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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02369

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Centaurin-₁ interacts directly with kinesin motor protein KIF13B

¹ Department of Pharmacology, School of Medical Sciences, The University of Bristol, University Walk, Bristol, BS8 1TD, UK
² Department of Pharmacology, UIC Cancer Center, University of Illinois College of Medicine, 900 S. Ashland Avenue, Chicago, IL 60607, USA

View larger version (79K): [in a new window]   Fig. 1. Schematic diagram of the domain structure of KIF13B and its deduced amino acid sequence. (A) KIF13B contains an N-terminal motor domain (aa 56-354), a middle stalk domain and a C-terminal CAP-GLY domain (aa 1661-1703). Amino acid residues are numbered from the first Met of KIF13B. KIF13B (aa 387-649) represents the cDNA clone isolated by two-hybrid screening. KIF13BC (aa 1-998) was used to study the interaction between KIF13B and centaurin-1 in vitro. KIF13BMD (aa 355-1767) and KIF13BCG (aa 1-1660) were used to study the importance of the motor domain and the CAP-GLY domain for the intracellular localization of KIF13B. (B) Predicted amino acid sequence of KIF13B (GenBank/EMBL/DDBJ accession number AJ605719) is shown. The motor domain, KIF13B sequence and the CAP-GLY are underlined with dashed line, solid line and double solid line, respectively.

View larger version (34K): [in a new window]   Fig. 2. Northern and western blotting of KIF13B. The KIF13B cDNA (789 base pairs, nucleotides 1159-1947) isolated by the two-hybrid screening was labelled with 32P and hybridized with rat multiple tissue poly(A)+ RNA, resolved by electrophoresis and transferred to a nylon membrane. Autoradiography of the blot is shown with the sizes of molecular weight markers in kb. A single band of KIF13B mRNA (9.5 kb) was detected in brain, kidney and testis. (B) Protein samples of various cell lines and rat brain were resolved by SDS-PAGE and immunoblotted using an affinity-purified anti-KIF13B polyclonal antibody. The expression analysis was repeated two further times with identical results.

View larger version (28K): [in a new window]   Fig. 3. Analysis of the specificity of interaction between centaurin-1 and KIF13B by yeast two-hybrid assay. Centaurin-1 or cytohesin 2 fused with the DNA binding domain of LexA (Bait) were co-transformed with KIF13B/IPCEF-1/IPCEF-2 fused to the activation domain of Gal4 (Prey) into yeast (L40) and then we analysed the ability of the transformants to grow on solid synthetic drop-out medium lacking histidine, and to produce ß-galactosidase as assessed by conversion of X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) into a blue-coloured product. The panels left to right show three independent clones of each transformant grown in the presence (+H) and absence (–H) of histidine, and the filter ß-galactosidase assay (ß-gal). This analysis was performed three times with three different yeast transformants, with identical results.

View larger version (22K): [in a new window]   Fig. 4. Centaurin-1 directly binds KIF13B in vitro. (A) 35S-radiolabelled KIF13BC (aa 1-991) was expressed using an in vitro translation system and incubated with glutathione beads immobilized with either GST or GST-centaurin-1. The beads were washed and bound protein was resolved by SDS-PAGE and detected by fluorography. (B,C) Purified recombinant centaurin-1 was incubated with beads linked to GST or GST-KIF13B. After washing the beads, protein bound to the beads was resolved by SDS-PAGE and analysed by Coomassie Blue staining (B) or immunostaining using an anti-centaurin-1 antibody (C). The experiment was repeated two further times with identical results.

View larger version (23K): [in a new window]   Fig. 5. In vivo interaction between centaurin-1 and KIF13B. (A) Co-immunoprecipitation of FLAG-centaurin-1 with GFP-KIF13B. COS cells transfected with the indicated expression vectors were lysed after 2 days and immunoprecipitated (IP) with an anti-GFP antibody. After washing, immunoprecipitates were resolved on SDS-PAGE, blotted onto PVDF membranes and probed with a monoclonal anti-FLAG antibody (IB) to detect FLAG-tagged centaurin-1. One-twentieth of the input (cell lysates) was also immunoblotted (IB) with anti-GFP (lower panel) and anti-FLAG (upper panel) antibodies to ensure that GFP-KIF13B and FLAG-centaurin-1, respectively, were expressed. (B) Co-immunoprecipitation of endogenous centaurin-1 and KIF13B. The lysates of brain were immunoprecipitated (IP) with control pre-immune serum (lane 2) or an anti-centaurin-1 antiserum (lane 3) and endogenous KIF13B visualized in the precipitate and cell lysate (lane 1) by immunoblotting with an anti-KIF13B antibody (I). The lysates of brain were also immunoprecipitated (IP) with control pre-immune serum (lane 2) or an anti-KIF13B antiserum (lane 3) and endogeonous centaurin-1 detected in the precipitate and cell lysate (lane 1) by immunoblot (IB) using an anti-centaurin-1 antibody (II). These experiments were repeated a further two times with similar results.

