Aurora A–TPX2 complex (Bayliss et al., 2003; Eyers et al., cell death or sensitizes the cells to other mitotic poisons. Further 2003; Tsai et al., 2003), and, paradoxically, PP1 is therefore u-n work will be necessary to test these ideas, but preliminary data able to act upon the active form of Aurora A. As shown here, support this hypothesis (unpublished data). The strategy pre- PP6 is able to act on the Aurora A–TPX2 complex and does not sented here is also likely to have general relevance for other suffer this limitation. PP6 is also the major Aurora A T-loop classes of enzyme important for cell cycle control and cell-sig phosphatase activity in cell lysates, and PP1 contributes little, if naling pathways. Although it is notoriously difficult to identify any, activity consistent with the finding that PP1 is inactivated enzyme substrates, the biochemical method used here provides by inhibitory CDK1 phosphorylations in mitosis (Dohadwala, a simple way to find the specific enzyme-catalyzing removal of 1994; Kwon, 1997). This raises the question of how PP6 has any labile modification, such as phosphorylation, ubiquitylation, been overlooked until now. There may be three reasons for this. sumoylation, and others. First, PP6 is inhibited by the toxins okadaic acid, microcystin- LR, and calyculin A used to study PP1 and PP2A, and, depend- Materials and methods ing on the model substrate used, is actually more sensitive than PP2A to these toxins (Prickett and Brautigan, 2006). In fact, the Reagents and antibodies residues in PP2A required for very high affinity toxin binding Standard laboratory reagents were obtained from Thermo Fisher Scientific and Sigma-Aldrich. Antibodies to Eg5 (1–450 aa) were raised in sheep and are conserved in PP6, including the hydrophobic cage–forming rabbit using hexahistidine-tagged human proteins expressed in and purified residues glutamine 122 and histidine 191 (Xing et al., 2006). from bacteria. Specific antibodies were purified using the antigens conjugated These residues are not conserved in PP1, explaining the reduced to Affigel-15. Commercially available antibodies were used to -tubulin (mouse DM1A; Sigma-Aldrich), Plk1 (mouse SC-17783; Santa Cruz Biotech- inhibition of PP1 by okadaic acid and microcystin-LR (Xing nology, Inc.), Aurora A (rabbit AB12875; Abcam), Aurora A pT288 (rabbit et al., 2006). Therefore, inhibitor studies do not unambiguously 3079; Cell Signaling Technology), PPP6C (rabbit A300-844A; Bethyl Labo- define which PPP family member is acting on a specific target. ratories, Inc.), SAPS1–3 (rabbit A300-968A, A300-969A, and A300-972A; Bethyl Laboratories, Inc.), ANKRD28–52 (rabbit A300-974A and A302- Second, inhibitor-2 (I-2) was previously used to implicate PP1 372A; Bethyl Laboratories, Inc.), CREST (human auto-antiserum; Europa as the Aurora A phosphatase (Tsai et al., 2003). However, it was Bioproducts Ltd.), KID (rabbit AKIN12-A; Cytoskeleton, Inc.), NuMa (mouse subsequently shown that I-2 can directly stimulate Aurora A AB5673; Abcam), PP1- (rabbit 2582; Cell Signaling Technology), PP1- (rabbit A300-905A; Bethyl Laboratories, Inc.), PP1- (goat SC-6108; Santa autophosphorylation activity (Satinover et al., 2004), suggesting Cruz Biotechnology, Inc.), PP2A (mouse 610555; BD), TPX2 (mouse that the effects of I-2 are independent of any inhibition of PP1. ab32795; Abcam), Hec1 (mouse GTX70268; GeneTex, Inc.), CenpE Third, previous studies have used the commercially available (mouse ab5093; Abcam), CenpA (mouse ab13939; Abcam), pericentrin (rabbit ab4448; Abcam), and cyclin B1 (mouse GNS3; Millipore). Aurora A trypsin-resistant catalytic fragment of PP1, which lacks the reg- kinase inhibitor was obtained from Selleck Chemicals (MLN8237; 10 mM ulatory subunits and, therefore, does not have the same substrate of 1,000,000× stock). Secondary antibodies raised in donkey to