Research Article 4847 β-Dystrobrevin interacts directly with kinesin heavy chain in brain

P. Macioce*, G. Gambara, M. Bernassola, L. Gaddini, P. Torreri, G. Macchia, C. Ramoni, M. Ceccarini and T. C. Petrucci Laboratory of , Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy *Author for correspondence (e-mail: [email protected])

Accepted 28 July 2003 Journal of Cell Science 116, 4847-4856 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00805

Summary β-Dystrobrevin, a member of the dystrobrevin α- and β-dystrobrevin, indicating that this interaction is not family, is a -related and -associated protein restricted to the β-dystrobrevin isoform. The site for Kif5A restricted to non-muscle tissues and is highly expressed in binding to β-dystrobrevin was localized in a carboxyl- kidney, liver and brain. Dystrobrevins are now thought to terminal region that seems to be important in heavy chain- play an important role in intracellular , mediated kinesin interactions and is highly homologous in in addition to providing a membrane scaffold in muscle, but all three Kif5 isoforms, Kif5A, Kif5B and Kif5C. Pull-down the precise role of β-dystrobrevin has not yet been and immunofluorescence experiments also showed a direct determined. To study β-dystrobrevin’s function in brain, interaction between β-dystrobrevin and Kif5B. Our we used the yeast two-hybrid approach to look for findings suggest a novel function for dystrobrevin as a interacting . Four overlapping clones were motor protein receptor that might play a major role in the identified that encoded Kif5A, a neuronal member of the transport of components of the dystrophin-associated Kif5 family of proteins that consists of the heavy chains of protein complex to specific sites in the cell. conventional kinesin. A direct interaction of β-dystrobrevin with Kif5A was confirmed by in vitro and in vivo association assays. Co- with a Key words: Dystrobrevin, Kinesin, Yeast two-hybrid, Brain, monoclonal kinesin heavy chain antibody precipitated both

Introduction homologous proteins, α- and β-dystrobrevin (Ambrose et al., In muscle, the dystrophin-associated protein complex (DPC) is 1997; Loh et al., 1998). Several distinct transcripts are derived generally thought to link the cortical cytoskeleton to the from each by or initiation sites, extracellular matrix, and of dystrophin or other generating a large family of dystrobrevin isoforms (Blake components in the complex result in a variety of muscular et al., 1996; Peters et al., 1997b; Blake et al., 1998). α- dystrophies (Straub and Campbell, 1997). The DPC is Dystrobrevin is expressed predominantly in , expressed in muscle and non-muscle tissues and is now heart, lung and brain (Blake et al., 1996; Sadoulet-Puccio et considered to be not only a mechanical component of cells, but al., 1996) and is thought to be involved in synaptic transmission also a highly dynamic multifunctional structure that can serve at the (Sanes et al., 1998; Grady et al., as a scaffold for signaling proteins (Rando, 2001). The DPC 2000) and in intracellular signaling (Grady et al., 1999). α- can be subdivided into three distinct sub-complexes: the Dystrobrevin knockout mice develop a mild form of muscular dystroglycan complex, the complex and the dystrophy and display a phenotype similar to that of null cytoplasmic complex (for a review, see Blake and Davies, dystrophin mutants, although the integrity of the DPC is largely 1997). The cytoplasmic complex consists of maintained (Grady et al., 1999). Since they have reduced levels (Adams et al., 1993; Ahn et al., 1994; Yang et al., 1994) and of cGMP and have less nNOS at the , it has been dystrobrevins, both of which are intracellular proteins that bind proposed that the muscle degeneration observed in these mice directly to dystrophin (Ahn and Kunkel, 1995; Suzuki et al., is caused by a deficiency in intracellular signaling mediated by 1995; Sadoulet-Puccio et al., 1997). α-dystrobrevin (Grady et al., 1999). The second member of the Dystrobrevins are members of the dystrophin family with dystrobrevin family, β-dystrobrevin, is restricted to non-muscle unique extreme carboxyl termini (Roberts, 2001) and a tissues, is most abundantly expressed in kidney and brain and significant sequence homology with the cysteine-rich forms complexes with dystrophin isoforms and in carboxyl-terminal region of dystrophin (Wagner et al., 1993; liver and brain (Peters et al., 1997a; Blake et al., 1998; Puca et Ambrose et al., 1997). This region of similarity can be divided al., 1998; Blake et al., 1999). In brain β-dystrobrevin associates into several functional domains, namely two EF hands (Koenig with dystrophin isoforms in the cortex, and et al., 1988), a ZZ domain (Ponting et al., 1996), and two Purkinje neurons and is highly enriched in post-synaptic coiled-coil regions (Blake et al., 1995). Dystrobrevins are the densities (Blake et al., 1998; Blake et al., 1999). Since product of two different coding for two highly approximately one third of Duchenne muscular dystrophy 4848 Journal of Cell Science 116 (23) patients suffer from mental retardation and cognitive deficits Full-length human Dp71 cDNA, obtained as reported previously (Hodgson et al., 1992; Lidov, 1996), it has been proposed that, (Ceccarini et al., 1997), was utilized as a template to amplify the by analogy to the situation in muscle, a neuronal DPC-like region