Laboratory of Cell Biology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
* Author for correspondence (e-mail: macioce{at}iss.it)
Accepted 28 July 2003
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Summary |
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Key words: Dystrobrevin, Kinesin, Yeast two-hybrid, Brain, Dystroglycan
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Introduction |
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Dystrobrevins are members of the dystrophin family with unique extreme carboxyl termini (Roberts, 2001) and a significant sequence homology with the cysteine-rich carboxyl-terminal region of dystrophin (Wagner et al., 1993
; Ambrose et al., 1997
). This region of similarity can be divided into several functional domains, namely two EF hands (Koenig et al., 1988
), a ZZ domain (Ponting et al., 1996
), and two coiled-coil regions (Blake et al., 1995
). Dystrobrevins are the product of two different genes coding for two highly homologous proteins,
- and ß-dystrobrevin (Ambrose et al., 1997
; Loh et al., 1998
). Several distinct transcripts are derived from each gene by alternative splicing or initiation sites, generating a large family of dystrobrevin isoforms (Blake et al., 1996
; Peters et al., 1997b
; Blake et al., 1998
).
-Dystrobrevin is expressed predominantly in skeletal muscle, heart, lung and brain (Blake et al., 1996
; Sadoulet-Puccio et al., 1996
) and is thought to be involved in synaptic transmission at the neuromuscular junction (Sanes et al., 1998
; Grady et al., 2000
) and in intracellular signaling (Grady et al., 1999
).
-Dystrobrevin knockout mice develop a mild form of muscular dystrophy and display a phenotype similar to that of null dystrophin mutants, although the integrity of the DPC is largely maintained (Grady et al., 1999
). Since they have reduced levels of cGMP and have less nNOS at the sarcolemma, it has been proposed that the muscle degeneration observed in these mice is caused by a deficiency in intracellular signaling mediated by
-dystrobrevin (Grady et al., 1999
). The second member of the dystrobrevin family, ß-dystrobrevin, is restricted to non-muscle tissues, is most abundantly expressed in kidney and brain and forms complexes with dystrophin isoforms and syntrophin in liver and brain (Peters et al., 1997a
; Blake et al., 1998
; Puca et al., 1998
; Blake et al., 1999
). In brain ß-dystrobrevin associates with dystrophin isoforms in the cortex, hippocampus and Purkinje neurons and is highly enriched in post-synaptic densities (Blake et al., 1998
; Blake et al., 1999
). Since approximately one third of Duchenne muscular dystrophy patients suffer from mental retardation and cognitive deficits (Hodgson et al., 1992
; Lidov, 1996
), it has been proposed that, by analogy to the situation in muscle, a neuronal DPC-like complex may be involved in the establishment of these CNS defects (Blake et al., 1999
). In mdx3Cv mice, which lack all known dystrophin isoforms, neither ß-dystrobrevin expression and localization at the membrane, nor the assembly of the DPC are affected in brain and kidney, whereas in muscle this complex is destabilized. It has therefore been speculated that ß-dystrobrevin may be a key regulator in the assembly and maintenance of DPC-like complexes in non-muscle tissues (Blake et al., 1999
; Loh et al., 2000
). Recent studies with ß-dystrobrevin-deficient mice have shown, however, that although ß-dystrobrevin is required for the membrane localization of the short dystrophin isoform Dp71 and syntrophin in kidney and liver, its presence is not necessary for normal function (Loh et al., 2001
).
In the search for new insights into the functions of dystrobrevins, new partners have recently been identified using the yeast two-hybrid system (Benson et al., 2001; Mizuno et al., 2001
; Newey et al., 2001
). Two of these partners, syncoilin and desmuslin, are novel members of the intermediate filament family, and are expressed mainly in skeletal and cardiac muscle. They interact with
-dystrobrevin, and are reported to be concentrated at the neuromuscular junction (Newey et al., 2001
) and at the Z-lines (Mizuno et al., 2001
), respectively. They also interact directly with desmin (Mizuno et al., 2001
; Poon et al., 2002
), suggesting that
-dystrobrevin may tether the intermediate filament network to the DPC (Blake and Martin-Rendon, 2002
). A third novel dystrobrevin-interacting protein, dysbindin, is a 40 kDa coiled-coil-containing protein that binds to
- and ß-dystrobrevin in muscle and brain (Benson et al., 2001
). In the brain, dysbindin was detected primarily in the axons of many types of neurons, in which it co-localized with ß-dystrobrevin, suggesting their association in a protein complex (Benson et al., 2001
).
