Analysis of the weak interactions of ADP-Unc104 and ADP-kinesin with microtubules and their inhibition by MAP2c.
Microtubule based motors like conventional kinesin (Kinesin-1) and Unc104 (Kinesin-3), and classical microtubule associated proteins (MAPs), including MAP2, are intimately involved in neurite formation and organelle transport. The processive motility of both these kinesins involves weak microtubule interactions in the ADP-bound states. Using cosedimentation assays, we have investigated these weak interactions and characterized their inhibition by MAP2c. We show that Unc104 binds microtubules with five-fold weaker affinity and two-fold higher stoichiometry compared with conventional kinesin. Unc104 and conventional kinesin binding affinities are primarily dependent on positively charged residues in the Unc104 K-loop and conventional kinesin neck coiled-coil and removal of these residues affects Unc104 and conventional kinesin differently. We observed that MAP2c acts primarily as a competitive inhibitor of Unc104 but a mixed inhibitor of conventional kinesin. Our data suggest a specific model in which MAP2c differentially interferes with each kinesin motor by inhibiting its weak attachment to the tubulin C-termini. This is reminiscent of the defects we have observed in Unc104 and kinesin mutants in which the positively charged residues in K-loop and neck coiled-coil domains were removed.
Al-Bassam
Jawdat
J
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Roger
Benoit
B
Halpain
Shelley
S
Milligan
Ronald A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008956:Models, Chemical; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; C489788:MAP2 protein, human; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C423186:UNC-104 protein, C elegans; D000244:Adenosine Diphosphate; D016547:Kinesins