coding for amino acids 285-622 using the appropriate primers. complex may be involved in the establishment of these CNS Dp71285-622 fragment was sub-cloned into pCRII-TOPO, to produce defects (Blake et al., 1999). In mdx3Cv mice, which lack all pCRII-TOPO/Dp71285-622. The fragment was then EcoRI-restricted known dystrophin isoforms, neither β-dystrobrevin expression and sub-cloned into the EcoRI site of pGADT7 to generate pGADT7/ Dp71285-622. and localization at the membrane, nor the assembly of the DPC A previously described rabbit β-dystroglycan (β-DG) cDNA (Rosa are affected in brain and kidney, whereas in muscle this et al., 1996) was used to amplify the region coding for the cytoplasmic complex is destabilized. It has therefore been speculated that portion of β-dystroglycan (amino acids 774-895), and pGBKT7/β- β-dystrobrevin may be a key regulator in the assembly and DG774-895 was obtained basically as described for β-DB. maintenance of DPC-like complexes in non-muscle tissues HA-Kif5A#18 DNA fragment coding for amino acids 611-1027 of (Blake et al., 1999; Loh et al., 2000). Recent studies with Kif5A and the hemagglutinin (HA) epitope tag upstream was β-dystrobrevin-deficient mice have shown, however, that amplified by PCR from pACT2-Kif5A#18 template, and sub-cloned although β-dystrobrevin is required for the membrane into pCRII-TOPO to create pCRII-TOPO/HA-Kif5A#18. localization of the short dystrophin isoform Dp71 and Kif5A771-1027, Kif5A804-1027, Kif5A804-934, and Kif5A934-1027 were syntrophin in kidney and liver, its presence is not necessary for also obtained by PCR using pACT2-Kif5A#18 as a template, and sub- cloned into pCRII-TOPO vector. After EcoRI digestion, the Kif5A normal function (Loh et al., 2001). fragments were inserted into the EcoRI site of pGADT7 vector, In the search for new insights into the functions of to create pGADT7/Kif5A deletion mutant constructs (pGAD- dystrobrevins, new partners have recently been identified using T7/Kif5A771-1027, pGADT7/Kif5A804-1027, pGADT7/Kif5A804-934, the yeast two-hybrid system (Benson et al., 2001; Mizuno et and pGADT7/Kif5A934-1027). al., 2001; Newey et al., 2001). Two of these partners, The correct orientation of cDNA inserts was verified by restriction and desmuslin, are novel members of the intermediate filament enzyme analysis, and sequence analysis was used to check that they family, and are expressed mainly in skeletal and cardiac were in-frame. muscle. They interact with α-dystrobrevin, and are reported to A fully encoding human Kif5B cDNA (Navone et al., 1992), be concentrated at the neuromuscular junction (Newey et al., subcloned into pCB6 (Brewer and Roth, 1991), was a kind gift from 2001) and at the Z-lines (Mizuno et al., 2001), respectively. Dr Francesca Navone, CNR, Milano. They also interact directly with (Mizuno et al., 2001; Poon et al., 2002), suggesting that α-dystrobrevin may tether Yeast two-hybrid screen the intermediate filament network to the DPC (Blake and Two-hybrid screening was carried out by yeast mating, using Martin-Rendon, 2002). A third novel dystrobrevin-interacting the Matchmaker Gal4 Two-Hybrid System 3 (Clontech). Both protein, , is a 40 kDa coiled-coil-containing protein pGBKT7/β-DB and pGADT7/β-DB tested negative for auto- that binds to α- and β-dystrobrevin in muscle and brain activation of reporter gene activity in the yeast two-hybrid reporter (Benson et al., 2001). In the brain, dysbindin was detected strains, Saccharomyces cerevisiae AH109 (MATa, trp1-901, leu2-3, ∆ ∆ primarily in the axons of many types of neurons, in which it 112, ura3-52, his3-200, gal4 , gal80 , LYS2:: GAL1UAS – GAL1TATA β – HIS3, GAL2UAS – GAL2TATA – ADE2, URA3:: MEL1UAS – MEL1TATA co-localized with -dystrobrevin, suggesting their association α in a protein complex (Benson et al., 2001). – lacZ MEL1) and S. cerevisiae Y187 (MAT , ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4∆, gal80∆,met–, URA3:: To improve our understanding of the role of β-dystrobrevin GAL1UAS – GAL1TATA – lacZ MEL1). The Gal4 DNA binding domain in brain, we searched for new interacting proteins using the construct pGBKT7/β-DB was used to transform the MATa yeast strain yeast two-hybrid system (2-HS). Among the cDNA clones AH109. AH109[pGBKT7/β-DB] was then employed as a bait strain isolated we characterized four overlapping clones of mouse to screen a mouse brain cDNA library (Clontech), which was cloned neuronal kinesin heavy chain Kif5A. We confirmed the into the activation domain vector pACT2, and pre-transformed in the interaction of β-dystrobrevin with Kif5A by in vitro co- MATα yeast strain Y187. The yeast mating screening was performed immunoprecipitation and pull-down experiments and found according to the manufacturer’s instructions. Diploids were selected that kinesin heavy chain also co-immunoprecipitated with by culture on minimal synthetic dropout medium (SD) lacking Trp, dystrobrevin from rat brain extracts. We also found, through Leu, His and Ade (–TLHA), and including 5-bromo-4-chloro-3- indolyl-α-D-galactopyranoside (X-α-Gal) for 8-16 days. pull-down and transfection experiments, a direct interaction + + + β Ade , His , Mel1 colonies were isolated and then re-tested for between -dystrobrevin and ubiquitous kinesin heavy chain lacZ (β-Gal) activity by colony lift filter assay. lacZ+ colonies were Kif5B. re-streaked at least twice onto SD/-TL/X-α-Gal