Huntington-Associated Phosphorylation of Kinesin-1 Enhances Autoinhibition in a Phosphomimic

Abstract

One of the consequences of the triplet expansion in Huntington's disease is inhibition of fast axonal transport (FAT). Phosphorylation of Ser176 in human kinesin-1 by JNK3 has been implicated in this inhibition (Morfini, et al., Nature Neruo. 12, 866 (2009)). To investigate the molecular basis for the inhibition of FAT, we have generated the S182E phosphomimic of the homologous residue in Drosophila kinesin-1. When introduced into short dimer of motor domains, the S182E mutation produces a 30% decrease in the maximum rate of microtubule-stimulated ATPase rate in solution and a similar reduction in the sliding rate of axonemes in a multimotor sliding assay. This only modest decrease suggests that direct inhibition of motility is not likely to be the principal cause of the pronounced inhibition of FAT. However, free kinesin is known to be autoinhibited through the binding of a tail domain to a dimer of motor domains (heads) and the Ser182 phosphorylation site is near the tail binding site on the heads (Kaan, et al., Science 333, 883 (2011)) where it could influence autoinhibition. One possibility is that the increased negative charge on the heads due to phosphorylation of Ser182 could produce a stronger interaction with the positively charge tail domain that would strengthen autoinhibition and inhibit FAT. To test the effect of the phosphomimic on autoinhibition, the binding of a monomeric tail domain to a dimer of motor domains was determined using a FRET assay. In 100 mM KCl, monomeric tail domains bind to mutant S182E heads three-fold more tightly than to wild type heads. This suggest that inhibition of FAT may principally be due to enhanced tail binding and accompanying autoinhibition of kinesin-1 following phosphorylation.Supported by NIH Grant NS058848.