View larger version (55K): [in a new window]   Fig. 6. Mapping of the KIF13B binding site within centaurin-1 using yeast two-hybrid analysis. (A) Schematic representation of the domain structure (GAP domain, aa 1-126; N-PH, aa 130-230; C-PH, aa 253-356) and various deletion mutants of centaurin-1. (B) Interaction of KIF13B with the full-length (FL) and deletion constructs of centaurin-1. The full-length and deletion mutants of centaurin-1 fused to the DNA binding domain of LexA (bait) pBTM, were co-transformed with KIF13B fused to the activation domain of Gal4 (prey) into the L40 yeast strain, and the transformants analysed for their ability to grow on medium lacking histidine, and to metabolise X-gal by ß-galactosidase. Panels, left to right, show three clones of each transformant grown on medium with (+H) and without (–H) histidine, and the filter ß-galactosidase assay (ß-gal). The experiment was performed three times with three different yeast transformations with identical results.

View larger version (16K): [in a new window]   Fig. 7. Mapping of the centaurin-1 and KIF13B binding sites using a GST pull-down assay. (A) Lysates of COS cells expressing GFP or GFP-tagged centaurin-1 (cent1) or its deletion mutants were incubated with either GST or GST-KIF13B coupled to glutathione beads. (B) The lysates of COS cells expressing GFP-tagged KIF13B or KIF13B were incubated with either GST or GST-centaurin-1 coupled to glutathione-beads. After washing the beads, the bound proteins were separated on SDS-PAGE, blotted onto PVDF membranes, and probed with an anti-GFP antibody. 5% of the inputs (cell lysates) are also shown for each of the sets of experiments. These experiments were repeated three times with similar results.

View larger version (24K): [in a new window]   Fig. 8. Interaction with KIF13B is not conserved among ARF GAPs. COS cells were transiently transfected with the indicated expression vectors. After 2 days, cells were lysed and incubated with GST-tagged KIF13B coupled to glutathione beads. After washing the beads, the bound proteins were analysed by immunoblotting using an anti-GFP antibody (right-hand panel). 5% of the input (cell lysates) was also immunoblotted using an anti-GFP antibody to ensure that the indicated GFP-tagged ARF GAPs were expressed (left-hand panel). Positions of molecular mass standards (kDa) are shown. This analysis was performed three times with identical results.

View larger version (47K): [in a new window]   Fig. 9. Centaurin-1 colocalizes with KIF13B at the leading edges of the cell. HeLa cells were transiently transfected with either FLAG-centaurin-1 or GFP-KIF13B, or both. After 2 days, the cells were fixed with paraformaldehyde and then immunostained with an anti-FLAG primary antibody and a Cy3-labelled secondary antibody, mounted onto glass slides and imaged using a confocal microscope. Where indicated, the transfected cells were incubated with 3.5 µM nocadazole for 60 minutes at 37°C prior to the fixation. The images are representative of 85-90% of 90-110 transfected cells from five different cell preparations. Arrows indicate regions within the cell where KIF13B is localised. Magnification 100 x.

View larger version (58K): [in a new window]   Fig. 10. KIF13B inhibits the ARF6 GAP activity of centaurin-1. HeLa cells were transfected with HA-ARF6, GFP or GFP-centaurin-1, and Myc-KIF13B or Myc-KIF13B. After 2 days, the cells were serum starved for 2 hours, incubated with or without EGF (200 ng/ml) for 5 minutes and fixed with paraformaldehyde. The fixed cells were then immunostained using anti-HA monoclonal and anti-Myc polyclonal primary antibodies, and Cy3- and Cy5-labelled secondary antibodies. The immunostained cells were imaged by confocal microscopy. The images are representative of 80-90% of 40-50 transfected cells from five similar experiments. Arrows indicate regions within the cell where KIF13B is localised. Magnification 100 x.

View larger version (21K): [in a new window]   Fig. 11. Biochemical confirmation of KIF13B inhibition of ARF6 GAP activity of centaurin-1. (A) COS cells were transfected with the indicated expression plasmids. 2 days later cells were serum starved, incubated with or without EGF (200 ng/ml). The cells were then lysed and ARF6GTP precipitated using the GST-MT2A coupled to glutathione beads. The precipitates were then immunoblotted (IB) with an anti-HA antibody. The cell lysates (input) were also immunoblotted using anti-HA antibody, anti-GFP and anti-FLAG antibodies to determine HA-ARF6, FLAG-centaurin1 and GFP-KIF13B levels respectively. (B) Quantification of data obtained from three similar experiments as shown in A above. Data are the means±s.e.

View larger version (15K): [in a new window]   Fig. 12. Effect of centaurin-1 and KIF13B on hGH secretion. PC12 cells were co-transfected with pXGH5 and the indicated test plasmids. After 2 days, the cells were incubated under basal or stimulatory (0.3 mM ATP) conditions for 10 minutes. The amount of secreted hGH in the assay medium as well as unsecreted hGH in the cells was determined by ELISA, and the percentage of total hGH secreted was calculated. Data are means±s.e. of three independent experiments performed in triplicate.

View larger version (13K): [in a new window]   Fig. 13. Co-immunoprecipitation of GFP-KIF13B and FLAG-centaurin-1 with HA-ARFGTP. COS cells transiently transfected with the indicated expression constructs were lysed after 2 days of transfection and immunoprecipitated (IP) with an anti-HA antibody polyclonal antibody. The precipitates were then immunoblotted (IB) with anti-HA, anti-FLAG and anti-GFP monoclonal antibodies to detect HA-ARF6, FLAG-centaurin-1 and GFP-KIF13B, respectively. One-twentieth of the cell lysates (Input) were also immunoblotted (IB) with the anti-HA, anti-FLAG and anti-GFP antibodies to ensure that HA-ARF6GTP, FLAG-centaurin-1 and GFP-KIF13B, respectively, were expressed. The experiment was repeated two further times with identical results.