to allow segregation, Our data suggest a new role for dystrobrevin as a motor then transferred to SD/-TLHA/X-α-Gal to verify that they protein receptor that may be involved in the traffic and maintained the correct phenotype. Library plasmids from positive targeting of components of the DPC to specific sites in the cell. colonies were isolated and rescued using E. coli strain DH5α on ampicillin-resistant plates. AD/library inserts were then amplified by PCR and analyzed by restriction digestion. Unique inserts were Materials and Methods sequenced and DNA and protein sequence analyses were performed Plasmid constructs with the BLAST algorithm at the National Center for Biotechnology The coding region of β-dystrobrevin (β-DB) was cloned by RT-PCR Information (NCBI). from mouse brain, into pCRII-TOPO vector (Invitrogen). After EcoRI After isolation of the pACT2 plasmids encoding the library clones, digestion, the β-DB fragment was inserted into the EcoRI site of these plasmids were tested for auto-activation of the reporter gene in pGEX-6P-1 (Amersham Pharmacia Biotech), pGBKT7 and pGADT7, yeast by both co-transformation, using either the empty bait plasmid and pCMV-HA vectors (Clontech) to create pGEX-6P-1/β-DB, pGBKT7 or the same plasmid encoding an unrelated negative control pGBKT7/β-DB and pGADT7/β-DB, and pCMV-HA/β-DB, (lamin C), and yeast mating, using Y187[pGBKT7] as the empty bait respectively. strain, or Y187[pGBKT7-lamin C] as a negative control. Isolates Kinesin/β-dystrobrevin interaction 4849 growing in SD/-TLHA/X-α-Gal and developing blue staining without Immunoprecipitation the presence of the β-DB bait were excluded from further Tissue from rat cerebellum was homogenized (100 mg/ml) in 0.25 M investigation. Activation of the reporter genes in the positive colonies sucrose, 5 mM EDTA, 5 mM EGTA, 7.5 mM phosphate buffer, pH was confirmed in the same experiments. 7.4, containing protease inhibitors. After centrifugation at 600 g at 4°C, the pellet was solubilized 1:1 in 2× 10 mM phosphate buffer, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, In vitro transcription and translation 0.5% sodium deoxycholate and protease inhibitors, incubated on ice In vitro transcription and translation of pCRII-TOPO/HA-Kif5A#18, for 30 minutes and centrifuged at 20,000 g for 30 minutes at 4°C. The pGADT7/Kif5A deletion mutant constructs, pGADT7/ Dp71285-622, supernatant was used for co-immunoprecipitation experiments. To pGBKT7/β-DB and pGBKT7/β-DG774-895 was carried out using the minimize non-specific binding, the sample was pre-incubated for 3 TNT SP6 or T7 Quick Coupled Transcription/Translation System hours at 4°C with 25 µl of protein G-agarose-conjugated mouse IgG (Promega) in the presence of PRO-MIX (70% L-[35S]methionine; (Pierce) (50% slurry). After centrifugation, 2 µg of dystrobrevin 30% L-[35S]cysteine; Amersham Pharmacia Biotech) according to the monoclonal antibody (Clone 23, Transduction Laboratories) or 5 µg manufacturer’s protocol. Newly synthesized proteins were separated of kinesin heavy chain monoclonal antibody (Clone H2, Chemicon by SDS-PAGE and analyzed with an Instant-Imager (Packard). International) were added to the supernatant, and the sample was incubated overnight at 4°C. 40 µl of Protein G-Agarose (50% slurry) were added to the tube and incubated for 3 hours at 4°C. The resin Co-immunoprecipitation assay was washed three times with ice-cold TBS-T and immunoprecipitates Co-immunoprecipitation of in vitro translated proteins was carried were separated by SDS-PAGE, transferred to a nitrocellulose out as follows. 5 µl of reticulocyte lysate containing labeled HA- membrane and incubated with the kinesin heavy chain monoclonal tagged protein and 5 µl containing labeled c-Myc-tagged protein antibody (Clone H2) and either a polyclonal β-dystrobrevin antibody were mixed together and incubated at 30°C for 1 hour. After or the dystrobrevin monoclonal antibody (Clone 23). Bound incubation, 460 µl of TBS-T (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, antibodies were visualized using the ECL system (Pierce). 0.1% Tween 20) supplemented with 1 mM DTT and protease Polyclonal β-dystrobrevin antibody was prepared as follows. After inhibitors, 20 µl of 50% slurry of protein G-agarose beads (Pierce) removal of GST by digestion with pre-scission protease (80 U/ml; and 10 µl HA-tag polyclonal antibody (Clontech) were added. The Amersham Pharmacia Biotech), recombinant β-dystrobrevin was mixture was rocked gently at 4°C for 2 hours, the beads were injected into rabbits according to a previously described protocol collected by brief centrifugation and washed three times with TBS- (Rosa et al., 1996). The affinity-purified antibody was obtained by T. The pellets were resuspended in 15 µl of Laemmli’s loading buffer, standard methods, using GST-β-DB coupled to Sepharose-CNBr the proteins were eluted and denatured by boiling for 2 minutes and beads (Amersham Pharmacia Biotech). then separated by SDS-PAGE. The gel was subsequently fixed, dried, analyzed with an Instant-Imager (Packard) and exposed on Kodak X- OMAT-AR film. Cell culture and transient transfections Cos-7 cells were grown and maintained (5% CO2, 37°C) in DMEM supplemented with 10% fetal bovine serum. Cells seeded on sterile, Pull-down experiments untreated glass coverslips were transfected using Fugene 6 (Roche) pGEX-6P/β-DB and pGEX-6P were used