JCB: Article Y G O L O I B L L E C F O L A N R U O J E H T Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphor ylation state of Aurora A bound to its activator TPX2 Kang Zeng, Ricardo Nunes Bastos, Francis A. Barr, and Ulrike Gruneberg University of Liverpool, Cancer Research Centre, Liverpool L3 9TA, England, UK M served regulatory step involving T-loop phos- any protein kinases are activated by a con- phorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regula- tory subunits interferes with spindle formation and chro- mosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A–TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A–TPX2 complexes in vitro, whereas catalytically inactive mu- tants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phos- phatase regulating Aurora A activity during spindle for- mation and suggest the general importance of this form of regulation. Introduction Dynamic protein phosphorylation, mediated by a conserved co- hort of protein kinases, controls the profound changes in cell-u lar organization required for mitosis and cytokinesis (Nigg, 2001). Many of these kinases share a common activation mech- anism involving phosphorylation of a threonine residue within the activation or T loop and binding to a coactivator protein (Fig. 1 A; Gold et al., 2006).These events promote the position- ing of key residues required for the phosphotransfer reaction from ATP bound in the kinase active site to the acceptor residue in the substrate protein (Huse and Kuriyan, 2002). T-loop phos- phorylation can be autocatalytic or mediated by an upstream kinase and generally increases kinase activity by several orders of magnitude (Adams, 2003). Recent studies on the activation of Aurora A exemplify the importance of T-loop phosphoryl-a tion as a regulatory mechanism (Bayliss et al., 2003; Eyers et al., 2003). Aurora A is localized to the centrosomes and spindle poles from late S phase throughout mitosis, which is consi-s tent with its function in organizing mitotic spindle formation F.A. Barr and U. Gruneberg contributed equally to this paper. Correspondence to Francis A. Barr: fabarr@liv.ac.uk; or Ulrike Gruneberg: u.gruneberg@liv.ac.uk Abbreviations used in this paper: PPP, phosphoprotein phosphatase; SAPS, SIT4 phosphatase–associated protein. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 191 No. 7 1315–1332 www.jcb.org/cgi/doi/10.1083/jcb.201008106 (Glover et al., 1995; Giet et al., 2002). Localization to the sp-in dle is achieved through the association of Aurora A with its binding partner TPX2 (Kufer et al., 2002). Besides this target- ing function, TPX2 is critically important for autocatalytic phosphorylation of threonine 288 in the T loop of Aurora A and, hence, Aurora A activation (Bayliss et al., 2003; Eyers et al., 2003). Furthermore, TPX2 also prevents the dephosphorylation of this residue (Bayliss et al., 2003; Eyers et al., 2003). Other interaction partners of Aurora A, such as PAK1, Ajuba, and Bora, have also been reported to facilitate T288 phosphoryl-a tion, although the structural basis for these effects is not yet known (Hirota et al., 2003; Zhao et al., 2005; Hutterer et al., 2006). Consistent with the function of Aurora A in spindle pole maturation and separation, T288-phosphorylated and, hence, activated Aurora A can be detected at the spindle poles (Ohashi et al., 2006). Several potential Aurora A substrates on mitotic spindles have been described previously, including the BimC family kinesin KIF11/Eg5 (Giet et al., 1999, 2002; Kinoshita et al., 2005). Because KIF11/Eg5 is critically required for spindle © 2010 Zeng et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- lication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 1315