Cep68 can be regulated by Nek2 and SCF complex.

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ARTICLE IN PRESS European Journal of Cell Biology xxx (2015) xxx–xxx

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Cep68 can be regulated by Nek2 and SCF complex Xiaohui Man a,b , Timothy L. Megraw b , Yoon Pin Lim a,c,d,∗ a

Cancer Science Institute, National University of Singapore, Singapore 117456, Singapore Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA c Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore d Bioinformatics Institute, Agency for Science, Technology and Research, Singapore b

a r t i c l e

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Article history: Received 24 November 2014 Received in revised form 28 January 2015 Accepted 28 January 2015 Keywords: Cep68 Nek2 Centrosome separation SCF Cell cycle

a b s t r a c t Centrosome cohesion maintains centrosomes in close proximity until mitosis, when cell cycle-dependent regulatory signaling events dissolve cohesion and promote centrosome separation in preparation for bipolar spindle assembly at mitosis. Cohesion is regulated by the antagonistic activities of the mitotic NIMA-related kinase 2 (Nek2), protein phosphatase 1, the cohesion fiber components rootletin, centrosomal Nek2-associated protein 1 (C-Nap1) and Cep68. The centrosomal protein Cep68 is essential for centrosome cohesion and dissociates from centrosomes at the onset of mitosis. Here, our cell line studies show the C-terminal 300–400 amino acids of Cep68 are necessary to localize Cep68 to interphase centrosomes while C-terminal 400–500 amino acids might regulate Cep68 dissociation from centrosomes at mitotic onset. In addition, Nek2 was demonstrated to phosphorylate Cep68 in vivo and this phosphorylation appears to promote Cep68 degradation in mitosis. We further show that the SCF complex destroys Cep68 at mitosis through recognition by the beta-Trcp F box component of SCF. Together, the findings provide a new insight into the control of centrosome separation by Cep68 during mitosis. © 2015 Elsevier GmbH. All rights reserved.

Introduction The centrosome is the major microtubule-organizing center (MTOC) of animal cells and is involved in essential processes such as organelle transport, cell shape, cell motility, cell polarity, and cell division. Centrosomes also play an important role in maintaining the fidelity of chromosome segregation, thereby assuring genomic stability during cell division (Fukasawa, 2007). Centrosome separation at the onset of mitosis is important to the formation of bipolar spindle. The loss of cohesion initiates this separation while failure to complete this separation/cleavage results in monopolar spindles (Meraldi and Nigg, 2001). The centriole pair that is inherited by each daughter cell following cell division maintains close proximity throughout most of the cell cycle. It is suggested after the metaphase–anaphase transition, the centriole pair first separates, a process known as disengagement (Sluder, 2013; Tsou et al., 2009). Each centriole then fosters assembly of a new daughter centriole during the ensuing cell cycle,

∗ Corresponding author at: Department of Biochemistry, Yong Loo Lin School of Medicine, 8 Medical Drive, Block MD4A, #02-05, Singapore 117597, Singapore. Tel.: +65 66011891. E-mail address: [email protected] (Y.P. Lim).

all the while maintaining cohesion between the two “mothers” during the growth of each new daughter at the proximal base of each mother centriole. In late G2 phase, centrosome maturation occurs by recruiting extra pericentriolar proteins. Throughout these steps, the mother centrioles remain tethered by cohesion fibers. As cells enter mitosis, centrosome separation occurs. The separated centrosomes then drive microtubule assembly into a bipolar spindle apparatus at mitosis. Centrosome cohesion and separation are tightly regulated during the cell cycle (Nigg and Raff, 2009). “Centrosome separation” describes the cell cycle-regulated separation of centrosomes at the onset of mitosis (Mayor et al., 2000). Centrosomes are connected through a proteinaceous structure called the centrosomal linker between the two parental centrioles, a fibrous interconnection of filaments comprised of the structural component rootletin that connects to mother centriole proximal ends by C-Nap1 (Bahe et al., 2005; Fry et al., 1998). Cell-cycle-dependent phosphorylation is required to regulate centrosome separation (Meraldi and Nigg, 2001). C-Nap1 and rootletin are the major structural components of the linker that keeps centrioles together during interphase. Rootletin assembles into filaments that bind to C-Nap1, which localizes to the proximal ends of centrioles and binds directly to rootletin, anchoring the interconnecting fibers (Bahe et al., 2005; Yang et al., 2006).

http://dx.doi.org/10.1016/j.ejcb.2015.01.004 0171-9335/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Man, X., et al., Cep68 can be regulated by Nek2 and SCF complex. Eur. J. Cell Biol. (2015), http://dx.doi.org/10.1016/j.ejcb.2015.01.004