to transform E. coli BL21 according to the manufacturer’s instructions. For some cells, cells. Overnight cultures were diluted 1:10, grown at 37°C until A600 nocodazole (Sigma) was used at a final concentration of 20 µM for 4 was 0.7-1.0, and induced for 3 hours with 1 mM isopropyl-β-D- hours before fixation. Wash-out was performed by washing thiogalactoside (IPTG) at 30°C. Recombinant proteins were purified nocodazole-treated cells three times with culture medium, followed from the soluble cell lysates by glutathione-Sepharose column by incubation at 37°C in nocodazole-free medium for 2 hours. following the procedure described by Kennedy et al. (Kennedy et al., 1991). GST (glutathione S-transferase)-β-DB or GST bound to Immunofluorescence glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) Twenty-four to 66 hours after transfection, the cells were washed with were equilibrated in binding buffer (20 mM Hepes, pH 7.4, 0.15 M TBS, fixed with 3.7% formaldehyde in TBS for 15 minutes at room NaCl, 1 mM DTT, 0.2% Triton X-100, 5 µM MgSO4, and protease temperature and permeabilized for 5 minutes in TBS containing 0.5% inhibitors). For binding assays, 10 µl of in vitro-translated reaction Triton X-100. After washing, cells were blocked for 30 minutes in products were incubated with 20 µl of 50% slurry bead-bound TBS containing 3% normal goat serum and then incubated for 1 to 3 GST-fusion proteins overnight at 4°C on a rotator. After six washes hours with primary antibodies diluted in TBS containing 1% normal with 450 µl binding buffer, bound radioactive proteins were re- goat serum. Kif5B and β-dystrobrevin were stained by the kinesin suspended in Laemmli’s loading buffer and separated by SDS- heavy chain monoclonal antibody (Clone H2) and the polyclonal β- PAGE. Gels were dried and radiolabeled proteins were detected by dystrobrevin antibody, respectively. autoradiography. Cells were then incubated with the appropriate secondary For GST pull-down assays with COS-7 extracts, semi-confluent antibodies conjugated with either FITC or TRITC for 1 hour and cells from a 100-mm plate were harvested, washed twice with TBS, mounted with VectaShield (Vector Laboratories). re-suspended in binding buffer and lysed by passing through a 25- The images were examined under a Leica TCS 4D confocal gauge needle, on ice. The lysates were cleared by centrifugation at microscope. Image acquisition and processing were performed using 15,000 g for 15 minutes at 4°C, and the supernatants were incubated the SCANware and Multicolor Analysis (Leica Lasertechnik Gmbh) overnight at 4°C on a rotator with 100 µl of 50% slurry bead-bound and Adobe Photoshop (Adobe Systems) software programs. GST-fusion proteins. The beads were washed as described above and bound proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and incubated with the kinesin polyclonal Results anti-uKHC antibody, which reacts specifically with ubiquitous kinesin β heavy chain Kif5B (Niclas et al., 1994), a kind gift from Dr Francesca Identification of Kif5A as a potential binding partner of - Navone, CNR, Milan. Bound antibodies were visualized using the dystrobrevin by yeast two-hybrid screening ECL system (Pierce). In order to identify proteins that associate with β-dystrobrevin 4850 Journal of Cell Science 116 (23)

(β-DB) in the brain, we used a two-hybrid mating strategy Table 1. Interactions between β-dystrobrevin and Kif5A using a high level of stringency to identify proteins that interact clones in α-galactosidase assays β strongly with our bait protein. The full-length -dystrobrevin pACT2 (AH109) coding sequence was cloned by RT-PCR from mouse brain pGBKT7 RNA. Brain transcripts predominantly lack exon 10 and may (Y187) Kif5A#18 Kif5A#69 Kif5A#75 Kif5A#81 or may not have exons 17 and 18 (Loh et al., 1998). Sequence Empty – – – – β β-DB +++ +++ +++ +++ analysis of the pCRII-TOPO/ DB clone revealed that exons 10 Lamin C – – – – and 17 were both spliced out. The mouse β-dystrobrevin coding sequence was fused in frame to the DNA-binding The numbers of plus signs represent the relative rates at which diploid domain of Gal4 (pGBKT7) and transformed into yeast strain strains turned blue after incubation at 30°C on –TLHA/X-α-Gal plates; +++, AH109. The bait strain was used to screen a mouse brain 1-3 hours; –, no growth. A diploid strain derived from mating AH109 [pGBKT7-53] and Y187[pTD1-1], which started to turn blue in less than 1 cDNA library sub-cloned into the Gal4 activation domain hour on –TLHA/X-α-Gal plates, was used as a positive control (not shown); vector pACT2 pre-transformed into yeast strain Y187. Y187[pGBKT7-lamin C] provided a negative control. At least four Approximately 106 independent clones were screened for independent diploid colonies were tested for each pair. expression of the reporter genes HIS3, ADE2 and Mel1/lacZ, and 204 positive clones were found. These clones could contain more than one AD/library plasmid, so we allowed pACT2, and pGBKT7/β-DB, carrying a c-Myc epitope tag, to segregation and recollected them on SD/-TLHA/X-α-Gal perform in vitro transcription and translation. In vitro co- plates. We selected 37 diploids strongly activating Mel1/lacZ immunoprecipitation confirmed our 2-HS results, showing that reporter genes, rescued AD/library plasmids via transformation the interactions between bait and prey proteins were specific of E. coli and sorted