Oncogene (2008) 27, 6141–6150 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE TSPY and its X-encoded homologue interact with cyclin B but exert contrasting functions on cyclin-dependent 1 activities

Y Li and Y-F Chris Lau

Department of Medicine, VA Medical Center, University of California, San Francisco, San Francisco, CA, USA

Testis-specific Y-encoded (TSPY) is the putative in XY sex-reversed patients and intersex individuals to for the gonadoblastoma locus on the Y gonadoblastoma development (Page, 1987; Lau, 1999). (GBY). TSPY and an X-homologue, TSPX, harbor a The 2.8-kbTSPY transcriptional unit is embedded in a conserved domain, designated as SET/NAP domain, but 20.4-kbDNA fragment that is repeated tandemly at the differ at their C termini. Ectopic expression of TSPY critical region for GBY on the short arm, proximal to accelerates cell proliferation byabbreviating the G 2/M the centromere, of the human Y chromosome (Skaletsky stage, whereas overexpression of TSPX retards cells et al., 2003). The number of TSPY repeats varies from at the same stage of the cell cycle. Previous studies B23 to 64 among phenotypically normal individual demonstrated that the SET oncoprotein is capable males (Repping et al., 2006), but could change under of binding to cyclin B. Using various protein interaction certain disease conditions (Vijayakumar et al., 2006), techniques, we demonstrated that TSPY and TSPX suggesting the possibility of genomic instability in this indeed bind competitivelyto cyclinB at their SET/NAP region of the chromosome. TSPY is normally expressed domains in vitro and in vivo. TSPY colocalizes with in early gonocytes and prespermatogonia in fetal testis cyclin B1 during the cell cycle, particularly on the and in spermatogonia, spermatocytes and round sper- mitotic spindles at metaphase. TSPY enhances while matids in adult testis (Honecker et al., 2004; Kido and TSPX represses the cyclin B1–CDK1 phosphorylation Lau, 2005), and has been postulated to serve normal activity. The inhibitory effect of TSPX on the cyclin functions in stem germ cell proliferation and meiotic B1–CDK1 complex has been mapped to its carboxyl division (Schnieders et al., 1996; Lau, 1999). TSPY is acidic domain that is absent in TSPY, suggesting that ectopically expressed in tumor cells in various types of TSPX could serve a normal function in modulating germ cell tumors, including gonadoblastoma, testicular cell-cycle progression at the G2/M stage, whereas germ cell tumors and male intracranial germ cell tumors, TSPY has acquired a specialized function in germ cell and has been considered as a key marker that is renewal and differentiation. Epigenetic dysregulation of important in the pathogenesis of these types of human TSPY in incompatible germ or somatic cells could tumors (Lau et al., 2000; Kersemaekers et al., 2005; promote cell proliferation and predispose susceptible cells Hoei-Hansen et al., 2006; Oram et al., 2006; Li et al., to tumorigenesis. 2007b). Significantly, it is also aberrantly expressed in Oncogene (2008) 27, 6141–6150; doi:10.1038/onc.2008.206; prostate cancer and numerous somatic cancers, includ- published online 30 June 2008 ing hepatocellular carcinoma and melanoma of male origins (Lau and Zhang, 2000; Lau et al., 2003; Keywords: TSPY; TSPX; sex ; cyclin Gallagher et al., 2005; Yin et al., 2005), suggesting that B–CDK1; kinase activities; protein–protein interaction the TSPY tandem arrays are hot spots for epigenetic dysregulation. Currently, the exact mechanism(s) by which TSPY exerts its putative oncogenic functions on these somatic cancers is uncertain. Introduction The highly conserved 2.8-kbTSPY transcriptional unit is capable of producing a variety of transcripts with The testis-specific protein Y-encoded (TSPY) is the alternative splicing mechanisms (Krick et al., 2003; Lau putative gene for the gonadoblastoma locus on the Y et al., 2003). The predominant TSPY (type 1) transcript chromosome (GBY) that predisposes dysgenetic gonads encodes a 38-kDa phosphoprotein of 308 amino acids. The open reading frames of variant transcripts, how- ever, are in-frame with that of the type 1 TSPY Correspondence: Dr Y-F Chris Lau, Department of Medicine, transcript and encode polymorphic TSPY with VA Medical Center, University of California, San Francisco, 111C5, various interstitial in-frame deletions between exons 4150 Clement Street, San Francisco, CA 94121, USA. E-mail: [email protected] 1 and 2. Significantly, all TSPY isoforms harbor the Received 14 March 2008; revised 30 May 2008; accepted 30 May 2008; entire or part of a conserved domain of B160 amino published online 30 June 2008 acids, termed SET/NAP domain initially identified in TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6142 the human SET oncoprotein and nucleosome assembly Results protein-1 (NAP-1) (Tsuchiya et al., 1995; Vogel et al., 1998). The SET/NAP proteins are structurally TSPY and TSPX bind cyclin B competitively at their related and serve a wide range of biological functions, SET/NAP domain including cell-cycle regulation, histone chaperone, Previously we demonstrated that overexpression of chromatin organization, transcription modulation and TSPY accelerated cell proliferation in vitro and in vivo steroid receptor co-regulation (Adachi et al., 1994; by shortening the G2/M stage of the cell cycle (Oram Kellogg et al., 1995; Estanyol et al., 1999; Compagnone et al., 2006). As SET/NAP