© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs238931. doi:10.1242/jcs.238931

RESEARCH ARTICLE RACK1 regulates duplication through promoting the activation of polo-like kinase 1 by Aurora A Yuki Yoshino1,2,3,*, Akihiro Kobayashi1,2,*, Huicheng Qi1,2, Shino Endo1,2, Zhenzhou Fang1,2, Kazuha Shindo1,3, Ryo Kanazawa1,3 and Natsuko Chiba1,2,3,‡

ABSTRACT consist of a pair of , termed the mother Breast cancer gene 1 (BRCA1) contributes to the regulation of centriole and the daughter centriole, and proteinaceous number. We previously identified receptor for activated C surrounding the mother centriole (Conduit kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold et al., 2015). The proper number of centrosomes is stably protein that interacts with multiple proteins through its seven WD40 maintained by a regulatory network that ensures duplication only domains, directly binds to BRCA1 and localizes to centrosomes. once per (Conduit et al., 2015; Fujita et al., 2016; Nigg RACK1 knockdown suppresses centriole duplication, whereas and Holland, 2018). At entry into , a cell has two RACK1 overexpression causes centriole overduplication in a subset centrosomes, each containing a pair of tightly engaged centrioles. of mammary gland-derived cells. In this study, we showed that During mitosis, centrioles lose their connection in a process termed RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and disengagement, and the two centrioles are joined by a flexible promotes the Aurora A–PLK1 interaction. RACK1 knockdown linker. In late of the following cell cycle, a new daughter decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal centriole is generated on the side wall of each mother centriole. The localization of Aurora A and p-PLK1 in S phase, whereas RACK1 daughter centrioles grow during S phase, resulting in the formation overexpression increased p-PLK1 level and the centrosomal localization of two pairs of centrioles in two centrosomes in . In late G2 of Aurora A and p-PLK1 in interphase, resulting in an increase of cells phase, the linker is degraded and the two centrosomes separate to with abnormal centriole disengagement. Overexpression of cancer- form bipolar spindles. derived RACK1 variants failed to enhance the Aurora A–PLK1 During centriole duplication, the newly formed daughter centrioles interaction, PLK1 phosphorylation and the centrosomal localization of suppress the formation of additional daughter centrioles to prevent p-PLK1. These results suggest that RACK1 functions as a scaffold multiple rounds of duplication (Tsou and Stearns, 2006a; Nigg, 2007). protein that promotes the activation of PLK1 by Aurora A in order to Although the mechanism underlying this suppression is poorly promote centriole duplication. understood, disengagement is thought to release the suppression (Tsou and Stearns, 2006a; Nigg, 2007; Shukla et al., 2015). Therefore, This article has an associated First Person interview with the first author the timing of disengagement is important for the regulation of of the paper. centriole duplication to maintain the proper number of centrosomes. Centrosome amplification, a common condition in many cancers, KEY WORDS: Aurora A, PLK1, Cancer, Centriole duplication, including breast cancer (Chan, 2011), is associated with chromosomal Centrosome instability and aggressive phenotypes (Denu et al., 2016; Godinho et al., 2014; Schneeweiss et al., 2003). Breast cancer gene 1 (BRCA1) INTRODUCTION mutations are responsible for hereditary breast and ovarian cancer The centrosome is a major microtubule organizing center and syndrome, and loss or mutation of BRCA1 causes centrosome facilitates proper bipolar spindle formation (Conduit et al., 2015). amplification (Starita et al., 2004; Ko et al., 2006). Certain BRCA1 An abnormal number of centrosomes causes aberrant variants found in familial breast cancers are associated with (e.g. monopolar and multipolar division) and chromosome deficiencies in the regulation of centrosome number (Kais et al., segregation errors, leading to carcinogenesis (Milunovic-Jevtić ́ 2012). BRCA1 germline mutation and negative expression of BRCA1 et al., 2016; Cosenza et al., 2017; Levine et al., 2017). Therefore, the are significantly associated with centrosome amplification in breast regulation of centrosome number is important for the maintenance cancer tissues (Shimomura et al., 2009; Watanabe et al., 2018). of chromosomal stability and tumor suppression. BRCA1 forms multi-protein complexes with BRCA1-associated RING domain protein 1 (BARD1) and other proteins that function in a number of cellular processes, including DNA repair and 1Department of Cancer Biology, Institute of Aging, Development, and Cancer, centrosome regulation (Takaoka and Miki, 2018). In previous Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. studies, we identified Obg-like ATPase 1 (OLA1) (Matsuzawa et al., 2Department of Cancer Biology, Tohoku University Graduate School of Medicine, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan. 3Laboratory of Cancer Biology, 2014; Yoshino et al., 2018) and receptor for activated C kinase 1 Graduate School of Life Sciences, Tohoku University, 4-1 Seiryomachi Aoba-ku, (RACK1) (Yoshino et al., 2019) as components of the BRCA1– Sendai 980-8575, Japan. BARD1 complex. OLA1 localizes to centrosomes and directly *These authors contributed equally to this work binds to BRCA1, BARD1 and γ-tubulin. Knockdown or ‡Author for correspondence ([email protected]) overexpression of OLA1 increases the fraction of cells with centrosome amplification caused by centriole overduplication in Y.Y., 0000-0003-0029-3467; N.C., 0000-0001-6504-1290 mammary tissue-derived cells (Matsuzawa et al., 2014; Yoshino Handling Editor: David Glover et al., 2018). RACK1 also localizes to centrosomes and directly

Received 7 September 2019; Accepted 29 July 2020 binds to BRCA1, OLA1 and γ-tubulin, and interacts with BARD1. Journal of Cell Science