To improve our understanding of the role of ß-dystrobrevin in brain, we searched for new interacting proteins using the yeast two-hybrid system (2-HS). Among the cDNA clones isolated we characterized four overlapping clones of mouse neuronal kinesin heavy chain Kif5A. We confirmed the interaction of ß-dystrobrevin with Kif5A by in vitro co-immunoprecipitation and pull-down experiments and found that kinesin heavy chain also co-immunoprecipitated with dystrobrevin from rat brain extracts. We also found, through pull-down and transfection experiments, a direct interaction between ß-dystrobrevin and ubiquitous kinesin heavy chain Kif5B.
Our data suggest a new role for dystrobrevin as a motor protein receptor that may be involved in the traffic and targeting of components of the DPC to specific sites in the cell.
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Materials and Methods |
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Full-length human Dp71 cDNA, obtained as reported previously (Ceccarini et al., 1997), was utilized as a template to amplify the region coding for amino acids 285-622 using the appropriate primers. Dp71285-622 fragment was sub-cloned into pCRII-TOPO, to produce pCRII-TOPO/Dp71285-622. The fragment was then EcoRI-restricted and sub-cloned into the EcoRI site of pGADT7 to generate pGADT7/Dp71285-622.
A previously described rabbit ß-dystroglycan (ß-DG) cDNA (Rosa et al., 1996) was used to amplify the region coding for the cytoplasmic portion of ß-dystroglycan (amino acids 774-895), and pGBKT7/ß-DG774-895 was obtained basically as described for ß-DB.
HA-Kif5A#18 DNA fragment coding for amino acids 611-1027 of Kif5A and the hemagglutinin (HA) epitope tag upstream was amplified by PCR from pACT2-Kif5A#18 template, and sub-cloned into pCRII-TOPO to create pCRII-TOPO/HA-Kif5A#18.
Kif5A771-1027, Kif5A804-1027, Kif5A804-934, and Kif5A934-1027 were also obtained by PCR using pACT2-Kif5A#18 as a template, and sub-cloned into pCRII-TOPO vector. After EcoRI digestion, the Kif5A fragments were inserted into the EcoRI site of pGADT7 vector, to create pGADT7/Kif5A deletion mutant constructs (pGAD-T7/Kif5A771-1027, pGADT7/Kif5A804-1027, pGADT7/Kif5A804-934, and pGADT7/Kif5A934-1027).
The correct orientation of cDNA inserts was verified by restriction enzyme analysis, and sequence analysis was used to check that they were in-frame.
A fully encoding human Kif5B cDNA (Navone et al., 1992), subcloned into pCB6 (Brewer and Roth, 1991
), was a kind gift from Dr Francesca Navone, CNR, Milano.
Yeast two-hybrid screen
Two-hybrid screening was carried out by yeast mating, using the Matchmaker Gal4 Two-Hybrid System 3 (Clontech). Both pGBKT7/ß-DB and pGADT7/ß-DB tested negative for auto-activation of reporter gene activity in the yeast two-hybrid reporter strains, Saccharomyces cerevisiae AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4, gal80
, LYS2:: GAL1UAS - GAL1TATA - HIS3, GAL2UAS - GAL2TATA - ADE2, URA3:: MEL1UAS - MEL1TATA - lacZ MEL1) and S. cerevisiae Y187 (MAT
, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4
, gal80
,met-, URA3:: GAL1UAS - GAL1TATA - lacZ MEL1). The Gal4 DNA binding domain construct pGBKT7/ß-DB was used to transform the MATa yeast strain AH109. AH109[pGBKT7/ß-DB] was then employed as a bait strain to screen a mouse brain cDNA library (Clontech), which was cloned into the activation domain vector pACT2, and pre-transformed in the MAT
yeast strain Y187. The yeast mating screening was performed according to the manufacturer's instructions. Diploids were selected by culture on minimal synthetic dropout medium (SD) lacking Trp, Leu, His and Ade (-TLHA), and including 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-
-Gal) for 8-16 days.