domain proteins had been et al., 2000; Chai et al., 2001; Seo et al., 2001; Canela demonstrated to bind type B cyclins (Kellogg et al., et al., 2003; Ozbun et al., 2003; Gamble et al., 2005; 1995), which is critical for G2/M progression, we Kandilci and Grosveld, 2005; Vera et al., 2005; Kido surmised that TSPY could exert its cell-cycle regulatory and Lau, 2006). Currently, it is uncertain if these TSPY effects by interacting with the mitotic cyclins. To explore isoforms possess the same or different functions, this possibility, various recombinant glutathione S- retention of the SET/NAP domain suggests that (GST) fusion proteins were synthesized in this conserved domain could be important for their bacteria and used in GST pull-down assays with the biological action(s). respective 35S-labeled interactive candidate proteins. Ectopic expression of TSPY accelerates proliferation Using GST fusion proteins of human cyclin A, B1, B2 in cultured cells and promotes tumorigenesis in (Pines and Hunter, 1992) and B3 (transcript 2 coding for athymic mice (Oram et al., 2006). TSPY mediates such the short version of this meiotic cyclin (Tschop et al., proliferative properties by, either directly or indirectly, 2006)) as baits, the type 2 TSPY variant protein was upregulating pro-growth and repressing apoptosis consistently retained with GST–cyclin B1, B2 and B3 genes and growth inhibitors. Specifically, cells over- proteins, but not that of cyclin A (Figure 1a). The expressing TSPY transit the G2/M phase of the cell cycle cDNA for type 2 TSPY was originally isolated in our more rapidly than those without TSPY expression. laboratory (Zhang et al., 1992). It differs from the These observations are in contrast to those of the SET predominant type 1 transcript at its 30 ends due to oncoprotein and cell division autoantigen-1 (CDA1) utilization of a cryptic splice acceptor site 11 nucleotides (Chai et al., 2001; Canela et al., 2003). CDA1 and the ahead of that for type 1 transcript at the fifth exon and differentially expressed nucleolar TGF-b1 target encodes a protein of 294 amino acids (Lau et al., 2003). (DENTT) (Ozbun et al., 2001, 2003) are isoforms TSPY proteins encoded by both type 1 and 2 transcripts encoded by the X-homologue of TSPY, which has harbor an identical SET/NAP domain but differ slightly recently been designated as TSPX (Delbridge et al., at their C termini with divergent amino acids at residue 2004). CDA1 differs from DENTT by 60 additional no. 279 and beyond. To confirm the cyclin B interaction amino acids at its N terminus. Overexpression of either with other TSPY variants, GST–cyclin B1 fusion TSPX (CDA1) or SET retards/arrests cell-cycle progres- protein was used as bait in binding assays with similarly sion at G2/M stage (Chai et al., 2001; Canela et al., labeled TSPY isoforms. Our results showed that the 2003). Further studies on the SET oncoprotein suggest GST–cyclin B1 bait pulled down all TSPY isoforms, that it could mediate such cell-cycle regulatory func- for example, type 1, Exon1A, Exon1C and Intron4 tion(s) by interacting with cyclin B, the mitotic cyclin (Figure 1b). As the X-homologue, TSPX, and TSPY are important for G2/M progression (Canela et al., 2003). highly conserved at the SET/NAP domain (Delbridge To address the probable mechanism(s) by et al., 2004; Lau et al., 2007), we had explored the which TSPY exerts its cell-cycle regulatory functions, possibility that TSPX could also bind cyclin B in similar we had conducted a series studies designed to investigate assays. Using GST–cyclin B1 and B2 and labeled TSPX, the possible TSPY–cyclin B interaction, and we were able to confirm the interactions between cyclin consequences thereof on the cyclin-dependent kinase B and TSPX (Figure 1c) in similar pull-down experi- activities. TSPX and SET were studied in parallel ments. Interestingly, GST–TSPY binding to labeled experiments as references. Our results demonstrated cyclin B1 could be effectively abolished with increasing that TSPY interacted with all three types of cyclin amounts of recombinant TSPX protein (Figure 1d), B, that is, cyclin B1, B2 and B3, at its SET/NAP suggesting that their bindings to cyclin B1 are domain. It enhanced the phosphorylation activities competitive in nature. of an activated cyclin B1–CDK1 complex on its To define the cyclin B-binding domain on TSPY, targets in vitro and in vivo. TSPY colocalized with the various deletion mutants were generated with the type 1 endogenous cyclin B1 in transfected cells at various TSPY cDNA (Figures 2A, a–f) and analysed with pull- cell-cycle stages, including mitosis. Significantly, down assays using GST–cyclin B1 fusion protein as TSPX and SET also bound cyclin B1, but exerted bait. The results showed that there were two potential inhibitory effects on cyclin B–CDK1 activities in sites (residue nos. 152–180 and 181–265) within the the same assay. The inhibitory domains of TSPX and SET/NAP domain on TSPY that were capable of SET were mapped to their respective acidic carboxyl binding to cyclin B1 (Figure 2B). Pull-down assays with domain, which is absent in TSPY. Such structural labeled cyclin B1 and GST fusion proteins harboring the differences between TSPY and TSPX/SET could be N terminus (residue nos. 1–120), SET/NAP domain responsible for the respective effects on cell-cycle (residue nos. 121–265) and C terminus (residue nos. progression. 266–308) of TSPY confirmed the SET/NAP domain as