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Knockdown of RACK1 suppresses centriole duplication in some each cell cycle phase. MCF7 cells were transfected with control or breast cancer cells. Conversely, overexpression of RACK1 increases HA–RACK1 vector and synchronized to the G1/S transition by a the fraction of cells with centrosome amplification caused by double thymidine block (Fig. 1B, Protocol 1). Then, cells were centriole overduplication in mammary tissue-derived cells. released into fresh medium and fixed for immunocytofluorescence Adequate RACK1 expression levels contribute to the proper with anti-HA and γ-tubulin antibodies after 0, 4, 8, and 12 h of localization of BRCA1 to centrosomes. Alterations in the incubation, which represented G1/S transition (G1/S), S, G2, and expression levels or missense mutations of OLA1 and RACK1 are G1 phases, respectively (Fig. 1C). RACK1-OE cells were identified associated with various cancers, as reported in the COSMIC as HA-positive cells and subjected to analysis of centrosome database (https://cancer.sanger.ac.uk/cosmic). These data suggest number. Cells with amplified (more than two) centrosomes that the centrosomal BRCA1–BARD1 complex containing OLA1 were counted, and their percentage was calculated. RACK1 and RACK1 is important for the regulation of centrosome number overexpression significantly increased the proportion of cells with and tumor suppression. However, the underlying molecular centrosome amplification in S and G2 cells, but not in G1 and G1/S mechanism remains unknown. cells (Fig. 1D,E). These data indicate that the centrosome Centriole duplication is regulated by several mitotic kinases (Wang amplification observed in RACK1-OE cells occurred during S et al., 2014; Nigg and Holland, 2018). Polo-like kinase 1 (PLK1) is a and G2 phases. critical regulator of centriole disengagement during mitosis (Tsou To confirm that RACK1 overexpression induces centriole et al., 2009; Shukla et al., 2015), and its activity is tightly regulated overduplication in MCF7 cells, as previously observed in spatially and temporally throughout the cell cycle. Aurora A kinase Hs578T cells (Yoshino et al., 2019), we stained the centriole phosphorylates PLK1 at Thr210 under the control of CDK1 and Bora with anti-CP110 antibody, the mother centriole with anti-CEP152 to activate PLK1 in late G2 phase and initiate mitotic entry (Macurek antibody, and the daughter centriole with anti-SAS6 antibody in et al., 2008; Schmucker and Sumara, 2014; Seki et al., 2008; RACK1-OE MCF7 cells (Fig. S1A). RACK1 overexpression Bruinsma et al., 2014). In centrosomes, PLK1 activation by Aurora A increased the fraction of cells with a CP110 foci/CEP152 foci ratio and the scaffold protein CEP192 is essential for centrosome of >2 (Fig. S1B,D) and that of cells with extra SAS6 foci that did maturation and bipolar spindle formation during G2 and mitotic not pair with CEP152 foci (Fig. S1C,D). These results suggest that phases (Joukov et al., 2014; Meng et al., 2015). In addition, PLK1 centriole overduplication is also induced in some RACK1-OE contributes to centrosome amplification induced by S phase or G2 MCF7 cells. arrest or DNA damage (Loncareǩ et al., 2010; Zou et al., 2014). PLK4, another member of the PLK family, is essential for centriole PLK1, Aurora A and PLK4 activities contribute to duplication (Zitouni et al., 2014). PLK4 activation in late G1 phase centrosome amplification induced by RACK1 overexpression triggers cartwheel formation, which is the initial step of centriole To identify the signaling pathway involved in centrosome duplication (Kleylein-Sohn et al., 2007; Kim et al., 2013; amplification upon RACK1 overexpression, RACK1-OE cells Bettencourt-Dias et al., 2005). Although the function of these were treated with inhibitors of kinases important for centriole mitotic kinases in centriole duplication has been studied extensively, duplication, namely, MLN8054, BI6727 and centrinone B, the mechanism by which the regulation of centriole duplication by which are inhibitors of Aurora A, PLK1, and PLK4, these kinases is perturbed in cancer is not well understood. respectively (Wong et al., 2015; Manfredi et al., 2007; In the present study, we investigated the effect of RACK1 Rudolph et al., 2009). These inhibitors block cell cycle overexpression on centriole overduplication in breast cancer progression; treatment with MLN8054 or BI6727 causes G2/M cells. The results showed that RACK1 overexpression-induced arrest, and treatment with centrinone B causes G1 arrest. To centrosome amplification involved the activities of Aurora A and minimize the effect on the cell cycle, cells were synchronized at PLK1. RACK1 directly bound to Aurora A and PLK1, promoted G1/S by a double thymidine block and released into fresh their interaction, and was involved in the centrosomal localization of medium containing inhibitors for 4 h (Fig. 2A, Protocol 2). As Aurora A and the activation of PLK1 at the centrosome. These results shown in Fig. 2B,C, treatment with BI6727 and centrinone B, but suggest that RACK1 functions as a scaffold involved in the regulation not MLN8054, decreased the proportion of cells with centrosome of the Aurora A/PLK1 signaling axis and centriole duplication. amplification in RACK1-OE cells. Because Aurora A is the upstream activator of PLK1, we speculated that Aurora A might RESULTS contribute to centrosome amplification at an earlier cell cycle RACK1 overexpression increases the fraction of cells with phase than that affected by PLK1. To verify this, MLN8054 centrosome amplification during S and G2 phases treatment was started at 12 h before the release from a double We previously reported that RACK1 overexpression increases the thymidine block, and centrosome number was analyzed at 4 h proportion of cells with centrosome amplification by inducing after the release (Fig. 2A, Protocol 3). Under these conditions, centriole overduplication (Yoshino et al., 2019). Cell cycle arrest at MLN8054 treatment significantly decreased the fraction of cells S or G2 phase causes centriole overduplication (Balczon et al., with centrosome amplification in RACK1-OE cells (Fig. 2D,E). 1995; Loncareǩ et al., 2010). To examine the effect of RACK1 These results suggest that activation of Aurora A, followed by overexpression on cell cycle progression, MCF7 cells were PLK1 and PLK4 activation contributes to centrosome transfected with empty vector (control) or HA-tagged RACK1 amplification induced by RACK1 overexpression. (HA–RACK1) expression vector, and analyzed by flow cytometry. No significant differences in cell cycle distribution were observed RACK1 binds to PLK1 and Aurora A and promotes their between HA–RACK1-overexpressing MCF7 cells (RACK1-OE interaction cells) and control cells (Fig. 1A), as reported previously in RACK1- Overexpression of RACK1 increases the fraction of cells with knockdown cells (Yoshino et al., 2019). centriole overduplication (Yoshino et al., 2019; Fig. S1). Activation The effect of RACK1 overexpression on inducing centrosome of PLK4 initiates centriole duplication at centrioles licensed via amplification was investigated by measuring centrosome number in disengagement (Kleylein-Sohn et al., 2007; Kim et al., 2013; Journal of Cell Science

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Fig. 1. Centrosome amplification occurs in S and G2 phases in a subset of RACK1-OE cells. (A) MCF7 cells were transfected with control or HA–RACK1 vector. At 72 h after transfection, cells were analyzed by flow cytometry. At least 10,000 cells were analyzed. (B) Protocol 1 for the analysis of centrosome amplification. TF, transfection. (C) MCF7 cells were treated according to Protocol 1, harvested at the indicated time points, and analyzed by flow cytometry. At least 10,000 cells were analyzed. (D) MCF7 cells were transfected with control or HA–RACK1 vector and treated according to Protocol 1. Cells were fixed and stained with anti-HA and anti-γ-tubulin (a centrosome marker) antibodies, and DAPI. Scale bars: 10 µm. (E) Quantification of centrosome amplification from the experiments shown in D. Cells with more than two centrosomes were counted, and the percentages were calculated from more than 100 cells in each sample. G1/S, S, G2 and G1 represent samples incubated for 0, 4, 8, and 12 h after the second release, respectively. In RACK1-OE cells, only cells with HA staining were counted. The mean±s.e.m. of three individual experiments is shown. *P<0.05, **P<0.01 (two-tailed Welch’s test).

Bettencourt-Dias et al., 2005). Disengagement requires activation of immunoprecipitation between the kinase domain or polo-box the PLK1 signaling pathway (Shukla et al., 2015; Tsou et al., 2009; domains of PLK1 and RACK1, which revealed that both the Tsou and Stearns, 2006b). Therefore, we presumed that RACK1 kinase domain and polo-box domains interacted with RACK1 overexpression activates PLK1 and Aurora A to induce centriole (Fig. S2C,D). overduplication upstream of PLK4 activation. To explore how RACK1 is composed of seven WD domains and functions as a PLK1 and Aurora A contribute to centrosome amplification induced scaffold protein that binds to numerous proteins, resulting in their by RACK1 overexpression, the interaction of RACK1 with PLK1 activation (Ron et al., 2013). Because RACK1 interacted with and Aurora A was analyzed in immunoprecipitation experiments. both PLK1 and Aurora A, we determined whether RACK1 Endogenous RACK1 associated with endogenous PLK1 and co-expression affects the interaction between PLK1 and Aurora Aurora A (Fig. 3A,B). These associations were confirmed using A. The interaction between PLK1 and Aurora A was markedly exogenously expressed tagged proteins (Fig. S2A,B). To determine strengthened by RACK1 co-overexpression in a dose-dependent which domain of PLK1 interacts with RACK1, we performed manner (Fig. 3C; Fig. S2E). Consistent with this, knockdown of Journal of Cell Science

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Fig. 2. The Aurora A/PLK1 signaling axis and PLK4 contribute to centrosome amplification in RACK1-OE cells. (A) Protocols 2 and 3 for the analysis of the effects of kinase inhibitor treatments on centrosome amplification. TF, transfection. (B) MCF7 cells were treated according to Protocol 2. The final concentrations of inhibitors were 1 µM for MLN8054, 100 nM for BI6727 and 500 nM for centrinone B. Cells were stained with anti-HA and anti-γ-tubulin antibodies and DAPI. (C) Quantification of centrosome amplification from the experiments shown in B. The percentage of cells with more than two centrosomes was calculated from more than 100 cells in each sample. The mean±s.e.m. of more than three individual experiments is shown. (D) MCF7 cells were treated with MLN8054 according to Protocol 3. (E) Quantification of the samples shown in D. The mean±s.e.m. of three individual experiments is shown. Scale bars: 10 µm. **P<0.01; n.s., not significant (two-tailed Welch’s test).

