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Dyrk2-Associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation* 1 2

Hae-Yun Jung1, Xin Wang1, Sohee Jun1, Jae-Il Park1,2 3 4

1From Department of Experimental Radiation Oncology, 2Program in Cancer Biology, The University of 5 Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. 6

7 *Running title: Telomerase inhibition by Dyrk2-associated E3 ligase 8

9 To whom correspondence should be addressed: Jae-Il Park, Department of Experimental Radiation 10 Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, 11 TX 77030, USA; tel.: (713) 792-3659, fax.: (713) 794-5369, email: jaeil@mdanderson.org. 12 13 Key words: telomerase, TERT, Dyrk2, ubiquitination, E3 ligase 14 15 Background: Telomerase is an essential enzyme 16 for the immortalization of stem and cancer cells. 17 Results: Dyrk2-associated E3 ligase targets 18 telomerase reverse transcriptase, a catalytic 19 subunit of telomerase. 20 Conclusion: Dyrk2-E3 ligase is necessary to 21 negatively regulate telomerase activity. 22 Significance: Learning how telomerase is 23 regulated is crucial for understanding telomerase 24 regulatory mechanism in cancer and stem cells. 25 26 Summary 27 28

Telomerase maintains the telomere, a 29 specialized chromosomal end structure that is 30 essential for genomic stability and cell 31 immortalization. Telomerase is not active in 32 most somatic cells, but its reactivation is one of 33 the hallmarks of cancer. In this study, we found 34 that dual-specificity tyrosine-(Y)- 35 phosphorylation-regulated kinase 2 (Dyrk2) 36 negatively regulates telomerase activity. Dyrk2 37 phosphorylates TERT protein, a catalytic 38 subunit of telomerase. Phosphorylated TERT is 39 then associated with the EDD-DDB1-VprBP E3 40 ligase complex for subsequent ubiquitin- 41 mediated TERT protein degradation. During 42 the cell cycle, Dyrk2 interacts with TERT at the 43 G2/M phase and induces degradation. In 44 contrast, depletion of endogenous Dyrk2 45 disrupts the cell cycle-dependent regulation of 46 TERT and elicits the constitutive activation of 47 telomerase. Similarly, a Dyrk2 nonsense 48 mutation identified in breast cancer 49

compromises ubiquitination-mediated TERT 50 protein degradation. Our findings suggest the 51 novel molecular mechanism of kinase- 52 associated telomerase regulation. 53

54

In stem and regenerating cells, maintenance of 55 genomic stability is essential for self-renewal and 56 the subsequent transmission of accurate genetic 57 information to progenitor cells. In eukaryotes, 58 telomerase overcomes the end-replication problem 59 by adding the telomeric repeat sequence 60 (TTAGGG) to the ends of chromosomes (1,2). In 61 somatic cells, the absence of telomerase results in 62 the gradual loss of telomeric repeats with every 63 cell division, which is referred to as a telomere 64 crisis. These cells typically undergo growth arrest 65 and cellular senescence (3-5). However, genetic 66 and epigenetic defects in oncogenic pathways can 67 induce abnormal cell proliferation, resulting in 68 cancer (6,7). Importantly, the re-activation of 69 telomerase in most cancers prevents a telomere 70 crisis and leads to cellular immortality. 71

Telomerase is composed of two 72 subunits—telomerase RNA template (TERC), an 73 RNA template, and telomerase reverse 74 transcriptase (TERT) (8). The results of several 75 studies suggest that telomerase activity is 76 modulated at various steps, including TERC 77 biosynthesis, TERT transcription, TERT protein 78 modification, telomerase holoenzyme formation, 79 and telomere-associated proteins (9-17). Recent 80 studies have shown that Wnt/β-catenin signaling 81 participates in transactivating TERT in cancer and 82

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.416792The latest version is at JBC Papers in Press. Published on January 28, 2013 as Manuscript M112.416792

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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stem cells (18,19), highlighting the importance of 1 the regulatory mechanism of TERT in controlling 2 telomerase activity. Although telomerase’s pivotal 3 roles in cancer have been extensively studied, its 4 regulatory mechanism in such pathological 5 conditions is still unclear. In this study, we 6 determined an important telomerase regulatory 7 mechanism. We found that the dual-specificity 8 tyrosine-(Y)-phosphorylation-regulated kinase 2 9 (Dyrk2)-associated E3 ligase complex is involved 10 in degradation of TERT protein and subsequent 11 inhibition of telomerase activity. 12 13 Experimental Procedures 14 Mammalian cell culture - HeLa, 293T, and MCF-7 15 cells were maintained with Dulbecco’s modified 16 Eagle medium (DMEM) containing 10% fetal 17 bovine serum (FBS). Mouse embryonic stem cells 18 were maintained in DMEM, 20% FBS, and 19 leukemia inhibitory factor (1000 units per ml) on 20 feeders that were isolated from 13.5-day-old 21 mouse embryos. HeLa cells that stably expressed 22 either FLAG-TERT or HA-Dyrk2 were 23 established by transduction with 3FLAG-TERT- 24 pMGIB and 3HA-Dyrk2-pWZL retroviruses. 25 26 Constructs - Full-length or fragments of TERT, 27 Dyrk2, and FOXO3A were cloned into HA-tagged 28 pcDNA3.1, FLAG-tagged pcDNA3.1, HA-tagged 29 pWZL (retrovirus), FLAG-tagged pMIGB 30 (retrovirus), and pGEX-6T vectors. Myc-tagged 31 EDD, Myc-tagged VprBP, S- 32 protein/FLAG/streptavidin-binding protein-tagged 33 DDB1, Cul4A, and Roc1 constructs were provided 34 by J. Chen (The University of Texas MD 35 Anderson Cancer Center, Houston, TX) and R. 36 Maddika (Centre for DNA Fingerprinting and 37 Diagnostics, Hyderabad, India). The Dyrk2 38 K178R and TERT S457A mutants were generated 39 via polymerase chain reaction (PCR). Dyrk2 40 shRNA lenti-viral plasmids (Sigma) and retroviral 41 plasmids (pMGIB and pWZL) were packaged into 42 293T cells with standard protocols. 43 44 Small-scale kinase screening - 23 genes encoding 45 kinases in various signaling pathways were 46 subcloned into pcDNA3.1 and pCS2 expression 47 vector. Then, HeLa cells stably expressing FLAG- 48 TERT were transiently transfected with each 49 construct. 36 hours after transfection, cell lysates 50 were analyzed for immunoblotting with anti- 51