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Depletion of C-Nap1 or rootletin results in centrosome splitting. Nek2 phosphorylates C-Nap1 and rootletin, triggering their release from the centrosome at the onset of mitosis and initiating centrosome separation (Bahe et al., 2005; Fry et al., 1998; Mi et al., 2007; Yang et al., 2006). Recent evidence suggests that ␤-catenin also localizes in the intercentrosomal linker region of interphase centrosomes and is involved in centrosome separation. Stabilized ␤-catenin decreased centrosome cohesion and increased intercentrosome distance in interphase cells, while loss of ␤-catenin increased cohesion in mitosis (Bahmanyar et al., 2008). ␤-Catenin has been reported to be phosphorylated by Nek2 to regulate centrosome separation (Bahmanyar et al., 2008). The current model for centrosome cohesion therefore proposes that phosphorylation of substrates, including C-Nap1, rootletin and ␤-catenin by Nek2 in late G2 leads to the breakdown of the intercentriolar linkage that holds centrosomes together and promote subsequent centrosome separation. Increasing evidence shows ubiquitin-dependent proteolysis of centrosomal proteins playing important roles in the regulation of centrosome structure and function (Castro et al., 2002; Crasta et al., 2006; Guderian et al., 2010; Kasbek et al., 2010; Kitagawa et al., 2011; Mori et al., 2007; Song and Lim, 2004; Strnad et al., 2007; Wu et al., 2007). Specificity in proteolysis by the Ub-proteasome system is controlled by the action of Ub-protein ligases (Ubls), multisubunit complexes that recognize “degrons” in substrate proteins and bring them to the ubiquitination machine (Pray et al., 2002). SCF (SKP1–CUL1–F-box protein) and APC/C (Anaphase Promoting Complex/cyclosome) are two well-characterized mammalian cullin RING ubiquitin ligases. Most SCF substrates are recognized and bound by the F box protein subunit only when they are post-translationally modified by phosphorylation at specific sites (Ang and Wade Harper, 2005), and the F box protein provides the substrate targeting specificity of the complex. The APC/C is structurally similar to the SCF complex, and consists of invariable core components – APC11 (RBX1-related RING-finger protein), APC2 (CUL1-related scaffold protein) and at least 11 other components without a defined role and a variable component known as an activator. There are two such variable components in mitotically cycling cells, cell division cycle 20 (CDC20) and CDH1, and they confer substrate specificity in the same way that F-box proteins do in the SCF complex (Irniger et al., 2000; Pray et al., 2002; Vodermaier, 2004). Previous studies also demonstrated that the proteasome is located at centrosomes (Freed et al., 1999; Fukasawa, 2007; Wojcik et al., 2000). The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus (Freed et al., 1999). In addition to SCF complex, APC/C is also present on centrosomes and spindle microtubules. It is suggested that during mitosis, APC/C may be locally activated at centrosomes by CDK1 and PLK1 (both enriched there), and APC/C substrates may have to localize to this site in order for them to be ubiquitinated (Castro et al., 2005). SCF and APC/C target many proteins that regulate centrosome separation for proteolysis (Vodermaier, 2004). Cep68 was first identified as a centrosomal protein in a proteomics study of human centrosomes (Andersen et al., 2003). The Cep68 gene is located on chromosome 2p14 and encodes at least two products, a long and a short isoforms. Recent evidence suggests that Cep68 may also be an essential component of the intercentrosomal linker structure (Graser et al., 2007). Cep68 decorated fibers emanating from the proximal ends of parental centrioles (Graser et al., 2007). Depletion of Cep68 caused a loss of rootletin from centrioles and vice versa, and depletion of C-Nap1 caused a loss of Cep68. Like C-Nap1 and rootletin (Bahe et al., 2005; Graser et al., 2007; Yang et al., 2006), Cep68 was displaced from centrosomes at the onset of mitosis and absent from mitotic spindle poles,

consistent with the notion that a linker structure needs to be dismantled for centrosome separation at the onset of mitosis (Graser et al., 2007). Fang et al. (2014) further showed Cep68 is a novel substrate of Nek2 and form a complex with C-Nap1 and centlein to maintain centrosome cohesion. In this study we show phosphorylation of Cep68 by Nek2 creates a potential phosphodegron that directs Cep68 destruction through the activity of SCF-beta-Trcp. We propose that such a mechanism promotes the loss of Cep68 from the centrosome and subsequent centrosome separation. Materials and methods DNA constructs Polymerase chain reaction was used to amplify full-length human Cep68 from KIAA0582 clone (Kazusa DNA Research Institute, Kisarazu, Japan). The Cep68 cDNA was then subcloned into pcDNA-cmv-tag2a (Stratagene) and pAcGFP (Clontech) vectors, respectively. pAcGFP-Cep68 construct was used to yield the deletion mutants of Cep68. All constructs were confirmed by sequencing. Nek2 and Plk1 cDNA were amplified from HeLa cell cDNA and inserted into pDsRed-C2 (Stratagene). The construct pDsRed-C2-K37R (inactive Nek2 kinase) was produced using site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing. beta-Trcp cDNA was amplified from HeLa cell cDNA and cloned into pcDNA3.1-FLAG and pcDNA3.1-Myc. Aurora A and Aurora B plasmids were the kind gifts of Dr Shen Kiat Lim (Cancer Science Institute, Singapore). Myc-tagged Ubiquitin was a gift from Mr Kah Yap Tan (Cancer Science Institute, Singapore). Cep68 antibody Immunized Cep68 serum (Ni-Cep68) was kindly provided by Professor Erich A. Nigg (Graser et al., 2007). Cell culture and treatment HeLa and HEK293 Cells were obtained from ATCC and grown at 37 ◦ C under 5% CO2 . DMEM medium was used, supplemented with 10% FBS and penicillin-streptomycin (100 IU/ml and 100 ␮g/ml, respectively). HeLa cells were treated where indicated with 2 mM thymidine (Sigma), 1 ␮g/ml aphidicolin (Sigma), 1 ␮M nocodazole (Sigma) or 20 ␮M MG132 (Sigma). For synchronization at G1/S, dishes with 20% confluent cells were treated for 14 h with thymidine, washed three times with PBS, re-incubated with normal medium for 9 h and, finally, treated for 14 h with aphidicolin. For synchronization in mitosis, cells were released for 10 h from the G1/S block, treated with nocodazole for 4.5 h and collected by mechanical shake off. Alternatively, mitotic cells were collected by mechanical shake off after 16 h of nocodazole treatment. For transfection study, HeLa or HEK293 cells were transfected using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were analyzed 48 h after transfection. Immunofluorescence and immunoblotting Immunofluorescence procedure was as described (Bahe et al., 2005). Briefly, coverslips were removed from culture dish and washed in PBS. −20 ◦ C methanol was added and the cells fixed at −20 ◦ C for 10 min. Following removal of methanol, the coverslips were washed in PBS and blocked for 1 h with 3% BSA followed by primary antibody incubation for 1 h at room temperature. Following washing for 3 min × 5 min with PBS, secondary antibody was applied for 1 h at room temperature. Cells were washed again for 3 min × 5 min with PBS before the coverslips were mounted