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6143 and used as baits in pull-down experiments. The results showed that cyclin B1 bound to all three peptides (Figure 2D). To address whether the same regions within the TSPX protein could also be interactive domains for cyclin B1, peptides corresponding to residue nos. 271–286 and 310–327 of TSPX were synthesized and used in similar binding assays with 35S-labeled cyclin B1 protein. Our results showed that indeed these TSPX peptides were capable of retaining the labeled cyclin B1 (Figure 2E), suggesting that cyclin B1 bound to TSPY and TSPX in two specific sites, respectively at residue nos. 152–180 and 203–220 of TSPY and residue nos. 271–286 and 310–327 of TSPX. To demonstrate similar interactions in vivo between cyclin B1 and TSPY and TSPX, FLAG epitope-tagged expression vectors for respective proteins were transi- ently transfected to human HEK293 cells. The expressed proteins were immunoprecipitated with a specific anti- body against FLAG and analysed by western blotting using specific antibodies against the FLAG fusion proteins, cyclin B1 and CDK1. Epitope-tagged proteins were detected only in cells transfected with the respective gene-expression vectors (Figure 3, lanes a2, 4; lanes b2, 3, 5, 6), but not in those transfected with cloning vector alone (Figure 3, lanes a1, 3; lanes b1, 4), in both the total cell lysates and those immunoprecipitated with FLAG antibody. Reprobing the filters with antibodies specifi- cally against cyclin B1 and CDK1 showed that both endogenous proteins were expressed in the transfected cells, as detected in the total cell lysates (Figure 3, lanes a1, 2; lanes b1–3). Significantly, among the protein samples initially immunoprecipitated with the FLAG antibody, only those that had been transfected with Figure 1 In vitro interactions between testis-specific protein gene-specific expression vectors showed positive signals Y-encoded (TSPY) variants and type B cyclins. (a–b) In vitro for both cyclin B1 and CDK1 (Figure 3, a4 (for FLAG- synthesized and 35S-labeled TSPY variant proteins were reacted TSPY); b5 (FLAG-SET), b6 (FLAG-TSPX)), whereas independently with glutathione S-transferase (GST)-cyclin B1, B2 those transfected with the vector alone did not show any or B3 recombinant protein, and the bound proteins were analysed signals (Figure 3, a3; b4). The results demonstrated that with SDS–polyacrylamide gel electrophoresis (PAGE) and auto- radiography. All TSPY variants harbored a conserved SET/NAP the endogenous cyclin B1 interacted with TSPY, TSPX domain at their C termini (Supplementary Figure S1) and were and SET and was co-immunoprecipitated with these retained by the respective Sepharose-bound GST–cyclin B bait. interactive proteins in the respective transfected 293 cell (c) Pull-down of 35S-labeld TSPX by GST–cyclin B1 and GST– populations. The fact that CDK1 was co-immuno- cyclin B2 fusion proteins. (d) Binding of 35S-labeled human cyclin B1 to GST–TSPY could be competitively displaced by increasing precipitated with cyclin B1-TSPY/TSPX/SET suggested amount of His-tagged TSPX recombinant protein (His-TSPX). that it was also part of complexes consisting of these Input ¼ 10% of labeled protein used for each binding reaction; SET/NAP domain-containing proteins and cyclin B1. GST, GST alone; þ , present; À, absent of the respective protein. Hence, TSPY, TSPX and SET not only interacted with cyclin B1, but also with an activated cyclin B1–CDK1 complex. the cyclin B-binding domain for TSPY (Figure 2C). As TSPY binding to cyclin B could be effectively abolished TSPY colocalizes with cyclin B1and CDK1in interphase by excess TSPX, we surmised that TSPY and TSPX and mitotic cells could possess cyclin B-binding domains at their SET/ We had previously demonstrated a colocalization of NAP regions. Using the ClustalW Protein Alignment TSPY and cyclin B1 in carcinoma in situ (CIS)/ program on the two proteins, we had identified two intratubular germ cell neoplasia unclassified (ITGCNU) regions, that is, residue nos. 152–180 and 203–220, (Li et al., 2007a, b) and tumor cells of gonadoblastoma within this conserved domain of TSPY that shared and seminoma specimens (Supplementary Figures S2 significant homologies with the corresponding regions of and S3 respectively). To provide more details on TSPX (Figure 2F). To further define the cyclin B- possible subcellular locations of these proteins, transi- binding domain of TSPY, three peptides (that is, g-152- ently transfected cells were analysed with specific 180, h-165-180 and i-203-220) corresponding to these antibodies against TSPY and cyclin B1. The subcellular regions were synthesized, conjugated to Sepharose resin locations of cyclin B1 are cell-cycle stage dependent. It is