Ade+, His+, Mel1+ colonies were isolated and then re-tested for lacZ (ß-Gal) activity by colony lift filter assay. lacZ+ colonies were re-streaked at least twice onto SD/-TL/X--Gal to allow segregation, then transferred to SD/-TLHA/X-
-Gal to verify that they maintained the correct phenotype. Library plasmids from positive colonies were isolated and rescued using E. coli strain DH5
on ampicillin-resistant plates. AD/library inserts were then amplified by PCR and analyzed by restriction digestion. Unique inserts were sequenced and DNA and protein sequence analyses were performed with the BLAST algorithm at the National Center for Biotechnology Information (NCBI).
After isolation of the pACT2 plasmids encoding the library clones, these plasmids were tested for auto-activation of the reporter gene in yeast by both co-transformation, using either the empty bait plasmid pGBKT7 or the same plasmid encoding an unrelated negative control (lamin C), and yeast mating, using Y187[pGBKT7] as the empty bait strain, or Y187[pGBKT7-lamin C] as a negative control. Isolates growing in SD/-TLHA/X--Gal and developing blue staining without the presence of the ß-DB bait were excluded from further investigation. Activation of the reporter genes in the positive colonies was confirmed in the same experiments.
In vitro transcription and translation
In vitro transcription and translation of pCRII-TOPO/HA-Kif5A#18, pGADT7/Kif5A deletion mutant constructs, pGADT7/Dp71285-622, pGBKT7/ß-DB and pGBKT7/ß-DG774-895 was carried out using the TNT SP6 or T7 Quick Coupled Transcription/Translation System (Promega) in the presence of PRO-MIX (70% L-[35S]methionine; 30% L-[35S]cysteine; Amersham Pharmacia Biotech) according to the manufacturer's protocol. Newly synthesized proteins were separated by SDS-PAGE and analyzed with an Instant-Imager (Packard).
Co-immunoprecipitation assay
Co-immunoprecipitation of in vitro translated proteins was carried out as follows. 5 µl of reticulocyte lysate containing labeled HA-tagged protein and 5 µl containing labeled c-Myc-tagged protein were mixed together and incubated at 30°C for 1 hour. After incubation, 460 µl of TBS-T (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% Tween 20) supplemented with 1 mM DTT and protease inhibitors, 20 µl of 50% slurry of protein G-agarose beads (Pierce) and 10 µl HA-tag polyclonal antibody (Clontech) were added. The mixture was rocked gently at 4°C for 2 hours, the beads were collected by brief centrifugation and washed three times with TBS-T. The pellets were resuspended in 15 µl of Laemmli's loading buffer, the proteins were eluted and denatured by boiling for 2 minutes and 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.
Pull-down experiments
pGEX-6P/ß-DB and pGEX-6P were used to transform E. coli BL21 cells. Overnight cultures were diluted 1:10, grown at 37°C until A600 was 0.7-1.0, and induced for 3 hours with 1 mM isopropyl-ß-D-thiogalactoside (IPTG) at 30°C. Recombinant proteins were purified from the soluble cell lysates by glutathione-Sepharose column following the procedure described by Kennedy et al. (Kennedy et al., 1991).
GST (glutathione S-transferase)-ß-DB or GST bound to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) were equilibrated in binding buffer (20 mM Hepes, pH 7.4, 0.15 M NaCl, 1 mM DTT, 0.2% Triton X-100, 5 µM MgSO4, and protease inhibitors). For binding assays, 10 µl of in vitro-translated reaction products were incubated with 20 µl of 50% slurry bead-bound GST-fusion proteins overnight at 4°C on a rotator. After six washes with 450 µl binding buffer, bound radioactive proteins were re-suspended in Laemmli's loading buffer and separated by SDS-PAGE. Gels were dried and radiolabeled proteins were detected by autoradiography.
For GST pull-down assays with COS-7 extracts, semi-confluent cells from a 100-mm plate were harvested, washed twice with TBS, re-suspended in binding buffer and lysed by passing through a 25-gauge needle, on ice. The lysates were cleared by centrifugation at 15,000 g for 15 minutes at 4°C, and the supernatants were incubated overnight at 4°C on a rotator with 100 µl of 50% slurry bead-bound 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 anti-uKHC antibody, which reacts specifically with ubiquitous kinesin heavy chain Kif5B (Niclas et al., 1994), a kind gift from Dr Francesca Navone, CNR, Milan. Bound antibodies were visualized using the ECL system (Pierce).