mouse, specificity as the PP1 holoenzyme (Johnson et al., 1987; Tóth rabbit, sheep/goat, and human conjugated to HRP, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 568, and Alexa Fluor 647 were obtained from Invit- et al., 2000; Terrak et al., 2004). The high degree of similarity in rogen and Jackson ImmunoResearch Laboratories, Inc. PPP family catalytic subunits also means that in the absence of Molecular biology regulatory subunits they will have limited specificity (Xing Full-length clones were amplified with the KOD polymerase (Merck) from et al., 2006). Fitting with this view, catalytic subunits alone of total HeLa RNA extracted using TRIZOL (Invitrogen). Point mutations were either PP1 or PP2A can dephosphorylate free Aurora A (Bayliss introduced using the QuickChange system (Agilent Technologies) accord- ing to the manufacturer’s protocol. All primers were obtained from Meta- et al., 2003; Eyers et al., 2003; Tsai et al., 2003). bion GmbH. Expression constructs were produced in pcDNA3 vectors modified to encode the chicken -actin promoter to reduce expression Aurora A and PP6 in cancer levels, the required tags (EGFP, FLAG, or mCherry), and the geneticin, Finally, our findings have relevance beyond the biochemistry puromycin, or blasticidin resistance selection markers. The siRNA library targeting human phosphatase subunits and single duplexes corresponding and cell biology of Aurora A regulation. Aurora A is amplified to the individual pools were obtained from Thermo Fisher Scientific and are in many cancers and is currently the focus of much interest as a listed in Table S1. drug target for cancer therapy (Sen et al., 1997; Bischoff et al., Cell culture 1998; Zhou et al., 1998; Karthigeyan et al., 2010). Intriguingly, HeLa and MRC-5 cells were cultured in growth medium (DME containing PPP6C is mutated at an absolutely conserved histidine lying 10% fetal bovine serum) at 37°C and 5% CO2. For plasmid transfection within the catalytic site in melanoma (Bamford et al., 2004; and siRNA transfection, LT1 (Mirus Bio, LLC) and Oligofectamine (Invitro- gen), respectively, were used according to the manufacturer’s instruc- Forbes et al., 2010). As we show here, loss of PP6 function will tions. For making stable cell lines, constructs were transfected into HeLa lead to increased Aurora A activity similar to the effects of S3 cells. After a 24-h selection, drugs were added as follows: 700 µg/ml Aurora A amplification. PPP6C could therefore be considered geneticin, 1.0 µg/ml puromycin, and 2.0 µg/ml blasticidin. Colonies were picked after a 14-d selection, and expression was checked by a novel tumor suppressor gene. Recent work suggests that the Western blotting. mitotic machinery is an Achilles’ heel for cancer cells carrying mutant Ras (Luo et al., 2009), and innovative ways of targeting Fixed and live-cell microscopy key mitotic regulators are therefore of great interest. Our work Fixed samples on glass slides were imaged using a 60× 1.35 oil immer- sion objective on a standard upright microscope system with filter sets for suggests additional ways in which Aurora A activity can be tar- DAPI, EGFP/Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 568, and Alexa geted through inactivation of PP6. PP6 inhibitors may be useful Fluor 647 (Chroma Technology Corp.), a camera (CoolSNAP HQ2; Roper for targeting cancers with amplified or in some other way per- Industries), and imaging software (MetaMorph 7.5; Molecular Dynamics, Inc.). For live-cell imaging, cells were plated in 35-mm dishes with a 14-mm turbed Aurora A activity. Under these conditions, PP6 inhibition 1.5 thickness coverglass window on the bottom (MatTek Corporation). For might push the system into a runaway state that directly promotes imaging, the dishes were placed in a 37°C and 5% CO2 environment Protein phosphates 6 regulates Aurora A • Zeng et al. 1329