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. 2005 Sep;16(9):4225-30.
doi: 10.1091/mbc.e05-02-0116. Epub 2005 Jun 22.

Abl tyrosine kinase and its substrate Ena/VASP have functional interactions with kinesin-1

MaryAnn Martin  1 , Shawn M Ahern-Djamali, F Michael Hoffmann, William M Saxton
Affiliations

Abl tyrosine kinase and its substrate Ena/VASP have functional interactions with kinesin-1

MaryAnn Martin et al. Mol Biol Cell. 2005 Sep.

Abstract

Relatively little is known about how microtubule motors are controlled or about how the functions of different cytoskeletal systems are integrated. A yeast two-hybrid screen for proteins that bind to Drosophila Enabled (Ena), an actin polymerization factor that is negatively regulated by Abl tyrosine kinase, identified kinesin heavy chain (Khc), a member of the kinesin-1 subfamily of microtubule motors. Coimmunoprecipitation from Drosophila cytosol confirmed a physical interaction between Khc and Ena. Kinesin-1 motors can carry organelles and other macromolecular cargoes from neuronal cell bodies toward terminals in fast-axonal-transport. Ena distribution in larval axons was not affected by mutations in the Khc gene, suggesting that Ena is not itself a fast transport cargo of Drosophila kinesin-1. Genetic interaction tests showed that in a background sensitized by reduced Khc gene dosage, a reduction in Abl gene dosage caused distal paralysis and axonal swellings. A concomitant reduction in ena dosage rescued those defects. These results suggest that Ena/VASP, when not inhibited by the Abl pathway, can bind Khc and reduce its transport activity in axons.

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Figures

Figure 1.
Figure 1.
Ena and Khc yeast two-hybrid interaction. (A) A cartoon of Drosophila Ena (684 amino acids). EVH1 and EVH2 represent the Ena-VASP homology domains, and Proline represents the proline-rich region. The 243-amino-acid C-terminal region used to screen for Ena-interacting proteins in a yeast two-hybrid assay is indicated with a black line. The region of Ena necessary for immunoprecipitation of Khc (see Figure 2) is bounded by arrows. (B) A cartoon of Drosophila Khc (975 amino acids) that notes structural features and the prey fragment (black line) that interacted with the Ena-C-terminal bait.
Figure 2.
Figure 2.
Khc and Ena coimmunoprecipitate from Drosophila cytosol. (A) Western blots of unfractionated cytosol or immunoprecipitate (IP) fractions from normal S2 cells (lanes N) or from S2 cells cotransfected with full-length Ena and Khc expression vectors (lanes X). A blot of cytosol was stained first with anti-Khc R1.5 (top panel), then stripped and reprobed with anti-Ena-carboxy-terminal antibody (bottom panel). Separate blots of the immunoprecipitates (Ena-IP or Khc-IP) were stained with anti-Khc R1.5 (top panel) or anti-Ena-carboxy-terminal antibody (bottom panel). The immunoprecipitations were done with anti-Ena-carboxy-terminal antibody or anti-Khc Flyk2 antibody. (B) Western blots of unfractionated cytosol or anti-Khc Flyk2 immunoprecipitate (Khc-IP) from cells cotransfected with expression constructs for Khc and one of the following: full-length Ena (lanes 1), Ena lacking the C-terminal 52 amino acids (lanes 2), or Ena lacking the C-terminal 243 amino acids (lanes 3). Staining was done with an anti-Ena-amino-terminal antibody. Note that despite the abundance of truncated Ena, detectable amounts were not precipitated by the Khc antibody (lanes 3). This suggests a specific Khc binding site between amino acids 441 and 632 of Ena (see arrows in Figure 1A). (C) Western blot of various immunoprecipitates from adult fly-head cytosol stained with anti-Khc Flyk2. Immunoprecipitation was done with beads alone (mock-IP), anti-GFP (IgG-IP), anti-Ena-carboxy-terminal (Ena-IP), or anti-Drosophila-Khc from Cytoskeleton, (Khc-IP). The positions of prestained standards are noted beside the blots with numbers corresponding to their molecular weights. In all Khc- and Ena-IP experiments, either duplicate blots or stripped original blots were stained with the homotypic antibody to confirm robust precipitation of the primary protein (unpublished data).
Figure 3.
Figure 3.
Ena and CSP localization in wild-type and Khc mutant nerves and motor-axon terminals. Scanning confocal fluorescence micrographs of (A and B) larval segmental nerves and (C and D) motor-neuron terminals in segment A4 of wild-type (+/+) or Khc6/Df(2R)Jp6 mutant (Khc6/Df) third instar larvae. Staining was with mouse anti-CSP antibody and rabbit anti-Ena-amino-terminal antibody. The punctate background staining in the Ena panels of C and D is in muscle cells 6/7. Scale bar, 10 μm.
Figure 4.
Figure 4.
Mapping a dominant enhancer of Khc to the Abl locus. Depicted at the top is a portion (bands 72E to 74A) of the left arm of chromosome 3 (from Bridges's polytene map: Lindsley and Zimm, 1992). On the left are the names of various deletions tested for EK activity in a Khc16/Khc+ genetic background. Black bars indicate the regions not deleted, while gaps indicate the deleted regions. Plus-symbols indicate definite EK activity. The genes Abl, Baldspot (blp), Galpha73B, zetaCOP, and CG13032 are likely to be in the smallest deletion interval that has EK activity (73B01-02) based on sequence data (Flybase, 2005). Single gene mutant alleles of Abl and Baldspot were tested for EK activity. Mutant alleles of the other genes were not available (na).
Figure 5.
Figure 5.
Genetic interactions among Khc, Abl, and ena. Genotypes are noted in each panel: Khc = Khc16, Abl = Abl1, and ena = ena210. (A) Single video frames of third-instar larvae crawling on hard agar. Anterior is to the left. The arrow shows posterior paralysis or “tail flipping,” a classic axonal-transport phenotype, in the Khc Abl double heterozygote. Larvae heterozygous for Khc16 alone (top) or Abl1 alone (unpublished data) showed no tail flipping. (B) Immunofluorescence micrographs of third-instar larval segmental nerves (n) in abdominal segment A4 of larval neuromuscular preparations. Anti-synaptotagmin shows the locations of axonal swellings (e.g., arrow). These images are representative for each genotype, as determined by two observers scoring coded images. Larvae were ∼3 mm in length. Scale bar for (B), 10 μm.
Figure 6.
Figure 6.
A model for kinesin-1 regulation by Abl and Ena. Kinesin-1 is represented as a tetramer of two elongated Khc molecules (orange globular domains connected by black coiled-coil helix) plus two Klc molecules (gray crescents). It is shown linked to a cargo by unknown proteins (dark red ellipses). Activation pathways for Abl and Ena are not certain, as noted by white question marks. Inhibition effects are noted with a line and cross-bar. Activation effects are noted with a line and arrowhead.
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