Immunoprecipitation
Tissue from rat cerebellum was homogenized (100 mg/ml) in 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA, 7.5 mM phosphate buffer, pH 7.4, containing protease inhibitors. After centrifugation at 600 g at 4°C, the pellet was solubilized 1:1 in 2x 10 mM phosphate buffer, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors, incubated on ice for 30 minutes and centrifuged at 20,000 g for 30 minutes at 4°C. The supernatant was used for co-immunoprecipitation experiments. To minimize non-specific binding, the sample was pre-incubated for 3 hours at 4°C with 25 µl of protein G-agarose-conjugated mouse IgG (Pierce) (50% slurry). After centrifugation, 2 µg of dystrobrevin monoclonal antibody (Clone 23, Transduction Laboratories) or 5 µg of kinesin heavy chain monoclonal antibody (Clone H2, Chemicon 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 was washed three times with ice-cold TBS-T and immunoprecipitates were separated by SDS-PAGE, transferred to a nitrocellulose membrane and incubated with the kinesin heavy chain monoclonal antibody (Clone H2) and either a polyclonal ß-dystrobrevin antibody or the dystrobrevin monoclonal antibody (Clone 23). Bound antibodies were visualized using the ECL system (Pierce).
Polyclonal ß-dystrobrevin antibody was prepared as follows. After removal of GST by digestion with pre-scission protease (80 U/ml; Amersham Pharmacia Biotech), recombinant ß-dystrobrevin was injected into rabbits according to a previously described protocol (Rosa et al., 1996). The affinity-purified antibody was obtained by standard methods, using GST-ß-DB coupled to Sepharose-CNBr beads (Amersham Pharmacia Biotech).
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, untreated glass coverslips were transfected using Fugene 6 (Roche) according to the manufacturer's instructions. For some cells, nocodazole (Sigma) was used at a final concentration of 20 µM for 4 hours before fixation. Wash-out was performed by washing nocodazole-treated cells three times with culture medium, followed by incubation at 37°C in nocodazole-free medium for 2 hours.
Immunofluorescence
Twenty-four to 66 hours after transfection, the cells were washed with TBS, fixed with 3.7% formaldehyde in TBS for 15 minutes at room temperature and permeabilized for 5 minutes in TBS containing 0.5% Triton X-100. After washing, cells were blocked for 30 minutes in TBS containing 3% normal goat serum and then incubated for 1 to 3 hours with primary antibodies diluted in TBS containing 1% normal goat serum. Kif5B and ß-dystrobrevin were stained by the kinesin heavy chain monoclonal antibody (Clone H2) and the polyclonal ß-dystrobrevin antibody, respectively.
Cells were then incubated with the appropriate secondary antibodies conjugated with either FITC or TRITC for 1 hour and mounted with VectaShield (Vector Laboratories).
The images were examined under a Leica TCS 4D confocal microscope. Image acquisition and processing were performed using the SCANware and Multicolor Analysis (Leica Lasertechnik Gmbh) and Adobe Photoshop (Adobe Systems) software programs.
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Results |
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In vitro association of Kif5A and ß-dystrobrevin
We next performed immunoprecipitation and pull-down experiments to investigate whether the interaction between ß-dystrobrevin and Kif5A also takes place in vitro.
We used pCRII-TOPO/HA-Kif5A#18, which encodes the longest Kif5A polypeptide plus the HA epitope tag from pACT2, and pGBKT7/ß-DB, carrying a c-Myc epitope tag, to perform in vitro transcription and translation. In vitro co-immunoprecipitation confirmed our 2-HS results, showing that the interactions between bait and prey proteins were specific (Fig. 2A). The dystroglycan cytoplasmic domain did not interact with kinesin (Fig. 2A), as expected on the basis of previous 2-HS results (data not shown).
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As an additional demonstration of interaction between ß-dystrobrevin and Kif5A, we performed GST-ß-DB pull-down experiments. Recombinant GST-ß-DB, or GST alone, bound to glutathione-Sepharose beads were allowed to interact with 35S-labeled Kif5A#18. GST-ß-DB binds 35S-labeled Kif5A#18 protein, as well as a Dp71285-622 positive control (Sadoulet-Puccio et al., 1997), but GST protein does not (Fig. 2B).
In vivo association of kinesin heavy chain and ß-dystrobrevin
To ascertain whether the interaction between ß-dystrobrevin and Kif5A occurs in vivo, we performed co-immunoprecipitation experiments using rat brain extracts. Lysates from rat cerebellum were pre-cleared and incubated with a dystrobrevin or a kinesin antibody, respectively. Protein G-agarose beads selectively precipitated the immuno-complexes, which were subsequently separated by SDS-PAGE and immunoblotted with dystrobrevin and kinesin antibodies (Fig. 3). The monoclonal dystrobrevin antibody used for immunoprecipitation is reactive to all dystrobrevin isoforms: -dystrobrevin 1 (78 kDa),
-dystrobrevin 2 (55 kDa) and ß-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 to immunoprecipitate kinesin, we detected the presence of co-immunoprecipitated dystrobrevin isoforms. Fig. 3B shows an upper band, corresponding to
-dystrobrevin 1, and a lower band, corresponding to
-dystrobrevin 2 and ß-dystrobrevin, also including IgG heavy chain. Taken together, our data show that kinesin heavy chain and dystrobrevins specifically interact in vivo.
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Association with ß-dystrobrevin involves Kif5A domain spanning amino acids 804 to 934
In order to characterize further the structural requirements of the interaction between Kif5A and ß-dystrobrevin, we generated a set of progressively smaller constructs for Kif5A (Fig. 4A) and tested them for interactions with ß-dystrobrevin by pull-down assay. The minimum region of overlap for binding between the two proteins was found to be between amino acids 804 and 934 of Kif5A (Fig. 4B). This region corresponds to GluR2-interacting protein (GRIP1) minimal binding site of all Kif5s (Kif5A, Kif5B and Kif5C) (Setou et al., 2002) and overlaps with the binding domain for kinectin (Ong et al., 2000
) and the cargo-binding domain of the Neurospora kinesin (Seiler et al., 2000
), indicating that this part of the molecule might play a major role in heavy chain-mediated kinesin interactions.
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In vitro association of Kif5B and ß-dystrobrevin
To clarify whether ß-dystrobrevin interacts specifically with Kif5A or whether the interaction includes other isoforms of conventional kinesin heavy chain, we performed a GST pull-down assay on COS-7 cell lysates. While not expressing neuronal-specific Kif5A, COS-7 cells do express ubiquitous kinesin heavy chain isoform Kif5B, which showed a specific association with GST-ß-DB fusion protein (Fig. 5). These data clearly demonstrate a direct interaction between ß-dystrobrevin and Kif5B.
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Immunolocalization of kinesin and ß-dystrobrevin in transfected COS cells
We decided to investigate further the in vivo association of ß-dystrobrevin with kinesin by over-expressing ß-dystrobrevin and Kif5B in COS-7 cells. Prior to these studies, we determined the distribution of ß-dystrobrevin and of Kif5B by single transfection of pCMV-HA/ß-DB and of pCB6/Kif5B. Immunofluorescence labeling using either a polyclonal ß-dystrobrevin antibody or a polyclonal anti-HA antibody (not shown) showed that cells transiently transfected with ß-dystrobrevin displayed an intense, punctate staining pattern distributed throughout the cell, which often concentrated around the nucleus (Fig. 6A), as also described by Benson et al. (Benson et al., 2001). The transfected Kif5B, as visualized by the monoclonal kinesin antibody showed a reticular distribution throughout the entire cytoplasm (Fig. 6B); superimposed on this pattern, a filamentous staining which coincided with microtubules (not shown) was also evident. These immunostaining results are consistent with those observed by Navone et al. (Navone et al., 1992
) in CV-1 cells expressing the same Kif5B cDNA, as well as by other authors in COS cells (Verhey et al., 1998
). When the constructs encoding ß-dystrobrevin (pCMV-HA/ß-DB) and ubiquitous kinesin (pCB6/Kif5B) were co-transfected, immunofluorescence microscopy of the two co-expressed proteins showed that the punctate organization of ß-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 ß-dystrobrevin and kinesin distribution in nocodazole-treated cells. The extent of microtubule depolymerization, or restoration, after treatment with nocodazole, or nocodazole wash-out, was verified by tubulin staining (data not shown). We observed a dramatic re-localization of ß-dystrobrevin and kinesin, with a nearly identical distribution in vesicular aggregates (Fig. 6F-H), which varied in size and arrangement within different cells, but always showed a high level of co-localization of the two co-expressed proteins. The patch-like pattern of ß-dystrobrevin and kinesin reverted to a tubulovesicular network after nocodazole wash-out (not shown).