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6144

Figure 2 Mapping of the cyclin B interactive domain in testis-specific protein Y-encoded (TSPY). (A) Various TSPY deletion proteins (a–f) were synthesized and 35S-labeled and used in pull-down assays with glutathione S-transferase (GST)-cyclin B1 fusion protein (B). The cyclin B1 interactive domain was mapped to a region between residue nos. 152–265 within the SET/NAP domain of TSPY (predominant type 1 isoform). (C) Pull-down analysis of 35S-labeled human cyclin B1 and GST-fusion proteins harboring the N- terminal portion (GST-N), SET/NAP (GST-NAP) or C-terminal portion (GST-C) of TSPY. (D and E) Pull-down assays with 35S- labeled cyclin B1 and resin-bound peptides derived from TSPY (D) and TSPX (E) proteins. (F) Alignment of homologous amino-acid sequences from TSPY and TSPX encompassing the respective peptides used for pull-down assays in d and e. *, identical, and similar amino acid.

primarily located in the cytoplasm during most stages of immunofluorescence. Our results showed that TSPY the cell cycle, migrates to the nucleus in upon initiation was indeed located at the a-tubulin-containing micro- of cell division and is degraded upon mitotic exit by the tubules of the mitotic spindles in metaphase and anaphase-promoting complex ubiquitination system. In anaphase cells (Figures 4g and h). transfected cells, TSPY was localized with cyclin B1 in the cytoplasm of cells presumably at interphase TSPY and TSPX exert opposite effects on the (Figure 4a). When cyclin B1 was localized at the nucleus phosphorylation activities of a cyclin B1–CDK1 complex during prophase, similar nuclear location of TSPY in vitro and in vivo could also be observed (Figure 4b). At mitosis, both The co-immunoprecipitation of the endogenous CDK1 proteins were preferentially located at the microtubules and cyclin B1 with TSPY, SET and TSPX suggested of the mitotic spindles (Figure 4c). Significantly, similar that these SET/NAP domain-containing proteins could colocalization of TSPY and CDK1 could also be interact with an activated cyclin B1–CDK1 complex in observed at prophase and metaphase cells (Figures 4d the host cells. This postulation raised the possibility that and e respectively). Selected mitotic cells at prometa- these SET/NAP proteins could exert their effects on phase, metaphase, anaphase and cytokinesis showed cyclin-dependent kinase activities by their interactions that TSPY was abundantly distributed in these cells with cyclin B1 of the active complex. To validate (Figure 4f). Again, preferential location of TSPY could our hypothesis, we had investigated the effects of each of be observed in metaphase cells (for example, Figure 4f, three SET/NAP proteins in a reconstituted protein middle panel). To confirm its mitotic spindle location, phosphorylation system consisting of activated cyclin these cells were analysed with both TSPY and a-tubulin B1–CDK1 enzyme complex, its substrate histone H1

