Differentiation of Cytoplasmic and Meiotic Spindle Assembly MCAK Functions by Aurora B-dependent Phosphorylation

Abstract

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylationdependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly. INTRODUCTIONAssembly of the microtubule-based bipolar spindle, the apparatus that powers chromosome segregation during mitosis and meiosis (M phase), is driven by the inherent dynamic instability of microtubules, as well as by proteins that alter microtubule dynamics (Hyman and Karsenti, 1996;Wittmann et al., 2001). Accordingly, M-phase onset triggers global, cytoplasmic changes in microtubule dynamics that expedite spindle assembly, most notably an increase in the frequency of catastrophes, the transition from microtubule growth to shrinkage (Belmont et al., 1990;Verde et al., 1992;Rusan et al., 2001;Kinoshita et al., 2002). In Xenopus egg extracts, the KinI kinesin MCAK, classified by its internally located motor domain (Wordeman and Mitchison, 1995;Walczak et al., 1996), is essential for governing this change in catastrophe frequency (Walczak et al., 1996;Tournebize et al., 2000). Biochemical studies indicate that MCAK likely accomplishes catastrophe induction directly by catalytically depolymerizing microtubules from their protofilament ends (Desai et al., 1999b;Hunter et al., 2003).Although spindle assembly demands global regulation of microtubule dynamics, proper spindle function also requires precise control of microtubule dynamics regulation at defined locations within the spindle. Because accurate chromosome segregation requires that each kinetochore of a pair of sister chromatids be attached to microtubules derived from opposing spindle poles (biorientation), it is particularly important to control the interaction of spindle microtubules with kinetochores (Rieder and Salmon, 1...

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

“…This is consistent with the complex localization of MCAK in the spindle and the highly complicated phosphorregulatory schemes that regulate its localization and activity at centromeres and at spindle poles (Andrews et al, 2004;Gorbsky, 2004;Lan et al, 2004;Ohi et al, 2004;Knowlton et al, 2006;Zhang et al, 2007Zhang et al, , 2008. This suggests that MCAK is responsible for preferentially altering the dynamics individual MT subclasses and raises the important question of how other MT dynamics effectors contribute to the control of spindle MT subclasses.…”

Functional Analysis of Human Microtubule-based Motor Proteins, the Kinesins and Dyneins, in Mitosis/Cytokinesis Using RNA Interference

“…MCAK has been shown to colocalize with the Aurora-B kinase to centromeres. Aurora-B is a chromosomal passenger protein that regulates chromosome movement and spindle dynamics, and MCAK is one of its critical downstream targets (Shannon and Salmon, 2002;Andrews et al, 2004;Lan et al, 2004;Ohi et al, 2004). Phosphorylation by Aurora-B regulates the localization of MCAK to centromeres and also its MT depolymerase activity.…”

Centromere Targeting of the Chromosomal Passenger Complex Requires a Ternary Subcomplex of Borealin, Survivin, and the N-Terminal Domain of INCENP

“…Consequently, knockdown or inhibition of Aurora B or any of the other subunits of the CPC results in chromosome congression defects, kinetochore-microtubule attachment errors, lagging chromosomes in anaphase, and failure of cytokinesis (Vagnarelli and Earnshaw, 2004;Tanaka, 2005). Consistent with the idea that the localization of the CPC is important for target recognition by its kinase subunit, Aurora B has been demonstrated to phosphorylate proteins at the centromere, such as CENP-A and mitotic centromere-associated kinesin (MCAK) (Zeitlin et al, 2001;Andrews et al, 2004;Lan et al, 2004;Ohi et al, 2004), and at the central spindle and midbody, e.g., Mklp1, MgcRacGap, and vimentin (Goto et al, 2003;Minoshima et al, 2003;Guse et al, 2005). Yet, the molecular details of how phosphorylation by Aurora B influences its different substrates and thus regulates the different stages of the cell cycle are still not clear.…”