them using PCR followed by restriction (Fig. 2A). The dystroglycan cytoplasmic domain did not analysis, obtaining 12 different types of insert. Sequencing of interact with kinesin (Fig. 2A), as expected on the basis of the junctions with Gal4 AD and homology search (BLAST) previous 2-HS results (data not shown). revealed that four of them (see Fig. 1) were overlapping As an additional demonstration of interaction between β- fragments of the same gene, Kif5A (GenBank Accession no. dystrobrevin and Kif5A, we performed GST-β-DB pull-down NM_008447). The specificity of the interaction between Kif5A experiments. Recombinant GST-β-DB, or GST alone, bound to and β-dystrobrevin in yeast was confirmed by mating different glutathione-Sepharose beads were allowed to interact with bait and prey strains (Table 1). Kif5A clones interact with β- 35S-labeled Kif5A#18. GST-β-DB binds 35S-labeled Kif5A#18 dystrobrevin but not with the unrelated protein lamin C. Kif5A protein, as well as a Dp71285-622 positive control (Sadoulet- clones do not transactivate reporter gene expression when Puccio et al., 1997), but GST protein does not (Fig. 2B). mated with the haploid bait strain pre-transformed with the empty plasmid pGBKT7 (Table 1). In vivo association of kinesin heavy chain and β- dystrobrevin In vitro association of Kif5A and β-dystrobrevin To ascertain whether the interaction between β-dystrobrevin We next performed immunoprecipitation and pull-down and Kif5A occurs in vivo, we performed co- experiments to investigate whether the interaction between β- immunoprecipitation experiments using rat brain extracts. dystrobrevin and Kif5A also takes place in vitro. Lysates from rat cerebellum were pre-cleared and incubated We used pCRII-TOPO/HA-Kif5A#18, which encodes the with a dystrobrevin or a kinesin antibody, respectively. Protein longest Kif5A polypeptide plus the HA epitope tag from G-agarose beads selectively precipitated the immuno- complexes, which were subsequently separated by SDS-PAGE and immunoblotted with dystrobrevin and kinesin antibodies 1330 907 1027 (Fig. 3). The monoclonal dystrobrevin antibody used for Kif5A immunoprecipitation is reactive to all dystrobrevin isoforms: α-dystrobrevin 1 (78 kDa), α-dystrobrevin 2 (55 kDa) and β- motor head coiled-coil stalk globular tail dystrobrevin isoforms (59 kDa). Our results showed that monoclonal anti-dystrobrevin co-immunoprecipitates kinesin with dystrobrevins from rat cerebellum lysates (Fig. 3A). When we used a monoclonal anti-kinesin heavy chain antibody 611 1027 Clone 18 to immunoprecipitate kinesin, we detected the presence of co- immunoprecipitated dystrobrevin isoforms. Fig. 3B shows an 654 1027 Clone 75 upper band, corresponding to α-dystrobrevin 1, and a lower band, corresponding to α-dystrobrevin 2 and β-dystrobrevin, 743 1027 Clone 69 also including IgG heavy chain. Taken together, our data show that kinesin heavy chain and dystrobrevins specifically interact 768 1027 in vivo. Clone 81

Fig. 1. Schematic representation of Kif5A clones found to interact β with β-dystrobrevin in the two-hybrid screening of a mouse brain Association with -dystrobrevin involves Kif5A domain cDNA library. A diagram of full-length Kif5A is aligned at the top. spanning amino acids 804 to 934 The positions of the amino acids are indicated. In order to characterize further the structural requirements of Kinesin/β-dystrobrevin interaction 4851

Fig. 3. Direct interaction of dystrobrevin and kinesin heavy chain in rat brain. (A) The anti-dystrobrevin antibody (Clone 23) co- immunoprecipitates kinesin and dystrobrevin isoforms. Pre-cleared Fig. 2. Direct in vitro interaction of β-dystrobrevin and neuronal rat cerebellum lysate (input), mouse IgG control immunoprecipitate kinesin heavy chain. (A) Co-immunoprecipitation: cMyc-β- (IP: control IgG) and anti-dystrobrevin immunoprecipitate (IP: dystrobrevin and HA-kinesin (Kif5A clone 18) were translated in dystrobrevin) were separated by SDS-PAGE, and transferred to vitro in the presence of [35S]methionine and [35S]cysteine, and mixed nitrocellulose. The blot was probed with anti-kinesin (Clone H2) and together. The mixtures were immunoprecipitated with polyclonal the polyclonal anti-β-dystrobrevin antibody, and revealed by ECL. anti-HA, and the immunoprecipitated proteins visualized by Besides detecting β-dystrobrevin (59 kDa), the polyclonal anti-β- autoradiography after SDS-PAGE. Non-specific IgG were used as dystrobrevin antibody cross-reacts with α-dystrobrevin isoforms. negative control. 35S-labeled cMyc-β-dystroglycan (amino acids 774- (B) The anti-kinesin antibody (Clone H2) co-immunoprecipitates 895) did not co-immunoprecipitate with kinesin, showing the dystrobrevin isoforms and kinesin. Rat cerebellum lysate (input), specificity of the β-dystrobrevin interaction with kinesin. (B) Pull- anti-kinesin immunoprecipitate (IP: kinesin) and mouse IgG control down assay: purified GST-β-dystrobrevin (GST-β-DB) or GST- immunoprecipitate (IP: control IgG), were separated by SDS-PAGE protein (GST), pre-bound to glutathione-Sepharose beads, was and transferred to nitrocellulose. The blot was probed with anti- incubated with in vitro translated [35S]kinesin (Kif5A clone 18) or dystrobrevin (Clone 23) and anti-kinesin (Clone H2) antibodies, and 35 [ S]Dp71 (Dp71285-622, used as a positive control). After extensive revealed by ECL. The anti-dystrobrevin