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on slides with Prolong Gold Antifade reagent with DAPI (Invitrogen). The primary antibodies used were: anti-␥-tubulin mouse monoclonal antibody (1:1000, GTU-88, Sigma), anti-Cep68 serum Ni-Cep68 (1:1000), and anti-GFP polyclonal antibody (1:1000, Clontech). Secondary antibodies used were either Alexa Fluor 488 or 568-conjugated donkey IgGs (1:2000, Invitrogen). Immunofluorescence microscopy was performed using a Zeiss Axioplan II microscope (Carl Zeiss, Jena, Germany) equipped with an Apochromat 100 × oil immersion objective, and images were acquired using a Micromax charge coupled device (CCD) camera (model CCD1300-Y; Princeton Instruments) and MetaView software (Visitron Systems). Immunoblotting experiments were performed as described previously (Chen et al., 2007). Briefly, membranes were blocked with 1% BSA in PBS before incubating with primary antibodies for 1 h at room temperature. Following washing with PBS + 1% Tween 20 for 3 min × 5 min, secondary antibodies were incubated for another 1 h at room temperature. After washing 3 min × 10 min, blots were developed with Enhance Chemiluniscence (ECL) from GE Biosciences. Primary antibodies were used at the following concentrations: Ni-Cep68 (1:5000), mouse monoclonal anti-␣ tubulin antibody (1:5000, Abcam), mouse monoclonal anti-FLAG antibody (1:5000, Sigma), rabbit polyclonal anti-cyclinB1 antibody (1:1000, Santa Cruz), rabbit polyclonal anti-cyclinA antibody (1:1000, Santa Cruz), rabbit polyclonal antiGFP antibody (1:5000, Clontech), rabbit polyclonal anti-Cdc25c antibody (1:1000, Santa Cruz), mouse monoclonal anti-cullin-1 antibody (1:1000, Santa Cruz), goat polyclonal anti-DsRed antibody (1:1000, Santa Cruz), rabbit polyclonal anti-Nek2 antibody (1:1000, Santa Cruz) and mouse monoclonal anti-Myc antibody 9E10 (1:1000, Santa Cruz). Secondary antibodies were HRP-conjugated goat anti-rabbit (1:30,000, Sigma) or anti-mouse (1:30,000, Sigma) IgGs.

Co-immunoprecipitation Transfected HEK293 Cells were removed from culture dishes and collected by centrifugation (300 × g). They were then resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP40 and 2 mM EDTA) supplemented with 2 ␮g/ml Aprotinin, 50 mM NaF, 1 × protease inhibitor cocktail (Sigma), 1 mM orthovanadate, 1 mM PMSF, 0.1 mM benzamidine-HCl, and 1 ␮g/ml phenanthroline and incubated on an orbital shaker at 4 ◦ C for 1 h. The lysates were transferred to 1.5 mL eppendorf tubes and centrifuged at 20,000 × g for 30 min. The lysates were pre-cleared using mouse IgG agarose conjugate beads on an orbital shaker at 4 ◦ C for 3 h and then centrifuged for 5 min at 1000 × g. Lysates were then transferred to 50 ␮L of anti-FLAG M2-agarose beads (Sigma) equilibrated in lysis buffer and rotated on an orbital shaker at 4 ◦ C for 3 h. The anti-FLAG M2-agarose beads were collected via centrifugation and washed three times by resuspension and recentrifugation in lysis buffer. The agarose was then subjected to a final wash in 1 × TE buffer, pH 7.4 plus protease inhibitors. 50 ␮L of 2 × SDS-PAGE loading buffer (117 mM Tris-HCl pH 6.8, 10% glycerol, 3% SDS, 2% ␤-mercaptoethanol and 0.2% bromophenol blue) were added into beads and boiled at 95 for 10 min, then centrifuged at 2500 × g for 2 min to remove agarose beads and 25 ␮L of each supernatant was loaded on duplicate gels. 30 ␮L of whole cells lysate was mixed with 30 ␮L of 2 × SDS-PAGE loading buffer mixed, then divided in half and loaded in duplicate gels. Proteins were resolved on duplicate 7% SDS-PAGE gel for 1 h at 180 V, constant voltage. Immunoblotting was carried out as above described. Antibodies and IgGs used for immunoprecipitations were: mouse monoclonal anti-FLAG antibody agarose conjugate (Sigma), rabbit polyclonal anti-GFP antibody (Clontech), mouse monoclonal anti-FLAG

