Figure 1. Identification of human phosphatases required for normal mitosis. (A) A model for the T loop–mediated kinase activation. Below is a schematic of the human protein phosphatase superfamily covering the phosphoprotein phosphatases (PPP), the metallophosphatases (PPM), and the phosphotyrosine protein (PTP) and dual-specificity phosphatases (DUSPs) modified from Chen et al. (2007). The number of phosphatase subunits screened in each sub- family is indicated. (B) The screening procedure and the phenotypes expected are categorized using Roman numerals together with a brief description of the underlying causes. (C) HeLa cells were transfected with siRNA pools for 78 human phosphatase subunits, fixed after 48 h, and stained with DAPI to reveal the DNA and nuclear morphology. Abnormal nuclear morphology was scored according to the categories (I–V) described and is expressed as a histogram sorted from high to low (n = 3). The dotted line indicates twice the median value for nuclear abnormalities. For verification, HeLa cells were transfected with the four siRNA duplexes (06–09) making up the PPP6C siRNA pool, fixed after 48 h, and then stained with DAPI to reveal the DNA and nuclear morphology. Numbers in the top left corner correspond to the siRNA library pool used. Con., control. Bar, 10 µm. pole separation and bipolar spindle formation, a potential u-p stream regulatory role for Aurora A coordinating KIF11/Eg5 activity with that of other spindle assembly factors is an attrac- tive model (Clarke and Zhang, 2008; Eckerdt et al., 2008). Despite the recent advances in understanding how T-loop phosphorylation of the major mitotic kinases is accomplished (Gold et al., 2006), the opposing phosphatases remain largely elusive. The prevailing view is that dephosphorylation during mitosis is performed by phosphatase complexes of the phospho- protein phosphatase (PPP) family (Bollen et al., 2009), making them good candidates for T-loop phosphatases. These enzymes consist of a catalytic subunit in complex with one or more regul-a tory subunits (Shi, 2009). The regulatory subunits confer loca-l ization and substrate specificity to these holoenzyme complexes, both increasing activity toward true substrates and reducing ac- tivity toward other phosphorylated proteins (Johnson et al., 1996; Hirano et al., 1997; Tóth et al., 2000; Terrak et al., 2004). To achieve a clearer understanding of the regulation of mitosis, it is therefore necessary to identify the specific phosphatase holoenzyme com- plexes opposing the key mitotic kinases. The recent identification of specific protein phosphatase 1 (PP1) and PP2A complexes regulating chromosome condensation and sister chromatid co- hesion, respectively, has highlighted the importance of local d-e phosphorylation of kinase substrates during mitosis (Kitajima et al., 2006; Tang et al., 2006; Vagnarelli et al., 2006). In this study, we focus on the kinases themselves. Using an unbiased two-stage screen of protein phosphatase subunits, we search for T-loop phosphatase activities important for normal mitosis. 1316 JCB • VOLUME 191 • NUMBER 7 • 2010