FLAG antibody (M2; Sigma). 52 53 Pulse chase labeling assay - HeLa-S3 cells that 54 stably expressed FLAG-TERT were washed and 55 pre-incubated for 1 hour with 10% Met- and Cys- 56 free DMEM in FBS. The medium was then 57 replaced with fresh Met- and Cys-free medium 58 containing 1.48×106 Bq of [35S]-Met or –Cys, and 59 the cells were incubated for 1 hour before being 60 washed and incubated in complete medium for 61 each time point. Two milligrams of cell lysates 62 were immunoprecipitated with anti-FLAG agarose 63 (Sigma). The samples were subjected to 8% 64 sodium dodecyl sulfate-polyacrylamide gel 65 electrophoresis (SDS-PAGE), followed by 66 autoradiography. 67 68 Immunoblotting and immunoprecipitation - Whole 69 cell lysates were prepared using Nonidet P-40 70 (NP-40) lysis buffer (0.5% NP-40, 1.5 mM MgCl2, 71 25 mM HEPES, 150 mM KCl, 10% glycerol, 1 72 mM phenylmethanesulfonyl fluoride (PMSF), 12.7 73 mM benzamidine HCl, 0.2 mM aprotinin, 0.5 mM 74 leupeptin, and 0.1 mM pepstatin A) for 20 minutes 75 at 4°C, followed by centrifugation (14,000 rpm, 10 76 minutes). Supernatant fractions were denatured 77 with 5× SDS sample buffer (200 mM Tris-Cl, pH 78 6.8; 40% glycerol; 8% SDS, 200 mM 79 dithiothreitol; and 0.08% bromophenol blue) at 80 95°C for 5 minutes, followed by SDS-PAGE. For 81 immunoblot blocking and antibody incubation, 82 0.1% non-fat dry milk in Tris-buffered saline with 83 Tween-20 (25 mM Tris-HCl, pH 8.0; 125 mM 84 NaCl; and 0.5% Tween-20) was used. Super 85 Signal West Pico and Femto (Pierce 86 Biotechnology) reagents were used to detect 87 horseradish peroxidase-conjugated secondary 88 antibodies. For immunoprecipitation, cells on 10- 89 cm plates were lysed with 0.3 ml NP-40 lysis 90 buffer for 20 minutes at 4°C, followed by spinning 91 at 14,000 rpm for 10 minutes. One hundred 92 microliters of extract (10% of input) was saved for 93 later immunoblotting. The cell lysates were 94 incubated with 20 µl of HA agarose or FLAG- 95 magnetic beads at 4°C overnight. 96 Immunoprecipitates were washed with NP-40 lysis 97 buffer four times, eluted using SDS sample buffer, 98 and analyzed by immunoblotting. The following 99 antibodies were used: anti-Dyrk2 (Abcam), FLAG 100 (M2, Sigma), HA (12CA5, HA-7, and 3F10), Myc 101 (9E10), Brg-1 (Santa Cruz), BAF57 (Santa Cruz), 102

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PCNA (Santa Cruz), TPP1 (Bethyl), TRF2 (Santa 1 Cruz), and tubulin (Sigma). 2 3 Telomerase rapid amplification protocol (TRAP) 4 assay - Cells (293T or MCF-7) were lysed with 5 100 µl of NP-40 lysis buffer containing 1 mM 6 PMSF, 12.7 mM benzamidine HCl, 0.2 mM 7 aprotinin, 0.5 mM leupeptin, 0.1 mM pepstatin A, 8 and 400 U RNase inhibitor. The cell lysates were 9 analyzed using the TRAP reaction assay with the 10 TRAPEZE telomerase detection kit (Chemicon) or 11 real-time PCR (20). 12 13 Cell synchronization - HeLa cells with FLAG- 14 tagged TERT and HA-tagged Dyrk2 were 15 synchronized using a double thymidine block. 16 Cells were harvested, washed with phosphate- 17 buffered saline, and fixed with ice-cold 70% 18 ethanol. Cells were then treated with 5 µg/µl 19 RNase A and 50 µg/µl propidium iodide for 30 20 minutes at 37°C and analyzed on a C6 flow 21 cytometer (Accuri). 22 23 Transfection - HeLa S3 and 293T cells were 24 transfected with plasmids and siRNAs using 25 Lipofectamine 2000 (Invitrogen) in accordance 26 with the manufacturer’s protocol. Control siRNA, 27 VprBP siRNAs (siRNA1, 28 GGAGGGAAUUGUCGAGAAU; siRNA2, 29 CCACAGAAUUUGUUGCGCA; siRNA3, 30 GGAAUGACACUGUGCGCUU; siRNA4, 31 CGGAGUUGGAGGAGGACGA), and Dyrk2 32 siRNAs (siRNA1, 33 CAAAUGGGCUUACAACAGU; siRNA2, 34 UCACGUGGCUUACAGGUAU; siRNA3, 35 GGUGCUAUCACAUCUAUAU; siRNA4, 36 GGCCUACGAUCACAAAGUC) were purchased 37 from Dharmacon. 38 39 Semiquantitative PCR - One microgram of RNA 40 extracted from each cell was used to generate 41 cDNA samples (Superscript II; Invitrogen). Next, 42 cDNA was analyzed using standard PCR. The 43 sequence information for each primer is available 44 upon request. 45 46 GST pull-down assay - GST-enhanced green 47 fluorescent protein (EGFP; control) and Dyrk2 48 proteins were purified from an Escherichia coli 49 BL21 strain using a standard procedure. The 50 purified protein (0.1 µg) was incubated with 51

HeLa-FLAG-TERT cell lysates for 30 min and 52 precipitated using Glutathione Sepharose 4B beads 53 (GE Healthcare). Then precipitates were analyzed 54 using IB with an anti-FLAG antibody. 55 56 Ubiquitination assay - For in vivo ubiquitination 57 assay, HeLa S3 cells were transiently co- 58 transfected with pCMV-HA-tagged UBC and 59 3FLAG-tagged TERT-pcDNA3.1, and 60 subsequently immunoprecipitated with HA- 61 agarose (HA-7). The precipitates were then 62 immunoblotted with an anti-FLAG antibody. In 63 vitro ubiquitination reactions were performed at 30 64 °C for 8 h in 30 µl of ubiquitination reaction 65 buffer (40 mM Tris-HCl, pH 7.6, 2 mM 66 dithiothreitol, 5 mM MgCl2, 0.1 M NaCl, 2 mM 67 ATP) containing 100 µM ubiquitin, 20 nM E1 68 (UBE1), 100 nM UbcH5b (all from Boston 69 Biochem), and EDVP E3 ligase components (50 70 ng each of EDD, DDB1, VprBP, and Dyrk2). A 71 FLAG-TERT substrate was generated using a 72 TNT-coupled reticulocyte lysate system 73 (Promega). After the ubiquitination reaction, 74 samples were boiled in an SDS-PAGE loading 75 buffer. Ubiquitination of TERT was monitored 76 using IB with anti-ubiquitin and anti-FLAG 77 antibodies. 78 79 In vitro kinase assay - Wild-type and mutant 80 (S457A) TERT expressed in bacterial cells (BL21) 81 were purified using Glutathione Sepharose 4B 82 beads. FLAG-tagged Dyrk2 expressed in 293T 83 cells was immunoprecipitated using anti-FLAG- 84 agarose and used as a kinase source. The Dyrk2 85 kinase and TERT substrate were incubated in a 86 kinase reaction buffer (10 mM HEPES, pH 7.5, 50 87 mM NaCl, 10 nM MgCl2, 10 mM MnCl2, 1 mM 88 EGTA, 1 mM dithiothreitol, 10 mM NaF, 10 µCi 89 [gamma-32P]) for 60 min at 30 °C. Kinase 90 reactions were resolved using SDS-PAGE and 91 autoradiography. 92 93 94 Results 95 96 Dyrk2-induced TERT ubiquitination and 97 degradation 98 To understand the regulatory mechanism of 99 telomerase, we first compared the level of TERT 100 mRNA transcript with telomerase activity in 101 various cells, including human embryonic stem 102