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antibody (Sigma), mouse monoclonal anti-myc antibody 9E10 (Santa Cruz). siRNA experiments beta-Trcp (5 -GTGGAATTTGTGGAACATC-3 ), Nek2 siRNA(5 GGAGGGGAUCUGGCUAGUG-3 ) and Cullin-1(5 -AATAGACATTGGGTTCGCCGT-3 ) were purchased from Sigma. Luciferase siRNA (5 -CGUACGCGGAAUACUUCGA-3 ) (Invitrogen) served as negative control. Lipofectamine 2000 (Invitrogen) was used for the transfection following the manufacturer’s instructions. For the knockdown of Cullin-1 and Nek2, HeLa cells were transfected with a final concentration of 5 nM of each siRNA. For beta-Trcp1 and beta-Trcp2 knockdown, HeLa cells were cotransfected with 2 ␮g of FLAG-Trcp1 or FLAG-Trcp2 and a final concentration of 5 nM of beta-Trcp siRNA per 35 mm plate. The cells were incubated for 24 h and then serumfree medium was replaced with complete medium including 10% FBS. Seventy-two hr after siRNA transfection, cells were harvested for immunoblotting analysis to confirm knockdown of target protein. In vivo ubiquitination HeLa cells were cotransfected with ubiquitin-Myc expressing plasmid and GFP-Cep68 and incubated for 48 h. Nocodazole (1 ␮M) was added for last 18 h and MG132 (20 ␮M) was added for last 6 h. Cells was lysed with 100 ␮l cell lysis buffer (2% SDS, 500 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 × protease inhibitors cocktail (Sigma)) per 6 cm dish. Cells were collected with a cell scraper and transferred to a 1.5 mL eppendorf tube followed by boiling for 10 min at 95 ◦ C. The cell lysates were sheared with a sonication device. 900 ␮l of dilution buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% Triton) were added into samples. Samples were then incubated at 4 ◦ C for 30–60 min with rotation and centrifuged at 20,000 × g for 30 min. The resulting supernatant was transferred to a new eppendorf tube. The cleared lysates were used for immunoprecipitation with antiMyc antibody. The beads were washed extensively using washing buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM LiCL) before the bound proteins were eluted and analyzed by immunoblotting. Phosphatase treatment HeLa cells transiently transfected with indicated plasmid constructs were lysed with lysis buffer as above described, however sodium fluoride (NaF, 50 mM) and sodium vanadate (1 mM) were omitted. Cell lysates were left untreated or treated with 1 unit of calf intestinal phosphatase (CIP, New England Biolabs) per gram of cell lysates. Reaction was performed in the reaction buffer provided for 1 h at 37 ◦ C and stopped on addition of 2 × SDS-PAGE buffer. Results Cep68 associates with interphase centrosome via its amino acids 300–400 Cep68’s association with interphase centrosome has been reported previously (Graser et al., 2007). To assess the region of Cep68 that is important for targeting it to the interphase centrosome, we transiently expressed a series of GFP-Cep68 deletion mutants lacking C-terminal acids in HeLa cells (Fig. 1A). Wild-type GFP-Cep68 localized to interphase centrosomes, as did the deletion mutants GFP-400 (1–400 amino acids of Cep68), GFP-500 (1–500 amino acids of Cep68), and GFP-600 (1–600 amino acids of Cep68). In contrast, the deletion mutants GFP-300 (1–300 amino acids of Cep68), GFP-200 (1–200 amino acids of Cep68) and GFP-100 (1–100



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Fig. 1. Cep68 localizes in interphase centrosome through N-terminal 300–400 amino acids. (A) Diagrammatic representation of the Cep68 deletion mutants. (B) GFP tagged Cep68 wild type and deletion mutants were transfected into HeLa cells and ␥-tubulin was used to indicate the centrosome. Scale bar: 10 ␮m.

amino acids of Cep68) could not localize to centrosomes, indicating that residues within the 300–400 amino acid region are necessary for centrosomal targeting of Cep68 (Fig. 1B). Cell cycle analysis of Cep68 protein As the centrosomal localization of Cep68 fluctuated with the cell cycle, we next asked whether the total cellular Cep68 protein levels oscillate during cell cycle. For this experiment we used a previously characterized anti-Cep68 serum Ni-Cep68 (Graser et al., 2007) that could detect long and short isoforms of endogenous Cep68 on Western blots. The functional role of short isoform in

centrosome separation remains elusive while long isoform of Cep68 has been established in centrosome separation (Fang et al., 2014; Graser et al., 2007). Our unpublished results indicated overexpressed short isoform of Cep68 was also degraded in mitosis and these two isoforms are differentially expressed in clinical human cancer samples. While expression of the Cep68 long isoform was detected in asynchronous HeLa cell lysates, no detectable expression was observed in extracts of cells that were arrested in M phase with nocodazole, even after long exposures of the blots (Fig. 2A). Since only the long isoform of Cep68 was detected in the current study, we decided to focus on the long isoform of Cep68 (hereafter referred to as Cep68) for the rest of the study. Substantial decrease




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Fig. 2. Cep68 is a cell cycle regulated protein. (A) Endogenous Cep68 using Ni-Cep68 antibody (Left), GFP-Cep68 (middle), and FLAG-Cep68 (right) were detected in adherent and shaken-off mitotic HeLa cells after nocodazole (Noc) treatment. Anti-␣-tubulin blot was used as loading control. (B) Endogenous Cep68 protein expression during cell cycle was evaluated by probing with Ni-Cep68 antibodies on lysates from Noc synchronized cells. M phase exit was indicated by a decrease in cyclin B1 level and M phase entry indicated by phosphorylated form of Cdc25c (arrow). (C) HeLa cells were synchronized by double thymidine block and release into Noc. Cells were harvested at indicated times and cell lysates were used for immunoblotting. Entry of mitosis was indicated by phosphorylated form of Cdc25c (arrow) and accumulated cylcinB1.

of Cep68 expression at the onset of mitosis was also observed with GFP- and FLAG-tagged Cep68 proteins driven by the CMV promoter upon transient transfection of plasmid constructs. To further examine the regulation of Cep68 expression during cell cycle, we measured Cep68 protein levels in synchronized HeLa cells. In a nocodazole-induced cell cycle block, Cep68 levels were strongly reduced, whereas cyclin B1 was stabilized as expected due to the ability of nocodazole to activate the spindle checkpoint (Fig. 2B). These events were concomitant with the appearance of a mitotic marker: phosphorylated form of Cdc25c (indicated by a molecular size shift). When HeLa cells were released from the nocodazole block, cells exited mitosis and progressed through G1 phase. During this time-course, Cep68 levels rose as cells entered G1 and S phase (from 6 h to 13 h). Cyclin B1 and Cdc25c also exhibited the expected trend following release from mitotic block. Using a second cell cycle arrest paradigm, when cells were released from a double thymidine block into nocodazole, Cep68 was stable through S phase (0–4 h), but destroyed in early mitosis (8 h) (Fig. 2C). Therefore, through two different synchronization protocols, we found that Cep68 protein levels oscillated during the cell cycle, accumulating in late G1 phase and disappearing in early mitosis. When overexpressed, Cep68 appeared as two bands in mitotic cell lysates (Fig. 2A middle and right). One band was at the same position as the interphase protein, and the other migrated more slowly on SDS-PAGE, suggesting a mitosis-associated modification of Cep68. This suggests that modification of Cep68 at mitosis might be involved in regulating its expression. Since endogenous Cep68 was undetectable in mitotic cell lysates with available antibodies, we used overexpression of Cep68 to probe this possibility further. We employed GFP-Cep68 transient over-expression for the ensuing experiments where we investigated the mechanism for Cep68