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Discussion |
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In neurons, conventional kinesin (kinesin-I) exists as a tetramer of two heavy chains (KHC: Kif5), which contain the amino-terminal motor domain, as well as two light chains (KLC), which bind to the heavy chain tail (Vale et al., 1985; Hirokawa, 1998
) and are known to interact with the cargo (Verhey et al., 2001
). In addition to binding proteins through its light chains, kinesin can interact with proteins such as myosin-Va (Huang et al., 1999
), kinectin (Ong et al., 2000
) or GRIP1 (Setou et al., 2002
) through its heavy chains. Several studies in the filamentous fungus Neurospora crassa, which lacks KLC, have also indicated that kinesin heavy chain alone binds to cargo (Steinberg and Schliwa, 1995
; Kirchner et al., 1999
; Seiler et al., 2000
). In mouse, three different Kif5 components have been described (Xia et al., 1998
; Kanai et al., 2000
). Kif5B is ubiquitous in its expression, whereas Kif5A and Kif5C are expressed only in neuronal tissue, the former showing pan-neuronal distribution, the latter enriched in lower motor neurons (Xia et al., 1998
; Kanai et al., 2000
). Each kinesin is thought to share a conserved motor domain that reversibly binds to microtubules and converts chemical energy into motion, and a variable tail domain that interacts with different cellular binding partners, directly or through accessory light chains (Goldstein, 2001
). The identification and characterization of proteins that bind to tail domains of kinesin motors have suggested that there are at least two mechanisms of linkage. Kinesin may bind directly to vesicles through transmembrane proteins such as APP and syd/JIP-3 (Bowman et al., 2000
; Kamal et al., 2000
), or it may bind to them indirectly, through scaffolding proteins linked in their turn to transmembrane vesicle proteins (Nakagawa et al., 2000
; Setou et al., 2000
; Verhey et al., 2001
). Scaffolding proteins are multifunctional proteins with several protein-protein interaction modules that can assemble large protein-protein complexes at the plasma membrane. Since they are implicated in signal transduction cascades they are now thought to play a role in connecting the organization of intracellular traffic with that of cell signaling (Klopfenstein et al., 2000
; Verhey and Rapoport, 2001
). Based on work carried out on DPC in skeletal muscle, we know that
-dystrobrevin may be involved in intracellular signaling, either directly or through its association with the syntrophins (Grady et al., 1999
). It has been speculated that ß-dystrobrevin may act as a scaffold for signaling molecules in a similar manner (Loh et al., 2001
). Since scaffolding proteins may assemble multi-protein complexes at great distances from their final locations, it seems advantageous for neurons, but equally for all cells, to assemble the components of the DPC in the neuronal cell body, load them onto a motor protein, and then carry the complex to the appropriate location. Recent evidence suggests that functionally related axonal components may travel together along the axon to ensure precise delivery (Zhai et al., 2001
), although how this traffic is temporally and spatially regulated is still unclear. Our data suggest that Kifs could transport the DPC, or a part of it, to the cell membrane by binding with the ß-dystrobrevin receptor. In Torpedo electrocyte it has been shown that two components of the DPC, the integral glycoprotein ß-dystroglycan and the peripheral cytoplasmic protein syntrophin, are transported together. They are targeted to the post-synaptic membrane in post-Golgi vesicles, associated with the acetylcholine receptor and rapsyn, but dystrophin and dystrobrevin are absent from these vesicles (Marchand et al., 2001
). Since dystrophin and dystrobrevin eventually associate with dystroglycan and/or syntrophin at the post-synaptic membrane, a model for separate targeting of the various components of the DPC and a step-by-step assembly at the post-synaptic membrane has been proposed (Marchand et al., 2001
). Delivery of dystrophin and dystrobrevin would depend only on the presence of specific binding sites at the post-synaptic membrane, but how these proteins travel and which are their partners during intracellular transport remained an open issue. Part of this question is answered by our findings, which suggest that ß-dystrobrevin travels in association with Kifs. Our findings also shed new light on ß-dystroglycan interactions. Although it has been reported that full-length dystroglycan interacts with
-dystrobrevin (Chung and Campanelli, 1999
) and we observed that a dystroglycan molecule consisting of part of
-dystroglycan and the whole ß-dystroglycan (amino acids 467-895) could interact with ß-dystrobrevin in pull-down experiments (data not shown), we found that the ß-dystroglycan cytoplasmic domain alone does not interact with ß-dystrobrevin either in immunoprecipitation and pull-down assay or in the yeast 2-HS (data not shown). Taken together, our results indicate that dystroglycan and dystrobrevin are not directly associated in the DPC and may be transported separately, as cargoes of different vesicles.
