Kinesin-5: Cross-bridging mechanism to targeted clinical therapy

Highlights

• Kinesin-5 is primarily a mitotic microtubule motor that exhibits complex regulation.

• Biophysical studies of the motor core alter the paradigm of kinesin catalysis.

• Toolbox of small-molecule inhibitors opens basic and clinical research avenues.

• Kinesin-5 inhibitors provide translational lessons about targeted patient therapies.

Abstract

Kinesin motor proteins comprise an ATPase superfamily that works hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on multi-faceted analyses of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.

Introduction

Kinesins are the smallest and most abundant nanomotor in cells and the only canonical motor protein that is ubiquitous in all eukaryotes. Like dynein and myosin, these proteins hydrolyze ATP and convert chemical energy into mechanical energy. This permits transport and movement along the cytoskeletal track. Of the > 16 different kinesin isoforms (Wickstead and Gull, 2006), Kinesin-5 (BimC/Eg5/N-2/Kif11) family members were the first identified to be essential in mitosis. Genetic analysis provided the initial insight into the key mitotic role of this motor family. In the search for strains that were defective in cellular division at a restrictive temperature, screens of fungal libraries and fission yeast uncovered BimC (Enos and Morris, 1990) and Cut7 (Hagan and Yanagida, 1990), respectively, in the early 1990s. These mutants had malfunctions in spindle pole body separation and nuclear division and were unable to undergo mitosis. Cloning and sequencing of these mutants (Hagan and Yanagida, 1990, Kashina et al., 1997) revealed that the gene product encoded a 130 kDa protein with high similarity to the conventional kinesin (KHC/Kinesin-1) involved in motility in squid and mammalian brains. Similarly, simultaneous loss of function in Cin8p and Kip1p in Saccharomyces cerevisiae revealed a redundant function of these Kinesin-5 family members for spindle assembly (Hoyt et al., 1992, Roof et al., 1992).

The gene family has expanded from the early days of the molecular and bioinformatics era. Many groups (Bannigan et al., 2007, Bishop et al., 2005, Blangy et al., 1995, Chauviere et al., 2008, Heck et al., 1993, Hoyt et al., 1992, Sawin et al., 1992, Tihy et al., 1992) initially identified orthologs in Xenopus, S. cerevisiae, Drosophila melanogaster, Homo sapiens, Mus musculus, and Caenorhabditis elegans (Table 1). Currently, there are over 70 different Kinesin-5 proteins identified by sequence homology in 66 eukaryotes (Fig. 1). Subsequently classified as the Kinesin-5 family (Lawrence et al., 2004), this group of related kinesins localizes to spindle microtubules and structures present at spindle poles.

The Kif11 gene product has four domains (Fig. 2A), three of which are assigned to roles resulting in differentiation of kinesin function. An N-terminal globular motor domain performs conserved functions of binding microtubules (MTs) and nucleotide hydrolysis. Variations in sequence in the tail, interrupted coiled-coil region, and neck-linker/cover neck (Hesse et al., 2013) are thought to dictate how these motor proteins bind specific cargo, have particular oligomerization states, and control directionality of net motion of the motor domain, respectively. Unlike the canonical dimeric kinesin motors, electron microscopy showed that native Kinesin-5 proteins are bipolar homotetramers (Blangy et al., 1995, Cole et al., 1994), with their motor domains positioned at the ends of the tetramer's long axis. The arrangement of two sets of antiparallel dimers is hypothesized to result in Kinesin-5 motors crosslinking and sliding antiparallel microtubules (Fig. 2B).

The human Eg5 gene product (HsEg5) is of particular interest amongst the Kinesin-5 proteins, because of its potential as a therapeutic target for cancer treatment. It is sensitive to a battery of small-molecule inhibitors (DeBonis et al., 2004, Hotha et al., 2003, Luo et al., 2007) that allosterically block Eg5 activity (DeBonis et al., 2003, Maliga et al., 2002). The specificity of these synthetic inhibitors is exceptional: although all eukaryotic organisms examined to date contain at least one Kinesin-5 protein, not all Kinesin-5 proteins are sensitive to these compounds (DeBonis et al., 2003, Maliga et al., 2002). Of the inhibitors most frequently employed, monastrol (Mayer et al., 1999) and S-trityl-l-cysteine [STC; (DeBonis et al., 2003)], were uncovered from independent chemical screens, inclusive for mitotic inhibition and exclusive for microtubule interactions. They induced mitotic arrest in human tissue culture cells by inhibiting Eg5-dependent MT motility (Kapoor et al., 2000, Mayer et al., 1999) and resulted in a spectacular reorganization of the mitotic spindle to aberrant monoastral form with no apparent effect on interphase MT arrays (Fig. 3). Failure either in chromosome segregation or in the timing of cell division events can result in aneuploidy that is strongly linked with developmental defects and cancer (Kops et al., 2005).

The discovery of these chemical inhibitors of HsEg5 is important on two fronts. First, they can be used as tools to dissect mechanotransduction in this mitotic kinesin and provide answers to still open questions of how catalysis is used and converted into force and motion. Second, numerous small-molecule agents that solely target this human mitotic Kinesin-5 protein with high specificity are leads for anti-cancer therapy; several are in trials as clinical anti-cancer agents [for example, see (Carol et al., 2009, Kathman et al., 2007, Purcell et al., 2010)].