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cells, mouse embryonic stem cells, mouse 1 embryonic fibroblasts, and HeLa cells. 2 Interestingly, we observed that the level of the 3 TERT mRNA transcript was not correlated with 4 telomerase activity in several cell lines 5 (Supplemental Fig. 1), indicating the existence of 6 an additional layer of telomerase activity 7 regulation beyond TERT transcription. Among 8 several interpretations of this discrepancy between 9 TERT transcription and telomerase activity, we 10 hypothesized that post-transcriptional TERT 11 regulation determines telomerase activity. Then, 12 we measured TERT protein half-life using a pulse 13 chase labeling assay. HeLa cells stably expressing 14 FLAG-tagged TERT (HeLa-FLAG-TERT) were 15 metabolically labeled with 35S-methioinine and 16 immunoprecipitated with FLAG antibody for 17 subsequent autoradiography. .We found that the 18 half-life of TERT protein was ~2.1 hours (Figs. 19 1A and B), suggesting that telomerase activity 20 might be determined by TERT protein stability. 21 22

Protein stability is generally controlled by 23 the ubiquitin-mediated proteasome pathway (21). 24 Specific posttranslational modifications of a target 25 protein are recognized by the E1-E3 ubiquitination 26 cascade, which initiates proteasome-mediated 27 degradation of the target protein (22). Of the 28 various types of posttranslational modifications, 29 we focused on kinase-induced target protein 30 phosphorylation and ubiquitination. To identify 31 kinases involved in downregulation of TERT 32 protein, we performed small-scale kinase 33 screening. Briefly, we transiently overexpressed 34 plasmids encoding individual kinases in HeLa- 35 FLAG-TERT cells, and analyzed the level of 36 TERT protein using immunoblotting (IB) for 37 FLAG-TERT. We found that a member of Dyrk 38 family proteins significantly downregulated TERT 39 (Fig. 1C). Dyrk proteins are categorized into two 40 groups: 1) Dyrk1A and Dyrk1B, and 2) Dyrk2, 41 Dyrk3, and Dyrk4 (23). We observed that Dyrk1A 42 did not downregulate TERT protein level, but 43 Dyrk2 and Dyrk3 did, indicating substrate 44 specificity among the Dyrk proteins. Additionally, 45 ectopic expression of Dyrk2 decreased the level of 46 TERT protein in a dose-dependent manner (Fig. 47 1D). Next, we examined whether Dyrk2-induced 48 downregulation of TERT protein is independent of 49 transcriptional downregulation of TERT. Indeed, 50 ectopic expression of Dyrk2 did not downregulate 51

endogenous TERT transcripts in HeLa cells. To 52 further analyze the effects of Dyrk2 on TERT 53 transcription, we established TERT knock-in 54 mouse embryonic stem cells (mESCs) that harbor 55 tdTomato fluorescent protein in place of TERT 56 allele (TERTtdTomato/+). tdTomato expression 57 represents the transcriptional level of TERT. 58 Consistently, Dyrk2 expression did not affect 59 endogenous TERT promoter activity 60 (Supplemental Fig. 2). These data suggest that 61 Dyrk2 downregulates TERT protein at post- 62 transcriptional level. Next, we sought to determine 63 whether Dyrk2-mediated TERT inhibition affects 64 telomerase activity. We ectopically expressed 65 Dyrk2 in 293T and HeLa cells, and performed 66 telomerase rapid amplification protocol (TRAP) 67 assay. Similar to IB results, the overexpression of 68 Dyrk2 decreased telomerase activity. To 69 compensate Dyrk2 gain-of-function analysis, we 70 also employed Dyrk2 knockdown approaches. 71 Depletion of endogenous Dyrk2 using either small 72 interfering RNA (siRNA) or short hairpin RNA 73 (shRNA) increased the level of TERT protein in 74 HeLa-FLAG-TERT cells (Fig. 1F). Moreover, 75 Dyrk2 knockdown (shRNA)-induced TERT 76 stabilization was reverted by introducing shRNA 77 non-targetable Dyrk2, confirming the specific on- 78 target effects of Dyrk2 shRNA on TERT 79 stabilization (Fig. 1G). Additionally, we examined 80 whether Dyrk2 depletion affects TERT protein 81 half-life using cycloheximide (CHX) treatment in 82 HeLa-FLAG-TERT cells (shGFP control vs. 83 shDyrk2). We found that Dyrk2 depletion 84 increased TERT protein half-life in HeLa cells 85 (Figs. 1H and I). These data suggest that Dyrk2 86 specifically de-stabilizes TERT protein and 87 subsequently inhibits telomerase activity. 88 89 Dyrk2 binds to TERT and phosphorylates its 90 serine residue 91 We next explored the molecular mechanism of 92 Dyrk2-mediated TERT protein downregulation. 93 First, we tested whether Dyrk2 is associated with 94 TERT. Coimmunoprecipitation (co-IP) of 95 ectopically expressed TERT and Dyrk2 exhibited 96 the reciprocal protein interaction in 293T cells 97 (Fig. 2A). Additionally, a glutathione S-transferase 98 (GST) pull-down assay of GST-Dyrk2 from HeLa- 99 FLAG-TERT cell lysates showed that TERT 100 protein bound directly to Dyrk2 (Fig. 2B). 101 Moreover, we examined the endogenous 102

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interaction between TERT and Dyrk2. Due to 1 absence of antibody detecting endogenous TERT 2 protein (24), we utilized triple-HA epitope 3 knockin mESCs (TERTHA/+) for co-IP assays. By 4 gene targeting in mESCs, HA epitope was inserted 5 into start codon of TERT to express HA-TERT 6 fusion protein (25). Indeed, endogenous TERT 7 protein is associated with Dyrk2 in mESCs (Fig. 8 2C). To further analyze TERT-Dyrk2 protein 9 interaction, we performed binding domain 10 mapping using truncated mutants of TERT and 11 Dyrk2. We observed that the reverse transcriptase 12 (RT) domain and C-terminal regions in TERT and 13 the kinase domain in Dyrk2 are responsible for 14 TERT-Dyrk2 interaction (Fig. 2D, E). These data 15 suggest that TERT protein is physically associated 16 with Dyrk2 protein. 17 18

Dyrk2 belongs to a serine/threonine 19 protein kinase family (26), implying that Dyrk2 20 might phosphorylate TERT protein and lead to 21 degradation. To address this, we examined 22 whether Dyrk2’s kinase activity is required for 23 TERT protein destabilization. A point mutation 24 (K178R) in the kinase domain of Dyrk2 results in 25 loss of its kinase activity (27). We ectopically 26 expressed wild-type or K178R Dyrk2 in HeLa- 27 FLAG-TERT cells and performed IB for TERT. 28 Intriguingly, a K178R kinase-inactive mutant of 29 Dyrk2 failed to induce TERT protein degradation 30 (Fig. 2F), suggesting that Dyrk2-induced TERT 31 phosphorylation is required for TERT degradation. 32 Nonetheless, co-IP assays showed that Dyrk2 33 K178R can still bind to TERT (Fig. 2F). Next, we 34 examined whether Dyrk2 phosphorylates TERT 35 protein using in vitro kinase assay. As shown in 36 Fig. 2G, Dyrk2 specifically phosphorylated TERT 37 protein. Then, we located the evolutionarily 38 conserved Dyrk2 consensus phosphorylation 39 sequence (RKXX[X]S/TP)(28) at Ser457 in 40 TERT’s RNA-interacting domain 2 (Fig. 2H). To 41 verify Ser457 as a Dyrk2 target serine residue, we 42 utilized a site-specific TERT mutant (Ser to Ala at 43 457) as a substrate for kinase assay. Indeed, 44 S457A mutant was not phosphorylated by Dyrk2, 45 while wild-type TERT was phosphorylated (Fig. 46 2I). These data showed that Dyrk2 specifically 47 phosphorylated TERT at Ser457. These results 48 suggested that Dyrk2 induces TERT 49 phosphorylation, which is required for TERT 50 protein downregulation. 51

