Figure 9. Spindle assembly factors are misregulated after PPP6C depletion. (A) HeLa cells were transfected for 48 h with control or PPP6C si08 duplexes before fixation. Cells were stained using DAPI to detect DNA and antibodies to -tubulin (Tub), Eg5, and NuMa. (B) Control and PPP6C-depleted cells treated with solvent (DMSO) or 10 µM MLN8237 for 10 min were fixed and then stained for Eg5, NuMa, tubulin (not depicted), and with DAPI to detect DNA. Bars, 10 µm. In both panels, KIF11/Eg5, NuMa, and tubulin signals were measured for the spindle region in ImageJ. KIF11/Eg5 and NuMa sig- nals were normalized for tubulin and are plotted in the graph (n = 12). Error bars indicate the standard error of the mean. Arrowheads mark fragmented spindle poles. and S phase may have unique substrates determined by the ANKRD28/44 and 52 subunits, and this will be an important avenue for further investigation. Exactly what constitutes a PP6 substrate recognition de- terminant is unclear. In the future, it should be possible to define this determinant by comparing the mitotic PP6 substrate Aurora A and the closely related kinase Aurora B, which is not a su-b strate (unpublished data). High quality crystal structures have been solved for both the Aurora A–TPX2 and the Aurora B– INCENP (inner centromere protein) complexes (Bayliss et al., 2003; Sessa et al., 2005). These reveal that Aurora A pT288 is exposed to solvent in the absence of TPX2, reorients and hydro- gen bonds with Aurora A–arginine 255, and, thus, is protected from the bulk solution in the presence of TPX2. Here, we show that PP6 is a physiologically relevant T-loop phosphatase in human cells, which recognizes the active, TPX2-complexed form of Aurora A. This suggests that PP6 can access the buried pT288 residue, possibly by altering the conformation of the Aurora A– TPX2 complex. An intriguing possibility is that the Aurora A– specific activator TPX2 also acts as a recognition determinant for the PP6 T-loop phosphatase. This would allow PP6 to sp-e cifically recognize and act upon the Aurora A–TPX2 complex. Previous studies have reported that Aurora A can be dephosphorylated by PP1 or PP2A in vitro (Andrésson and Ruderman, 1998; Walter et al., 2000; Bayliss et al., 2003; Eyers et al., 2003; Tsai et al., 2003). However, this is prevented in the and CREST to mark centromeres. Micronucleation is quantitated in the graph (n = 3). (C) HeLa cells were transfected for 48 h with control, PPP6C si08, and TPX2 duplexes alone or in combination before fixation. Cells were stained using DAPI to detect DNA and CREST to mark centromeres. The graph shows the percentages of normal and micronucleated cells for the various conditions (n = 3). (B and C) Arrowheads indicate CREST staining outside the main nucleus defined by the DAPI staining. Bars, 10 µm. Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. Protein phosphates 6 regulates Aurora A • Zeng et al. 1327