Results the pool were able to deplete PPP6C and cause the same altered nuclear morphology (Fig. 1 C, Western blot and bottom images). PP6 may therefore be a cell cycle regulator and a candidate T-loop Identification of phosphatases required for phosphatase in human cells, and it was investigated further. normal mitosis Loss of T-loop phosphatase function for the essential mitotic kinases should cause defective spindle formation, chromosome Analysis of the PP6 holoenzyme in mitosis alignment, and cytokinesis and, therefore, give rise to cells with Like other PPP family members, PP6 is a multisubunit enzyme, characteristic nuclear abnormalities (Fig. 1 B, stage 1). To iden- comprising a single catalytic subunit and one of each of the tify nonredundant protein phosphatases required for normal three SIT4 phosphatase–associated protein (SAPS) domain mitosis, a visual screen for cells with abnormal nuclei after 48-h regulatory subunits and three ankyrin repeat domain subunits siRNA-mediated depletion of phosphatase subunits was per- (Stefansson and Brautigan, 2006; Stefansson et al., 2008). PP6 formed (Fig. 1, A–C). For this purpose, siRNA pools were used also interacts with 4, which has a chaperone-like activity re- containing four different duplexes against the same target gene quired to maintain active PP2A, PP4, and PP6 (Prickett and (Table S1). Of the phosphatase subunits tested, only seven gave Brautigan, 2006; Kong et al., 2009). To define which of these rise to nuclear morphology defects with a frequency above a subunits are important for mitosis, PP6 holoenzyme complexes threshold set at twice the median value (Fig. 1 C). The most were isolated from mitotic cells stably expressing FLAG-tagged striking of these was the PP6 catalytic subunit (PPP6C), in which PPP6C and then analyzed by SDS-PAGE and mass spectrome- >70% of cells showed abnormally shaped fragmented nuclei try. This revealed that HeLa cells express all known PP6 s-ub but otherwise normal cell morphology (Fig. 1 C). Interestingly, units and that these are present in PPP6C complexes during a previous screen of phosphatases required for mitosis in Dro- mitosis (Fig. 2 A). To test whether the PP6 SAPS domain regu- sophila melanogaster found that PP6 depletion caused a two- latory subunits and ankyrin repeat domain subunits are required fold increase in the mitotic index, but its function was not for normal mitosis, they were depleted using siRNA. Western investigated further in this study (Chen et al., 2007). Mutations blotting revealed that these subunits could be depleted singly or in PP6 were recently also found to cause spindle position and in combination, but this did not alter the amount of the PP6 cat- cell contractility defects inCaenorhabditis elegans early em- alytic subunit present (Fig. 2 B). When all three SAPS domain bryonic cell divisions (Afshar et al., 2010). However, no su-b regulatory subunits were depleted together, altered nuclear strates were identified. Of the other phosphatase subunits morphology similar to that seen after PPP6C depletion was identified, PPP1R12A (MYPT1) has been reported to function observed (Fig. 2 C). Single depletions showed partially altered as part of a PP1 holoenzyme complex in mitosis by opposing nuclear morphology (Fig. 2 C), supporting the idea that there Plk1 (Yamashiro et al., 2008). Because the initial 48-h screen is redundancy between these subunits. Depletion of the ANKRD52 failed to pick the PP1 catalytic subunits, these were examined in subunit had no effect on nuclear morphology, whereas ANKRD28 more detail. At 72 h, the PPP1C-B catalytic subunit gave a nu- and ANKRD44 ankyrin repeat subunit depletion altered nuclear clear morphology defect similar to PPP1R12A F(ig. S1 A), morphology (Fig. 2 C). Collectively, these findings suggest that which was consistent with previous findings that MYPT1 binds a PP6 holoenzyme, comprising PPP6C catalytic, SAPS1–3 regu- to PPP1C-B (Hartshorne et al., 2004; Yamashiro et al., 2008). latory, and ANKRD28–44 subunits, is required for normal m-i Although no obvious change was observed with PPP1C-A, totic progression. PPP1C-C depletion resulted in alterations to the microtubule cytoskeleton and cell shape change without causing nuclear PP6 activity is required for normal mitosis abnormalities (Fig. S1 A), suggesting that PP1 catalytic subunits The function of PP6 in mitosis could be explained by either a have distinct functions. structural role or as an active phosphatase. To discriminate The PP2A regulatory subunit PPP2R5A was also found in between these two possibilities, stable cell lines expressing the screen. PP2A regulatory (PPP2R5/B56/B isoform) and cat- either wild type (si08ResPPP6CWT) or catalytically inactive alytic subunits are present in the shugoshin complexes contro-l (si08ResPPP6CPD) mutant forms of the PP6 catalytic subunit were ling centromeric cohesion (Kitajima et al., 2006). In this case, generated (Fig. 3 A). To enable depletion of the endogenous redundancy between the different PP2 catalytic subunits was PP6 catalytic subunit, these constructs were engineered to be observed (Fig. S1 B), explaining why they were not found in the resistant to the PPP6C si08 duplex while retaining sensitivity to initial screen. Depletion of both the A and B catalytic subunits si07, to allow depletion of all PPP6C. When siR0e8sPPP6CWT of PP2A was necessary to cause cells to arrest in mitosis with cells were depleted of endogenous PPP6C using si08, the wild- scattered chromosomes (Fig. S1 B). Because the functions of type transgene was able to rescue the phenotype, and cells r-e PPP1R12A and PPP2R5A as part of PP1 and PP2A complexes, tained normal-shaped nuclei (Fig. 3 B). Depletion of all PPP6C respectively, are already known (Kitajima et al., 2006;Yamashiro using si07 gave the expected altered nuclear morphology. et al., 2008), it was therefore decided to focus on the category I In contrast, when si08ResPPP6CPD cells were depleted of endo-g phenotype seen with PPP6C. enous PPP6C using si08, the phosphatase-dead mutant trans- To test whether the defect observed for PPP6C was both gene was unable to rescue the phenotype, and cells displayed specific and indicative of a general function for this phosphatase abnormally shaped nuclei similar to wild-type HeLa cells in mitosis, additional verification steps were performed. When treated with the same duplex (Fig. 3 B). Depletion of endoge- tested in isolation, all four of the single siRNA duplexes used in nous PPP6C by si07 and si08 and of the transgene-expressed Protein phosphates 6 regulates Aurora A • Zeng et al. 1317