RACK1 decreased the interaction between PLK1 and Aurora A To confirm the effect of RACK1 on promoting the Aurora (Fig. S2F). Co-overexpression of RACK1 increased PLK1 A–PLK1 interaction, recombinant Aurora A, PLK1 and RACK1 phosphorylation at Thr210 (Fig. 3C). These results indicate proteins expressed in E. coli were purified for pull-down assays. that RACK1 contributes to the formation of the Aurora A–PLK1 RACK1 directly bound to Aurora A and both the kinase domain and complex and may activate PLK1. polo-box domains of PLK1 (Fig. 3D,E; Fig. S2G). As shown in Journal of Cell Science

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Fig. 3. RACK1 enhances the interaction between PLK1 and Aurora A. (A) Cycling HEK-293T cells were lysed with hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 5% glycerol, and 0.5% NP-40) supplemented with protease inhibitors, and then cleared by centrifugation at 1000 g for 10 min to yield a cytosolic lysate. The lysate was immunoprecipitated (IP) with polyclonal anti-t-PLK1 antibody or control IgG. Short and long exposures of the same blots of PLK1 are shown. (B) The cytosolic lysate obtained from cycling HEK-293T cells was immunoprecipitated with polyclonal anti-Aurora A antibody or control IgG. Short and long exposures of the same blots of Aurora A are shown. The arrow and arrowhead indicate non-specific bands and AuroraA, respectively. (C) HEK-293T cells were transfected with the indicated vectors. At 48 h after transfection, cells were harvested and immunoprecipitated with anti- FLAG antibody. Short and long exposures of the same blots of immunoprecipitated Aurora A are shown. (D) His-NusA (Nus)–Aurora A was pulled down by GST or GST–RACK1. His-Nus protein was used as the negative control. His-Nus and His-Nus–Aurora A were loaded as the input. (E) His-Nus–Aurora A and His–RACK1 were sequentially pulled down by GST or GST–PLK1. In the A→R sample, GST–PLK1 was incubated with His-Nus– Aurora A for 1 h and washed, and beads were incubated with His–RACK1 for 1 h. In the R→A sample, GST–PLK1 was incubated with His–RACK1 for 1 h and washed, and beads were incubated with His-Nus–Aurora A for 1 h. His-Nus, His-Nus–Aurora A, and His–RACK1 were loaded as the input. Journal of Cell Science

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Fig. 3E, PLK1 weakly interacted with Aurora A in the absence of were treated with BI6727 according to Protocol 2 in Fig. 2A without RACK1, whereas pre-binding of PLK1 to RACK1 markedly transfection, and cells were fixed at 4 h after the release. Centrinone increased the Aurora A–PLK1 interaction. Reversing the reaction B was used as a control for suppressing centriole duplication. As sequence (i.e. first Aurora A and then RACK1) did not increase the shown in Fig. 5C, BI6727 treatment decreased the number of centrin interaction of PLK1 with Aurora A. These data suggest that RACK1 foci per cell, as observed previously for RACK1 knockdown functions as a scaffold protein to enhance the Aurora A–PLK1 (Yoshino et al., 2019). These results suggest that the kinase activity interaction. of PLK1 in G1/S and S phase is involved in centriole duplication. RACK1 binding to Aurora A and PLK1 was further investigated by identifying the domains of RACK1 responsible for binding to RACK1 overexpression increases the localization of Aurora A and to the kinase and polo-box domains of PLK1 using phosphorylated PLK1 and Aurora A to centrosomes recombinant maltose binding protein (MBP)-tagged RACK1 To determine the effect of RACK1 overexpression on PLK1 and fragments. Each fragment contained the two indicated WD Aurora A at the centrosome, the localization of p-PLK1, t-PLK1 and domains (Fig. S3A). Aurora A bound strongly to the WD23 and Aurora A to centrosomes was analyzed in RACK1-OE cells. WD34 fragments of RACK1 and weakly to the WD67 fragment RACK1 overexpression increased the fraction of cells with (Fig. S3B). The kinase domain of PLK1 bound to WD23 and centrosomal p-PLK1 signals (Fig. 6A,B), whereas it had no effect weakly to the WD34 and WD67 fragments of RACK1 (Fig. S3C). on the centrosomal localization of t-PLK1 (Fig. 6C,D). Consistent The polo-box domains of PLK1 bound strongly to WD23 and with these results, RACK1 overexpression increased the weakly to WD12, WD34, and WD56 (Fig. S3D). fluorescence intensity of p-PLK1 at centrosomes (Fig. S6A), but not that of t-PLK1 (Fig. S6B). The RACK1 overexpression-induced RACK1 is involved in the localization of Aurora A to the increase in the fraction of cells with centrosomal p-PLK1 signals centrosome and PLK1 phosphorylation to promote centriole was confirmed in another breast cancer cell line, T47D (Fig. S6C,D). duplication in S phase Western blot analysis showed increased p-PLK1 levels in RACK1- To examine the physiological role of RACK1 in PLK1 and Aurora overexpressing HEK-293T cells, whereas no significant changes of t- A activities at the centrosome, we analyzed the effect of RACK1 PLK1 expression were observed (Fig. 6E). Cell cycle synchronization knockdown on the localization of phosphorylated and total PLK1 revealed that the proportion of cells with centrosomal p-PLK1 signals (denoted p-PLK1 and t-PLK1, respectively) and Aurora A to was increased in G1, G1/S and S phases in RACK1-OE cells (Fig. 6F; centrosomes. Western blot analysis confirmed that RACK1 siRNA Fig. S7A). The fraction of cells with a centrosomal Aurora A signal downregulated RACK1 without affecting Aurora A and t-PLK1 was also increased in G1, G1/S and S phases in RACK1-OE cells expression (Fig. S4A). RACK1 knockdown decreased the fraction (Fig. 6G; Fig. S7B). RACK1 overexpression also significantly of cells containing centrosomal p-PLK1 in S phase but not that in increased the fluorescence intensity of Aurora A at centrosomes in G1 or G1/S phase (Fig. 4A,B). RACK1 knockdown also decreased asynchronized cells (Fig. S7C). the fraction of cells with centrosomal p-PLK1 signals in G2 phase, although to a smaller extent than in S phase (Fig. S4B,C). The RACK1 overexpression induces premature centriole fluorescence intensity of p-PLK1 at centrosomes in S phase cells disengagement through the kinase activities of PLK1 and was also decreased significantly (Fig. S4D). The centrosomal Aurora A localization of t-PLK1 was not affected by RACK1 knockdown, The kinase activity of PLK1 is critical for centriole disengagement, which was confirmed by examining the fluorescence intensity of t- and PLK1 overactivation causes premature centriole disengagement PLK1 at centrosomes in S phase (Fig. 4C,D and Fig. S4E). (Loncareǩ et al., 2010; Kong et al., 2014). We therefore examined Consistent with these results, immunoprecipitation followed by centriole disengagement in RACK1-OE cells using C-Nap1 (also western blotting revealed a markedly decreased p-PLK1 level in known as CEP250) as a marker of disengagement in G1, G1/S and S RACK1-knockdown cells under asynchronized and S-phase- phases. C-Nap1 localizes to the basal part of the mother centriole synchronized conditions (Fig. 4E). These results suggest that and disengaged daughter centrioles (Mayor et al., 2000). Engaged RACK1 is involved in the phosphorylation of PLK1 at the centrioles have one C-Nap1 focus, whereas disengaged centrioles centrosome in S phase. have two C-Nap1 foci (Tsou and Stearns, 2006b). Therefore, the RACK1 knockdown also decreased the fraction of cells with presence of more than two C-Nap1 foci in G1/S and S phases centrosomal Aurora A signals in S phase but not in G1 or G1/S indicates premature centriole disengagement (Fig. 7A). phase (Fig. 5A,B). We also observed that the fraction of S phase To visualize centrioles and identify transfected cells, MCF7 cells cells with high fluorescence intensity of centrosomal Aurora A was were co-transfected with the GFP–centrin2 vector and decreased by RACK1 knockdown, although the difference between HA–RACK1. Most control cells contained two C-Nap1 foci at RACK1-knockdown cells and the control cells was not statistically G1, G1/S and S phases (Fig. 7B,C). However, the fraction of cells significant (Fig. S5A). To exclude off-target effects of the siRNA, with more than two C-Nap1 foci increased in G1/S and S phases in the experiments in S phase were repeated using a different siRNA RACK1-OE cells, indicating premature disengagement. In G1 against RACK1, which yielded similar results (Fig. S5B–D). These phase, the fraction of cells with more than two C-Nap1 foci in results suggest that RACK1 is involved in the activation of PLK1 at RACK1-OE cells was small and comparable to that in control cells. the centrosome by promoting the localization of Aurora A to To test whether formation of excess C-Nap1 foci depended on centrosome in S phase. Aurora A or PLK1 kinase activities, cells were treated with We previously reported that knockdown of RACK1 decreases the MLN8054 or BI6727 according to Protocol 4 in Fig. 7D, and the number of centrioles per cell in S and G2 phases (Yoshino et al., number of C-Nap1 foci was counted. Both MLN8054 and BI6727 2019). We hypothesized that the RACK1 knockdown-induced significantly decreased the fraction of cells with more than two decrease in PLK1 activation in S phase was responsible for the C-Nap1 foci in RACK1-OE cells (Fig. 7E,F). These results suggest reduction of centriole number. To determine whether decreased that RACK1 overexpression causes centriole overduplication by