We show that dystrobrevins and kinesin can be co-immunoprecipitated from rat brain using a monoclonal kinesin heavy chain antibody broadly cross-reactive with mammalian Kifs, and a monoclonal dystrobrevin antibody reactive to all dystrobrevin isoforms. Our co-immunoprecipitation data confirm the two-hybrid and in vitro association results, and also suggest a more general interaction between different 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 are all co-immunoprecipitated with each of the anti-Kif5 antibodies (Kanai et al., 2000). This suggests that Kif5s can also form heterodimers in addition to forming homodimers (Kanai et al., 2000
) and may thus possibly bind to numerous different partners. In order to elucidate whether, in the same tissue, specific dystrobrevin isoforms interact with specific Kif5 components, we used COS cell lysates to perform pull-down experiments. COS-7 cells express Kif5B but not Kif5A, and our results show that ß-dystrobrevin also interacts directly with Kif5B.
We have localized the ß-dystrobrevin binding site on neuronal kinesin heavy chain, and found that it lies at the carboxyl terminus that comprises the end of the coiled-coil region and part of the globular tail and is highly homologous in the three Kif5s components. This domain is the same one that binds glutamate-receptor-interacting protein GRIP1 (Setou et al., 2002) and overlaps with the binding domain for kinectin (Ong et al., 2000
), as well as with the cargo-binding domain of Neurospora kinesin (Seiler et al., 2000
).
We found that ß-dystrobrevin and Kif5B co-localize in transfected COS cells in an intricate, interconnected tubulo-vesicular network. Since a substantial part of Kif5B immunostaining coincides with microtubules, it is likely that this network represents the kinesin fraction travelling with its cargoes, including ß-dystrobrevin. Indeed, the network seems to depend on the presence of microtubules, as indicated by its disappearance in nocodazole-treated cells and its restoration after nocodazole wash-out. The change in the distribution of ß-dystrobrevin and Kif5B, and their co-localization in aggregated vesicular structures in COS cells lacking a microtubule cytoskeleton may therefore indicate that ß-dystrobrevin and Kif5B travel together.
The direct interaction between ß-dystrobrevin and Kif5s sheds new light on the mechanisms of vesicle transport, giving at the same time one more example of a kinesin heavy chain binding to a vesicle receptor. We suggest that the interaction between dystrobrevins and Kif5s might be important for the transport of components of the DPC to the neuronal membrane. Since most of the other KHC binding partners previously reported, such as kinectin (Toyoshima et al., 1992; Kumar et al., 1995
), myosin-Va (Huang et al., 1999
) and GRIP1 (Setou et al., 2002
) are mainly dendritic, and most of the KLC partners, such as syd/JIP-3 (Bowman et al., 2000
; Verhey et al., 2001
) and APP (Kamal et al., 2000
) are mainly axonal, it has been suggested that kinesin transport can be divided into a KHC pathway with somato-dendritic prevalence, and a KLC pathway with somato-axonal prevalence (Setou et al., 2002
). Our data strengthen this hypothesis, since ß-dystrobrevin was found to be associated with neuronal somata, dendrites and nuclei in brain cortex, hippocampus and cerebellum, and has been reported to be particularly enriched in post-synaptic densities (Blake et al., 1999
).
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