antibody (Clone 23) is washing, bound radioactive proteins were separated by SDS-PAGE reactive to all dystrobrevin isoforms. In the anti-kinesin and detected by autoradiography. The extra band under [35S]kinesin immunoprecipitate (IP: kinesin) a band corresponding to α- in the first lane is a product present after the in vitro transcription and dystrobrevin 1 (78 kDa) is clearly visible. The 59 and 55 kDa bands translation. of dystrobrevin isoforms (see input) overlap with the co-migrating 55 kDa IgG heavy chain band (see IP: control IgG). Molecular masses β of size markers are indicated on the right-hand side (in kDa). WB, the interaction between Kif5A and -dystrobrevin, we western blot. generated a set of progressively smaller constructs for Kif5A (Fig. 4A) and tested them for interactions with β-dystrobrevin by pull-down assay. The minimum region of overlap for In vitro association of Kif5B and β-dystrobrevin binding between the two proteins was found to be between To clarify whether β-dystrobrevin interacts specifically with amino acids 804 and 934 of Kif5A (Fig. 4B). This region Kif5A or whether the interaction includes other isoforms of corresponds to GluR2-interacting protein (GRIP1) minimal conventional kinesin heavy chain, we performed a GST pull- of all Kif5s (Kif5A, Kif5B and Kif5C) (Setou et down assay on COS-7 cell lysates. While not expressing al., 2002) and overlaps with the binding domain for kinectin neuronal-specific Kif5A, COS-7 cells do express ubiquitous (Ong et al., 2000) and the cargo-binding domain of the kinesin heavy chain isoform Kif5B, which showed a specific Neurospora kinesin (Seiler et al., 2000), indicating that this association with GST-β-DB fusion protein (Fig. 5). These data part of the molecule might play a major role in heavy chain- clearly demonstrate a direct interaction between β- mediated kinesin interactions. dystrobrevin and Kif5B. 4852 Journal of Cell Science 116 (23)

Fig. 5. Direct interaction of β-dystrobrevin and ubiquitous kinesin heavy chain. COS-7 cell lysate was incubated with GST-β- dystrobrevin (GST-β-DB) or GST-protein (GST), pre-bound to glutathione-Sepharose beads. After extensive washing, bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with polyclonal anti-uKHC antibody, and revealed by ECL. Molecular masses of size markers are indicated on the right-hand side (in kDa). WB, western blot.

expressing the same Kif5B cDNA, as well as by other authors in COS cells (Verhey et al., 1998). When the constructs encoding β-dystrobrevin (pCMV-HA/β-DB) and ubiquitous kinesin (pCB6/Kif5B) were co-transfected, immunofluorescence microscopy of the two co-expressed proteins showed that the punctate organization οf β- dystrobrevin was changed by kinesin overexpression into a tubulovesicular network that extended across the cell, with significant co-localization (Fig. 6C-E). We next examined β- Fig. 4. Identification of β-dystrobrevin binding site on kinesin. (A) Schematic representation of the Kif5A truncation clones. The dystrobrevin and kinesin distribution in nocodazole-treated positions of the amino acids are indicated. The β-dystrobrevin- cells. The extent of microtubule depolymerization, or binding site on Kif5A was found to be between amino acids 804 and restoration, after treatment with nocodazole, or nocodazole 934, and is indicated in black. (B) Pull-down assay: each [35S]Kif5A wash-out, was verified by tubulin staining (data not shown). truncation protein was incubated with purified GST-β-dystrobrevin We observed a dramatic re-localization of β-dystrobrevin and pre-bound to glutathione-Sepharose beads. Radioactive proteins were kinesin, with a nearly identical distribution in vesicular separated by SDS-PAGE and detected by autoradiography. An aggregates (Fig. 6F-H), which varied in size and arrangement aliquot of labeled starting material is shown on the left panel (input); within different cells, but always showed a high level of the bound Kif5A truncated proteins are on the right (pull-down). co-localization of the two co-expressed proteins. The patch- Molecular masses of size markers are indicated on the left-hand side like pattern of β-dystrobrevin and kinesin reverted to a (in kDa). tubulovesicular network after nocodazole wash-out (not shown). Immunolocalization of kinesin and β-dystrobrevin in transfected COS cells We decided to investigate further the in vivo association of β- Discussion dystrobrevin with kinesin by over-expressing β-dystrobrevin Here we describe the interaction of β-dystrobrevin with kinesin and Kif5B in COS-7 cells. Prior to these studies, we heavy chain Kif5A and Kif5B. These proteins are members of determined the distribution of β-dystrobrevin and of Kif5B by the kinesin superfamily of proteins (Kifs), the molecular single transfection of pCMV-HA/β-DB and of pCB6/Kif5B. motors that generate the microtubule-based movement of a Immunofluorescence labeling using either a polyclonal β- wide variety of materials in eukaryotic cells (Hirokawa et al., dystrobrevin antibody or a polyclonal anti-HA antibody (not 1998; Miki et al., 2001). Using a combination of two-hybrid shown) showed that cells transiently transfected with β- analysis, in vitro and in vivo association assays, and dystrobrevin displayed an intense, punctate staining pattern transfection experiments, we obtained evidence that β- distributed throughout the cell, which often concentrated