Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

Abstract

(21) or an ERK-family kinase (22, 23). We have further noticed that this same sequence also includes the consensus phosphorylation site for the cell-cycle kinase p34cdc2 (consensus S/TPXK/R) (24). As yet, however, no evidence for phosphorylation of the bimC box by any serine(threonine) protein kinase has been obtained.To understand the mechanism by which Eg5 and related proteins work in mitosis, we would like to know (i) how they localize to the spindle, (ii) with what other spindle components they interact, and (iii) how their activity is regulated throughout the cell cycle and through the different stages of mitosis. Here we address the first of these questions, by mutating regions of the Eg5 protein that may be involved in targeting it to the spindle. We have found that elements of both the N-and C-terminal domains of Eg5 are required for spindle localization. Interestingly, a single point mutation in the most conserved part of the bimC box is able to abolish spindle localization completely, in a manner that is consistent with phosphorylation regulating the localization and/or activity of Eg5. MATERIALS AND METHODSPlasmid Construction. All constructs (Fig. 1) (see Results). The myc-tagged "near-full-length" plasmid pEM103 was constructed by cloning the 3.2-kb EcoRI fragment into the EcoRI site of mycHB, a vector derived from pcDNAI containing a cytomegalovirus promoter and an N-terminal c-myc

Interaction of Arabidopsis kinesin‐like calmodulin‐binding protein with tubulin subunits: modulation by Ca2+‐calmodulin

“…Recent studies show that both plus-end and minus-end kinesins interact with tubulin subunits and this interaction is modulated by polyglutamylation of tubulin subunits (Larcher et al, 1996;Walker, 1995). There is also evidence for mechanisms such as phosphorylation that modulate the interaction of motors with microtubules (Blangy et al, 1995;Liao et al, 1994;Sawin and Mitchison, 1995). Among motor proteins, the heavy chain of some actin-based myosin motors and light chain of microtubule-based kinesin motors show calcium and calmodulin-dependent regulation (Matthies etal., 1993;Wolenski, 1995).…”

Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

“…In the current study, microinjected antibody to the spindle-targeting phospho-tail domain on kinesin-5 (Sawin and Mitchison, 1995;Sharp et al, 1999a;Cheerambathur et al, 2008) rapidly dissociated KP61F from mitotic spindles. By utilizing stable transgenic embryos that express GFP-tagged KLP61F, we were able to directly visualize the extent of dissociation of the target antigen from spindles in different regions of the syncytium relative to the injection site.…”

“…Studies using transient transfection with Eg5 mutants in cultured Xenopus cells (Sawin and Mitchison, 1995), with KLP61F mutants in live Drosophila embryos (Cheerambathur et al, 2008) and using antibodies raised against phospho-and unphosphoepitopes in Drosophila embryos (Sharp et al, 1999a) suggest that the cyclin-dependent kinase-mediated phosphorylation of a conserved Thr-933 residue in the bimC box of kinesin-5 is critical for its localization to the spindle. We reasoned that antibodies to the phosphorylated form of this domain would competitively inhibit the binding of native phosphorylated KLP61F to the spindle and, by displacing the motor from its site of action, would inhibit its function.…”

Mechanistic Analysis of the Mitotic Kinesin Eg5

“…Homo sapiens Eg5/KSP, the human homologue of Xenopus Eg5, is a slow, plus-end-directed BimC kinesin that has been shown to localize along interpolar spindle microtubules and at the spindle poles (24,28,29). This spindle association is regulated by the cell cycle-dependent phosphorylation of a single threonine within a highly conserved region of the Eg5 tail domain by p34 cdc2 protein kinase (23,24,29). Monastrol, a small, cell-permeable molecule, can reversibly inhibit the function of Eg5 to arrest cells in mitosis, providing a useful model for anti-mitotic drug design (30 -35).…”