52 The Dyrk2-EDVP E3 ligase complex 53 ubiquitinates TERT for degradation 54 Previously, it was suggested that Dyrk2 acts as a 55 scaffolding protein for ubiquitination substrates 56 and the E3 ligase complex EDD, DDB1, and 57 VprBP (EDVP) (29). Thus, we hypothesized that 58 Dyrk2 and its associated EDVP E3 ligase complex 59 are involved in Dyrk2-mediated TERT protein 60 downregulation. First we questioned whether 61 Dyrk2 induces ubiquitination of TERT for protein 62 degradation. To address this, we performed in vivo 63 ubiquitination assays. Indeed, in HeLa cells, 64 ectopic expression of Dyrk2 moderately increased 65 TERT ubiquitination (Fig. 3A), suggesting that 66 Dyrk2 downregulates TERT protein via 67 ubiquitination. Next, we examined whether the 68 EDVP E3 ligase complex is also associated with 69 TERT protein. In the EDVP E3 ligase complex, 1) 70 EDD functions as a homologous to the E6-AP 71 carboxyl terminus (HECT) E3 ligase(30), 2) 72 DDB1 plays a dual role as a linker between the 73 cullin E3 ligase and a substrate receptor (31), and 74 3) VprBP, also known as a DDB1-cullin– 75 associated factor, serves as a substrate receptor for 76 target protein ubiquitination (32). As expected, in 77 co-IP experiments, we found that all components 78 of the EDVP E3 ligase interacted with the TERT 79 protein in HeLa cells (Fig. 3B, C). Interestingly, 80 similar to Dyrk2’s effects on telomerase, ectopic 81 expression of DDB1 or VprBP decreased 82 telomerase activity. However, EDD had little 83 effect on telomerase activity (Fig. 4D, E). Given 84 that only the DDB1-VprBP module specifies the 85 target recognition by physical binding, EDD alone 86 may be insufficient for destabilization of TERT 87 protein. Conversely, depletion of VprBP using 88 siRNA increased TERT protein (Fig. 3F), 89 suggesting that VprBP functions as a substrate 90 receptor for TERT in EDVP E3 ligase complexes. 91 Importantly, we found that TERT-EDD and 92 TERT-VprBP interactions were impaired upon 93 depletion of Dyrk2 (Fig. 3G), supporting the 94 previously identified role of Dyrk2 as a 95 scaffolding protein between substrates and the 96 EDVP E3 ligase (29). Also, in vitro ubiquitination 97 assays showed that Dyrk2 was indispensable for 98 EDVP-mediated TERT ubiquitination (Fig. 3H). 99 These data suggest that the Dyrk2-EDVP E3 100 ligase complex ubiquitinates TERT. 101 102

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Cell cycle-dependent association of Dyrk2 and 1 TERT 2 Telomerase activity is only detectable in self- 3 renewing cells, including stem cells, regenerating 4 cells, and germ cells (2,33), but its reactivation is 5 strongly associated with cell immortalization in 6 human cancer (34,35), suggesting that telomerase 7 activity should be tightly controlled during cellular 8 and tissue homeostasis. Therefore, we 9 hypothesized that Dyrk2 controls TERT protein 10 under normal physiological conditions, such as the 11 cell cycle. Because telomere synthesis mainly 12 occurs in the S phase of cell cycle (36), Dyrk2 13 likely plays a role in eliminating the TERT protein 14 after the S phase. Prior to analyzing Dyrk2’s 15 effects on TERT, we determined whether Dyrk2 16 affected the cell cycle. In our experimental setting, 17 we observed that Dyrk2 overexpression or 18 knockdown did not affect cell cycle (Supplemental 19 Fig. 3), which rules out the effects of Dyrk2- 20 induced cell cycle arrest on TERT protein 21 regulation. 22 23

Because the event that Dyrk2 binds to and 24 phosphorylates TERT seems to be a prerequisite 25 for TERT protein ubiquitination and degradation, 26 the TERT-Dyrk2 physical interaction may be a 27 key event in regulating telomerase activity during 28 the cell cycle. To address this, we induced cell 29 cycle arrest at each stage using a specific 30 treatment: thymidine for 24 hours, serum 31 starvation for 48 hours, and nocodazole for 14 32 hours in HeLa cells; we then analyzed the 33 interaction between TERT and Dyrk2 using co-IP 34 assays. Intriguingly, we found that the TERT- 35 Dyrk2 protein interaction mainly occurred in 36 G2/M phase-arrested cells. Moreover, the TERT- 37 Dyrk2 interaction was correlated with a decrease 38 in TERT protein levels (Fig. 4A, lane 5). 39 Conversely, TERT weakly interacted with Dyrk2 40 in S phase–arrested cells with the increase in 41 TERT protein levels (Fig. 4A, lane 3), indicating 42 the inversely proportional relationship between 43 TERT protein levels and TERT-Dyrk2 interaction. 44 These results suggest that Dyrk2 conditionally 45 binds to TERT protein only in the G2/M phase of 46 the cell cycle for ubiquitin-mediated TERT protein 47 degradation. To further validate these results, we 48 analyzed TERT-Dyrk2 interaction in each cell 49 cycle stage of synchronized HeLa-FLAG-TERT 50 cells (Fig. 4B). Consistent with the results using 51

cell cycle-arrested HeLa cells, the synchronized 52 HeLa cells displayed an increase in the TERT- 53 Dyrk2 association in the G2/M phase, with a 54 decreased level of TERT protein (Fig. 4C, lane 5). 55 This was also manifested by quantitative analysis 56 showing the increased TERT-Dyrk2 interaction at 57 G2/M phase (Fig. 4D). These results recapitulate 58 that TERT-Dyrk2 interaction is a prerequisite for 59 TERT protein degradation and strongly suggest 60 that the dynamic association of Dyrk2 with TERT 61 is an essential biological event that negatively 62 modulates telomerase activity in a cell-cycle 63 dependent manner. 64 65

Next, we validated our observation that 66 Dyrk2 specifically destabilizes TERT protein 67 during the G2/M phase of the cell cycle via a 68 Dyrk2 loss-of-function approach. Using shRNA- 69 mediated depletion of endogenous Dyrk2, we 70 determined whether Dyrk2 depletion compromises 71 cell cycle-dependent regulation of TERT protein 72 stability. Indeed, knockdown of endogenous 73 Dyrk2 disrupted G2/M phase-specific 74 downregulation of TERT protein, resulting in the 75 constitutive upregulation of TERT protein, 76 independently of cell cycle (Fig. 4E and F). Of 77 note, nocodazole treatment decreased TERT 78 protein levels (Fig. 4E, lane 7), as also shown in 79 Fig. 4A (lane 5) and Fig. 4C (lane 6). However, 80 Dyrk2 knockdown abolished the effects of 81 nocodazole-induced TERT downregulation (Fig. 82 4E, compare lane 7 and 14; 4F), indicating that 83 Dyrk2 is necessary to downregulate TERT protein 84 at the G2/M phase of the cell cycle. Although it is 85 evident that Dyrk2 targets TERT protein in a cell 86 cycle-dependent manner, our studies utilized cells 87 that ectopically expressed TERT. To validate the 88 effects of Dyrk2 on endogenous telomerase 89 activity, we examined whether depletion of 90 endogenous Dyrk2 enhances endogenous 91 telomerase activity in MCF-7 cells. shRNA- 92 mediated Dyrk2 knockdown markedly increased 93 telomerase activity. By contrast, introduction of 94 shRNA non-targetable Dyrk2 reverted telomerase 95 activity (Fig. 4G), verifying the specific effects of 96 Dyrk2 on telomerase activity. These results 97 suggest that Dyrk2 is a key molecule in controlling 98 TERT protein during the cell cycle. 99 100 Dyrk2 nonsense mutation compromises TERT 101 protein destabilization 102