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6145

Figure 3 Co-immunoprecipitation of endogenous cyclin B1 and CDK1 with testis-specific protein Y-encoded (TSPY) (a), SET and TSPX (b) from transfected HEK293 cells. Plasmids capable of expressing the FLAG-tagged proteins were transfected to HEK293 cells, which were lysed and immunoprecipitated with anti-FLAG antibody. The immunoprecipitated proteins were analysed by western blot with a TSPY monoclonal antibody (a) or anti-FLAG antibody (b). The expression of the respective proteins could be detected in total cell lysates of transfected cells (a, lane 2; b, lanes 2 and 3) and immunoprecipitated proteins (a, lane 4; b, lanes 5 and 6), but not cells transfected with FLAG vector alone without (a–b, lane 1) or with (a, lane 3; b, lane 4) immunoprecipitation procedure. Western blotting of parallel samples demonstrating the presence of endogenous cyclin B1 and CDK1 in total cell lysates (a, lanes 1 and 2; b, lanes 1, 2 and 3) and immunoprecipitated proteins derived from cells transfected with FLAG-TSPY (a, lane 4), FLAG-SET (b, lane 5) or FLAG-TSPX (b, lane 6). Some faint signals were also observed in the FLAG control, lane b4, and could be considered as background as their intensity was significantly lower than those of the real signals at the adjacent lanes. and 32P-g-ATP as phosphorous donor. Various amounts of recombinant SET/NAP proteins (Figure 5a) were added to the reconstituted system and the extent of phosphorylation of histone H1 was analysed with SDS– polyacrylamide gel electrophoresis (PAGE) and auto- radiography. Our results demonstrated that TSPY (Figure 5b) enhanced whereas TSPX (Figure 5c) and SET (Figure 5e) repressed the CDK1 kinase activities at dosage-dependent manners. Significantly, the enhance- ment and repression effects of TSPY and TSPX/SET were competitive in nature, such that the respective effects could be neutralized by those of opposite action (Figure 5d for TSPY and TSPX; Figure 5f for TSPY Figure 4 Colocalization of testis-specific protein Y-encoded and SET respectively). (TSPY) with cyclin B1, CDK1 and tubulin in various stages of As structurally the significant differences between the cell cycle. (a–c) Cyclin B1 (red) colocalized with TSPY (green) in TSPY and TSPX/SET are located at the acidic domains the cytoplasm of interphase cell (a), in the nucleus in late G2/early mitosis (b); and enhanced at the mitotic spindles during metaphase of both SET and TSPX, which is absent in TSPY (Lau (c). (d–e) CDK1 (red) colocalized with TSPY (green) in late et al., 2007), we had addressed the probable roles of G2/early mitosis (d), and enhanced at the mitotic spindles these acidic tails in modulating the cyclin B–CDK1 during metaphase (e). (f) Distribution of TSPY protein (red) in kinase activities. Recombinant proteins for TSPY, reconstituted stages of mitosis, prophase, prometaphase, meta- TSPX and SET truncated immediately beyond the phase, anaphase/telophase, cytokinesis (left to right). (g–h) Colocalization of TSPY (red) with tubulin (green) at the mitotic respective SET/NAP domains were synthesized spindles at metaphase (g) and anaphase (h). Merged, merged (Figure 5g) and used in the in vitro kinase activity images of the respective fluorescence staining to the left. DAPI assays. Truncation of the small carboxyl tail of TSPY (4-6-diamidino-2-phenylindole; blue), DNA staining (see online did not affect its enhancement on the activity of an version for colour figure). activated cyclin B1–CDK1 complex (Figure 5h). Trun- cation of the respective acidic domains at the C termini protein, our observations suggested that this N-terminal of both TSPX and SET reversed their repression domain probably did not contribute to the TSPX functions, resulting in various degrees of enhancement repression of cyclin B1–CDK1 functions. Significantly, of cyclin B1–CDK1 kinase activities on the histone H1 replacing the short carboxyl tail of TSPY with the long substrate (Figures 5i and j, respectively). As the proline- acidic tail of TSPX resulted in severe repression of cyclin rich domain was present in the TSPXN truncated B1–CDK1 kinase function (Figure 5k), as observed with

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6146

Figure 5 In vitro phosphorylation of histone H1 by an activated cyclin B1–CDK1 complex. (a) diagrammatic illustration of testis- specific protein Y-encoded (TSPY), SET, TSPX and (g) the respective variant recombinant proteins used in the cyclin B1–CDK1 kinase assays. TSPY (b) enhanced and TSPX (c) and SET (e) repressed the CDK1 kinase activities on its substrate in dosage-specific manners. The TSPY enhancement of CDK1 activities could be suppressed by addition of TSPX (d) or SET (f) in the reactions. Deletion of the small C terminus beyond the SET/NAP domain in TSPY showed minimal effects on its enhancement of the cyclin B1–CDK1 phosphorylation (h), whereas similar deletions of the acidic domains of TSPX (i) or SET (j) reversed the respective repression to moderate enhancement of the same kinase complex. Fusion of the TSPX acidic tail to the C terminus of TSPY resulted in significant repression of the cyclin B1–CDK1 kinase activities (k), similar to the same effects by intact TSPX protein (c). The relative fold changes were derived from densitometry scanning and calculated with reference to the average of cyclin B1–CDK1 activities in the control reactions without the respective recombinant protein(s) in each experiment.

the intact TSPX (Figure 5c). These results clearly To determine the in vivo effects of these SET/NAP demonstrated that the domain responsible for TSPX domain containing proteins on cyclin B1–CDK1 repression on activated cyclin-dependent kinase func- functions, we had analysed the cyclin B1–CDK1 kinase tion is located at its acidic carboxyl tail. activities in transfected cells using the CycLex

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6147 Cdc2-Cyclin B Kinase Assay Kit. This kit utilized an anti-phospho-Cdc7-Threonine376 monoclonal antibody to detect the CDK1/cyclin B phosphorylation of its specific substrate, Cdc7. Hence, detection of a phos- phorylated form of Cdc7 was proportional to the relative amount of activated cyclin B1–CDK1 kinase in the transfected and control cells (Masai et al., 2000). Our results demonstrated that HEK293 cells transfected with the vector alone showed moderate cyclin B1–CDK1 kinase activities. Transfection of the same cells with expression vectors for TSPY type 1 and 2 individually elevated the in vivo cyclin B1–CDK1 kinase activities whereas expression of SET and TSPX repressed such activities in the same assay (Figure 6a). Inclusion of roscovitine, a specific inhibitor for cyclin B1–CDK1 (Whittaker et al., 2007), in parallel assays reduced the detectable cyclin B1–CDK1 kinase activities to uniformly minimal levels in all samples, supporting the specificity of the cyclin B1–CDK1 activities in this system. To confirm that these effects were consequences of the transfected gene-expression vectors, western blot was performed on total cell lysates of transfected and control cells with various specific antibodies. Using a monoclonal antibody against the FLAG epitope, the respective epitope-tagged SET/NAP proteins could be Figure 6 In vivo effects of testis-specific protein Y-encoded detected in all cells transfected with the corresponding (TSPY), SET and TSPX on cyclin B–CDK1 activities in transfected expression vector (Figure 6b, lanes 2–5), except those cells. (a) The in vivo cyclin B1–CDK1 activities were analysed by the CycLex Cdc2-Cyclin B kinase assays using total cell lysates transfected with vector alone (Figure 6b, lane 1). To from respective HEK293 transfected cells. Expression of FLAG- confirm that the endogenous cyclin B1–CDK1 com- tagged TSPY type 1 (TSPY1) or type 2 (TSPY2) protein in plexes were present in these cell lysates, parallel western HEK293 cells enhanced the endogenous cyclin B1–CDK1 activ- blots were probed independently with specific antibodies ities, whereas similar expression of SET or TSPX protein repressed against cyclin B1 and CDK1. The results confirmed the the same kinase complex. In parallel assays, the presence of roscovitine, an inhibitor of cyclin B1–CDK1 kinase, abolished the presence of endogenous cyclin B1–CDK1 complexes in cyclin B–CDK1 activities to the same level among the same cell the respective cell lysates (Figure 6c), used in the cyclin lysates. (b) Western blotting of total cell lysates showing the B-dependent kinase assays. Our findings, taken together, expression of TSPY, SET and TSPX in the respectively transfected demonstrated that TSPY exerted an enhancing function, cell populations. (c) Western blotting of the corresponding total cell lysates demonstrating the presence of endogenous cyclin B1 whereas its X-homologue, TSPX and the related SET (top row) and CDK1 (middle row) proteins and tubulin control phosphoprotein exerted various repression functions (bottom row). on cyclin B1–CDK1 complex activities, leading to an expedition and retardation of G2/M phase transit respectively in the host cells. CASK-interacting nucleosome assembly protein (Chai et al., 2001; Ozbun et al., 2001; Wang et al., 2004a). Discussion Recent studies on the gene organization and encoded proteins showed that its gene structure is closely related TSPY is a unique member of the SET/NAP protein to that of TSPY, including conservation of intron–exon family and does not harbor an acidic C terminus junctions and sequences, particularly at SET/NAP frequently present in other members (Lau et al., domain, but with exceptions at their amino and C 2007). Significantly, TSPY-like genes have been termini that encode a proline-rich and acidic domain, identified throughout the mammalian genomes (Vogel respectively (Delbridge et al., 2004; Lau et al., 2007). et al., 1998; Lau et al., 2007). These TSPY-like genes These studies suggest that TSPY-, TSPX- and TSPY- are mostly functional, that is, capable of producing like genes could constitute a subfamily of SET/NAP transcripts, although they might possess various spatio- domain-containing proteins (Lau et al., 2007). Currently temporal patterns of expression. In most cases, they are the exact functions of this gene family are uncertain. results of retrotransposition events of TSPY-derived Mutation that truncates the SET/NAP domain in transcripts as they are single exon genes whose open TSPY-L1 gene are associated with the sudden infant reading frames encode proteins similar to TSPY without death with dysgenesis of the testis syndrome on the acidic domain. TSPX, on the other hand, was chromosome 6 (Puffenberger et al., 2004), suggesting initially considered as a TSPY-like gene, termed TSPY- that the SET/NAP domains are important for like 2. It has been independently isolated in various the respective functions of TSPY subfamily of proteins. systems and has been designated as CDA1, DENTT and Results of the present study further substantiate