dystrobrevin interacts directly with Kif5A and Kif5B. Our data around the nucleus (Fig. 6A), as also described by Benson et suggest a novel function for dystrobrevin as a motor protein al. (Benson et al., 2001). The transfected Kif5B, as visualized receptor that may be involved in the traffic and targeting of by the monoclonal kinesin antibody showed a reticular components of the DPC to specific sites in cells. distribution throughout the entire (Fig. 6B); In neurons, conventional kinesin (kinesin-I) exists as a superimposed on this pattern, a filamentous staining which tetramer of two heavy chains (KHC: Kif5), which contain the coincided with microtubules (not shown) was also evident. amino-terminal motor domain, as well as two light chains These immunostaining results are consistent with those (KLC), which bind to the heavy chain tail (Vale et al., 1985; observed by Navone et al. (Navone et al., 1992) in CV-1 cells Hirokawa, 1998) and are known to interact with the cargo Kinesin/β-dystrobrevin interaction 4853

Fig. 6. Co-localization of β-dystrobrevin and ubiquitous kinesin heavy chain in transfected COS-7 cells. The sub-cellular distribution of β- dystrobrevin and Kif5B was determined by transfecting COS-7 cells with pCMV-HA/β-DB (A) and pCB6/Kif5B (B). β-Dystrobrevin is located in intensely staining punctae throughout the cell, more concentrated around the nucleus (A), whereas kinesin immunoreactivity appears reticular within the COS cell cytoplasm (B). Co-transfection of β-dystrobrevin and Kif5B resulted in a tubulo-vesicular pattern of the two co-expressed proteins, β-dystrobrevin (C) and kinesin (D), with a significant co-localization (E). Nocodazole treatment of co-transfected cells resulted in a dramatic redistribution of β- dystrobrevin (F) and kinesin (G) into vesicular aggregates, with precise co-localization (H). β- Dystrobrevin was detected using the polyclonal β-dystrobrevin antibody, followed by a goat anti-rabbit IgG secondary antibody conjugated with TRITC (A,C,F). Kinesin was detected using a monoclonal kinesin antibody (Clone H2), followed by a goat anti-mouse IgG secondary antibody conjugated with FITC (B,D,G). E and H are the merged images from each experiment: co-localization areas appear yellow. Scale bar, 10 µm.

(Verhey et al., 2001). In addition to binding proteins through in signal transduction cascades they are now thought to play a its light chains, kinesin can interact with proteins such as role in connecting the organization of intracellular traffic with -Va (Huang et al., 1999), kinectin (Ong et al., 2000) or that of cell signaling (Klopfenstein et al., 2000; Verhey and GRIP1 (Setou et al., 2002) through its heavy chains. Several Rapoport, 2001). Based on work carried out on DPC in skeletal studies in the filamentous fungus Neurospora crassa, which muscle, we know that α-dystrobrevin may be involved in lacks KLC, have also indicated that kinesin heavy chain alone intracellular signaling, either directly or through its association binds to cargo (Steinberg and Schliwa, 1995; Kirchner et al., with the syntrophins (Grady et al., 1999). It has been 1999; Seiler et al., 2000). In mouse, three different Kif5 speculated that β-dystrobrevin may act as a scaffold for components have been described (Xia et al., 1998; Kanai et al., signaling molecules in a similar manner (Loh et al., 2001). 2000). Kif5B is ubiquitous in its expression, whereas Kif5A Since scaffolding proteins may assemble multi-protein and Kif5C are expressed only in neuronal tissue, the former complexes at great distances from their final locations, it seems showing pan-neuronal distribution, the latter enriched in lower advantageous for neurons, but equally for all cells, to assemble motor neurons (Xia et al., 1998; Kanai et al., 2000). Each the components of the DPC in the neuronal cell body, load kinesin is thought to share a conserved motor domain that them onto a motor protein, and then carry the complex to reversibly binds to microtubules and converts chemical energy the appropriate location. Recent evidence suggests that into motion, and a variable tail domain that interacts with functionally related axonal components may travel together different cellular binding partners, directly or through along the axon to ensure precise delivery (Zhai et al., 2001), accessory light chains (Goldstein, 2001). The identification and although how this traffic is temporally and spatially regulated characterization of proteins that bind to tail domains of kinesin is still unclear. Our data suggest that Kifs could transport the motors have suggested that there are at least two mechanisms DPC, or a part of it, to the cell membrane by binding with the of linkage. Kinesin may bind directly to vesicles through β-dystrobrevin receptor. In Torpedo electrocyte it has been transmembrane proteins such as APP and syd/JIP-3 (Bowman shown that two components of the DPC, the integral et al., 2000; Kamal et al., 2000), or it may bind to them β-dystroglycan and the peripheral cytoplasmic indirectly, through scaffolding proteins linked in their turn to protein syntrophin, are transported together. They are targeted transmembrane vesicle proteins (Nakagawa et al., 2000; Setou to the post-synaptic membrane in post-Golgi vesicles, et al., 2000; Verhey et al., 2001). Scaffolding proteins are associated with the acetylcholine receptor and rapsyn, but multifunctional