PLK1 activation reduces centriole number in S phase, MCF7 cells promoting premature centriole disengagement in some cells, Journal of Cell Science

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Fig. 4. Knockdown of RACK1 decreases the centrosomal localization of p-PLK1. (A–D) MCF7 cells were transfected with siRNA against RACK1 (RACK1-1), and treated according to Protocol 1 in Fig. 1B. Cells were fixed at the indicated time points and stained with anti-p-PLK1 (A,B) or anti-t-PLK1 (C,D) antibodies. Representative images stained with anti-p-PLK1 or anti-t-PLK1 antibody are shown in A and C, respectively. Scale bars: 10 µm. In B and D, cells positive for p-PLK1 or t-PLK1 were counted and the mean±s.e.m. of three individual experiments is shown. **P<0.01 (two-tailed Welch’s test). (E) Asynchronous or S-phase synchronized MCF7-tet-shRACK1 cells were subjected to immunoprecipitation (IP) using anti-p-PLK1 antibody. Synchronization was performed according to Protocol 1 in Fig. 1B and cells were harvested 4 h after the second release. Doxycycline (DOX) (1.0 ng/ml) was added to the medium at the first release. Band intensity of p-PLK1 was quantified by densitometry and normalized to that of actin, and the normalized values are shown below each band. Western blot shown is representative of at least three experiments. and this effect is mediated by abnormal activation of Aurora A that of wild-type RACK1 (Yoshino et al., 2019). We therefore and PLK1. examined the effect of these RACK1 variants on enhancing the interaction between Aurora A and PLK1. The RACK1 variants Missense variants of RACK1 fail to enhance Aurora A–PLK1 T94A, Y302F and T303M caused a significantly lower level of signaling enhanced interaction between Aurora A and PLK1 compared with We previously reported that several missense variants of RACK1 the effect of wild-type RACK1 in our repeated experiments fail to increase the proportion of cells with centrosome amplification (Fig. 8B; Fig. S8A). To examine whether these variants lose when overexpressed (Fig. 8A; Table S1) (Yoshino et al., 2019). The centrosome regulating activity through a defect in PLK1 centrosomal localization of these RACK1 variants was similar to phosphorylation, T94A and T303M variants were overexpressed Journal of Cell Science

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Fig. 5. Knockdown of RACK1 decreases the centrosomal localization of Aurora A and suppresses centriole duplication. (A,B) MCF7 cells were transfected with siRNA against RACK1 (RACK1-1), and treated according to Protocol 1 in Fig. 1B. Cells were fixed at the indicated time points and stained with anti-Aurora A antibody. (A) Representative images are shown. Scale bars: 10 µm. (B) Cells positive for Aurora A were counted, and the mean±s.e.m. of three individual experiments is shown. (C) MCF7 cells were synchronized to G1/S phase by a double thymidine block according to Protocol 1. Then, cells were released into fresh medium containing 500 nM BI6727 or 500 nM centrinone B and incubated for 4 h. After fixation, cells were stained with anti-centrin and anti-γ-tubulin antibodies. The histogram shows the mean±s.e.m. from three independent experiments in which at least 100 cells were analyzed. *P<0.05, **P<0.01 (two-tailed Welch’s test). in MCF7 cells, and p-PLK1 localization to centrosomes was block. In another breast cancer cell line, Hs578T, centrosome analyzed. Wild-type RACK1 and the K280E variant were used as amplification was observed in more than 30% of RACK1-OE cells controls. The K280E variant decreases binding to BRCA1, at 36 h after transfection (Yoshino et al., 2019), and most cells died localization of BRCA1 to the centrosome, and centrosome within 3 days after transfection. RACK1 overexpression induced amplification induced by its overexpression in cells derived from cleavage of caspases and poly(ADP-ribose) polymerase (PARP) normal mammary epithelium (Yoshino et al., 2019). As shown in (markers of apoptosis) in Hs578T cells (Fig. S8B). Thus, it was Fig. 8C,D, the T94A and T303M variants did not increase the difficult to perform cell cycle synchronization in RACK1-OE fraction of cells with centrosomal p-PLK1 signals, whereas wild- Hs578T cells. By contrast, in MCF7 cells, RACK1 overexpression type RACK1 and the K280E variant significantly increased the caused centrosome amplification in 18% of cells at 72 h after fraction of cells with centrosomal p-PLK1 signals. Consistent with transfection, and a small fraction of cells underwent cell death by these results, western blot analysis confirmed that the T94A and apoptosis (Fig. S8C). RACK1 overexpression increased the fraction T303M variants failed to increase p-PLK1 in HEK-293T cells of cells with centriole overduplication in MCF7 cells as well as in (Fig. 8E). Hs578T cells (Fig. S1). Therefore, we used MCF7 cells for cell cycle synchronization experiments in this study. DISCUSSION An increase in the fraction of cells with centrosome amplification We previously reported that adequate expression levels of RACK1 was observed at 4 and 8 h after the release, indicating that are important for normal centriole duplication in mammary tissue- centrosome overduplication in RACK1-OE cells occurs during S derived cells; RACK1 knockdown suppresses centriole duplication, and G2 phases (Fig. 1D,E). In the following G1 phase at 12 h after whereas its overexpression causes centriole overduplication in a the release, the increase in the fraction of cells with centrosome subset of mammary gland-derived cells (Yoshino et al., 2019). In amplification was not observed (Fig. 1D,E). We presume that most this study, we showed that RACK1 contributes to the regulation of cells with extra centrosomes died during mitosis, possibly by centriole duplication by modulating the Aurora A/PLK1 signaling apoptosis (Fig. S8C). As shown in Fig. 2, the activity of both PLK1 axis (Fig. 8F). and PLK4 was involved in centrosome amplification in RACK1-OE To investigate how RACK1 regulates centriole duplication, cells during G1/S and S phases, whereas Aurora A inhibition before we analyzed centrosome amplification induced by RACK1 G1/S phase was required to suppress centrosome amplification in overexpression in cells synchronized by a double thymidine RACK1-OE cells. These results are consistent with the role of Journal of Cell Science

