enzyme close to the cytoskeleton. Keywords: actin • cytoskeleton • GAPDH • immunoprecipitation • scaffold

Introduction MAP1B, a major component of the neuronal cytoskeleton, is a 320 kDa fibrous protein predominantly expressed in the developing nervous system and essential in newly forming axons, during neurite elongation, and in growth cone plasticity (for review, see refs 1 and 2). MAP1B consists of 2464 amino acids and is cleaved near amino acid 2100 into a heavy and a light chain, termed MAP1B-HC and MAP1B-LC1.3 HC and LC1 may form a complex to bind and stabilize microtubules via a noncovalent binding with the N-terminal region of the HC.4 Both proteins contain a microtubule binding and an actin binding site.4,5 It is believed that the HC acts as regulatory unit of the active site in the MAP1B-LC1. Furthermore, they differ in the control of neurite growth dynamics in that MAP1B-HC promotes axon elongation and LC1 is related to filopodia,6 possibly due to a differential reaction with microtubules and microfilaments, respectively. MAP1B may have an important cross-linking function between cytoskeletal structures and may therefore interact with a variety of proteins such as gigaxonin, a protein that links microtubules and intermediate filaments,7,8 with gamma-aminobutyric acid (GABA)c receptor rho1 subunit * To whom correspondence should be addressed. Dr. B.M. Riederer, DBCM, Universite´ de Lausanne, Rue du Bugnon 9, 1005 Lausanne, Switzerland. E-mail, [email protected]. † De´partement de Biologie Cellulaire et de Morphologie. ‡ Centre des Neurosciences Psychiatriques.

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linking GABA receptors to the cytoskeleton,9 and with myelinassociated glycoprotein (MAG) typical for glial cells.10,11 However, MAP1B is such a large protein that it is likely to find additional interactions with other proteins. Here, we have used MAP1B immunoprecipitation to identify potential MAP1B-HC interacting proteins that are precipitated together with MAP1B-HC. Associated proteins were identified by two-dimensional gel electrophoresis and MALDI-TOF analysis. In particular, we have investigated one of these proteins, the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), for its binding properties and its relation to the cytoskeleton. GAPDH is a protein of 37 kDa, the key enzyme of the glycolytic pathway and essential for the energy production. It catalyzes the most important reaction in glycolysis and energy metabolism, i.e., the dehydrogenation of phospho-glyceraldehyde into diphospho-1-3-glyceride. However, the role of GAPDH is more complex, because it was found associated with microtubules,12 and the enzyme is involved in multiple other functions such as endocytosis, membrane fusion, vesicular transport, nuclear tRNA transport, DNA replication and repair, translational control, apoptosis, and neurodegenerative diseases.13,14

Materials and Methods Brain Tissue Preparation. Brain tissue of a mouse strain NOR (kept in house) was used for this study. Experiments including animals were authorized by the federal veterinary 10.1021/pr070081z CCC: $37.00

 2007 American Chemical Society

MAP1B-GAPDH Interaction

service of Switzerland. Animals were anaesthetized with pentobarbital, perfused with PBS, and brains removed and immediately frozen in liquid nitrogen. Immunoprecipitation of MAP1B from Mouse Brain. The antibody used for immunoprecipitation is a well-characterized antibody and showed no crossreactivity to other proteins.5,15 The immunoprecipitation was previously described.15 Immunoglobulins of anti-MAP1B1404 rabbit (MAP1B-HC) and preimmune sera were purified by ammonium sulfate precipitation and diaylised against PBS. Amounts of 2.6 mg anti-MAP1B immunoglobulins (Ig) and nonspecific Ig’s each were bound to 1 mL of CNBr-Sepharose 4B (according to the manufacturer’s protocol). This Sepharoses were then used to precipitate MAP1B from mouse brain tissues of newborn (P0) and adult animals. All steps were performed on ice. Five hundred mg of mouse brain was homogenized in 1.8 mL of immunoprecipitation buffer (20 mM Tris-HCl pH 8.3, 0.8 M NaCl, 5 mM MgCl2, 1 mM EGTA and 1% (V/V) Triton X-100 and protease inhibitor cocktail (Roche)) and spun for 45 min at 100 000× g and 4 °C. The supernatant was collected and incubated with 300 µL antiMAP1B Ig-Sepharose for 2 h at 4 °C under agitation. The samples were precipitated by centrifugation in an Eppendorf centrifuge at 1000 rpm. Pellets were washed first in 10 mM TrisHCl pH 7.5, 1% (V/V) Triton X-100 and 500 mM NaCl, followed by the same buffer but with 250 mM NaCl and finally without NaCl. The last pellet was resuspended in 180 µL isoelectrofocusing or SDS-electrophoresis sample buffer, for the separation on one-dimensional SDS-polyacrylamide gels or for twodimensional gel electrophoresis. For 2D electrophoresis, IPG ready strips of 11 cm length and pH 3-10 (BioRad, Reinach Switzerland) were used in the first dimension and 4-15% gradient SDS-PAGE was applied in the second dimension. Visible bands and spots in Coomassie blue stained electrophoresis gels were cut out of the gels and used for MALDITOF identification (see below). Proteins were also electrically transferred to nitrocellulose and proteins were identified by immuno-blots and ECL. Antibodies used are a mouse monoclonal anti-actin, dilution 1:6000 (Chemicon, VWR International, Lucern, Switzerland); rabbit anti-MAP1B1404, 1:100 000 diluted,15 a mouse monoclonal anti-tubulin 27b hybridoma supernatant was 1:10 diluted;16 and monoclonal anti phosphorylated MAP1B (SMI31) from Sternberger Incorporated Monoclonal was at a dilution of 1:250. A rabbit polyclonal antibody against the MAP1B C-terminal sequence 2100-2464, and corresponding to the MAP1B-LC1 was produced by injecting a New Zealand White rabbit with recombinant MAP1B-LC1 in Specol by fortnightly boosts. The antibody was used 1:3000 diluted on blots or 1:1000 diluted in immunohistochemistry and reacted specifically with the MAP1BLC1 and the full length MAP1B. Pull Down Assay. The MAP1B1404 or MAP1B C-terminus (LC1) was subcloned in pQE31 vector to obtain a 6-His N-tag fusion protein.15 COS-7 cell extracts were made in Tris-HCl 20 mM pH 7.4, Sucrose 0.27 M/l, EDTA 1 mM, EGTA 1 mM, Na3VO2 1 mM, NaF 50 mM, β-glycerophosphate 10 mM, DTT 1 mM, 1% (V/V) Triton X-100, and protease inhibitors (Roche Diagnostics, Rotkreuz, Switzerland). Fifty micrograms of 6-Hispurified MAP1404 were incubated with 20 µL of Ni 2+ beads in Tris-HCl 50 mM pH 7.5, NaCl 150 mM, and protease inhibitor cocktail. Extracts of PC12 cells, were either exposed to NGF or not, and prepared for biochemistry of immunocytochemistry. For pull down assay, 100 µg COS-7 or PC12 cell protein extracts were incubated with Ni2+ beads to pre-clear 4 h at

research articles 4 °C and then with nickel beads bound to 6-His-MAP1404, 6-His- MAP-LC1, or with 6-His nickel beads alone (as negative control) overnight at 4 °C in 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 2 µM GTP. After centrifugation (5000 rpm), pellets were washed three times in stringent buffer (NaCl 75 mM, TrisHCl 25 mM pH 7.5, EDTA 2.5 mM, 0.5% (V/V) Triton-X100, and 0.25% (W/V) desoxycholate), and resuspended in 25 µL of SDS sample buffer and subjected to SDS-PAGE. The stringent incubation buffer contained MgCl2 (5 mM) and GTP (20 µM) to maintain GAPDH in its active form. Proteins were detected by Western blots with anti-GAPDH, by peroxidase-conjugated secondary antibody and revealed by ECL (Amersham/GE Biosciences, Zurich Switzerland). 6-His fusion proteins were also incubated with 2 µg of GAPDH purified from rabbit muscle (Roche), instead of cell line lysates, to test a specific binding of GAPDH with MAP1B constructs. Protein Identification by Tandem Mass Spectrometry. Excised Coomassie blue stained spots were destained. Sample preparation and MALDI-TOF was previously described15 and was performed by the local Proteomics Analysis Facility, PAF. An in-gel proteolytic cleavage was performed by the robotic workstation Investigator ProGest (Genomic Solutions, Ann Arbor, Michigan). The instrument washes the gel pieces, performs reduction and alkylation, adds trypsin (Promega, Madison, WI), and finally extracts the peptides from each gel piece in 30% (V/V) acetonitrile, 0.5% (V/V) formic acid. The peptide extracts were then dried by centrifugal evaporation and subsequently resuspended in 4 µL of matrix (4 mg/mL alphacyanohydroxy cinnamic acid in 40% acetonitrile). One microliter of the obtained samples were deposed on a MALDI plate, rapidly dried, and analyzed on a 4700 Proteomics Analyzer MALDI-TOF instrument (Applied Biosystems, Framingham, MA). The analysis performed was peptide mass fingerprinting combined with MS/MS on the 10 most intense peptide signals with the exclusion of trypsin and keratine peaks. All the spectra were used for analysis. Database searches were performed with the MASCOT software (www.matrixscience.com). The identification parameters were set as follows: database, release 5.7 Uniprot (SWISSPROT + TrEMBL); species, human; enzyme, trypsin; mass tolerance, 25 ppm for PMF spectra and 0.5 Da for MS/MS spectra; the statistical threshold for protein identification by MASCOT was 41. Cell Culture and Immunofluorescence. PC12 cells were grown at 37 °C, 7.5% of CO2 in high glucose DMEM (GibcoBRL) supplemented with 5% (V/V) horse serum, and 5% (V/V) fetal calf serum in presence of antibiotics (0.5% (V/V) peniciline-streptomycine, Gibco, BRL, Life technologies, Basel, Switzerland). Cells were plated on coverslip coated with polylysine + laminin. NGF (Alomone labs, Jerusalem, Israel) is added at a concentration of 50 ng/mL for 48 h. Cells were fixed with formaldehyde 3.7% (V/V) for 30 min at room temperature. Antibodies used are: a rabbit anti-MAP1B1404 1:4000 diluted, followed by an incubation with Oregon green conjugated goat anti rabbit antibody, 1:200 diluted (Molecular Probes), a mouse monoclonal anti GAPDH 1:50 diluted (Calbiochem, VWR International, Lucern, Switzerland), followed by a Cy3-conjugated donkey anti-rat antibody (Jackson Immuno Research Milan Analytica, La Roche Switzerland) 1:200 diluted. Cells were examined in the local Imaging Facility, by using a confocal Zeiss LSM510 Meta in multitrack configuration. Confocal image stacks were processed using Imaris and Adobe Photoshop computer software. Journal of Proteome Research • Vol. 6, No. 7, 2007 2641

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H2O2, Colcemid, and Cytochalasin D Treatment. PC12 cells were incubated 5 h at 37 °C with or without 5 mM H2O2 added to the medium. Cells were fixed with 3.7% formaldehyde for 30 min at room temperature and stained with the same antibodies mentioned above, nuclei were stained with Dapi, 1:100 diluted (Molecular Probes, VWR Intewrnational, Lucern, Switzerland). In several experiments, cells were also exposed for 2-24 h to 10 µM colcemid or 10 µM cytochalasin D or dimethyl-sulfoxide (DMSO) alone (Sigma, Buchs, Switzerland) and then let recuperate for 2-4 h to test for the reconstruction of the cytoskeleton.

Results MAP1B Interacting Proteins. MAP1B and its binding proteins were immunoprecipitated from brain tissue from adult, P10 or new born mice with an antibody directed against the MAP1B-HC. This allowed an identification of various potentially interacting proteins. This antibody recognized specifically the heavy chain and not the light chain.5,15 Individual proteins coprecipitating with MAP1B heavy chain were cut out from Coomassie blue stained 1D (Figure 1A) and 2D electrophoresis gels (Figure 1B) and analyzed by MALDI-TOF. High molecular weight proteins were clearly identified in 1D gradient gel with higher acrylamide concentrations (9-15%, W/V), whereas 2D gels were more advantageous in the identification of proteins between 100 and 10 kDa. Various immunoglobulin proteins also localized in the same molecular weight range but could be clearly separated from MAP1B coprecipitated proteins by their difference in charge. It is not surprising that some “degradation products” of MAP1B or MAP1B-HC and LC1 were identified. The presence of LC1 may suggest the formation of a macromolecular complex, because the antibody utilized is directed against the N-terminal part of MAP1B-HC and does not crossreact with MAP1B-LC1. Several other proteins coprecipitated with MAP1B heavy chain as listed in Table 1. Major proteins were several tubulin isoforms, the three actin isoforms, and GAPDH (Figure 1B2, spot no 8, 9, and 11). A variety of proteins were of minor stochiometry such as myosin, spectrin, a clathrin-related protein (MKIAA0034), heat shock proteins (HS7C, HSP8A), dihydropyrimidase related protein-2 and 3 (termed DPY2/3, ULIP2/3, CRMP2/3), protein-L-isoaspartate methyltransferase (PIMT) (Table 1). Among these proteins, GAPDH exhibited a striking difference between immunoprecipitates from newborn and adult brain tissue, in that only little GAPDH coprecipitated with anti-MAP1B from newborn tissue. The GAPDH in precipitates from adult brain was more abundant and revealed several modification forms, possibly due to differential phosphorylation. The Interaction MAP1B-GAPDH is Development-Dependent. A Western blot of P0 or adult mice brain with anti GAPDH indicated nearly equal levels of GAPDH in newborn brain on homogenate (Figure 2A, lane 1). GAPDH was present at lower levels in MAP1B precipitates from newborn brain than from adult (Figure 2B). This blot confirmed that indeed GAPDH is coprecipitated. Densitometry of Western blot and 2D gels indicated that 2-5 fold more GAPDH was associated with MAP1B in adult brain than in newborn nervous tissue, despite the fact that much GAPDH was present in newborn brain homogenates (starting material). Therefore, GAPDH may be more important for microtubules in mature brain tissue and may be present in different modified forms. Co-immunoprecipitated tubulin with MAP1B showed no significant difference in quantity (Figure 2C) between both ages. 2642

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Figure 1. Identification of proteins co-immunoprecipitating with MAP1B. (A) Control (immunoglobulins without brain homogenate) (panel 1), adult (panel 2), and newborn (panel 3) mouse brain homogenates were incubated with anti MAP1B heavy chain-sepharose, precipitate was separated on a 9-16% 1D polyacrylamide SDS-electrophoresis gel and stained with Coomassie blue. (B) Samples from immunoprecipitation from control (B1), adult (B2), and newborn (B3) mouse brain were submitted to 2D gel electrophoresis and stained with Coomassie blue. Molecular weights are indicated to the right in kDa. Note that despite a covalent binding of antibodies, several light (25 kDa) and heavy chain immunoglobulin molecules (55 kDa) are detached by electrophoresis sample buffer (B1). Individual proteins that were not serum proteins of the antibody used for the immunoprecipitation were cut out of the one- and two-dimensional gels and identified by MALDI-TOF and are listed in Table 1.

GAPDH Interacts Directly with the MAP1B-HC. Because GAPDH was able to interact directly with actin or tubulin,17-20 this coprecipitation could be explained by an indirect coprecipitation via MAP1B-actin or -tubulin complex. Therefore, we had to prove that MAP1B-HC can specifically bind GAPDH. To test this, we used recombinant and 6-His-tagged MAP1B1404, 6-His-tagged LC1, or 6-His-tag bound to nickel beads and incubated wtih COS-7 cell extracts. Extracts, 6-His-tag alone and bound proteins were separated on Western blots and tested for the presence of actin and GAPDH. Note that MAP1B1404 is able to bind actin (Figure 3A1) and GAPDH (Figure 3 A2) at similar ratios. The 6-His-tag alone had little specificity to bind