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Telomerase reactivation is observed in 90% of 1 human cancers (35). Having determined that 2 Dyrk2 negatively regulates telomerase activity via 3 ubiquitin-mediated protein degradation, we 4 reasoned that Dyrk2-mediated telomerase 5 regulation is associated with cancer. To evaluate 6 this, we determined whether any components of 7 the Dyrk2-EDVP E3 ligase complex were 8 transcriptionally suppressed or genetically 9 inactivated in human cancer using the Oncomine 10 database (www.oncomine.org). We found no 11 downregulation or genetic inactivation of EDVP 12 components, but we observed that Dyrk2 is 13 somatically mutated in human breast cancer from 14 the analysis of the COSMIC database 15 (www.sanger.ac.uk/genetics/CGP/cosmic/). These 16 Dyrk2 mutations were initially identified in 17 genome-wide analyses of various cancers (37-39) 18 (Supplemental Fig. 4). Of the Dyrk2 somatic 19 mutations, which are mostly missense mutations in 20 Dyrk2’s kinase domain, we focused on the Dyrk2 21 S471X nonsense mutation (X stands for the stop 22 codon generated by the frame-shift mutation) in 23 human breast cancer cells. Dyrk2-S471X mutant 24 lacks the Kelch motif, which may be responsible 25 for interaction with E3 ubiquitin ligase (Fig. 5A) 26 (40,41). Therefore, the Dyrk2-S471X nonsense 27 mutation likely comprises an endogenous 28 telomerase degradation mechanism. To test this, 29 we determined whether Dyrk2 S471X 30 downregulates TERT protein in HeLa-FLAG- 31 TERT cells. Dyrk2-S471X mutant failed to 32 decrease TERT protein levels, whereas wild-type 33 Dyrk2 induced TERT protein degradation (Fig. 34 5B). Consistent with the protein level of TERT, 35 TRAP assays also showed that ectopic expression 36 of Dyrk2 S471X mutant was unable to inhibit 37 telomerase activity (Fig. 5C). Because the Kelch 38 motif is missing in Dyrk2 S471X mutant, Dyrk2 39 S471X’s inability to target TERT protein may be 40 resulted from the loss of interaction with EDVP 41 E3 ligase components. Thus, we evaluated the 42 interaction between Dyrk2-S471X mutant and 43 EDVP E3 ligase components. Indeed, S471X 44 mutant no longer bound to the EDD and VprBP 45 proteins (Fig. 5D). These results suggest that 46 Dyrk2 mutant fails to target TERT protein and 47 leads to the constitutive hyperactivation of 48 telomerase. 49 50 51

52 Discussion 53 54 In this study, we identified an novel regulatory 55 mechanism of telomerase. Dyrk2 and its 56 associated EDVP E3 ligase complex specifically 57 target TERT protein via ubiquitination-mediated 58 degradation. Importantly, the genetic mutation in 59 Dyrk2 identified in breast cancer cells fails to 60 elicit TERT protein degradation, implying that 61 Dyrk2 mutation-induced telomerase deregulation 62 may contribute to tumorigenesis (Fig. 6). 63 64

Telomerase activity is strictly controlled at 65 multiple levels, including TERC biosynthesis, 66 TERT transcription, telomerase holoenzyme 67 formation, telomerase recruitment to telomere, and 68 telomere-associated proteins. Telomere elongation 69 is coupled with DNA replication and cell cycle 70 progression (36). In studies in budding yeast, cell 71 cycle-dependent telomere elongation was 72 regulated by various safeguards: (i) telomerase 73 recruitment to the telomere by Cdk1-induced 74 Cdc13 phosphorylation in yeast, (ii) changes in the 75 telomere’s chromosomal structure, and (iii) 76 telomere end processing (9,42-44). Accumulating 77 evidence indicates that various cell cycle-related 78 proteins are regulated at the protein level by 79 kinase-mediated ubiquitination and degradation at 80 different cell cycle checkpoints (45,46). In 81 addition, transcriptional activation of TERT by 82 Wnt/β-catenin signaling is required for telomerase 83 activity in cancer and stem cells (18,19). These 84 studies suggest that telomerase activity is tightly 85 controlled to allow full activation of telomerase 86 only in specific phases of the cell cycle. We found 87 that the Dyrk2-associated E3 ubiquitin ligase 88 complex negatively regulates telomerase in the 89 G2/M phase. However, Dyrk2 depletion stabilizes 90 TERT protein, resulting in constitutive activation 91 of telomerase, regardless of the cell cycle 92 checkpoint. Importantly, Dyrk2 knockdown also 93 reverses the effects of nocodazole on TERT 94 protein downregulation (see Fig. 4). These 95 findings underline the essential function of Dyrk2- 96 E3 ligase complex in regulating TERT protein 97 during cell cycle. 98 99

The EDVP components DDB1 and VprBP 100 are also part of the Cul4A-Roc1 E3 ligase complex 101 (29). In this complex, DDB1 functions as a linker 102

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protein between Cul4-Roc1 and VprBP (47). 1 DDB1 is relatively larger than the substrate 2 receptors for other proteins in the cullin family, 3 implying that it is versatile in protein-protein 4 interactions (31). Additionally, this suggests the 5 various roles of DDB1 in diverse physiological 6 events. In agreement with this, we found that 7 TERT was also associated with Cul4A and Roc1 8 E3 ligase (Supplemental Fig. 5). Nonetheless, 9 Cul4A and Roc1 failed to destabilize TERT 10 protein. Instead, Cul4A slightly enhanced 11 telomerase activity in HeLa cells (Supplemental 12 Fig. 6), suggesting that Cul4-mediated TERT 13 ubiquitination might provide an additional layer of 14 telomerase activity regulation via a 15 nondegradation mechanism. 16 17

Previous studies suggested that TERT 18 protein is ubiquitinated by several E3 ligases, 19 including MKRN1, CHIP, and HDM2 (48-50). 20 Although these studies suggest the effects of 21 ubiquitination of TERT on regulating telomerase 22 activity, their pathological relevance remains 23 elusive. In our study, we found that Dyrk2 24 mutation (S471X) in breast cancer disrupts the 25 interaction between Dyrk2 and the EDVP E3 26 ligase complex, which leads to interruption of 27 Dyrk2-mediated TERT protein degradation. Dyrk2 28 phosphorylates p53 at serine 46 and induces 29 apoptosis upon DNA damage (27). Therefore, 30 Dyrk2 may play a key role in cellular homeostasis 31 through two different mechanisms: (i) eliminating 32 irreparable DNA-damaged cells through p53- 33 induced cell death and (ii) limiting telomerase 34 activity in specific self-renewal cells and during 35 the S phase of the cell cycle. Thus, a truncated 36 mutation in Dyrk2 may compromise both the p53- 37 mediated DNA damage response and cell cycle- 38 dependent telomerase regulation, which may 39 contribute to tumorigenesis. Thus, it will be 40 necessary to further examine the pathological 41 association of telomerase deregulation with 42 Dyrk2’s genetic inactivation in human cancers. 43 Additionally, employing genetically engineered 44 mouse models for Dyrk2 knockout will address 45 the function of Dyrk2 in regulating p53 and 46 telomerase in vivo. 47 48

The Dyrk2 level remains constant during 49 the cell cycle, without intracellular re-distribution 50 (data not shown). These results suggest that the 51

unknown upstream regulator(s) may control 52 Dyrk2-TERT interaction. Previously, it was shown 53 that Dyrk2 phosphorylation by ATM controls 54 nuclear stabilization of Dyrk2 (51), implying 55 possible connection of telomerase regulation with 56 DNA damage response via Dyrk2. Therefore, it 57 will be interesting to identify the upstream 58 regulator of Dyrk2 in telomerase regulation. 59 Additionally, it is also necessary to evaluate the 60 effects of Dyrk2 on telomerase’s non-telomeric 61 functions in stem cells and cancer (25,52-57) (Fig. 62 6A). Taken together, our findings suggest that 63 Dyrk2-associated E3 ligase complex induces 64 TERT destabilization, and propose a novel 65 molecular mechanism of telomerase homeostasis. 66

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Acknowledgements 1 We thank Pierre McCrea, Lei Li, and Junjie Chen for their helpful comments on the manuscript and 2 Junjie Chen and Subbareddy Maddika for providing reagents. 3 4 Footnotes 5 *This work was supported by the University Cancer Foundation (IRG-08-061-01), a Center for Stem Cell 6 and Developmental Biology Transformative Pilot Grant (MD Anderson), an Institutional Research Grant 7 (MD Anderson), a New Faculty Award (MD Anderson Cancer Center Support Grant), and a Metastasis 8 Research Center Grant (MD Anderson). The DNA Analysis Core Facility was supported by the National 9 Institutes of Health through MD Anderson’s Cancer Center Support Grant, CA016672. 10 1To whom correspondence should be addressed: Jae-Il Park, Department of Experimental Radiation 11 Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, 12 TX 77030, USA; tel.: (713) 792-3659, fax.: (713) 794-5369, email: jaeil@mdanderson.org. 13 2The abbreviations used are Dyrk2: dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2, 14 TERT: telomerase reverse transcriptase, TERC: telomerase RNA template, TRAP: telomerase rapid 15 amplification protocol, EDVP: EDD, DDB1, and VprBP complex 16 17 18

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Figure Legends 1 2 Figure 1. TERT protein downregulation by Dyrk2-associated E3 ligase complex 3 (A and B) Instability of TERT protein. In HeLa cells that stably expressed FLAG-TERT (HeLa-TERT), 4 total translated proteins were labeled with 35S-Met and chased for each time point (0, 2, 4, and 6 hours). 5 Cell lysates were immunoprecipitated (FLAG) and analyzed using autoradiography (A). Solid arrow, full- 6 length of TERT; empty arrows, cleaved TERT fragments. The half-life (T1/2) of TERT protein was 7 calculated using an ImageJ analysis (B). The plot was derived from the intensity of the TERT protein on 8 autoradiography (A). N=3; error bars, standard deviation. 9 (C) Dyrk2 downregulation of TERT protein expression. HeLa (GFP-expressing control) and HeLa-TERT 10 cells were transiently transfected with three plasmids (Dyrk2, Dyrk3, and FOXO3A; 24 h) and harvested 11 to quantify TERT protein level using IB (FLAG). FOXO3A was used as a negative control. The 12 expression of HA-Dyrk2, Dyrk3, and HA-FOXO3A was confirmed using IB (HA). 13 (D) Dyrk2 downregulation of TERT protein expression in a dose-dependent manner. HeLa-TERT cells 14 were transfected with HA-Dyrk2 at different concentrations (0.5, 1, and 2 µg of plasmids) and then 15 analyzed using IB. Brg-1 and BAF57 were used as negative controls. 16 (E) Dyrk2 inhibition of telomerase activity. 293T and HeLa cells were transiently transfected (24 h) with 17 Dyrk2 for TRAP assays. An RNase A-treated sample of cell lysates (lane 5) was used as a negative 18 control. 19 (F) HeLa-TERT cells were transfected with Dyrk2 siRNA (36 h) and analyzed using IB (TERT and 20 Dyrk2). 21 (G) HeLa-TERT cells were stably transduced with shRNA against Dyrk2 (clones #3 and #4) and analyzed 22 using IB (lanes 2 and 4). Dyrk2-depletion-induced upregulation of TERT protein was rescued by shRNA 23 nontargetable Dyrk2-pMGIB retroviral infection (lanes 3 and 5). 24 (H and I) Increased TERT protein stability by Dyrk2 depletion. HeLa-TERT stably expressing shGFP or 25 shDyrk2 cells were treated with cycloheximide (CHX) at each time point. Then cell lysates were analyzed 26 for IB (H) and quantified using ImageJ (I). 27 28 Figure 2. Dyrk2 binds to TERT and phosphorylates its serine residue. 29 (A) Binding of Dyrk2 to TERT. 293T cells were transiently transfected with FLAG-TERT and HA-Dyrk2 30 plasmids. Twenty-four hours later, 293T cell lysates were analyzed using reciprocal IP with either anti- 31 HA or anti-FLAG antibodies. Dyrk2-TERT interaction was detected (lane 4, upper panel; lane 8, lower 32 panel). 33 (B) Direct interaction of Dyrk2 with TERT. Purified GST-EGFP (control) and GST-Dyrk2 proteins were 34 used for a GST pull-down assay by incubating each protein with HeLa-TERT cell lysates. The resulting 35 GST-protein complex was resolved using IB (FLAG). 36 (C) Association of endogenous TERT with Dyrk2. Endogenous TERT-Dyrk2 interaction was analyzed 37 using IP (HA) and IB (Dyrk2) with parental (TERT+/+) and TERT knockin (TERTHA/+) mouse embryonic 38 stem cells. 39 (D and E) Binding domain mapping of the TERT-Dyrk2 interaction. 40 (D) Various FLAG-TERT fragments and full-length HA-Dyrk2 plasmids were cotransfected into 293T 41 cells. After 36 h, each lysate of these cells was analyzed using IP (FLAG) and IB (HA). Of note is that 42 T4, T5, and T6 deletion mutants interacted with Dyrk2, indicating that a C-terminal domain including the 43 RT domain is necessary for TERT-Dyrk2 interaction. RID1 and 2, RNA-interacting domain 1 and 2; N, 44 N-terminus; C, C-terminus; ΔC, C-terminus deleted mutant. 45 (E) Each FLAG-Dyrk2 fragment with full-length HA-tagged TERT (HA-TERT) was cotransfected into 46 293T cells. Lysates of these cells were analyzed using IP (FLAG) and IB (HA). D1 fragment lacking a 47 kinase domain failed to interact with TERT protein. Also, deletion of the C-terminus in Dyrk2 was 48 dispensable for TERT interaction, indicating that the Dyrk2 kinase domain is sufficient for TERT 49 interaction. N, N-terminus; KD, kinase-dead; DC, ΔC-terminus deleted mutant; ΔN, N-terminus deleted 50 mutant. 51