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Fig. 6. Overexpression of RACK1 increases the centrosomal localization of p-PLK1 and Aurora A. (A) MCF7 cells were co-transfected with control or HA–RACK1 vector, and GFP–centrin2 vector. At 72 h after transfection, cells were fixed and stained with anti-p-PLK1 antibody and DAPI. (B) Quantification of the samples in A. Cells showing GFP–centrin foci with a positive p-PLK1 signal were counted, and the percentage of cells was calculated from more than 100 GFP-centrin-positive cells in each sample. The mean±s.e.m. of three individual experiments is shown. (C) Samples were prepared as in A and stained with anti-t-PLK1 antibody and DAPI. (D) Quantification of the samples in C. The mean±s.e.m. of three individual experiments is shown. (E) HEK-293T cells were transfected with empty vector or Myc–RACK1 expression vector. At 72 h after transfection, cells were lysed and subjected to immunoprecipitation. Band intensity of p-PLK1 was quantified by densitometry and normalized to Actin, and the normalized values are shown below each band. The arrow and arrowhead indicate endogenous RACK1 and Myc–RACK1, respectively. Western blot shown is representative of at least three independent experiments. (F) MCF7 cells were co-transfected with control or HA–RACK1 vector and GFP–centrin2 vector, and treated according to Protocol 1 shown in Fig. 1B. Cells were fixed and stained with anti-p-PLK1 antibody and DAPI. The mean±s.e.m. of three individual experiments is shown. (G) Samples were prepared as in F. Cells were stained with anti-Aurora A antibody. The mean±s.e.m. of three individual experiments is shown. *P<0.05; **P<0.01; n.s., not significant (two-tailed Welch’s test). Scale bars: 10 µm.

Aurora A as an upstream activator of PLK1. Collectively, these Aurora A requires a scaffold protein such as Bora, Furry or results indicated that the Aurora A/PLK1 signaling axis and PLK4 CEP192 to activate PLK1 (Bruinsma et al., 2014; Seki et al., 2008; activity contribute to centrosome amplification induced by RACK1 Joukov et al., 2014; Macurek et al., 2008; Ikeda et al., 2012). The overexpression. Because centriole disengagement mediated by present finding that RACK1 binds to Aurora A and PLK1 suggests PLK1 is followed by the initiation of centriole duplication governed that RACK1 also acts as a scaffold for the Aurora A–PLK1 by PLK4, these results suggest that RACK1 overexpression leads to interaction. This was supported by the finding that RACK1 centrosome amplification associated with PLK1 overactivation enhances the interaction between Aurora A and PLK1 in a dose- mediated by Aurora A in some cells. dependent manner in cells (Fig. 3C; Fig. S2E). Moreover, RACK1 Journal of Cell Science

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Fig. 7. Overexpression of RACK1 induces premature centriole disengagement in some cells. (A) Schematic illustration of C-Nap1 number during the cell cycle and premature centriole disengagement. (B) MCF7 cells were co-transfected with control or HA–RACK1 vector and GFP–centrin2 vector, and treated as indicated in Protocol 1. Cells were stained with anti-C-Nap1 antibody and DAPI. (C) Quantification of the samples in B. The percentage of cells with more than two C-Nap1 foci was calculated from more than 100 GFP-centrin-positive cells in each sample. The mean±s.e.m. of three individual experiments is shown. (D) Protocol 4 for the analysis of the effects of kinase inhibitor treatments on premature disengagement. (E) MCF7 cells were co-transfected with control or HA-RACK1 vector and GFP-centrin2 vector and treated with MLN8054 or BI6727, as indicated in Protocol 4. Cells were stained with anti-C-Nap1 antibody and DAPI. (F) Quantification of the samples shown in E. The mean±s.e.m. of three individual experiments is shown. **P<0.01; n.s., not significant (two-tailed Welch’s test). Scale bars: 10 µm. knockdown decreased the interaction between Aurora A and PLK1 a scaffolding role for the interaction between Aurora A and PLK1. (Fig. S2F). In addition, a pulldown assay showed that Aurora A RACK1 bound to both the kinase domain and polo-box domains of bound to the PLK1–RACK1 complex more efficiently than to PLK1. Because Thr210 is located in the kinase domain, the

PLK1 alone (Fig. 3E). These data support the idea that RACK1 has interaction between RACK1 and the kinase domain of PLK1 may Journal of Cell Science

