by cell lysis on ice for 2 h in the absence of phosphatase inhib-i standard lysis procedures resulted in the loss of Aurora A pho-s tors (outlined in Fig. 1 B, stage 2). phorylation in control cell lysateFsi(g. 7 A, lanes 1–4).Consis- Cell lysates prepared from control or phosphatase su-b tent with this, TPX2 did not interact with Aurora A under these unit–depleted cells were therefore Western blotted with an-ti conditions (Fig. 7 A, lane 6). Depletion of the PP6 catalytic su-b bodies to Aurora A and the phosphorylated T288 T-loop residue unit using si08 or the addition of excess phosphatase inhibitors (Ohashi et al., 2006). Because the visual screen indicated some stabilized the upshifted form of Aurora A and its interaction redundancy between PP1 and PP2 catalytic subunits, these were with TPX2 (Fig. 7 A, compare lanes 8 and 9 with lanes 5 and 6). depleted alone and in combination. As expected, Aurora A Kinase assays on these complexes showed that PPP6C depl-e phosphorylation was lost during lysate preparation from control tion stabilized Aurora A kinase activity toward TPX2 and the cells using standard lysis procedures without phosphatase i-n model substrate histone H3 under conditions in which it is lost hibitors (Fig. 6 A, red bars). Notably, it was retained in PPP6C- in the control samples (Fig. 7 A, compare lane 9 with lane 6). depleted cell lysates but not under the other conditions tested Similarly, depletion of PPP6C using a second duplex, si07, or of (Fig. 6 A, duplex 44), suggesting that PP6 is the major Aurora A the PP6 SAPS1/2/3 regulatory subunits stabilized the upshifted T-loop phosphatase. This shift was more clearly seen on gels form of Aurora A and its interaction with TPX2 (Fig. 7 B, com- cast with the Phos-tag reagent (Fig. 6 B, duplex 44), which pare lanes 7 and 8 with lanes 10 and 11 and 13 and 14). In contrast, slows the mobility of phosphorylated proteins and, thus, better depletion of PP2A or PP1 catalytic subunits did not stabilize resolves them from their nonphosphorylated counterpart. either the upshifted pool of Aurora A or its interaction with TPX2 Several criteria suggest that Aurora A is a specific target of PP6. (Fig. 7, C and D, compare lanes 7 and 8 or 20 and 21 with lanes No other phosphatase tested stabilized the phosphorylated form 10 and 11, 13 and 14, and 23 and 24). This is in agreement with of Aurora A (Fig. 6, A and B). Both the Aurora A pT288 signal previous findings that these phosphatases can only act on free and the upshifted phosphorylated form of Aurora A were Aurora A and not Aurora A in complex with TPX2 (Bayliss protected after depletion of either the PP6 catalytic or SAPS et al., 2003; Eyers et al., 2003). regulatory subunits (Fig. 6, C and D, respectively), which was To provide further evidence that PP6 acts directly on consistent with a requirement for the PP6 holoenzyme. Plk1 is Aurora A–TPX2 complexes, phosphatase assays using purified phosphorylated on T210 (Jang et al., 2002), and, in contrast to proteins were performed. When Aurora A–TPX2 complexes Aurora A T288 phosphorylation, this was not altered by PP6 were incubated with the catalytically active PP6 holoenzyme catalytic or SAPS regulatory subunit depletion (Fig. 6, C and D). purified from mitotic cells, Aurora A was dephosphorylated Levels of other phosphatases, such as PP1 and PP2, were un- (Fig. 7 E, lane 2). This effect was not seen when buffer, catalyt-i altered under these conditions (Fig. 6 C), suggesting altered cally inactive PP6, or a mock enzyme purification was used Aurora A phosphorylation was primarily caused by the loss of (Fig. 7 E, lanes 1, 3, and 4). The nonspecific phosphatase activ- PP6 activity. Addition of excess phosphatase inhibitor cocktail ity of calf intestinal phosphatase was unable to dephosphorylate to the buffer stabilized Aurora A T288 phosphorylation and the Aurora A–TPX2 complexes under these conditions (Fig. 7 E, upshifted form of Aurora A and revealed that there is slightly lane 5), which was consistent with previous observations that more T288-phosphorylated Aurora A in PP6 catalytic or regul-a the phosphorylated T288 T-loop residue is protected in the tory subunit–depleted cells than in the control cells (Fig. 6, TPX2 complex. This suggests that PP6 specifically recognizes C and D). Depletion of the PP1 and PP2 catalytic subunits the Aurora A–TPX2 complex and gains access to the protected alone (Fig. 6 E) or in combination (Fig. 6 A) did not stabilize phosphorylated T288 residue (Bayliss et al., 2003). These o-b Aurora A phosphorylation. Together, these observations indicate servations are summarized in a model in which the PP6 holo- that the PP6 holoenzyme comprising both catalytic and regul-a enzyme acts on the Aurora A–TPX2 complex, whereas PP1 and tory subunits is an Aurora A T-loop phosphatase and that the PP2A act on free Aurora A (Fig. 7 F). underlying defect after the loss of PP6 function is misregulation of Aurora A and not Plk1. Aurora A inhibition rescues the PPP6C depletion phenotype Previous studies have suggested that activated Aurora A in com- PP6 acts on the Aurora A–TPX2 complex Activated Aurora A forms a complex with the spindle assembly plex with TPX2 accumulates at the spindle (Kufer et al., 2002; factor TPX2 (Kufer et al., 2002; Bayliss et al., 2003; Eyers and Bayliss et al., 2003; Eyers et al., 2003; Ozlü et al., 2005; Ohashi Maller, 2004), and the effects of PP6 inactivation on the Aurora A– et al., 2006). Loss of PP6 function, which stabilizes the Aurora A– TPX2 complex were therefore investigated. As already shown, TPX2 complex, should, therefore, result in increased levels of phosphorylated/total Aurora A (n = 2). The red line indicates the median value for Aurora A phosphorylation. (B) HeLa cells were transfected with siRNA pools for 73 human phosphatase subunits for 54 h, treated for 18 h with nocodazole, and then lysed in the absence of phosphatase inhibitors for 2 h on ice. In addition, control and PPP6C-depleted samples were lysed in the presence or absence of phosphatase inhibitors. All samples were analyzed on Mn2+ Phos-tag gels to better resolve phosphorylated from nonphosphorylated Aurora A and then Western blotted for total Aurora A and actin as a loading control. Red boxes highlight siRNA 44 (PPP6C) in which Aurora A phosphorylation is retained and an adjacent sample in which it is not. (C–E) HeLa cells were transfected for 48 h with control, PPP6C si08, SAPS1–3, PP1C A, PP1C B, PP1C C, and PP2C A siRNA duplexes. Lysates were then prepared from these cells in immunoprecipitation buffer containing or lacking phosphatase inhibitor cocktail and Western blotted. Graphs show phosphorylated Aurora A levels (n = 3). Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. Where present, the red and black lines indicate the phosphorylated and nonphosphorylated forms of Aurora A. White lines indicate that intervening lanes have been spliced out. Protein phosphates 6 regulates Aurora A • Zeng et al. 1323