Roles for the opposing phosphatases are more poorly defined. delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to USL311 the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores. and (Chan et al., 2012; Redli et al., 2016). Two isoforms of PP1 (PP1 and PP1) are concentrated at kinetochores and bind Knl1 and Ska1 (Liu et al., 2010; Sivakumar et al., 2016; Trinkle-Mulcahy et al., 2003, 2006). Kinetochore-associated PP1 appears to play important roles in stabilizing kinetochore-microtubule attachments and opposing spindle checkpoint signaling (Liu et al., 2010; Pinsky et al., 2006; Sivakumar et al., 2016; Vanoosthuyse and Hardwick, 2009). The PP2A holoenzyme is a hetero-trimer composed of a scaffolding A subunit, regulatory B subunit and catalytic C subunit (Janssens et al., 2008). The B subunits are classified into three sub-families termed B (PR55/B55), B(PR61/B56) and B(PR72) (Bollen et al., 2009; Janssens et al., 2008). Plk1 phosphorylation of BubR1 recruits PP2A-B56 to kinetochores in prometaphase (Foley et al., 2011; Suijkerbuijk et al., 2012). At metaphase, PP2A-B56 levels diminish at kinetochores while PP1 increases, suggesting that kinetochore-microtubule interactions are stabilized by PP2A-B56 in prometaphase and by PP1 at metaphase. In agreement with this idea, depletion of PP2A shows stronger impairment of chromosome alignment compared to depletion of PP1 (Foley et al., 2011; Liu et al., 2010). In this study, we show that PP1 and PP2A phosphatases promote Ska recruitment to kinetochores. These results corroborate and extend previous work (Redli et al., USL311 2016). Forced targeting USL311 of PP1 to kinetochores partially rescues defects caused by Ska3 depletion. We propose a feedback mechanism in which the Ska complex recruits PP1 to kinetochores at metaphase which further recruits Ska to stabilize kinetochore-microtubule attachments and initiate anaphase. RESULTS AND DISCUSSION Phosphatases promote accumulation of Ska at kinetochores We and others have shown that Ska binds to kinetochores at prometaphase and maximally accumulates there at metaphase (Chan et al., 2012; Redli et al., 2016; Sivakumar et al., 2014). Inhibition of Aurora B kinase increased Ska accumulation on kinetochores lacking microtubule attachment (Chan et al., 2012). Correspondingly, expression of phosphomimetic mutants of Ska inhibited recruitment (Chan et al., 2012). These findings and recent data from Redli et al. (2016) indicate phosphatases likely regulate Ska binding to kinetochores. PP1 and PP2A are the major phosphatases implicated in mitotic transitions. PP1, principally the PP1 isoform, localizes to kinetochores and is CASP8 implicated in spindle checkpoint inactivation (Liu et al., 2010; Trinkle-Mulcahy et al., 2003). PP2A also accumulates at kinetochores and plays a role in promoting kinetochore-microtubule attachment in prometaphase (Foley et al., 2011). To test the role of the phosphatases in Ska recruitment, we depleted PP1 or PP2A A subunit. We analyzed recruitment of Ska to kinetochores using immunofluorescence with antibody to Ska3. Ska begins to concentrate at kinetochores before microtubule attachment but reaches maximum levels on bioriented metaphase chromosomes. In cells progressing through mitosis with intact spindles, we found that depletion of USL311 PP1 or PP2A A reduced Ska3 kinetochore levels (Fig.?S1A-C). However, depletion of phosphatases has direct effects on spindle microtubule stability (Foley et al., 2011; Liu et al., 2010). To eliminate the complication of varying spindle microtubule stability after depletion of phosphatases in experiments designed to quantify Ska accumulation on kinetochores, we measured Ska levels on kinetochores of nocodazole-treated cells and found that depletion of PP1 or PP2A phosphatase significantly decreased Ska3 accumulation (Fig.?1A,B). Previous work has shown that Plk1 and BubR1 promote PP2A recruitment to kinetochores (Foley et al., 2011; Suijkerbuijk et al., 2012). Depletion of Plk1 or BubR1 with siRNA caused the expected reduction of PP2A at kinetochores in cells with intact spindle microtubules (Fig.?S1D-F) and also resulted in lower levels of kinetochore-associated Ska3 in nocodazole-treated mitotic cells (Fig.?1C,D). Open in a separate window Fig. 1. Phosphatases PP1 and PP2A promote Ska recruitment and normal progression through mitosis. (A) HeLa cells grown on coverslips were transfected with control, PP1 or PP2A A siRNA. 45?h after transfection, cells were treated with 3.3?M nocodazole for 3?h and then prepared for immunofluorescence. Ska3 at kinetochores was quantified. PP1 or PP2A A depletion.