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6148 the essential role(s) of this conserved domain in their suggest that TSPX could maintain a house-keeping respective biological functions. function(s) in modulating cyclin B–CDK1 activities, and Although the TSPY array is located at the critical hence, cell-cycle progression, especially on G2/M transi- region and is the putative gene for GBY locus on the tion, whereas TSPY has acquired various special human Y chromosome (Salo et al., 1995; Tsuchiya et al., functions pertaining to male stem germ cell proliferation 1995), its exact oncogenic mechanism(s) in the disease and meiotic division. Significantly, male meiotic division process is still uncertain. The present studies have involves two rounds of chromosome divisions without demonstrated that TSPY exerts such cell-cycle regula- an intermediate interphase (Rajesh and Pittman, 2006; tory functions by directly interacting with type B cyclins Bowles and Koopman, 2007). TSPY interaction with and enhancing the cyclin B–CDK1 activities, resulting in cyclin B and enhancement of the cyclin B-dependent an accelerated progression through G2/M of the cell kinase activities could potentially be important for such cycle (Oram et al., 2006). Significantly, cyclin-dependent consecutive chromosome divisions. When ectopically kinase activity is essential for maintaining the integrity expressed in somatic cells incapable of male meiosis, the of the mitotic spindle checkpoint. The apparent TSPY actions on cyclin B and CDK1 might accelerate preferential colocalization of TSPY protein with cyclin cell proliferation (Oram et al., 2006), affect G2/M B1, CDK1 at the mitotic spindles of the metaphase cells checkpoints and increase in chromosome nondisjunction suggests that TSPY could affect the cyclin B1–CDK1 and genome instability (Lobrich and Jeggo, 2007; functions on the spindles (Pines and Hunter, 1992; Schmit and Ahmad, 2007; Decordier et al., 2008). Richter, 2001; Smits and Medema, 2001; Porter and Currently, it is uncertain how such cell-cycle dysfunction Donoghue, 2003; Wang et al., 2004b; Fukasawa, 2007), could lead to upregulation of selected oncogenes and thereby disrupting this mitotic checkpoint that could pro-growth genes observed in cells overexpressing potentially result in chromosome nondisjunction and TSPY. Conceivably, other oncogenic events could aneuploidy. Indeed, precursors of testicular germ cell exacerbate tumorigenesis associated with dysregulated tumors, that is, CIS/ITGCNU, could express TSPY at TSPY expression. The TSPY array possesses the highest relatively high levels, whereas the resulting TGCTs are mutation rate among the key regions, that is, TSPY usually aneuploid (Looijenga and Oosterhuis, 2002; gene array, AZFc, Yq heterochromatin and IR3/IR3 Oosterhuis and Looijenga, 2005; Rajpert-De Meyts, region, of the human Y chromosome (Repping et al., 2006), thereby supporting the notation that overexpres- 2006). Significantly, nonallelic homologous recombina- sion of TSPY could increase genomic instability. tion among its repeat units has been detected among Conversely, TSPX, the X-located homologue, and SET normal and disease populations (Repping et al., 2006; oncoprotein are also capable of interacting with cyclin B Vijayakumar et al., 2006). Hence, epigenetic dysregula- (Kellogg et al., 1995), but exert an inhibitory function tion and genomic instability, instead of specific muta- on the cyclin B1–CDK1 complex. As overexpression of tions of the coding sequences, of the TSPY array could TSPX (CDA1) or SET arrested cells at G2/M stage and constitute to the oncogenic and/or tumor predisposition retarded cell-cycle progression (Chai et al., 2001; Canela properties of this tandemly repeated gene on the human et al., 2003), their inhibitory actions on cyclin B–CDK1 Y chromosome. activities could contribute to such cell-cycle dysregula- tion. Using immunoprecipitated cyclin B–CDK1 com- plex from cultured cells, Canela et al. (2003) had also Materials and methods demonstrated similar inhibitory actions on cyclin B– CDK1 activities by SET protein in a dosage-dependent Plasmids and antibodies Various cDNAs for TSPY, TSPX, SET, cyclin A, cyclin B1, manner (Canela et al., 2003). Our deletion mapping and B2 and B3 (variant 2) were inserted in pGEX4T3 vector for domain swapping results further showed that the syntheses of GST fusion proteins in bacteria, in pET28b for carboxyl acidic tail of TSPX (and SET), which is absent in vitro protein syntheses, and in FLAG-epitope tagged in TSPY, is important in cell-cycle modulation. Indeed, p3XFLAG-CMV-7 vector (Sigma, St Louis, MO, USA) for truncation of the acidic domain in TSPX abolished such expression in mammalian cells. Anti-TSPY monoclonal (M7) inhibitory effects on cell-cycle progression and cyclin and polyclonal antibodies were previously described (Kido and B–CDK1 activities, thereby confirming the significance Lau, 2005). Other antibodies were purchased from commercial of this domain (or lack of it) in the biological functions vendors. of TSPX and TSPY. TSPY and TSPX have been postulated to evolve from Protein purification and GST pull-down assay the same ancestral gene on the proto-sex chromosomes GST recombinant proteins were synthesized in BL21(DE3) (Graves, 1995; Lau et al., 2007). TSPY is normally pLysS bacterial hosts and purified by affinity chromatography expressed in male early germ cells during embryogenesis, using glutathione-Sepharose beads, as previously described and spermatogonia, spermatocytes and, to certain (Oh et al., 2005; Li et al., 2006). GST pull-down assays were extent, the round spermatids, in adult testis (Zhang performed with respective GST fusion proteins and corre- sponding labeled proteins synthesized with the TnT T7 et al., 1992; Schnieders et al., 1996; Honecker et al., Quick Coupled Transcription/Translation System (Promega, 2004; Kido and Lau, 2005, 2006), whereas TSPX is Madison, WI, USA) in the presence of 35S-methionine expressed ubiquitously in numerous cells/tissues (Ozbun (Amersham Biosciences, Piscataway, NJ, USA). Specific et al., 2001, 2003, 2005; Delbridge et al., 2004; Wang polypeptides were conjugated to BrCN-activated Sepharose et al., 2004a; Lau et al., 2007). Such expression patterns 4B (Amersham Biosciences) or MicroLink Peptide Coupling