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MAP1B-GAPDH Interaction Table 1. Results of the MALDI-TOF Analysisa no

identification

1 2 3 4 5 6a 6b 7 8

P14873 Q62261 Q80U89 Q922D2 P08109 O08553 Q62188 P70333 P05217 P05218 Q9ERD7 Q80Y54 P19324 P02568 P60710 AAH03337 P16858 P23506 Q9D716

9 10 11 12 13

name

mass

score

Microtubule-associated protein 1B, MAP1B Spectrin beta chain, brain 1 MKIAA0034 protein (Fragment) Similar to myosin, heavy polypeptide 2, skeletal muscle, adult Heat shock cognate 71 kDa protein, HS7C Dihydropyrimidinase related protein-2, DPY2, CRMP2 Dihydropyrimidinase related protein-3, DPY3, CRMP3 Heterogeneous nuclear rbonucleoprotein H1 Tubulin beta-2 chain Tubulin beta-5 chain Tubulin beta-3 Tubulin beta-4 47kDa heat shock protein precursor, HS47 Actin, alpha skeletal muscle (Alpha-actin 1) Actin, cytoplasmic 1 (Beta-actin) Actin, gamma, cytoplasmic Glyceraldehyde 3-phosphate dehydrogenase, GAPDH Protein-L-isoaspartate(D-aspartate) O-methyltransferase, PIMT 231004H06 Rik protein

271556 275164 194123 183939 71055 62531 62296 51470 50255 50095 50842 50010 46674 42366 42052 42108 35941 24545 23806

140 166 285 240 222 193 232 415 1103 974 859 916 274 243 271 271 575, 368 258 434

a Pieces of Coomassie stained electrophoresis were cut out and analyzed by mass spectrometry. The identification of the protein, the hypothetical molecular mass, and the score found for each protein are listed in this table.

Figure 2. Less GAPDH is co-immunoprecipitating with MAP1B from newborn and adult mouse brain. Identification of GADPH on Western blots of brain homogenates from newborn (A1) and adult mice (A2). MAP1B was immunoprecipitated from brain homogenates of P0 and adult mice and separated by electrophoresis, electrically transferred to nitrocellulose and stained for GAPDH (B), β-tubulin (C), and MAP1B1404 (D).

GAPDH (lanes 2). The GAPDH bound to nickel resisted a wash with a stringent buffer. To exclude a secondary protein from the cell extract to interfere with GAPD-MAP1B binding. GAPDH, purified from rabbit muscle, was incubated in presence of MgCl2 and GTP together with MAP1B1404-nickel beads (Figure 3B1). In contrast, 6-His-tag MAP1B-LC1-nickel beads were unable to bind GAPDH (Figure 3B2 lane 3) and was even lower than GAPDH bound nonspecifically to nickel beads alone (lane 2). MAP1B-HC, GAPDH, and MAP1B-LC1 in PC12 Cells. The distribution of MAP1B-HC, MAP1B-LC1, and GAPDH in PC12 cells was investigated. PC12 cells grown without NGF showed a rather punctate and diffuse MAP1B-HC and GAPDH staining, with more GAPDH in the nucleus (Figure 4A1 and A2). The merge of the two micrographs showed some overlap in the cytosplasmic staining, mainly at the periphery of the cells (Figure 4A3) and suggests some overlap of staining, seen as yellow spots. In these cells, an exposure to nerve growth factor (NGF) induced neurite ourgrowth. MAP1B-HC showed a rather diffuse staining pattern in form of little dots (Figure 4A1) and with an intense staining in neurites (B1), whereas GAPDH was localized in nuclei and had disappeared from cytoplasm. H2O2induced stress resulted also in a disappearance of cytoplasmic GAPDH staining (Figure 4C2), as also observed with other