destruction and the role for Cep68 phosphorylation in regulating this process. Cep68 is degraded in mitosis by SCF complex Fig. 2 suggested that Cep68’s expression is regulated during cell cycle, presumably via proteolytic degradation. To test whether proteasome is involved, GFP-Cep68 overexpression in conjunction with M-phase arrest was performed in HeLa cells in the presence of the 26S proteasome inhibitor MG132. Anti-GFP immunoblots (Fig. 3A) showed that nocodazole-induced reduction in Cep68 expression could be prevented by MG132 pre-treatment. A control proteasome target cyclin A, was stabilized by MG132 treatment as expected, demonstrating the efficacy of proteasome inhibition (Fig. 3A). Cell cycle regulated proteins are often degraded through the proteasome pathway after being ubiquitinated (Ma et al., 2009). To test if Cep68 is a substrate for ubiquitinylation, GFPCep68 and Myc-tagged ubiquitin plasmids were co-transfected into HEK293 cells and the ubiquitin intermediates isolated by immunoprecipitation of myc-tagged proteins. The immunoprecipitates were then resolved on SDS-polyacrylamide gel and detected by immunoblotting against GFP to detect GFP-Cep68. The typical laddering of the polyubiquitin conjugates was specifically observed only when the anti-myc-ubiquitin immunoprecipitates from cells co-transfected with myc-tagged ubiquitin and GFP-tagged Cep68 were probed with anti-GFP antibody (Fig. 3B). The presence of anti-GFP signal in the immunoprecipitates from cells transfected with GFP-Cep68 alone is likely to be due to non-specific background noise or the dissociation of some GFP complexes. These results show that the disappearance of Cep68 in mitosis involved the ubiquitin-proteosome pathway.



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Because the APC/C and SCF complexes are both active at mitosis, we investigated if any of them is responsible for the mitotic degradation of Cep68. Cep68 was not ubiquitinated in a standard in vitro APC/C reaction (data not shown), so we suspect that Cep68 downregulation in early mitosis may occur through an ubiquitinand proteasome-dependent, but APC/C-independent, mechanism. To test whether depletion of SCF complex factors will affect Cep68 protein stability, we knocked down the beta-Trcp and cullin-1 components of SCF by RNAi, using previously published siRNA sequences (Shiraishi et al., 2007). Knockdown of beta-Trcp1&2 by siRNA was confirmed by depletion of FLAG tagged beta-Trcp1&2 (Fig. 3D) since endogenous beta-Trcp was hard to be detected on western blot. Stabilization of GFP-Cep68 in mitotic cell lysates was observed when cells were treated with the siRNAs against betaTrcp (Fig. 3C). Incomplete stabilization of Cep68 was observed

when cullin-1 was knocked down, suggesting a possible role of other adaptors in the degradation of Cep68. In contrast, silencing of beta-Trcp and Cullin-1 had no effect on cyclin A expression in the presence or absence of nocodazole because cyclin A was degraded by APC/C complex (Fig. 3C). For this experiment and sometimes others, we could not see the stabilization of phosphorylated band. It may be due to the fact that the stabilized band was too weak to be detected or that it is very labile. To test for a direct role of SCF in Cep68 degradation, we asked if Cep68 interacts with F-box protein beta-Trcp. By coexpressing Cep68 and beta-Trcp into HEK293 cells, reciprocal co-IP experiments showed interaction between Cep68 and beta-Trcp (Fig. 3E). A very small degree of myc signal was observed in the FLAG immunoprecipitates from cells transfected with myc-Trcp alone. This is probably due to the strong overexpression of myc-Trcp in lane 2

Fig. 3. SCF complex mediates the degradation of Cep68. (A) HeLa cells transfected with GFP-Cep68 were treated with Nocodazole in the presence or absence of MG132, shaken off mitotic cells were used for immunoblotting. Anti-cyclin A blot was used to indicate the effectiveness of MG132. (B) HEK293 cells were transfected with GFP-Cep68 and Myc-ubiquitin plasmid and treated with Noc and MG132. Cells were harvested and pulled down using Myc antibody and the immuonprecipitates were analyzed by western blotting. Lysates: whole cell lysates. (C) GFP-Cep68 and siRNA against beta-Trcp, cullin-1 or luciferase were cotransfected into HeLa cells, mitotic and interphase cells were harvested and probed with GFP. GFP-Cep68 expression levels as indicated are shown in a bar chart (mean ± SD and standard deviations of three independent experiments; *P < 0.05, student T test). (D) Efficiency of beta-Trcp1&2 siRNA were confirmed by knockdown of exogenous FLAG-beta-Trcp1&2 (Right). (E) Cep68 and beta-Trcp1 were overexpressed in HEK293 cells and Co-immunoprecipitation was done by using anti-FLAG-agarose. Bar graph shows relative immunoprecipitated Myc (IPed-myc) and GFP (IPed-GFP) signals as indicated (mean ± SD and standard deviations of three independent experiments; *P < 0.05, student T test).




and the cross reactivity of the primary antibodies used for IP since control IP using secondary antibodies conjugated to agarose beads alone without primary antibodies did not generate any background signal. The same is true for the reciprocal IP. From these data, we conclude that Cep68 downregulation in early mitosis is due to the SCF- and proteasome-dependent mechanism. Phosphorylation of Cep68 by Nek2 regulates its stability during mitosis Because protein phosphorylation generally precedes the degradation of SCF substrates, we suspected that Cep68 might be phosphorylated at the onset of mitosis. Fig. 2 showed that exogenous Cep68 appeared as a doublet in nocodazole-arrested cell lysates, suggesting that Cep68 maybe phosphorylated during mitosis. To test whether the mobility shift was due to phosphorylation, nocodazole-arrested cells transfected with GFP-Cep68 were treated with calf intestine phosphatase (CIP). Western blotting with GFP revealed a diminished signal of the slower-migrating Cep68 band in the presence of CIP (Fig. 4A). These findings support the notion that the mitosis-specific, upshifted form of Cep68 is phosphorylated. Nek2 has been shown to phosphorylate Cep68 in vivo and in vitro (Fang et al., 2014), so we overexpressed GFP-Cep68 and Nek2 or a panel of other mitotic kinases in HEK293 cells to investigate the potential kinase responsible for Cep68 phosphorylation during mitosis. As expected, Nek2 caused the most pronounced mobility shift in Cep68 (Fig. 4B). A kinase-dead mutant version of Nek2, K37R, failed to induce this shift (Fig. 4C). The Nek2-induced mobility shift of Cep68 could also be inhibited by CIP treatment of cell lysates harvested with buffer without phosphatase inhibitors (Fig. 4D). Together, these data indicate that the mobility shift of Cep68 at mitosis is due to Nek2 mediated phosphorylation. Because Nek2 is a known centrosomal protein kinase, GFPtagged Cep68 deletion mutants GFP-400, GFP-500, and GFP-600, all of which were able to localize to interphase centrosome, were cotransfected with Nek2 or K37R into HEK293 cells to map the potential region of Cep68 phosphorylation by Nek2. In the GFP500 and GFP-600 transfected cells, overexpression of Nek2 could induce an upper shift, which was absent in K37R transfected cells (Fig. 4E). While the GFP-400 deletion mutant also showed an upper shift, K37R was unable to inhibit the shift. It is not entirely clear why the GFP-400 mutant’s “phosphorylation” was not negated by the kinase dead K37R but it is possible that the GFP-400 fragment does not have structure-endowed inhibitory features that would otherwise prevent other kinases from acting on it. We consistently observed a stronger Nek2 signal in Nek2-overexpressing cells than in K37R-overexpressing cells (Fig. 4C–E). Since we excluded the possibility of poor plasmid preparation, this might be because of the inability of K37R to autophosphorylate itself thus failing to result in a more diffuse band seen in wild-type Nek2-overexpressing cells (Fry et al., 1995). We also tested the stability of wild-type Cep68 and its deletion mutants described above during nocodazol-induced mitosis block. Wild-type, GFP-500, and GFP-600 proteins were efficiently degraded when expressed in mitotic HeLa cells, whereas the GFP-400 deletion mutant was substantially stabilized (Fig. 4F). Taken together, the above data suggest that mitosis-induced Cep68 destruction is associated Nek2-mediated phosphorylation of Cep68 and amino acids from 400 to 500 of Cep68 is probably the minimal fragment that permits the fine regulation of Cep68 phosphorylation and stability by Nek2. To directly test the role of Nek2 in Cep68 degradation at mitosis, we obtained nocodazole-arrested lysates generated from HeLa cells transfected with GFP-Cep68 and Nek2 siRNA (Kokuryo et al., 2007) or control luciferase siRNA. Following depletion of Nek2, Cep68 was significantly stabilized in mitotic cells (Fig. 4G). Note that no Nek2 expression was observed

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in nocodazole- and control siRNA-treated cells because Nek2 itself is degraded prior to mitosis. As expected, cyclin A was destroyed in nocodazole treated cells. Next, we examined whether Nek2 overexpression could block the effect of MG132 in stabilizing Cep68 during mitosis. As seen in Fig. 4H, overexpression of Nek2 could substantially negate the MG132-induced stabilization of Cep68. Similarly, MG132 treatment increased Nek2 expression (compare lanes 2 and 4), since Nek2 is itself a substrate of degradation by APC/C during mitosis (Hames et al., 2001). Thus, Nek2 kinase activity is antagonistic to Cep68 accumulation and appears to phosphorylate Cep68 in mitosis to trigger its destruction by SCF prior to its own destruction at mitosis via APC/C. Nek2 dissociates Cep68 from interphase centrosomes Nek2 overexpression has been shown to induce centrosome separation in interphase cells by phosphorylating C-Nap1 and rootletin and dissociating them from centrosomes (Bahe et al., 2005; Faragher and Fry, 2003; Fry et al., 1998). Our above results showed that Nek2 promoted mitotic degradation of Cep68. We next asked whether Nek2 overexpression could cause Cep68 to dissociate from centrosomes in interphase cells. Endogenous Cep68 was localized to interphase centrosomes in HeLa cells as a focused signal (Fig. 5A). In contrast, Nek2 overexpression resulted in Cep68 appearing as diffused aggregated implying the dissociation of Cep68 from centrosomes (Fig. 5B). This suggests that Nek2 kinase activity interferes with the localization of Cep68 at centrosomes and favors the non-centrosomal localization of Cep68 to induce centrosome separation. This seems to be analogous to the displacement of C-Nap1 and rootletin from centrosome by Nek2 in previous studies (Bahe et al., 2005; Fry et al., 1998). Consistently with our hypothesis that Nek2-mediated phosphorylation is important for Cep68’s stability and hence localization to centrosomes, Cep68 in K37R kinase dead mutant-expressing cells colocalized with K37R which is known to localize in centrosome (Fig. 5C) (Faragher and Fry, 2003). We also observed aneuploidy caused by overexpression of K37R in agreement with previous results (Faragher and Fry, 2003). This may be caused by either failed mitosis or abortive cytokinesis when K37R is overexpressed. Combined, these results indicate that Nek2 promotes the dissociation of Cep68 from centrosomes by regulating its stability. Discussion Like rootletin and C-Nap1, Cep68 has to be removed from the centrosome linker structure when centrosome separation occurs (Bahe et al., 2005; Fry et al., 1998). Our results suggest that Cep68 associates with interphase centrosomes, requiring amino acids 300–400, and is unstable during mitosis. Our data also supports the notion exogenous Cep68 (exoCep68) is downregulated in early mitosis by SCF-Trcp dependent ubiquitin-mediated proteolysis. Endogeneous Cep68 is a cell cycle regulated centrosomal protein We demonstrated that the region including amino acids 300–400 targets Cep68 to interphase centrosomes. However, the adequacy of these 100 amino acids to target Cep68 to centrosomes remains to be shown. A deletion mutant lacking amino acid 300–400 is needed to evaluate the specificity of these 100 amino acids in centrosomal localization of Cep68. In addition, our data showing that Cep68 was displaced from centrosomes at the onset of mitosis is consistent with work shown by others (Graser et al., 2007). Moreover, our data support a mechanistic model whereby post-translational regulation of Cep68 regulates its stability during the cell cycle. From the Western blot data, total endogenous Cep68, N- and C-terminus-tagged Cep68 protein levels were markedly