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Fig. 8. Point mutation of RACK1 affects its activity in the regulation of the Aurora A/PLK1 axis. (A) Diagram of the locations of missense variants of RACK1. (B) HEK-293T cells were co-transfected with FLAG–PLK1, HA–Aurora A and expression vectors for HA-RACK1 variants. At 48 h after transfection, cells were harvested, and cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. Co-precipitated and Input HA–Aurora A were quantified by densitometry after immunoblotting against Aurora A. Quantified values of HA–Aurora A co-precipitated with PLK1 divided by that of Input Aurora A are shown. The mean±s.e.m. of four individual experiments is shown. (C) MCF7 cells were co-transfected with control vector or HA–RACK1 variants and GFP–centrin2 vectors. At 72 h after transfection, cells were fixed and stained with anti-p-PLK1 antibody and DAPI. Scale bar: 10 µm. (D) Quantification of the samples from C. Cells showing GFP–centrin foci with a positive p-PLK1 signal were counted, and the percentage of cells was calculated from more than 100 GFP–centrin- positive cells in each sample. The mean±s.e.m. of three individual experiments is shown. (E) HEK-293T cells were transfected with control, and HA–RACK1-WT, -T94A or -T303M expression vector. At 48 h after transfection cells were lysed and subjected to immunoprecipitation using anti-p-PLK1 antibody. Western blot shown is representative of at least three independent experiments. (F) Schematic of the regulation of the Aurora A/PLK1 signaling axis by RACK1. RACK1 is involved in the centrosomal localization of Aurora A in S phase. RACK1 enhances the interaction between Aurora A and PLK1, and promotes the phosphorylation of PLK1 by Aurora A at the centrosome. p-PLK1 functions in centriole duplication in S phase. RACK1 knockdown decreases the centrosomal localization of Aurora A and inhibits the phosphorylation of PLK1 at centrosomes in S phase, leading to the suppression of centriole duplication. By contrast, RACK1 overexpression increases the centrosomal localization of Aurora A and enhances the phosphorylation of PLK1 at the centrosome. Abnormal activation of PLK1 causes premature centriole disengagement, resulting in centrosome amplification. *P<0.05, **P<0.01, n.s., not significant (Dunnett’s test). Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs238931. doi:10.1242/jcs.238931 contribute to PLK1 phosphorylation, whereas the polo-box domains supported by RACK1 in S phase is required for centriole would contribute to interactions with PLK1 substrates (Zitouni duplication. Activation of PLK1 promotes centriole elongation et al., 2014). This suggests that RACK1 may be phosphorylated by and centriole maturation (Loncareǩ et al., 2010; Kong et al., 2014). PLK1; however, investigating this possibility exceeds the scope of However, the localization of SAS6 to the centrosome is not affected this paper. by PLK1 depletion (Loncareǩ et al., 2010), suggesting that PLK1 is RACK1 knockdown decreased the centrosomal localization of not essential for procentriole formation. By contrast, we showed that p-PLK1 in S phase, but not that of t-PLK1 (Fig. 4), which was knockdown of RACK1 decreases the fraction of cells with confirmed by measuring their fluorescence intensities at centrosomal localization of SAS6 in G2 phase (Yoshino et al., centrosomes (Fig. S4D,E). These results suggest that the 2019), indicating that RACK1 might be required for procentriole physiological activation of PLK1 in S phase is supported by formation. We showed that RACK1 knockdown decreased the RACK1 (Fig. 8F). Overexpression of RACK1 increased the localization of p-PLK1 to centrosomes only in S and G2 phases, and localization of p-PLK1, but not that of t-PLK1, to centrosomes not during the G1/S transition, the period during which procentriole (Fig. 6), indicating that RACK1 overexpression promoted PLK1 formation is initiated (Fig. 4A,B; Fig. S4B–D). Therefore, phosphorylation at the centrosome (Fig. 8F). The increase of RACK1 might be involved in procentriole formation in a centrosomal p-PLK1 was observed in G1, G1/S and S phases. manner that is independent of PLK1 activation. Future studies Increased expression of RACK1 induced PLK1 overactivation in should investigate the involvement of RACK1 and PLK1 in the wrong cell cycle phases, including G1/S and S phases, resulting centriole duplication. in an increase in cells with premature disengagement (Fig. 7). The We previously reported that missense variants of RACK1 affect level of p-PLK1 in cell lysates was decreased upon RACK1 its function in centrosomes. In this study, the T94A, Y302F and knockdown and increased upon RACK1 overexpression. This was T303A variants failed to enhance the interaction between Aurora A consistent with the change in the centrosomal localization and PLK1 (Fig. 8B). In addition, overexpression of the T94A and of p-PLK1. T303M variants did not cause the upregulation of p-PLK1 at the Similar to what was seen for p-PLK1, knockdown of RACK1 centrosome in contrast to that of wild-type RACK1 and the K280E decreased the centrosomal localization of Aurora A in S phase in some variant (Fig. 8C,D). The T94 residue is located in WD3, which cells (Fig. 5). The localization of Aurora A to centrosomes increased comprises RACK1 fragments that bind to Aurora A, and the kinase in G1, G1/S and S phases in some RACK1-OE cells (Fig. 6G). This domain and polo-box domains of PLK1 (Fig. S3). The Y302 and suggests that the recruitment of Aurora A to centrosomes by RACK1 T303 residues are located in WD7. The RACK1 fragment that contributes to the regulation of PLK1 activation at centrosomes includes WD7 showed weak binding to Aurora A and the kinase (Fig. 8F). Although RACK1 overexpression altered the localization of domain of PLK1. Thus, alteration of T94A, Y302F and T303 Aurora A, the effect was smaller than that on the localization of residues may affect the scaffolding activity of the RACK1 protein p-PLK1 to centrosomes (Fig. 6F,G). These results suggest that, in for the Aurora A–PLK1 interaction. These data suggest that addition to increasing the centrosomal localization of Aurora A, the mutations of RACK1 alter centrosome regulating activities in effect of RACK1 on the Aurora A–PLK1 interaction contributes to several distinct ways: proteins with T94A, Y302F and T303M PLK1 activation at the centrosome. mutations fail to regulate PLK1 phosphorylation by Aurora A, and Analysis of the number of C-Nap1 foci indicated that premature that with the K280E mutation fails to properly localize BRCA1 to disengagement occurred in G1/S and S phases more frequently in the centrosome. NetPhos 3.0 (http://www.cbs.dtu.dk/index.html) RACK1-OE cells than in control cells, and PLK1 and Aurora and NetworKIN (http://networkin.info/index.shtml/) predictions A activities were required for the increase in premature identified T94, Y302 and T303 as candidate residues for disengagement. The ratio of C-Nap1 to centrin foci is commonly phosphorylation; therefore, the phosphorylation of these residues used as a marker of disengagement (Tsou and Stearns, 2006b). may affect the activity of RACK1 in enhancing the Aurora A–PLK1 However, because RACK1 overexpression induces centriole interaction. T94A and T303M have been reported in colon overduplication and increases centriole number, the C-Nap1 to cancer (COSMIC database; https://cancer.sanger.ac.uk/cosmic), centrin ratio was not suitable for estimating disengagement status in suggesting that may contribute to carcinogenesis or induce a our experiments. As shown in Fig. 7A, the number of C-Nap1 foci cancer phenotype by dysregulating Aurora A–PLK1 signaling. The was sufficient to indicate premature disengagement from G1/S to S effect of the T97A/T98A, L206F, K245C and Y246F variants on phase. RACK1 overexpression increased the fraction of cells with altering the centrosome regulatory activity of RACK1 remains elevated numbers of C-Nap1 foci, and this effect was antagonized by unclear. In addition to further investigate these variants, future inhibition of PLK1 or Aurora A. These data suggest that RACK1 studies should examine the phosphorylation of these residues and overexpression induces premature disengagement by promoting the responsible kinases, and evaluate the association between the overactivation of the Aurora A/PLK1 signaling axis in some RACK1 variants and carcinogenesis. cells (Fig. 8F). In conclusion, RACK1 was identified as a new scaffold protein for Adequate spatio-temporal activation of PLK1 is regulated by the Aurora A–PLK1 interaction that regulates centriole duplication by several distinct mechanisms. At mitotic entry, Bora provides the activating the Aurora A/PLK1 signaling axis. Overactivation