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Β-Dystrobrevin Interacts Directly with Kinesin Heavy Chain in Brain

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Β-Dystrobrevin Interacts Directly with Kinesin Heavy Chain in Brain 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 Cell Biology, 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 protein α- and β-dystrobrevin, indicating that this interaction is not family, is a dystrophin-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 signal transduction, 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 proteins. 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-immunoprecipitation with a Key words: Dystrobrevin, Kinesin, Yeast two-hybrid, Brain, monoclonal kinesin heavy chain antibody precipitated both Dystroglycan 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 actin cytoskeleton to the from each gene by alternative splicing or initiation sites, extracellular matrix, and mutations 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 skeletal muscle, 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 neuromuscular junction (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 sarcoglycan 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 syntrophins 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 sarcolemma, 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 syntrophin 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, hippocampus 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 genes 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, syncoilin 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 desmin (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, dysbindin, 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.
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