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Fig. 4. Cell cycle regulated kinase Nek2 phosphorylates Cep68 and promotes its mitotic degradation. (A) HeLa cells transfected with GFP-Cep68 were treated with Noc for 16 h and the resulting mitotic cells were lysed in the absence of phosphatase inhibitors followed by incubation with calf intestine phosphatase (CIP) in the presence or absence of sodium fluoride(NaF, 50 mM). The graph shows the relative GFP-Cep68 expression level (mean ± SD and standard deviations of three independent experiments; *P < 0.05, student T test). (B) GFP tagged Cep68 and a panel of mitotic kinases were cotransfected into HEK293 cells and cell lysates was used for immunoblotting. Cotransfection of DsRed-Nek2 induced an upper shit of GFP-Cep68. (C) Cotransfection of DsRed-Nek2 also induces an upper shift in N-terminal FLAG-tagged Cep68 and this shift is absent in Nek2 kinase dead (DsRed-K37R) transfected cells. (D) DsRed-Nek2 induced shift is confirmed to be phosphorylation after CIP treatment. (E) GFP tagged Cep68 deletion mutants and DsRed tagged Nek2 or K37R were cotransfected into HEK239 cells and cell lysates was used for immunoblotting. (F) GFP tagged Cep68 deletion mutants was transfected into HeLa cells followed by treatment with nocodazole, mitotic and adherent cells were harvested for immunoblotting. (G) GFP-Cep68 transfected HeLa cells were treated with Luciferase or Nek2 siRNA and mitotic and adherent cells were collected after Nocodazole treatment and used for immunoblotting. Bar graph shows relative GFP-Cep68 expression level as indicated (mean ± SD and standard deviations of three independent experiments; *P < 0.05, student T test). (H) GFP-Cep68 and DsRed-Nek2 or control vector was cotransfected into HeLa cells and treated with nocodazole or MG132. Mitotic cells were harvested and used for immunoblotting.

reduced in mitotic cell extracts compared with levels in interphase cell extracts. Inhibition of proteosomal activity prevented mitotic loss of exoCep68 expression, implicating ubiquitin-dependent proteolysis as the mechanism for mitotic destruction of exoCep68. Additionally, in vivo ubiquitination experiments suggest that exoCep68 undergoes proteolysis during mitosis. Consistent with the hypothesis that phosphorylation of exoCep68 precedes its degradation, we detected a mobility shift of GFP-Cep68 caused by phosphorylation in mitotic cells. Phosphorylated GFP-Cep68 was substantially stabilized in the presence of proteosome inhibitor. We also showed that GFP-Cep68 was potentially phosphorylated in the presence of excess Nek2, suggesting that Cep68 is a target of Nek2 kinase. Whether Cep68 is a direct substrate of Nek2 remains to be investigated with in vitro kinase assay, which may not be definitive due to promiscuity of kinase action in vitro. Nevertheless, proteolysis of exoCep68 appeared to require the activity of SCF E3 ligase at the onset of mitosis.

Regulation of exoCep68 expression resembles that of many SCF substrates such as Emi1, Evi5, and beta-catenin in which priming of the degron sequence by phosphorylation facilitated degradation of these proteins (Eldridge et al., 2006; Reimann et al., 2001; Su et al., 2008). Cep68 is displaced from centrosomes in prophase, suggesting that phosphorylated Cep68 is degraded early in mitosis. We propose that the loss of centrosomal signal in early mitosis is a consequence of the degradation of Cep68 at centrosomes. This model is consistent with the localization of Nek2 at centrosomes, where it can act in close proximity to Cep68 and promote its destruction. We do not exclude the possibility that Nek2 may also act on Cep68 that is dispersed throughout the cytosol as well. However, no stabilized phosphorylated GFP-Cep68 was found when beta-Trcp was silenced. This could be due to the possibility that knockdown of SCF components affected the phosphorylation of Cep68 by Nek2 kinase if SCF components were necessary for Nek2 kinase activity




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Fig. 5. Nek2 dissociates Cep68 from interphase centrosomes. DsRed-Nek2 or DsRed-K37R was transfected into HeLa cells. Transfection of Nek2 (B) rather than K37R (C) dissociates Cep68 from centrosome. Endogenous Cep68 localizes at centrosomes (A). Cep68: green; DsRed: red; DAPI: Blue. Scale bar: 10 ␮m.

or its localization in centrosome. Our Co-IP study revealed interaction between GFP-Cep68 and beta-Trcp. This interaction may make Cep68 accessible to E3 ligase and subsequent degradation by proteasome machinery. Two known centrosomal proteins C-Nap1 and ninein contain putative F-box sequences. They may also function as F-box proteins or adaptors to promote recognition of Cep68 by E3 ligase and its subsequent degradation to promote centrosome separation (Fry et al., 1998). These possibilities remain to be tested. Nek2 phosphorylates exoCep68 to regulates its stability in mitosis Our data supported that Nek2 is a kinase for exoCep68 and can cause dissociation of endogenous Cep68 from centrosomes. This coincided with Cep68’s disappearance during mitosis and was consistent with previous findings (Fang et al., 2014; Graser et al., 2007). Hence, we propose that Nek2 phosphorylation of exoCep68 triggers its destruction at centrosomes. Nek2 is known to regulate centrosome separation and the APC/C is involved in its destruction at mitosis. It is conceivable that Nek2 destruction ensues after phosphorylation and activation of the degron of Cep68 to ensure the timely destruction of Cep68 prior to the elimination of Nek2. According to the current model regulating centrosome separation, increased Nek2 activity may lead to degradation or disassembly of

a molecular glue that holds the two centrosomes in close apposition during most stages of the cell cycle (Faragher and Fry, 2003). C-Nap1, rootletin, Cdk5rap2 and beta-catenin are four important proteins that regulate centrosome cohesion. Nek2 phosphorylates C-Nap1, rootletin and beta-catenin to regulate centrosome separation. Other proteins may reside within the linker structure and whose phosphorylation and degradation maybe important for disassembly of the linker structure. Previous data and our results indicated Cep68 may fulfill such a role and qualify Cep68 as a candidate component of this hypothetical centrosomal glue supporting previous results (Fang et al., 2014; Graser et al., 2007). In the current study, we observed that overexpression of Nek2 promoted the mitotic degradation of GFP-Cep68, while knockdown of Nek2 partially rescued its expression in mitotic cells. All these results indicate the involvement of Nek2 phosphorylation in dissociation of Cep68 from interphase centrosome and its degradation at the onset of mitosis. Cep68 deletion mutant GFP-400 is stabilized while GFP-500 are destroyed in mitotic cells, suggesting that mitotic degradation of exoCep68 depends on amino acids from 400 to 500. Kinase dead Nek2 failed to induce the phosphorylation of GFP-500 deletion mutants implying that the potential and physiological phosphorylation sites of exoCep68 by Nek2 reside between 400 and 500



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amino acids. Inspection of this 100 amino acid region revealed 11 serines and 7 threonines. We mutated each of these sites individually to alanine and performed nocodazole arrest to examine the requirement of each phosphorylation site for Cep68 stability. However, none of these mutations conferred stability to Cep68 in mitotic extracts (data not shown). Note however that we have only mutated the serine and threonine sites singly within the 400–500 amino acid region. It remains possible that multiple phosphorylation sites or sites beyond this region are involved in degron activation. In addition, we found one SCF substrate consensus sequence (DSGDVLDS) located a region from amino acid 332 to 338. However, since the GFP-400 Cep68 mutant was still stabilized in mitotic cell lysates, serine 332 and serine 338 were ruled out to be the potential phosphorylation sites. We also checked the eukaryotic linear motif resource (ELM) database for presence of phosphorylation sites in Cep68, and did not retrieve any possible sites. Also, we did not find any potential Nek2 phosphorylation consensus site LxxS/T in Cep68 protein sequence. Altogether, the data suggest the existence of other unidentified motif or phosphorylation residues in Cep68 that are important for its mitotic degradation. Notwithstanding the unresolved locality of the degron on Cep68, Nek2 phosphorylation appears to play an important role in exoCep68 degradation. In summary, our data show that Cep68 associates with interphase centrosomes and its expression is regulated in the cell cycle. During mitosis, SCF-dependent proteosomal degradation is likely to control the expression of exoCep68 through a degron located between amino acids 400 and 500. Nek2 may accelerate the phosphorylation and the destruction of exoCep68. Together, these data support a model whereby Nek2 regulates the dissociation of Cep68 from intercentriolar linkages at mitosis by phosphorylating it and triggering its destruction by SCF/Trcp. Mapping of the putative phosphodegron in Cep68 will provide further mechanistic insights into the regulated destruction of Cep68 during mitosis. Conflict of interest None. Acknowledgements We thank Dr Marito Araki for performing the in vitro ubiquitination assay. We also thank Dr Lim Shen Kiat and Mr Tan Kah Yap for critical and proof-reading this manuscript. This work was supported by the Biomedical Research Council (BMRC), Agency for Science, Technology and Research (A*STAR). References Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., Mann, M., 2003. Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426, 570–574. Ang, X.L., Wade Harper, J., 2005. SCF-mediated protein degradation and cell cycle control. Oncogene 24, 2860–2870. Bahe, S., Stierhof, Y.D., Wilkinson, C.J., Leiss, F., Nigg, E.A., 2005. Rootletin forms centriole-associated filaments and functions in centrosome cohesion. J. Cell Biol. 171, 27–33. Bahmanyar, S., Kaplan, D.D., Deluca, J.G., Giddings Jr., T.H., O’Toole, E.T., Winey, M., Salmon, E.D., Casey, P.J., Nelson, W.J., Barth, A.I., 2008. beta-Catenin is a Nek2 substrate involved in centrosome separation. Genes Dev. 22, 91–105. Castro, A., Arlot-Bonnemains, Y., Vigneron, S., Labbe, J.C., Prigent, C., Lorca, T., 2002. APC/fizzy-related targets Aurora-A kinase for proteolysis. EMBO Rep. 3, 457–462. Castro, A., Bernis, C., Vigneron, S., Labbe, J.C., Lorca, T., 2005. The anaphase-promoting complex: a key factor in the regulation of cell cycle. Oncogene 24, 314–325. Chen, Y., Low, T.Y., Choong, L.Y., Ray, R.S., Tan, Y.L., Toy, W., Lin, Q., Ang, B.K., Wong, C.H., Lim, S., et al., 2007. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells. Proteomics 7, 2384–2397.

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translocation of the cell cycle-regulated Nek2 kinase. J. Biol. Chem. 282, 26431–26440. Yang, J., Adamian, M., Li, T., 2006. Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells. Mol. Biol. Cell 17, 1033–1040.


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