of this scaffold for the interaction between Aurora A and PLK1 required for signaling pathway by RACK1 overexpression may cause centrosome PLK1 activation (Macurek et al., 2008; Seki et al., 2008; Bruinsma amplification. et al., 2014). After the entry into mitosis, PLK1 activation by Aurora A is amplified by the scaffold activity of CEP192 at centrosomes MATERIALS AND METHODS (Meng et al., 2015; Joukov et al., 2014). RACK1 is involved in the Cell lines and culture activation of PLK1 in S phase, thus acting earlier than Bora or MCF7 (ATCC HTB-22), T47D (ATCC HTB-133), and HEK-293T (ATCC CEP192. Inhibition of PLK1 during G1/S to S phase suppressed CRL-3216) cells and Hs578T cells (ATCC HTB-126) were purchased from centriole duplication (Fig. 5F), similar to the effect of RACK1 ATCC. MCF7 and HEK-293T cells were maintained in DMEM knockdown (Yoshino et al., 2019), suggesting that PLK1 activity supplemented with 8% fetal bovine serum (FBS). T47D cells were Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs238931. doi:10.1242/jcs.238931 maintained in RPMI-1640 supplemented with 10 µg/ml recombinant human medium without thymidine for 15 h, and treated with 2 mM thymidine in insulin and 8% FBS. Hs578T cells were maintained in DMEM medium for 24 h. After the second thymidine block, cells were washed with supplemented with 10 µg/ml recombinant human insulin and 8% FBS. PBS, released in fresh medium for the indicated duration, and fixed. All cells were incubated in an atmosphere containing 5% CO2. Cell line In experiments using kinase inhibitors, each chemical was added as identities were verified using the GenomeLab Human STR Primer set indicated in the protocols shown in the respective figures. (Beckman Coulter). Immunocytofluorescence Preparation of MCF7-tet-shRACK1 cells For immunostaining of centrosomes, cells were permeabilized in PBS To produce lentiviral particles, pLKO-tet-shRACK1 was co-transfected containing 0.2% Triton X-100, 1 mM EGTA and 1 mM MgCl2 for 1 min, with psPAX2 and pMD2.G (Addgene plasmids #12260 and #12259, and fixed in methanol for 15 min at −80°C. After washing and blocking, the respectively; deposited by Didier Trono) into HEK-293T cells. At 48 h after cells were incubated with primary antibodies in 1% BSA in PBS overnight. transfection, the supernatant was collected and filtered to remove cell debris. The following antibodies were used: anti-HA (3F10, 1:400, Roche), anti- MCF7 cells were infected by lentivirus in the supernatant and infected cells γ-tubulin (GTU-88, 1:500, Sigma-Aldrich), anti-p-PLK1 (D5H7, 1:200, were selected with puromycin for >7 days. Cell Signaling Technology), polyclonal anti-t-PLK1 (A300-251A, 1:2000, Bethyl Laboratories), polyclonal anti-Aurora A (A300-071A, 1:3000, Kinase inhibitors Bethyl Laboratories), anti-centrin (C7736, 1:1500, Sigma-Aldrich), MLN8054 and BI6727 were purchased from AdooQ Bioscience. polyclonal anti-CEP250/C-Nap1 (14498-1-AP, 1:2000, Proteintech), anti- Centrinone B was purchased from Tocris Bioscience. These inhibitors CP110 (140-195-5, 1:200, Merck), anti-CEP152 (A302-479A, 1:2000, were used at the concentrations of 1 μM for MLN8054, 100 nM for BI6727 Bethyl Laboratories) and anti-SAS6 (91.390.21, 1:200, SantaCruz and 500 nM for centrinone B. Biotechnology) antibodies. After incubation with primary antibodies, cells were washed with PBS containing 0.1% Tween-20 (PBS-T), and Transfection incubated with 1% BSA in PBS containing Alexa Fluor 488- and 568- Polyethylenimine MAX (Polysciences) was used for plasmid transfection. conjugated secondary antibodies (Thermo Fisher Scientific) for 30 min. After The TransIT-X2 Dynamic Delivery System (Mirus Bio) was used for washing with PBS-T, cells were mounted in mounting medium with DAPI siRNA transfection and siRNA and plasmid co-transfection. (Vector Laboratories). Immunocytofluorescence images were acquired with the BZ-X710 fluorescent microscope system (Keyence) equipped with a CFI Plasmid construction Plan Apo λ 100×H/1.45 NA oil immersion objective (Nikon) and The plasmids pCY4B-FLAG-RACK1, pCY4B-HA-RACK1, pCMV-Myc- monochrome cooled CCD camera (2/3 inches, 2.83 megapixels). RACK1 and pET-RACK1 were described previously (Yoshino et al., 2019). pCY4B-HA-RACK1 missense variants were generated by site-directed Western blotting mutagenesis (Yoshino et al., 2019). To construct pCY4B-FLAG- and Cells were lysed in 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% pCY4B-HA-PLK1, the coding sequences of PLK1 were amplified using SDS, 10% sucrose, and 5% 2-mercaptoethanol) supplemented with a primers containing XhoI and NotI sites using HeLa cDNA as the template. protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail The amplified PCR products were subcloned into the XhoI/NotI sites (5 mM sodium fluoride, 200 µM sodium orthovanadate, 1 mM sodium of the pCY4B-FLAG and pCY4B-HA vectors (Okano et al., 2000). The molybdate, 2 mM sodium pyrophosphate, and 2 mM disodium β- construction of pCY4B-FLAG- and pCY4B-HA-Aurora A was as described glycerophosphate as final concentrations), yielding whole-cell lysates. for PLK1 plasmids. To construct pET-Aurora A, the coding sequence of SDS-PAGE and western blotting were performed as previously described Aurora A was subcloned from pCY4B-HA-Aurora A into the XhoI/NotI (Yoshino and Ishioka, 2015). Primary antibodies were as follows: anti- sites of the pET-44a(+) plasmid (Merck). To construct pLKO-tet- FLAG (M2, 1:5000, Sigma-Aldrich), anti-HA (3F10, 1:3000, or 16B12, shRACK1-puro, annealed oligonucleotides (5′-CTAGCCAGGGATGAG- 1:3000, BioLegend, San Diego, CA, USA), polyclonal anti-t-PLK1 (A300- ACCAACTATGCTCGAGCATAGTTGGTCTCATCCCTGGTTTTTG-3′ 251A, 1:10,000), monoclonal anti-PLK1 (E-2, 1:1000, Santa Cruz and 5′-AATTCAAAAACCAGGGATGAGACCAACTATGCTCGAGC- Biotechnology), anti-p-PLK1 (D5H7, 1:3000), polyclonal anti-Aurora A ATAGTTGGTCTCATCCCTGG-3′) were subcloned into the NheI/Eco- (A300-071A, 1:10,000), anti-γ-actin (2F3, 1: 5000, Wako), anti-caspase 9 RI sites of EZ-Tet-pLKO-puro [Addgene plasmid #85966; deposited by (#9502, 1:1000, Cell Signaling Technology), anti-caspase 3 (#9662, 1:1000, Cindy Miranti (Frank et al., 2017)]. Cell Signaling Technology) and anti-PARP (#9532, 1:1000, Cell Signaling Technology) antibodies. Secondary antibodies were as follows: horseradish siRNA peroxidase (HRP)-tagged anti-mouse-IgG (GE Healthcare), HRP-tagged The sequences of the siRNAs designated as RACK1-1 and RACK1-2 were anti-rabbit-IgG (GE Healthcare) and HRP-tagged anti-mouse native form IgG 5′-CAGGCUAUCUGAACACGGU-3′ (Silencer™ Select siRNA, Thermo (TrueBlot, Rockland). Signals were detected using an ECL substrate (ATTO) Fisher Scientific) (Yoshino et al., 2019) and 5′-GAGGUUGUGGUGCU- on a CCD imager (Image Quant LAS 4000 mini, GE Healthcare). AGUUUCUCUdAdA-3′ (DsiRNA, Integrated DNA Technologies). The Silencer™ negative control siRNA template set (Thermo Fisher Scientific) Immunoprecipitation was used as the negative control. Cells were lysed in lysis buffer (10 mM HEPES pH 7.6, 250 mM NaCl, 0.5% NP-40, 5% glycerol, and 5 mM EDTA) supplemented with protease Cell cycle analysis inhibitor and phosphatase inhibitor cocktails, and then cleared by Cell cycle progression was analyzed by flow cytometry as described centrifugation at 5000 g for 10 min. The lysate was rotated with anti- previously (Yoshino and Ishioka, 2015). In brief, cells were trypsinized, FLAG antibody (2H8, Trans Genic) or anti-p-PLK1 antibody (K50-483, fixed with ethanol at 4°C and stored at −20°C until analysis. The fixed cells BD Pharmingen) at 4°C overnight. The lysate was rotated for another hour were suspended in phosphate-buffered saline (PBS) containing 0.1% Triton with Protein G–Sepharose (GE Healthcare). After vigorous washing, X-100, 200 µg/ml RNase A (Thermo Fisher Scientific) and 50 µg/ml proteins were eluted in 1× sample buffer and analyzed by western blotting. propidium iodide, and analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter). Pulldown assay His-tagged proteins were expressed in BL21-competent cells. The bacterial Cell cycle synchronization cell pellets were lysed with extraction buffer (50 mM phosphate buffer pH Cells were synchronized in G1/S transition using a double thymidine block 8.0, 300 mM NaCl, and 0.1% NP-40). His-tagged proteins were purified protocol (Ma and Poon, 2011) with minor modifications. In brief, cells were from the lysate using Ni-NTA-agarose (Qiagen). MBP-tagged proteins were incubated for 6 h after transfection and treated with 2 mM thymidine in obtained using the pMAL Protein Fusion and Purification System (New complete medium for 24 h. Cells were washed with PBS, released in fresh England Biolabs) according to the manufacturer’s instructions. Journal of Cell Science

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For the GST pulldown assay, GST-tagged proteins were expressed in synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary BL21 cells, and bacterial cell pellets were lysed in sonication buffer (20 mM cells. J. Cell Biol. 130, 105-115. doi:10.1083/jcb.130.1.105 Tris-OAc pH 7.9, 120 mM potassium acetate, 10% glycerol, 1% Triton X- Bettencourt-Dias, M., Rodrigues-Martins, A., Carpenter, L., Riparbelli, M., Lehmann, L., Gatt, M. K., Carmo, N., Balloux, F., Callaini, G. and Glover, D. M. 100, 1 mM EDTA and 1 mM DTT). After centrifugation at 12,000 g for (2005). SAK/PLK4 is required for centriole duplication and flagella development. 30 min, the supernatant was incubated with glutathione–Sepharose beads Curr. Biol. 15, 2199-2207. doi:10.1016/j.cub.2005.11.042 (GE Healthcare) at 4°C for 1 h. After vigorous washing, the beads were Bruinsma, W., Macurek, L., Freire, R., Lindqvist, A. and Medema, R. H. (2014). incubated with the purified His-tagged proteins in pulldown incubation Bora and Aurora-A continue to activate Plk1 in mitosis. J. Cell Sci. 127, 801-811. doi:10.1242/jcs.137216 buffer (10 mM HEPES pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM CaCl ) at 4°C or at room temperature for 1–4 h. The beads were washed and Chan, J. Y. (2011). A clinical overview of centrosome amplification in human 2 cancers. Int. J. Biol. Sci. 7, 1122-1144. doi:10.7150/ijbs.7.1122 analyzed by western blotting. Conduit, P. T., Wainman, A. and Raff, J. W. (2015). Centrosome function and For the MBP pulldown assay, purified MBP-tagged proteins were assembly in animal cells. Nat. Rev. Mol. Cell Biol. 16, 611-624. doi:10.1038/ incubated with amylose resin in column buffer (20 mM Tris-HCl pH 7.5, nrm4062 200 mM NaCl, 1 mM EDTA, 1 mM DTT) at 4°C for 1 h. After washing the Cosenza, M. R., Cazzola, A., Rossberg, A., Schieber, N. L., Konotop, G., resin, the indicated prey proteins were added and incubated in pulldown Bausch, E., Slynko, A., Holland-Letz, T., Raab, M. S., Dubash, T. et al. (2017). Asymmetric centriole numbers at spindle poles cause chromosome incubation buffer at room temperature for 1 h. After vigorous washing, the missegregation in cancer. Cell Rep. 20, 1906-1920. doi:10.1016/j.celrep.2017. beads were analyzed by western blotting. 08.005 Denu, R. A., Zasadil, L. M., Kanugh, C., Laffin, J., Weaver, B. A. and Burkard, Image processing and measurement of fluorescence intensity M. E. (2016). Centrosome amplification induces high grade features and is ImageJ v1.49 (http://imagej.nih.gov/ij/) was used for image analysis. For prognostic of worse outcomes in breast cancer. BMC Cancer 16, 47. doi:10.1186/ s12885-016-2083-x analysis of fluorescence intensity at centrosomes, integrated signal intensity Frank, S. B., Schulz, V. V. and Miranti, C. K. (2017). A streamlined method for the (ID) was measured in a fixed-size area around each centrosome. For analysis design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector. of p-PLK1 and t-Aurora A, the ID was used as the fluorescence intensity at BMC Biotechnol. 17, 24. doi:10.1186/s12896-017-0341-x centrosomes. For the analysis of t-PLK1, the adjusted fluorescent intensity Fujita, H., Yoshino, Y. and Chiba, N. (2016). Regulation of the centrosome cycle. was calculated by subtracting the ID of the background at cytoplasm from Mol. Cell. Oncol. 3, e1075643. doi:10.1080/23723556.2015.1075643 the ID at the centrosome. Godinho, S. A., Picone, R., Burute, M., Dagher, R., Su, Y., Leung, C. T., Polyak, K., Brugge, J. S., Théry, M. and Pellman, D. (2014). Oncogene-like induction of cellular invasion from centrosome amplification. Nature. 510, 167-171. doi:10. Statistical analysis 1038/nature13277 Statistical analysis was performed using JMP 12 software (SAS Institute Ikeda, M., Chiba, S., Ohashi, K. and Mizuno, K. (2012). Furry protein promotes Inc). Graphs were constructed using Excel 2016 (Microsoft). Statistical aurora A-mediated Polo-like kinase 1 activation. J. Biol. Chem. 287, 27670-27681. doi:10.1074/jbc.M112.378968 comparisons between two different samples were made using a two-tailed Joukov, V., Walter, J. C. and De Nicolo, A. (2014). The Cep192-organized aurora Welch’s test. Comparisons between more than two samples were made by A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly. ANOVA. When the result of ANOVA indicated a significant difference, Mol. Cell. 55, 578-591. doi:10.1016/j.molcel.2014.06.016 post-hoc comparisons were made using a Dunnett’s test to calculate P- Kais, Z., Chiba, N., Ishioka, C. and Parvin, J. D. (2012). Functional differences values. The P-value was calculated using Wilcoxon rank sum test to among BRCA1 missense mutations in the control of centrosome duplication. compare intensity data. P<0.05 was considered significant. Oncogene. 31, 799-804. doi:10.1038/onc.2011.271 Kim, T.-S., Park, J.-E., Shukla, A., Choi, S., Murugan, R. N., Lee, J. H., Ahn, M., Rhee, K., Bang, J. K., Kim, B. Y. et al. (2013). Hierarchical recruitment of Plk4 Acknowledgements and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and We thank Dr Akira Yasui and Dr Ryutaro Shirakawa, Institute of Aging, Development, Cep152. Proc. Natl. Acad. Sci. USA 110, E4849-E4857. doi:10.1073/pnas. and Cancer, Tohoku University, for useful suggestions and discussion, and 1319656110 Satoko Kaneko for technical assistance. Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Habedanck, R., Stierhof, Y.-D. and Nigg, E. A. (2007). Plk4-induced centriole biogenesis in human cells. Dev. Competing interests Cell 13, 190-202. doi:10.1016/j.devcel.2007.07.002 The authors declare no competing or financial interests. Ko, M. J., Murata, K., Hwang, D.-S. and Parvin, J. D. (2006). Inhibition of BRCA1 in breast cell lines causes the centrosome duplication cycle to be disconnected from Author contributions the cell cycle. Oncogene 25, 298-303. doi:10.1038/sj.onc.1209028 Conceptualization: Y.Y.; Methodology: Y.Y., A.K.; Validation: Y.Y., A.K.; Formal Kong, D., Farmer, V., Shukla, A., James, J., Gruskin, R., Kiriyama, S. and analysis: Y.Y., A.K.; Investigation: Y.Y., A.K., H.Q., S.E., Z.F., K.S., R.K.; Data Loncarek, J. (2014). Centriole maturation requires regulated Plk1 activity during curation: Y.Y., A.K., N.C.; Writing - original draft: Y.Y.; Writing - review & editing: Y.Y., two consecutive cell cycles. J. Cell Biol. 206, 855-865. doi:10.1083/jcb. N.C.; Supervision: N.C.; Project administration: N.C.; Funding acquisition: Y.Y., N.C. 201407087 Levine, M. S., Bakker, B., Boeckx, B., Moyett, J., Lu, J., Vitre, B., Spierings, D. C., Lansdorp, P. M., Cleveland, D. W., Lambrechts, D. et al. (2017). Funding Centrosome amplification is sufficient to promote spontaneous tumorigenesis in This study was supported by grants-in-aid from the Japan Society for the Promotion mammals. Dev. Cell 40, 313-322.e5. doi:10.1016/j.devcel.2016.12.022 of Science (JSPS) KAKENHI grant numbers JP16K18409 (to Y.Y.), JP18K15233 (to Lončarek, J., Hergert, P. and Khodjakov, A. (2010). Centriole reduplication during Y.Y.), JP16H04690 (to N.C. and Y.Y.), JP19H03493 (to N.C. and Y.Y.), the prolonged interphase requires procentriole maturation governed by plk1. Curr. Foundation for Promotion of Cancer Research in Japan, the Sasakawa Scientific Biol. 20, 1277-1282. doi:10.1016/j.cub.2010.05.050 Research Grant from The Japan Science Society (to Y.Y.), Friends of Leukemia Ma, H. T. and Poon, R. Y. C. (2011). Synchronization of HeLa cells. Methods Mol. Research Fund, Research Grant of the Princess Takamatsu Cancer Research Biol. 761, 151-161. doi:10.1007/978-1-61779-182-6_10 Fund, and Research Program of the Smart-Aging Research Center, Tohoku Macurek, L., Lindqvist, A., Lim, D., Lampson, M. A., Klompmaker, R., Freire, R., University (to N.C.). Clouin, C., Taylor, S. S., Yaffe, M. B. and Medema, R. H. (2008). Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature. 455, Supplementary information 119-123. doi:10.1038/nature07185 Manfredi, M. G., Ecsedy, J. A., Meetze, K. A., Balani, S. K., Burenkova, O., Chen, Supplementary information available online at W., Galvin, K. M., Hoar, K. M., Huck, J. J., LeRoy, P. J. et al. (2007). Antitumor https://jcs.biologists.org/lookup/doi/10.1242/jcs.238931.supplemental activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc. Natl. Acad. Sci. 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