Figure 3. MAP1B-HC but not MAP1B-LC1 interacts with GAPDH in vitro. (A) COS-7 lysates (lane 1) were incubated with purified 6-His tag alone (lane 2) or with 6-His MAP1404 (lane 3, in A1 and A2) or with 6 his MAP1B-LC1 (lane 3 in A3). 6-His-tag bound proteins were extracted with nickel beads, separated by electrophoresis, and electrically transferred to nitrocellulose. Western blots were stained with anti-actin (A1) and anti-GAPDH (A2). (B) Purified GADH (lane 1) was also used for incubation with 6-His (lanes 2), with 6-His MAP1404 (B1, lane 3), and with 6-His-tagged LC1 (B2, lane 2). Blots were stained with anti-GAPDH. The molecular weight is indicated to the right in kDa.

substances such as colcemid and cytochalasin D (not shown). The disappearance of cytoplasmic GAPDH staining after a colcemid or cytochalasin D exposure was reversible, because cultures incubated with medium only, revealed a return of GAPDH immunoreactivity in the cytoplasm within several hours. A localization of MAP1B-LC1 and GAPDH in PC12 cells did not show a co-localization (Figure 4D). A particular staining of the nuclear membrane was found with the LC1 antibody (Figure 4D1, arrow). Please note that the anti-MAP1B-LC1 staining was more granular. This confirms that GAPDH is not Journal of Proteome Research • Vol. 6, No. 7, 2007 2643

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Figure 4. Immunocytochemical localization of MAP1B-HC, GAPDH, and MAP1B-LC1. Localization of MAP1B-HC and GAPDH in PC12 cells without NGF (A and C) and in PC12 cells with NGFinduced neurite outgrowth (B). (A1, B1, and C1) MAP1B-HC staining in green; (A2. B2 and C2) GAPDH distribution in red; and panels A3, B3, and C3 represent the merge of the two staining patterns. The PC12 cell (C) was exposed to H2O2; note that in this cell the cytoplasmic staining of GAPDH disappeared with oxidative stress. A PC12 cell without NGF treatment (D) was stained for MAP1B-LC1 (D1) and for GAPDH (D2) and merge (D3). The arrow in D1 points to staining of the nuclear membrane. In C3 and D3, the nuclei are visualized with Dapi staining. Magnification bars ) 10 µm.

binding at all to the MAP1B-LC1. A pulldown with 6-His tag MAP1404 nickel beads from ( NGF-treated PC12 cell extracts indicated that recombinant MAP1404 was able to bind GAPDH only in extracts from cells that were not treated with NGF (data not shown). This may suggest that the nuclear GAPDH may be a different isoform that does not bind to MAP1B. The specific staining patterns of actin, tubulin, GAPDH, MAP1B-HC, and MAP1B-LC1 are compared in Figure 5. None of the patterns shows a striking similarity to GAPDH. Therefore, GAPDH may bind only partially to other proteins, if at all, and needs a confirmation by biochemical studies, as shown in Figures 2 and 3.

Discussion MAP1B Interactions with Other Proteins. A well-characterized antibody against MAP1B (comprising the N-terminal part of the MAP1B-HC) was used to immunoprecipitate MAP1B and associated proteins, followed by one- and two-dimensional gel electrophoresis and subsequent MALDI-TOF analysis. Several proteins were associated with MAP1B-HC. Tubulin and actin are major MAP1B interacting proteins, as already demonstrated,4,5 with tubulin and actin-binding sites at both ends of the full length MAP1B molecule and essential in the cross2644