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(F) Kinase activity of Dyrk2 is not required for TERT-Dyrk2 interaction but is necessary for TERT 1 protein degradation. In HeLa cells stably expressing HA-TERT, the plasmids FOXO3A, Dyrk2, and 2 Dyrk2-kinase dead mutant were transiently transfected (24 h) for assessment of TERT protein expression 3 using IB (HA) and TERT association with Dyrk2 (IP, FLAG; IB, HA). Of note, whereas the KD mutant 4 still bound to TERT (lanes 4, lower panel), it did not downregulate TERT protein expression (lane 4, top 5 panel). 6 (G) Dyrk2 phosphorylation of TERT protein. In vitro transcribed and translated TERT (substrate) and 7 Dyrk2 (kinase) were incubated with 32P-ATP. Dyrk2 phosphorylated TERT (lane 2, arrowhead). 8 (H) Evolutionary conservation of the Dyrk2 consensus substrate sequence (R/KXX(X)S/TP) in TERT in 9 vertebrates. Serine (red): candidate phosphorylation site for Dyrk2. 10 (I) Phosphorylation of TERT at Ser457 by Dyrk2. GST-EGFP (control), wild-type TERT, and Ser457Ala 11 (mutant) TERT fragments were incubated with Dyrk2 for in vitro kinase assay. GST-EGFP served as a 12 negative control for the kinase reaction. Wild-type TERT was phosphorylated by Dyrk2 (lane 4), but the 13 Ser457Ala mutant was not (lane 6). 14 15 Figure 3. The Dyrk2-EDVP E3 ligase complex ubiquitinates TERT for degradation. 16 (A) Dyrk2-increased TERT ubiquitination. HeLa cells were transiently transfected with UBC, TERT, and 17 Dyrk2 plasmids. After 36 h, cells were collected for IP (HA-tagged UBC) and IB (FLAG-TERT) 18 analysis. Dyrk2 downregulated the total level of TERT protein expression (lanes 2 and 3, input), and 19 increased TERT ubiquitination (lanes 2 and 3, IP). Blots were normalized by TERT input and quantified 20 using ImageJ. 21 (B) Association of the EDVP E3 ligase complex with TERT. 293T cells transfected with HA-TERT, 22 Myc-tagged EDD, FLAG-tagged DDB1, Myc-tagged VprBP, or FLAG-tagged Dyrk2 were analyzed 23 using IP (FLAG or Myc) and IB (HA). Of note is that each component of the EDVP complex bound to 24 TERT protein (lanes 4-7, top panel). 25 (C) Association of TERT with endogenous EDVP E3 ligase components. HeLa-TERT cell lysates were 26 analyzed using IP and IB. 27 (D and E) Inhibition of telomerase activity by the EDVP E3 ligase complex. 293T cells transiently 28 transfected with each EDVP component were analyzed for telomerase activity using TRAP assays (D). 29 To quantify the effects of Dyrk2-EDVP on telomerase activity, the TRAP assay results were quantified 30 using the ImageJ software program (E) and plotted (n = 3). The error bars indicate standard deviation. 31 RU, relative unit. 32 (F) VprBP depletion stabilizes TERT protein. HeLa-TERT cells transfected with VprBP siRNA were 33 analyzed using IB. Of note is that knockdown of VprBP expression increased the level of TERT protein, 34 indicating an endogenous function of VprBP in TERT destabilization. 35 (G) Mediation of the TERT-EDVP E3 ligase complex by Dyrk2. HeLa-TERT cells were transfected with 36 a control siRNA (si-Control) or Dyrk2 siRNA (si-Dyrk2), and analyzed using IP (FLAG) and IB. Under 37 Dyrk2-depleted conditions, both EDD and VprBP exhibited decreased binding to TERT. Of note is that 38 DDB1-TERT interaction was not affected by Dyrk2 shRNA, indicating an EDVP-independent DDB1 39 association with TERT. 40 (H) Requirement of Dyrk2 for EDVP E3 ligase-mediated TERT ubiquitination. For in vitro ubiquitination 41 assays of TERT, E1, E2, and E3 components, and Dyrk2 were incubated with TERT protein. Next, 42 ubiquitinated TERT was analyzed using IB (FLAG and ubiquitin). In the presence of Dyrk2, TERT was 43 ubiquitinated (lane 4). Of note, bacterially expressed GST-Dyrk2 failed to ubiquitinate TERT owing to a 44 lack of posttranslational modification of GST-Dyrk2 activity (tyrosine phosphorylation in the activation 45 loop of Dyrk2; lane 2). 46 47 Figure 4. Cell cycle-dependent TERT regulation by Dyrk2 48 (A) The cell cycle of HeLa (GFP-expressing control cells) and HeLa-FLAG-TERT + HA-Dyrk2 cells 49 was arrested by treatment with thymidine (2 mM, 24 hours), serum starvation (48 hours), or nocodazole 50 (100 μM, 14 hours). Cell lysates from each condition were analyzed by co-immunoprecipitation (HA) and 51

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immunoblotting (FLAG) assays. TERT protein level was inversely correlated with the TERT-Dyrk2 1 association (see lanes 3 and 5). Blots were quantified using ImageJ. 2 (B-D) HeLa-FLAG-TERT + HA-Dyrk2 cells were synchronized using thymidine double block (200 nM, 3 more than 17 hours each) and released for cell cycle progression. At each time point (0, 2, 4, 6, and 10 4 hours), cells were analyzed using flow cytometry (B) and co-immunoprecipitation (HA)-immunoblotting 5 (FLAG) assays (C). The physical association between TERT and Dyrk2 during the cell cycle was 6 quantified and plotted (ImageJ) (D) (N=2). When the TERT protein level decreased during the G2/M 7 phase, the TERT-Dyrk2 association increased. TERT:Dyrk2/TERT indicates the normalized TERT- 8 Dyrk2 interaction by total TERT protein level (asterisk). 9 (E and F) HeLa-TERT cells transiently transfected with siControl or siDyrk2 were synchronized by 10 thymidine double block and released for cell cycle progression. At different time points (0, 2, 4, 6, and 8 11 hours), HeLa-TERT cells were analyzed using immunoblotting assays for TERT protein (E). TERT 12 protein levels were quantified using ImageJ and plotted (N=3) (F). Compared with the control (lanes 2-8), 13 Dyrk2-depleted HeLa-TERT cells exhibited an overall increase in TERT protein levels (lanes 10-14). 14 Error bars = standard deviation; NC: nocodazole treatment (100 μM, 14 hours). 15 (G) Hyperactivation of endogenous telomerase activity by Dyrk2 knockdown. MCF-7 cells were stably 16 transduced with lentivirus expressing shGFP (control) or shDyrk2. shDyrk2 non-targetable Dyrk2 17 retrovirus was also stably transduced for reconstitution assay (lane 3). Cell lysates were prepared for 18 telomere rapid amplification protocol (TRAP) using real-time PCR. N=3; Student’s t-test; error bars, 19 standard deviation. 20 21 Figure 5. Dyrk2 mutant fails to induce TERT de-stabilization 22 (A) Three-dimensional structure of Dyrk2 protein. DOI: 10.2210/pdb3k2l/pdb. The protein kinase domain 23 (blue) is based on PFAM (PF00069). Y382 is in the activation loop of the kinase domain for 24 autophosphorylation. The S471X nonsense mutation results in the partial loss (two α-helices and two β- 25 sheets) of the kinase domain (red). 26 (B) Dyrk2 S471X had no effects on TERT protein downregulation. HeLa-TERT cells were transiently 27 transfected with Dyrk2 (wild-type) or S471X mutant. Twenty-four hours after transfection, cells were 28 analyzed for TERT quantification using an immunoblotting assay. (FLAG). Blots were normalized by 29 tubulin and quantified using ImageJ. 30 (C) Dyrk2 S471X mutant fails to inhibit telomerase activity. 293T cells were transiently transfected with 31 wild-type or S471X Dyrk2 plasmids and analyzed for telomerase activity using a TRAP assay. I.C.: 32 internal control. Ladders indicate telomeric repeats (TTAGGG), as represented by six-base pair 33 increments. Autoradiography was quantified using ImageJ. 34 (D) S471X nonsense mutation of Dyrk2 disrupts interaction with EDVP E3 ligase components. HeLa 35 cells were transiently transfected with wild-type or S471X mutant Dyrk2 plasmids and analyzed for 36 protein interaction by co-immunoprecipitation (EDD and VprBP) and immunoblotting assays. 37 38 Fig. 6. Illustration of Dyrk2-mediated telomerase regulation 39 (A) In normal cells expressing telomerase, Dyrk2 phosphorylates TERT protein (i). Then, phosphorylated 40 TERT is recognized by the Dyrk2-associated EDD-DDB1-VprBP E3 ligase complex (ii) for subsequent 41 ubiquitination and protein degradation of TERT. In contrast, Dyrk2 mutation (S471X) fails to recruit the 42 EDVP-E3 ligase to TERT protein, which did not induce TERT ubiquitination and degradation. Of note, 43 the upstream regulatory mechanism of Dyrk2 is still ambiguous. 44 (B) Cell cycle-dependent TERT regulation by Dyrk2-E3 ligase. Stable TERT protein is associated with 45 TERC, a RNA template of telomerase, and synthesizes a telomere repeat sequence at the early S phase of 46 the cell cycle. During the G2/M phase, the Dyrk2-E3 ligase targets TERT protein for degradation and 47 inhibits telomerase activity. 48