proteins with several protein-protein dystrophin and dystrobrevin are absent from these vesicles interaction modules that can assemble large protein-protein (Marchand et al., 2001). Since dystrophin and dystrobrevin complexes at the plasma membrane. Since they are implicated eventually associate with dystroglycan and/or syntrophin at the 4854 Journal of Cell Science 116 (23) post-synaptic membrane, a model for separate targeting of the aggregated vesicular structures in COS cells lacking a various components of the DPC and a step-by-step assembly microtubule cytoskeleton may therefore indicate that β- at the post-synaptic membrane has been proposed (Marchand dystrobrevin and Kif5B travel together. et al., 2001). Delivery of dystrophin and dystrobrevin would The direct interaction between β-dystrobrevin and Kif5s depend only on the presence of specific binding sites at the sheds new light on the mechanisms of vesicle transport, giving post-synaptic membrane, but how these proteins travel and at the same time one more example of a kinesin heavy chain which are their partners during intracellular transport remained binding to a vesicle receptor. We suggest that the interaction an open issue. Part of this question is answered by our findings, between dystrobrevins and Kif5s might be important for the which suggest that β-dystrobrevin travels in association with transport of components of the DPC to the neuronal membrane. Kifs. Our findings also shed new light on β-dystroglycan Since most of the other KHC binding partners previously interactions. Although it has been reported that full-length reported, such as kinectin (Toyoshima et al., 1992; Kumar et dystroglycan interacts with α-dystrobrevin (Chung and al., 1995), myosin-Va (Huang et al., 1999) and GRIP1 (Setou Campanelli, 1999) and we observed that a dystroglycan et al., 2002) are mainly dendritic, and most of the KLC molecule consisting of part of α-dystroglycan and the whole partners, such as syd/JIP-3 (Bowman et al., 2000; Verhey et al., β-dystroglycan (amino acids 467-895) could interact with β- 2001) and APP (Kamal et al., 2000) are mainly axonal, it has dystrobrevin in pull-down experiments (data not shown), we been suggested that kinesin transport can be divided into a found that the β-dystroglycan cytoplasmic domain alone does KHC pathway with somato-dendritic prevalence, and a KLC not interact with β-dystrobrevin either in immunoprecipitation pathway with somato-axonal prevalence (Setou et al., 2002). and pull-down assay or in the yeast 2-HS (data not shown). Our data strengthen this hypothesis, since β-dystrobrevin was Taken together, our results indicate that dystroglycan and found to be associated with neuronal somata, dendrites and dystrobrevin are not directly associated in the DPC and may nuclei in brain cortex, hippocampus and cerebellum, and has be transported separately, as cargoes of different vesicles. been reported to be particularly enriched in post-synaptic We show that dystrobrevins and kinesin can be co- densities (Blake et al., 1999). immunoprecipitated from rat brain using a monoclonal kinesin heavy chain antibody broadly cross-reactive with mammalian We are indebted to Dr Francesca Navone for helpful discussions Kifs, and a monoclonal dystrobrevin antibody reactive to and advice, and for kindly providing the human Kif5B cDNA clone all dystrobrevin isoforms. Our co-immunoprecipitation data and the anti-uKHC antibody. We also thank Margherita Grasso for her confirm the two-hybrid and in vitro association results, and also collaboration, and Bruno Ballatore for expert photographic assistance. This work was partially supported by grant Alz3 from the Ministero suggest a more general interaction between different della Salute, Italy. dystrobrevin and kinesin isoforms in the nervous system. Recent data have revealed a functional redundancy between the three Kif5s and shown that Kif5A, Kif5B and Kif5C References are all co-immunoprecipitated with each of the anti-Kif5 Adams, M. E., Butler, M. H., Dwyer, T. M., Peters, M. F., Murnane, A. A. antibodies (Kanai et al., 2000). This suggests that Kif5s can and Froehner, S. C. (1993). Two forms of mouse syntrophin, a 58 kd also form heterodimers in addition to forming homodimers dystrophin-associated protein, differ in primary structure and tissue (Kanai et al., 2000) and may thus possibly bind to numerous distribution. Neuron 11, 531-540. Ahn, A. H. and Kunkel, L. M. (1995). Syntrophin binds to an alternatively different partners. In order to elucidate whether, in the same spliced exon of dystrophin. J. Cell Biol. 128, 363-371. tissue, specific dystrobrevin isoforms interact with specific Ahn, A. H., Yoshida, M., Anderson, M. S., Feener, C. A., Selig, S., Kif5 components, we used COS cell lysates to perform pull- Hagiwara, Y., Ozawa, E. and Kunkel, L. M. (1994). Cloning of human down experiments. COS-7 cells express Kif5B but not Kif5A, basic A1, a distinct 59-kDa dystrophin-associated protein encoded on β 8q23-24. Proc. Natl. Acad. Sci. USA 91, 4446-4450. and our results show that -dystrobrevin also interacts directly Ambrose, H. J., Blake, D. J., Nawrotzki, R. A. and Davies, K. E. (1997). with Kif5B. Genomic organization of the mouse dystrobrevin gene: comparative analysis We have localized the β-dystrobrevin binding site on with the dystrophin gene. 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