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linking of microtubules and microfilaments. Another protein associated with MAP1B-HC at high levels in adult brain was GAPDH. This was briefly mentioned5 and was investigated in more detail in the current study. By several binding studies a direct binding of GAPDH to MAP1B-HC was demonstrated. GAPDH is the key enzyme in the glycolytic pathway, essential in the second phase of glycolysis and involved in the redox system with NAD+. Furthermore, together with actin it plays a role in axonal transport.21 Several proteins have been identified by two-hybrid screen to bind to MAP1B such as gigaxonin;7,8 GABAc receptor rho1 subunit linking GABA receptors to the cytoskeleton9 and MAG.10,11 However, none of these proteins were identified in MAP1B immunoprecipitates. Probably, due to a low stoichiometry such proteins escaped detection by MALDI-TOF. Other proteins of lower levels were identified, suggesting that MAP1B may form a macromolecular complex that extends beyond actin and tubulin. Brain spectrin is involved in the formation of the membrane skeleton and together with actin forms an essential structural stabilization.22,23 It could also provide an anchor for microtubule binding to the underlying plasma membrane. MKIAA0034, a clathrin-related protein, is essential in the synaptic vesicle recycling and endocytosis24,25 and is found together with heatshock proteins and actin in the slow component b fraction of axonal transport.26 Dihydropyrimidinase protein 2 and 3, also known as collapsin response mediator protein 2 and 3 (CRMP2 &3), belong to the Ulip family phosphoproteins and are involved in the development and in axonal guidance and regeneration.27,28 These proteins may be involved in morphological changes during development and may remodel microtubule organization.29 The protein-L-isoaspartate methyltransferase (PIMT) is an enzyme that repairs damaged proteins that have accumulated abnormal aspartyl residues, such as found in damaged tubulin.30 Several of these proteins, GAPDH,31,32 PIMT,33 CRMP234 are known to be implicated in some neurodegenerative diseases as well as MAP1B itself, such in schizophrenia35,36 or in Alzeihmer’s disease.37 However, the specificity and relevance of such binding needs further work. MAP1B Interaction with GAPDH. Despite the fact that MAP1B HC and LC have a tubulin and actin binding site, GAPDH is binding only to the MAP1B-HC and not to the MAP1B-LC1. However, GAPDH may bind indirectly to LC1 via actin and tubulin, because both proteins are known to bind also GAPDH.17 Tubulin, FtsZ, and GAPDH share a common fold of two domains connected by several central helices with sequence homology of the nucleotide-binding site.38 It remains to be seen whether this highly conserved nucleotide binding site of tubulin and GAPDH may interact with MAP1B. Coprecipitated GAPDH from adult brain was composed of several isoforms and was present in 4-5 fold higher amounts than in the pull down obtained from newborn brain. This suggests that post-translational modification of GAPDH occurs during development and may explain differences in the GAPDH binding to MAP1B. It should also be noted that MAP1B changes its phosphorylation state during development.39-41 Different GAPDH isoforms may differ in their interaction with other proteins. A 2D gel analysis revealed differences in intracellular GAPDH structure, in that cytoplasmic GAPDH had a pI of 7-7.5 whereas its nuclear counterpart displayed a pI of 8.7 and suggested that specific changes in its structure may be due to modifications like phosphorylation or glycosylation.42,43 Furthermore, phosphorylation seems also to influence immuno-

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MAP1B-GAPDH Interaction

Figure 5. Examples of the immunocytochemical distribution of actin, β-tubulin, GAPDH, MAP1B-HC, and MAP1B-LC1. Note that the distribution patterns are quite different, suggesting that a “colocalization” is not evident. Yet, MAP1B-HC and MAP1B-LC1 are both known to bind to tubulin and to actin. The magnification bar is ) 5 µm.

precipitation of GAPDH from PC12 cells. NGF induced an increase in phosphorylation activity and neurite outgrowth in PC12 cells44,45 and may therefore be the reason for a lesser GAPDH pull down with MAP1B. Furthermore, GAPDH is known to bind to actin and to microtubules and may promote actin polymerization and microtubule bundling (17-20) and modulated such binding by phosphorylation.46,47 One needs to consider that also GAPDH, next to MAP1B, may have a microtubule-stabilizing effect. We still ignore whether other proteins may interfere with MAP1B-GAPDH binding such as in juvenile brain tissue. GAPDH plays a role in a variety of cell functions. It displayed a distinct membrane, cytosolic and nuclear localization, and is involved in endocytosis and membrane fusion; vesicular secretory transport and translation control; nuclear tRNA transport, DNA replication and DNA repair.14 Furthermore, GAPDH was present as a tetramer in the cytoplasm where the enzyme catalyzed its glycolytic activity, whereas the uracil glycosylase activity of GAPDH was associated with the monomer form of GAPDH in the nucleus.48 Our results corroborate that GAPDH is disappearing from the cytoplasm when oxidative stress is involved or when cytoskeletal structures are destroyed by alkaloids such as by colcemid or cytochalasin D and thus putting cells under a structural stress. When colcemid treatment was stopped, microtubules and GAPDH reappeared in the cytoplasm. These preliminary results open new perspectives to investigate a correlation with GAPDH and the formation of microtubules and also indicate that GAPDH is not necessarily always a proapoptotic protein. Nuclear translocation of GAPDH appeared in non-neuronal and neuronal cells when subjected to various stress including dexamethasone treatment, NGF withdrawal, aging cultures, and oxidative stress.49-51 But the precise mechanism of the nuclear localization of GAPDH has not yet been clarified. Experiments using green fluorescent-GAPDH confirm movements of GAPDH fusion proteins from the cytosol to the nucleus shortly after exposure of cells to apoptosis-stimulating agents49,50 and demonstrated that the nuclear GAPDH translocation was an early event in apoptosis and preceding the chromatin alterations.51 Such observations are in-line with many results that point to a pro-apoptotic function of GAPDH. Initial in vitro studies found GAPDH associated to the cytoplasmic carboxyl terminal of beta amyloid precursor protein52 and immunofluorescence showed a presence of GAPDH in amyloid plaques from brains of patients with Alzheimer’s disease. There is growing evidence that GAPDH plays a critical role in some forms of neuronal apoptosis, because GAPDH is considered a pre-apoptotic protein and target protein for antiapoptotic drugs.53-55 The translocation of GAPDH initiates cell death and may play a role in neurodegerative diseases by interacting with Huntingtin, dentatorubralpallidoluysian atro-

phy proteins, or the E3-ubiquitin-ligase Siah1.56,57 GAPDH is also involved in ischemia, because it interacted with HSP70, known to play a major role in protection against ischemia.58,59 Indeed, the HSP70 and HSP47 were also among proteins that coprecipitated with MAP1B and could be therefore involved in development. GAPDH plays also a role in vesicular and membrane trafficking.60 The only membrane staining was visible with antiMAP1B-LC1, but there was no overlap in the immunocytochemical localization with GAPDH, nor was a direct binding between MAP1B-LC1 and GAPDH measured. Our experiments rather support the view that cytoplasmic GAPDH may interact via the MAP1B-HC and not the MAP1B-LC1. One possible explanation why some GAPDH is binding to MAP1B may represent a means to keep the key glycolytic enzyme close to cytoskeletal structures and ensure energy provision to the ATP- and GTPdependent and dynamic microfilaments and microtubules, respectively. For microtuble assembly and microfilament formation, the use of ATP and GTP is essential. Because MAP1B is a growth-associated protein, its role may be important during growth processes of dendrites and axons. Further experiments are necessary to corroborate this attractive hypothesis that GAPDH may be involved in the energy provision for cytoskeletal structures.

Conclusions MAP1B may have a central position between two cytoskeleton structures during growth process of axons and provide a link between growth of axons and local motility. It may harbor several key proteins involved in various functions and may act as a scaffold protein.2 It was shown that the N-terminal part of MAP1B can bind GAPDH and so keep the key protein of the energy metabolism close to cytoskeleton, a speculation that merits a closer look. Similar experiments need to be performed for the other MAP1B-associated proteins that were identified in this study. Their lower stoichiometry rather suggests an indirect binding to MAP1B via actin, tubulin, or GAPDH. It is likely that MAP1B together with these proteins forms a macromolecular complex that is involved in many cellular functions. Furthermore, the antibody against the MAP1B-LC1 may provide another tool to identify novel binding proteins or confirm some of the already reported MAP1B-LC1-binding proteins.

Acknowledgment. We thank Dr. M. Quadroni of the PAF for the MALDI-TOF identification. This work was supported by the Swiss National Science Foundation grant 3100067201.01. Journal of Proteome Research • Vol. 6, No. 7, 2007 2645

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