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TER

T pr

otei

n (a

rbitr

ary

unit)

0

2000

4000

6000

8000

10000

12000

14000

16000

0 2 4 6 8

S-Met/Cys-TERT35

T = 2.1 hr1/2

Hour

0 2 4 6 Hours175

83

62

48kD

S-TE

RT

35

A BJung_Fig1

-0.1499

-0.0685

shDyrk2shGFP

0 30 60 90 1200.0

0.2

0.4

0.6

0.8

1.0

1.2

TER

T pr

otei

n (a

rbitr

ary

unit)

CHX (min.)

I

H

FLAG-TERTDyrk2 (long exp.)

Tubulin

0 30 60 90 120 0 30 60 90 120shDyrk2 shGFP

Dyrk2 (short exp.)

TERTDyrk2

Dyrk2Brg-1

BAF57

Tubulin

Dyrk2RNaseA293T HeLa

- + - +

I.C.

(TTAG

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C

D

E

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CHX (min.)

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IP: FLAG IP: HA Input

TERTIB: HA

Dyrk2IB: FLAG

HA-TERTFLAG-Dyrk2

+ ++ +

+ ++ +

+ ++ +

1 2 3 4 5 6 7 8 9 10 11 12

IB: FLAGTERT (pull down)

InputTERT

GST-Dyrk2

GST-EGFP

GST-pull down

GST-Dyrk

2

GST-EGFP

A

B C

D E

T1 T2 T3 T4 T5 T6 : FLAGHA-Dyrk2

IB: HA

IB: FLAG

IB: HA

IB: FLAG

InputIP

: FLAG

IgG-H.C.

(Dyrk2)

(TERT)

(Dyrk2)

WTT1: NT2: RID1T3: RID2T4: RTT5: CT6: 6C

RID1 RID2 RTTERT (1132 aa)

---

+++

+++

+++Interaction

KDWTD1: ND2: KDD3: 6CD4: 6N

Dyrk2 (528 aa)

+++++++

+++-

Interaction

D1 D2 D3 D4HA-TERT

: FLAGIB: HA

IB: FLAG

IB: HA

IB: FLAG

InputIP

: FLAG

IgG-L.C.

(TERT)

(Dyrk2)

(TERT)IgG-H.C.

Contro

l

FLAG-F

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FLAG-D

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FLAG-D

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FoxO3A

Dyrk2Dyrk2-KD

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58

80

175

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+ +

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32Input

LLRQHSSPWQVYLLRQHSSPWQVYLLRQLSSPWQVYLLRQLSSPWQVYLLKQHSSIWQVYLLKSHSSPYRVY

Human CowRatMouse Frog Zebrafish

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F G

H I

Phospho-TERT

+ + GST-S457AGST-WTGST-EGFP

+++ +

GST-TERT

GST-EGFP

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(WT, S457A)

HA/++/+

IgG-H.C.Dyrk2

Dyrk2

TERT

Tubulin

N.S.

TERTIB: 3F10

InputIP

: HA

-7

TERT

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B CA

+ + Dyrk2FLAG-TERTHA-Ubc

+ ++ + + +

TERT

IgG-H.C.

Ub-TERT

Dyrk2

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: HA

-7

Tubulin

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0.0

0.2

0.4

0.6

0.8

1.0

Telo

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ase

activ

ity (R

U)

Con

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ED

DD

DB

1V

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IB: FLAG

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Ub-TERT

GST-Dyrk2Dyrk2 IP+

+

UbiquitinUBE1 (E1)UbcH5 (E2)EDDDDB1VprBPFLAG-TERT

175

175

kD

E

F

HeLa

HeLa-F

LAG-T

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EDD

DDB1

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IgG-H.C.

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DDB1

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D

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No

Ab

IgG

FLA

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AG

Myc

FLA

G

: IP

HA-TERTFLA

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1 2.6

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02

46

10

(Hrs)DNA content

Cel

l num

ber

Tubulin

PCNA

TPP1

TRF2

Dyrk2

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IgG-H.C.

Dyrk2

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Parental FLAG-TERT+HA-Dyrk2

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icle

Veh

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mid

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um

Noc

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IP: H

A-7

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C

D

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Thymidine release (hr)0

Dyrk2TERTDyrk2TERT

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Input

2 4 6 10 Nocodazole

HeLa-FLAG-TERT/HA-Dyrk2

Arb

itra

ry u

nit

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TERT:Dyrk2/TERT*

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8

10

12

14

G1 S MG2 G1

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0

1

2

3

4

5

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siControlsiDyrk2

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pro

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0 2 4 6 8 NC.

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k

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

GT

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tivity

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it)

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+ Dyrk

2

0

5

10

15

20

25 p<0.0001p=0.0006

Jung_Fig4

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S471

Y382

C

N

Kinase domainDeleted in S471X

(autophosphorylation)

B

C

TERT

Dyrk2 S471XTubulin

Dyrk2

FLAG-D

yrk2

FLAG-D

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471X

Contro

l

1 0.12 0.70

A

D

FLAG-D

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471X

FLAG-D

yrk2

Contro

l

- I.C.

(TTAGGG)n

1 0.87 0.28

EDD

Dyrk2

Dyrk2 S471X

Tubulin

FLAG-D

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FLAG-D

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471X

EDD

VprBPDyrk2

Dyrk2 S471XIgG-H.C.

VprBP

Contro

l

InputIP

: FLAG

Jung_Fig5

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TERT

VprBPDyrk2

DDB1

EDD

p

(Ub)n

i ii

TERT degradation

A B

TERT stabilization

S

G2

M

G1

TERT degradation

p

TERT

Dyrk2

TERC

Jung_Fig6

Dyrk2 mutation

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Hae-Yun Jung, Xin Wang, Sohee Jun and Jae-Il ParkDegradation

Dyrk2-Associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT

published online January 28, 2013J. Biol. Chem. 

  10.1074/jbc.M112.416792Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2013/01/28/M112.416792.DC1

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