Oncogene TSPY interacts and modulates cyclin B–CDK1 activities Y Li and Y-F Chris Lau 6149 kit (Pierce Biotechnology, Rockford, IL), and used in pull- in 20 ml buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,2mM down assays with 35S-methinione-labeled cyclin B1. The bound dithiothreitol, 1 mM ethylene glycol tetraacetic acid, 10 mM proteins were analysed in 10–15% SDS–PAGE gels and ATP and 2 mCi of [32P]ATP (Amersham Biosciences)) at 30 1C detected by autoradiography. for 30 min. Samples were resolved in 12% SDS–PAGE gels and analysed by autoradiography. Results were quantitated by Cell synchronization and Co-immunoprecipitation densitometry scanning and NIH ImageJ Program and Human HEK293 cells were transfected with p3 Â FLAG calculated as fold changes with respect to control reactions vector, p3 Â FLAG-TSPY, p3 Â FLAG-SET and p3 Â without recombinant proteins. FLAG-TSPX plasmids using FuGENE 6 reagents (Roche, In vivo CDK1 kinase activity was analysed using the Indianapolis, IN, USA), and synchronized at G2/M phase, reaction kit, CycLex Cdc2-Cyclin B Kinase Assay Kit (MBL as before (Oram et al., 2006). Co-immunoprecipitation was International, Woburn, MA, USA), according to the manu- performed with a monoclonal anti-FLAG RedView antibody facturer’s protocol. HEK293 cells were transfected with (Oh et al., 2005; Li et al., 2006). The immunocomplexes were various TSPY/TSPX/SET expression vectors for 36 h and analysed by SDS–PAGE electrophoresis and western blot with were then synchronized at G2/M by colcemid treatment rabbit anti-cyclin B, CDK1 or FLAG antibody. for 12 h and analysed with CycLex Cdc2-Cyclin B Kinase Assay Kit. The absorbance was measured by using a spectro- Immunofluorescence microscopy photometric plate reader (SpectraMAX 250; Molecular Immunofluorescence was performed on transfected HEK293 Devices, Sunnyvale, CA, USA). All assays were performed in or COS7 cells, using various primary antibodies, detected by triplicates. Individual protein levels were determined by respective Alexa 488 or Alexa 568- þ conjugated secondary western blot of the same samples with respective FLAG, antibodies, counterstained with DAPI, and analysed as cyclin B1, CDK1 and tubulin antibodies. described (Oh et al., 2005; Li et al., 2006). Acknowledgements Cyclin B–CDK1 activity in vitro and in vivo In vitro cyclin-dependent kinase assays were performed with We thank Dr Tatsuo Kido for assistance in immuno- respective recombinant proteins of TSPY, TSPX, SET and fluorescence of transfected cells presented in Figures 4f–h. TSPY-TSPX chimera and cyclin B1/CDK1 active complex This work was partially supported by a Merit Review (Calbiochem, San Diego, CA, USA). Various amounts (0.5, 1, grant and a Prostate Cancer REAP from the Department of 2, 4 mg) of purified recombinant proteins and two units of Veterans Affairs and an Idea Grant from the Department cyclin B1/CDK1 complex were used in kinase assay of 2 mgof of Defense to Y-FCL. Y-FC Lau is a research career scientist histone H1 (Upstate Biotechnology, Lake Placid, NY, USA) of the Department of Veterans Affairs.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene