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. 2013 Mar 8;288(10):7252-62.
doi: 10.1074/jbc.M112.416792. Epub 2013 Jan 28.

Dyrk2-associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation

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Dyrk2-associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation

Hae-Yun Jung et al. J Biol Chem. .
Free PMC article

Abstract

Telomerase maintains the telomere, a specialized chromosomal end structure that is essential for genomic stability and cell immortalization. Telomerase is not active in most somatic cells, but its reactivation is one of the hallmarks of cancer. In this study, we found that dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (Dyrk2) negatively regulates telomerase activity. Dyrk2 phosphorylates TERT protein, a catalytic subunit of telomerase. Phosphorylated TERT is then associated with the EDD-DDB1-VprBP E3 ligase complex for subsequent ubiquitin-mediated TERT protein degradation. During the cell cycle, Dyrk2 interacts with TERT at the G2/M phase and induces degradation. In contrast, depletion of endogenous Dyrk2 disrupts the cell cycle-dependent regulation of TERT and elicits the constitutive activation of telomerase. Similarly, a Dyrk2 nonsense mutation identified in breast cancer compromises ubiquitination-mediated TERT protein degradation. Our findings suggest the novel molecular mechanism of kinase-associated telomerase regulation.

Figures

FIGURE 1.
FIGURE 1.
TERT protein down-regulation by Dyrk2-associated E3 ligase complex. A and B, instability of TERT protein. In HeLa cells that stably expressed FLAG-TERT (HeLa-TERT), total translated proteins were labeled with [35S]Met and chased for each time point (0, 2, 4, and 6 h). Cell lysates were immunoprecipitated (FLAG) and analyzed using autoradiography (A). Solid arrow, full-length of TERT; empty arrows, cleaved TERT fragments. The half-life (T1/2) of TERT protein was calculated using an ImageJ analysis (B). The plot was derived from the intensity of the TERT protein on autoradiography (A). n = 3; error bars, S.D. C, Dyrk2 down-regulation of TERT protein expression. HeLa (GFP-expressing control) and HeLa-TERT cells were transiently transfected with three plasmids (Dyrk2, Dyrk3, and FOXO3A; 24 h) and harvested to quantify TERT protein level using IB (FLAG). FOXO3A was used as a negative control. The expression of HA-Dyrk2, Dyrk3, and HA-FOXO3A was confirmed using IB (HA). D, Dyrk2 down-regulation of TERT protein expression in a dose-dependent manner. HeLa-TERT cells were transfected with HA-Dyrk2 at different concentrations (0.5, 1, and 2 μg of plasmids) and then analyzed using IB. Brg-1 and BAF57 were used as negative controls. E, Dyrk2 inhibition of telomerase activity. 293T and HeLa cells were transiently transfected (24 h) with Dyrk2 for TRAP assays. An RNase A-treated sample of cell lysates (lane 5) was used as a negative control. F, HeLa-TERT cells were transfected with Dyrk2 siRNA (36 h) and analyzed using IB (TERT and Dyrk2). G, HeLa-TERT cells were stably transduced with shRNA against Dyrk2 (clones #3 and #4) and analyzed using IB (lanes 2 and 4). Dyrk2 depletion-induced up-regulation of TERT protein was rescued by shRNA nontargetable Dyrk2-pMGIB retroviral infection (lanes 3 and 5). H and I, increased TERT protein stability by Dyrk2 depletion. HeLa-TERT stably expressing shGFP or shDyrk2 cells was treated with cycloheximide (CHX) at each time point. Then cell lysates were analyzed for IB (H) and quantified using ImageJ (I).
FIGURE 2.
FIGURE 2.
Dyrk2 binds to TERT and phosphorylates its serine residue. A, binding of Dyrk2 to TERT. 293T cells were transiently transfected with FLAG-TERT and HA-Dyrk2 plasmids. Twenty-four hours later, 293T cell lysates were analyzed using reciprocal IP with either anti-HA or anti-FLAG antibodies. Dyrk2-TERT interaction was detected (lane 4, upper panel; lane 8, lower panel). B, direct interaction of Dyrk2 with TERT. Purified GST-EGFP (control) and GST-Dyrk2 proteins were used for a GST pull-down assay by incubating each protein with HeLa-TERT cell lysates. The resulting GST-protein complex was resolved using IB (FLAG). C, association of endogenous TERT with Dyrk2. Endogenous TERT-Dyrk2 interaction was analyzed using IP (HA) and IB (Dyrk2) with parental (TERT+/+) and TERT knockin (TERTHA/+) mouse embryonic stem cells. D and E, binding domain mapping of the TERT-Dyrk2 interaction. D, various FLAG-TERT fragments and full-length HA-Dyrk2 plasmids were cotransfected into 293T cells. After 36 h, each lysate of these cells was analyzed using IP (FLAG) and IB (HA). Of note is that T4, T5, and T6 deletion mutants interacted with Dyrk2, indicating that a C-terminal domain including the RT domain is necessary for TERT-Dyrk2 interaction. RID1 and 2, RNA-interacting domain 1 and 2; N, N terminus; C, C terminus; ΔC, C terminus-deleted mutant. E, each FLAG-Dyrk2 fragment with full-length HA-tagged TERT (HA-TERT) was cotransfected into 293T cells. Lysates of these cells were analyzed using IP (FLAG) and IB (HA). D1 fragment lacking a kinase domain failed to interact with TERT protein. Also, deletion of the C terminus in Dyrk2 was dispensable for TERT interaction, indicating that the Dyrk2 kinase domain is sufficient for TERT interaction. N, N terminus; KD, kinase-dead; ΔC, ΔC terminus-deleted mutant; ΔN, N terminus-deleted mutant. F, kinase activity of Dyrk2 is not required for TERT-Dyrk2 interaction but is necessary for TERT protein degradation. In HeLa cells stably expressing HA-TERT, the plasmids FOXO3A, Dyrk2, and Dyrk2-kinase dead mutant were transiently transfected (24 h) for assessment of TERT protein expression using IB (HA) and TERT association with Dyrk2 (IP, FLAG; IB, HA). Of note, whereas the KD mutant still bound to TERT (lanes 4, lower panel), it did not down-regulate TERT protein expression (lane 4, top panel). G, Dyrk2 phosphorylation of TERT protein. In vitro transcribed and translated TERT (substrate) and Dyrk2 (kinase) were incubated with [32P]ATP. Dyrk2 phosphorylated TERT (lane 2, arrowhead). H, evolutionary conservation of the Dyrk2 consensus substrate sequence (R/KXX(X)S/TP) in TERT in vertebrates. Serine (red): candidate phosphorylation site for Dyrk2. I, phosphorylation of TERT at Ser457 by Dyrk2. GST-EGFP (control), wild-type TERT, and S457A (mutant) TERT fragments were incubated with Dyrk2 for in vitro kinase assay. GST-EGFP served as a negative control for the kinase reaction. Wild-type TERT was phosphorylated by Dyrk2 (lane 4), but the S457A mutant was not (lane 6).
FIGURE 3.
FIGURE 3.
The Dyrk2-EDVP E3 ligase complex ubiquitinates TERT for degradation. A, Dyrk2-increased TERT ubiquitination. HeLa cells were transiently transfected with UBC, TERT, and Dyrk2 plasmids. After 36 h, cells were collected for IP (HA-tagged UBC) and IB (FLAG-TERT) analysis. Dyrk2 down-regulated the total level of TERT protein expression (lanes 2 and 3, input), and increased TERT ubiquitination (lanes 2 and 3, IP). Blots were normalized by TERT input and quantified using ImageJ. B, association of the EDVP E3 ligase complex with TERT. 293T cells transfected with HA-TERT, Myc-tagged EDD, FLAG-tagged DDB1, Myc-tagged VprBP, or FLAG-tagged Dyrk2 were analyzed using IP (FLAG or Myc) and IB (HA). Of note is that each component of the EDVP complex bound to TERT protein (lanes 4–7, top panel). C, association of TERT with endogenous EDVP E3 ligase components. HeLa-TERT cell lysates were analyzed using IP and IB. D and E, inhibition of telomerase activity by the EDVP E3 ligase complex. 293T cells transiently transfected with each EDVP component were analyzed for telomerase activity using TRAP assays (D). To quantify the effects of Dyrk2-EDVP on telomerase activity, the TRAP assay results were quantified using the ImageJ software program (E) and plotted (n = 3). The error bars indicate S.D. RU, relative unit. F, VprBP depletion stabilizes TERT protein. HeLa-TERT cells transfected with VprBP siRNA were analyzed using IB. Of note is that knockdown of VprBP expression increased the level of TERT protein, indicating an endogenous function of VprBP in TERT destabilization. G, mediation of the TERT-EDVP E3 ligase complex by Dyrk2. HeLa-TERT cells were transfected with a control siRNA (si-Control) or Dyrk2 siRNA (si-Dyrk2), and analyzed using IP (FLAG) and IB. Under Dyrk2-depleted conditions, both EDD and VprBP exhibited decreased binding to TERT. Of note is that DDB1-TERT interaction was not affected by Dyrk2 shRNA, indicating an EDVP-independent DDB1 association with TERT. H, requirement of Dyrk2 for EDVP E3 ligase-mediated TERT ubiquitination. For in vitro ubiquitination assays of TERT, E1, E2, and E3 components, and Dyrk2 were incubated with TERT protein. Next, ubiquitinated TERT was analyzed using IB (FLAG and ubiquitin). In the presence of Dyrk2, TERT was ubiquitinated (lane 4). Of note, bacterially expressed GST-Dyrk2 failed to ubiquitinate TERT because of a lack of post-translational modification of GST-Dyrk2 activity (tyrosine phosphorylation in the activation loop of Dyrk2; lane 2).
FIGURE 4.
FIGURE 4.
Cell cycle-dependent TERT regulation by Dyrk2. A, cell cycle of HeLa (GFP-expressing control cells) and HeLa-FLAG-TERT + HA-Dyrk2 cells was arrested by treatment with thymidine (2 mm, 24 h), serum starvation (48 h), or nocodazole (100 μm, 14 h). Cell lysates from each condition were analyzed by co-immunoprecipitation (HA) and immunoblotting (FLAG) assays. TERT protein level was inversely correlated with the TERT-Dyrk2 association (see lanes 3 and 5). Blots were quantified using ImageJ. B—D, HeLa-FLAG-TERT + HA-Dyrk2 cells were synchronized using thymidine double block (200 nm, more than 17 h each) and released for cell cycle progression. At each time point (0, 2, 4, 6, and 10 h), cells were analyzed using flow cytometry (B) and co-immunoprecipitation (HA)-immunoblotting (FLAG) assays (C), physical association between TERT and Dyrk2 during the cell cycle was quantified and plotted (ImageJ) (D) (n = 2). When the TERT protein level decreased during the G2/M phase, the TERT-Dyrk2 association increased. TERT:Dyrk2/TERT indicates the normalized TERT-Dyrk2 interaction by total TERT protein level (asterisk). E and F, HeLa-TERT cells transiently transfected with siControl or siDyrk2 were synchronized by thymidine double block and released for cell cycle progression. At different time points (0, 2, 4, 6, and 8 h), HeLa-TERT cells were analyzed using immunoblotting assays for TERT protein (E). TERT protein levels were quantified using ImageJ and plotted (n = 3) (F). Compared with the control (lanes 2–8), Dyrk2-depleted HeLa-TERT cells exhibited an overall increase in TERT protein levels (lanes 10–14). Error bars, S.D.; NC: nocodazole treatment (100 μm, 14 h). G, hyperactivation of endogenous telomerase activity by Dyrk2 knockdown. MCF-7 cells were stably transduced with lentivirus expressing shGFP (control) or shDyrk2. shDyrk2 non-targetable Dyrk2 retrovirus was also stably transduced for reconstitution assay (lane 3). Cell lysates were prepared for TRAP using real-time PCR. n = 3; Student's t test; error bars, S.D.
FIGURE 5.
FIGURE 5.
Dyrk2 mutant fails to induce TERT de-stabilization. A, three-dimensional structure of Dyrk2 protein. DOI: 10.2210/pdb3k2l/pdb. The protein kinase domain (blue) is based on PFAM (PF00069). Y382 is in the activation loop of the kinase domain for autophosphorylation. The S471X nonsense mutation results in the partial loss (two α-helices and two β-sheets) of the kinase domain (red). B, Dyrk2 S471X had no effects on TERT protein down-regulation. HeLa-TERT cells were transiently transfected with Dyrk2 (wild-type) or S471X mutant. Twenty-four hours after transfection, cells were analyzed for TERT quantification using an immunoblotting assay. (FLAG). Blots were normalized by tubulin and quantified using ImageJ. C, Dyrk2 S471X mutant fails to inhibit telomerase activity. 293T cells were transiently transfected with wild-type or S471X Dyrk2 plasmids and analyzed for telomerase activity using a TRAP assay. I.C.: internal control. Ladders indicate telomeric repeats (TTAGGG), as represented by six-base pair increments. Autoradiography was quantified using ImageJ. D, S471X nonsense mutation of Dyrk2 disrupts interaction with EDVP E3 ligase components. HeLa cells were transiently transfected with wild-type or S471X mutant Dyrk2 plasmids and analyzed for protein interaction by co-immunoprecipitation (EDD and VprBP) and immunoblotting assays.
FIGURE 6.
FIGURE 6.
Illustration of Dyrk2-mediated telomerase regulation. A, in normal cells expressing telomerase, Dyrk2 phosphorylates TERT protein (i). Then, phosphorylated TERT is recognized by the Dyrk2-associated EDD-DDB1-VprBP E3 ligase complex (ii) for subsequent ubiquitination and protein degradation of TERT. In contrast, Dyrk2 mutation (S471X) fails to recruit the EDVP-E3 ligase to TERT protein, which did not induce TERT ubiquitination and degradation. Of note, the upstream regulatory mechanism of Dyrk2 is still ambiguous. B, cell cycle-dependent TERT regulation by Dyrk2-E3 ligase. Stable TERT protein is associated with TERC, a RNA template of telomerase, and synthesizes a telomere repeat sequence at the early S phase of the cell cycle. During the G2/M phase, the Dyrk2-E3 ligase targets TERT protein for degradation and inhibits telomerase activity.

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