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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00805

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ß-Dystrobrevin interacts directly with kinesin heavy chain in brain

Laboratory of Cell Biology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

View larger version (13K): [in a new window]   Fig. 1. Schematic representation of Kif5A clones found to interact with ß-dystrobrevin in the two-hybrid screening of a mouse brain cDNA library. A diagram of full-length Kif5A is aligned at the top. The positions of the amino acids are indicated.

View larger version (14K): [in a new window]   Fig. 2. Direct in vitro interaction of ß-dystrobrevin and neuronal kinesin heavy chain. (A) Co-immunoprecipitation: cMyc-ß-dystrobrevin and HA-kinesin (Kif5A clone 18) were translated in vitro in the presence of [35S]methionine and [35S]cysteine, and mixed together. The mixtures were immunoprecipitated with polyclonal anti-HA, and the immunoprecipitated proteins visualized by autoradiography after SDS-PAGE. Non-specific IgG were used as negative control. 35S-labeled cMyc-ß-dystroglycan (amino acids 774-895) did not co-immunoprecipitate with kinesin, showing the specificity of the ß-dystrobrevin interaction with kinesin. (B) Pull-down assay: purified GST-ß-dystrobrevin (GST-ß-DB) or GST-protein (GST), pre-bound to glutathione-Sepharose beads, was incubated with in vitro translated [35S]kinesin (Kif5A clone 18) or [35S]Dp71 (Dp71285-622, used as a positive control). After extensive washing, bound radioactive proteins were separated by SDS-PAGE and detected by autoradiography. The extra band under [35S]kinesin in the first lane is a product present after the in vitro transcription and translation.

View larger version (14K): [in a new window]   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 rat cerebellum lysate (input), mouse IgG control immunoprecipitate (IP: control IgG) and anti-dystrobrevin immunoprecipitate (IP: dystrobrevin) were separated by SDS-PAGE, and transferred to nitrocellulose. The blot was probed with anti-kinesin (Clone H2) and the polyclonal anti-ß-dystrobrevin antibody, and revealed by ECL. Besides detecting ß-dystrobrevin (59 kDa), the polyclonal anti-ß-dystrobrevin antibody cross-reacts with -dystrobrevin isoforms. (B) The anti-kinesin antibody (Clone H2) co-immunoprecipitates dystrobrevin isoforms and kinesin. Rat cerebellum lysate (input), anti-kinesin immunoprecipitate (IP: kinesin) and mouse IgG control immunoprecipitate (IP: control IgG), were separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with anti-dystrobrevin (Clone 23) and anti-kinesin (Clone H2) antibodies, and revealed by ECL. The anti-dystrobrevin antibody (Clone 23) is reactive to all dystrobrevin isoforms. In the anti-kinesin immunoprecipitate (IP: kinesin) a band corresponding to -dystrobrevin 1 (78 kDa) is clearly visible. The 59 and 55 kDa bands of dystrobrevin isoforms (see input) overlap with the co-migrating 55 kDa IgG heavy chain band (see IP: control IgG). Molecular masses of size markers are indicated on the right-hand side (in kDa). WB, western blot.

View larger version (15K): [in a new window]   Fig. 4. Identification of ß-dystrobrevin binding site on kinesin. (A) Schematic representation of the Kif5A truncation clones. The positions of the amino acids are indicated. The ß-dystrobrevin-binding site on Kif5A was found to be between amino acids 804 and 934, and is indicated in black. (B) Pull-down assay: each [35S]Kif5A truncation protein was incubated with purified GST-ß-dystrobrevin pre-bound to glutathione-Sepharose beads. Radioactive proteins were separated by SDS-PAGE and detected by autoradiography. An aliquot of labeled starting material is shown on the left panel (input); the bound Kif5A truncated proteins are on the right (pull-down). Molecular masses of size markers are indicated on the left-hand side (in kDa).

View larger version (10K): [in a new window]   Fig. 5. Direct interaction of ß-dystrobrevin and ubiquitous kinesin heavy chain. COS-7 cell lysate was incubated with GST-ß-dystrobrevin (GST-ß-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.

View larger version (92K): [in a new window]   Fig. 6. Co-localization of ß-dystrobrevin and ubiquitous kinesin heavy chain in transfected COS-7 cells. The sub-cellular distribution of ß-dystrobrevin and Kif5B was determined by transfecting COS-7 cells with pCMV-HA/ß-DB (A) and pCB6/Kif5B (B). ß-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 ß-dystrobrevin and Kif5B resulted in a tubulo-vesicular pattern of the two co-expressed proteins, ß-dystrobrevin (C) and kinesin (D), with a significant co-localization (E). Nocodazole treatment of co-transfected cells resulted in a dramatic redistribution of ß-dystrobrevin (F) and kinesin (G) into vesicular aggregates, with precise co-localization (H). ß-Dystrobrevin was detected using the polyclonal ß-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.