JCB_201409129 1..11

Anaphase central spindle formation is controlled by a group of conserved microtubule-associated proteins and kinesin motors (Barr and Gruneberg, 2007; Glotzer, 2009; Brust-Mascher and Scholey, 2011; Duellberg et al., 2013). Key among these are the antiparallel microtubule-stabilizing factor PRC1 and a microtubule dynamic instability suppressing kinesin KIF4A, which work together to create microtubule overlaps of defined length (Bringmann et al., 2004; Bieling et al., 2010; Subramanian et al., 2010; Hu et al., 2011). These microtubule overlaps are further stabilized by the centralspindlin complex formed by the MKlp1/KIF23 kinesin-like motor protein and the Cyk-4/ MgcRacGAP Rho family GTPase regulator (Mishima et al., 2002; Douglas et al., 2010). Both PRC1 and centralspindlin are inhibited until anaphase onset by Cdk1–cyclin B phosphorylation (Jiang et al., 1998; Mollinari et al., 2002; Mishima et al., 2004; Zhu et al., 2006; Neef et al., 2007). At the onset of anaphase, PP2A–B55 removes the inhibitory Cdk1 phosphorylation on PRC1, triggering central spindle formation only after chromosome separation has commenced (Cundell et al., 2013). Together, these factors can explain the Cdk1-regulated decrease in microtubule catastrophe observed toward the cell equator in anaphase (Brust-Mascher et al., 2004; Cheerambathur et al., 2007). However, Cdk regulation of these components does not provide an obvious means to fine-tune microtubule dynamic instability in a spatially restricted manner and thereby promote anaphase B spindle elongation (Cheerambathur et al., 2007). In addition to negative regulation by Cdk1, all these components are subject to activating phosphorylations by either Aurora B or Plk1 (Polo-like kinase 1). PRC1 and centralspindlin phosphorylation by Plk1 are necessary for division plane specification (Neef et al., 2007; Petronczki et al., 2007; Wolfe et al., 2009). Aurora B phosphorylation of KIF4A at T799 activates its microtubule-stimulated ATPase activity and enhances KIF4A binding to PRC1, and the antiparallel microtubule overlap bundling activity of MKlp1 is promoted by Aurora B phosphorylation at S911 (Douglas et al., 2010; Nunes Bastos et al., 2013). KIF4A and MKlp1 are phosphorylated by a pool of Aurora B transported to the central spindle by the mitotic kinesin MKlp2 (Gruneberg et al., 2004; Neef et al., 2006; Ruchaud et al., 2007; Fuller et al., 2008; Brust-Mascher and Scholey, 2011; Nunes Bastos et al., 2013). Although, in both cases, previous studies The mitotic kinase Aurora B is concentrated at the anaphase central spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate central spindle length. In this paper, we identify a counteracting system in which PP2A–B56 and -, but not PP2A–B56, -, and -, are maintained at the central spindle by KIF4A. Biochemical assays show that PP2A–B56 can dephosphorylate the T799 Aurora B site on KIF4A and thereby counteract the Aurora B– and microtubule-stimulated ATPase activity of KIF4A. In agreement with these observations, combined silencing of PP2A–B56 and - resulted in increased phosphorylation of KIF4A T799 and decreased central spindle growth in anaphase B. Furthermore, reduced turnover of regulatory phosphorylation on another Aurora B substrate MKlp1 was observed, suggesting that PP2A–B56 and - play a general role opposing Aurora B at the central spindle. KIF4A and PP2A–B56 and - therefore create a spatially restricted negative feedback loop counteracting Aurora B in anaphase. KIF4A and PP2A–B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle