Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

Abstract

Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

Fig 1

C53 knockdown delays mitotic entry of HeLa cells. A. Experimental procedure for double thymidine arrest and C53 knockdown. B. Knockdown of endogenous C53 did not affect DNA synthesis. HeLa cells were transfected with shRNAs and synchronized at the G1/S transition as described in the methods. Cells were pulse labeled with BrdU (50 μM) for 30 min at indicated time points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of three independent experiments. C. shRNA-mediated knockdown of C53 protein delayed mitotic entry. Cell cycle progression of more than 1,000 cells were recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. D. siRNA-mediated C53 depletion led to delayed mitotic entry. Two C53 siRNAs (10 nM) were transfected into HeLa cells by HiPerfect transfection agent. The negative control was Allstar siRNA from Qiagen. Nocodazole (100 ng/ml) was added in the medium after the second release. Cells were harvested at indicated time points and subject to phospho-H3 staining and flow cytometry analysis. E. C53 knockdown causes accunluaiton of inactive Cdk1. Cell lysates were collected at indicated time points after release from the second block, and subjected to immunoblotting using indicated antibodies. F. C53 knockdown delayed Cdk1 activation. Cdk1 was precipitated from cell lysates at indicated time, and subject to in vitro kinase assay with histone H1 as a substrate. Relative intensity was measured with Openlab software and marked at the bottom of the gel. All data are represented as mean ± SEM.

Fig 2

C53 modulates checkpoint kinase-mediated DNA damage response. Ectopic expression of C53 suppressed the DNA damage checkpoint response. HeLa cells were transfected with the C53 or control vectors. At 24 hours after transfection, the cells were treated with etoposide (Etop, 20 μM, 18 hours), doxorubicin (Dox, 1 μM, 5 hours), hydroxyurea (HU, 4 mM, 18 hours). Cell lysates were subjected to SDS-PAGE and immunoblotting of indicated antibodies.

Fig 3

C53 interacts with Chk1 and antagonizes its activity. A. C53 co-immunoprecipitated with Chk1 and Chk2. Both C53-Flag and Myc-Chk1/Chk2 were overexpressed in HeLa cells. C53-Flag fusion protein was pulled down by Flag (M2) antibody-conjugated agarose beads, and Chk1/Chk2 were detected by immunoblotting using Myc antibody. B. Co-immunoprecipitation of endogenous C53 and Chk1. Endogenous C53 was pulled down with C53 antibody, and Chk1 was detected by immunoblotting using Chk1 antibody. IgG HC indicated IgG heavy chain. C. Mapping of C53’s Chk1-interacting domains. Myc-Chk1 and C53-Flag and its derivatives were overexpressed in HeLa cells. C53-Flag protein was pulled down by M2-conjugated agarose beads. The presence of Chk1 was detected by Myc immunoblotting. D. Full-length and C-terminal fragment of C53 protein antagonized Chk1-mediated Cdk1 inactivation. HeLa cells were transiently transfected with Myc-Chk1 and C53 constructs. Cells were collected at 24 hours after transfection. Total cell lysates were subjected to immunoblotting using indicated antibodies. E. Chk1 inhibitor UCN-01 prevented delayed Cdk1 activation induced by C53 knockdown. HeLa cells were synchronized by double thymidine block. UCN-01 (300 nM) was added into the medium at 5 hours after release from the second block. Cells were collected at 10 hours after release, and the total cell lysates were subjected to SDS-PAGE and immunoblotting using indicated antibodies. F. C53 constructs were used in this study.

Fig 4

A portion of C53 is localized at the centrosome. A. Immunoblotting of cell lysates with or without C53 knockdown using purified C53 rat polyclonal antibody. B. Immunostaining of endogenous C53. U-2 OS cells were stained with C53 polyclonal rat antibody and γ-tubulin monoclonal antibody (GTU-88, Sigma). For depletion, the primary antibodies were incubated with immobilized GST or GST-C53 fusion proteins. For shRNA knockdown, U-2 OS cells were transfected with C53 shRNA. After 4-day puromycin selection, the cells were subjected to immunostaining. C. C53 staining in HeLa cells. To better visualize the centrosomal staining of C53, HeLa cells were pre-extracted with 0.5% Triton in PBS for 5 min at RT. The prophase is indicated by centrosomal separation. All centrosomes were marked by arrowheads.

Figure 5

C53 regulates local activation of Cdk1 at the centrosomes. A. More centrosomes were decorated by inactive Cdk1 in C53-depleted cells. U-2 OS cells were infected with retroviruses expressing either control shRNA or C53 shRNA. After 4-day puromycin selection, U-2 OS cells were immunostained with p-Y15-Cdk1 and $\tilde{\gamma }$tubulin antibodies. The numbers of cells containing inactive Cdk1-decorated centrosomes were scored manually. More than 200 cells were examined in each of three independent experiments. Data are represented as mean ± SEM. B. Total p-Y15-Cdk1 was evaluated by immunoblotting. U-2 OS cells were infected with retroviruses expressing control shRNA or C53 shRNA. After 4-day drug selection (puromycin 2 μg/ml), cells were collected and subjected to immunoblotting using indicated antibodies. C. Centrosome-targeting C53 reduced the number of inactive Cdk1-decorated centrosomes in U-2 OS cells. C53-GFP-PACT or GFP-PACT constructs were transfected into U-2 OS cells. After 24 hours, cells were fixed and immunostained with p-Y15-Cdk1 antibody. p-Y15-Cdk1-decorated centrosomes were scored as described above. More than 200 cells were examined in each of three independent experiments. Data are represented as mean + SEM. D. Overexpression of centrosome-targeting C53 promoted overall Cdk1 activation. U-2 OS cells (6-well plate) were transfected with C53-GFP-PACT (200 ng) or GFP-PACT construct (200 ng). Cells were collected after 24 hours, and subjected to immunoblotting using indicated antibodies.