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. 2020 Nov;77(22):4693-4708.
doi: 10.1007/s00018-019-03436-3. Epub 2020 Feb 6.

A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis

Sihyeon An  1 , Oh Sung Kwon  1 , Jinbae Yu  1 , Sung Key Jang  2
Affiliations

A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis

Sihyeon An et al. Cell Mol Life Sci. 2020 Nov.

Abstract

During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58-a serine/threonine kinase abundantly expressed during M phase-represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from C. elegans to humans.

Keywords: CDK11/p58; Translation initiation; Translational repression in M phase; eIF3F.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
CDK11/p58 represses cap-dependent translation. a Schematic diagram of a bicistronic reporter mRNA (RCF) containing the cap structure (m7G) at the 5′ end, Renilla luciferase (Rluc) gene at the first cistron, cricket paralysis viral (CrPV) IRES at the intercistronic region, and firefly luciferase (Fluc) gene at the second cistron. b Cap- and CrPV IRES-dependent translation of the reporter mRNA (RCF) in cells ectopically expressing CDK11 isoforms were monitored by measuring Renilla and firefly luciferase activities. At 48 h post-transfection of the corresponding plasmids encoding for effector CDK11 isoforms, HeLa cells were transfected with the bicistronic reporter RNA (RCF) that was synthesized by in vitro transcription. After 4 h, cells were lysed, and luciferase activities were measured. The relative cap-dependent translation efficiencies, which were normalized to the CrPV IRES-dependent translation activity in each sample, are depicted. The cap-dependent translation activity in the control vector-transfected cells was set to 1 (lane 1). Experiments were repeated three times with duplicate samples. The columns and bars represent the means and ± standard deviations, respectively. Asterisks (**) depict P < 0.005, lanes 3, 4, and 6 compared with lane 1. c The levels of ectopically expressed proteins (CDK11 isoforms and CCND3) in cells were monitored by western blot using an anti-Flag antibody. GAPDH levels were also monitored by western blot using an anti-GAPDH antibody as an endogenous protein control
Fig. 2
Fig. 2
Translation repression in M phase is nullified by knockdown of CDK11. a Global translation in interphase or M phase-arrested cells was monitored by western blotting using SUnSET (left panel). Synchronization of cells in either interphase or M phase was achieved as described in “Materials and methods”. Puromycin was treated for 15 min after synchronization and the puromycin incorporation into newly synthesizing proteins was detected by western blotting using SUnSET. The band densities of newly synthesized proteins in interphase and M phase were measured and normalized to the amounts of total proteins measured by Coomassie blue staining (Fig. S1b). Relative values are depicted on the right panel. The sum of the band intensities from cells in interphase was set to 1 (lane 1). Experiments were repeated three times. Values are represented as the means and ± standard deviations, respectively. b Protein levels of CDK11/p110 and CDK11/p58 in interphase- and M phase-synchronized cells were monitored by western blotting using an anti-CDK11 antibody. c The effect of CDK11 knockdown on cell cycle-dependent translation. HeLa cells were transfected with either a control siRNA (lanes 1 and 2) or a siRNA against CDK11 (lanes 3 and 4) 3 h before synchronization. Translation efficiencies were measured as in a. Band densities of newly synthesized proteins in interphase and M phase were measured; relative values are depicted on the right panel. The sum of band intensities from cells in interphase treated with a control siRNA was set to 1 (lane 1). Experiments were repeated three times. The columns and bars represent the means and ± standard deviations, respectively. Asterisk (*) depicts P < 0.05, lane 4 compared with lane 2. d Protein levels of CDK11/p110 and CDK11/p58 in interphase- and M phase-synchronized cells treated with either a control siRNA (lanes 1 and 2) or an siRNA against CDK11 (lanes 3 and 4), were monitored by western blotting using an anti-CDK11 antibody
Fig. 3
Fig. 3
CDK11/p58 interacts with eIF3F. Flag-CDK11 isoforms (p110, p58, and p46) and myc-eIF3F were co-transfected to HeLa cells. The myc-eIF3F was immunoprecipitated using an anti-myc antibody-conjugated protein A agarose resin (lanes 6–10). Non-specific interactions were monitored using an anti-rabbit antibody-conjugated protein A agarose resin (control, lanes 11–15). Protein levels of CDK11 isoforms and eIF3F were monitored by western blotting using anti-Flag and anti-myc antibodies, respectively. GAPDH levels were monitored by western blotting using an anti-GAPDH antibody as an endogenous protein control
Fig. 4
Fig. 4
Endogenous eIF3F is phosphorylated by CDK11/p58 during M phase. a The phosphorylation levels of endogenous eIF3F in interphase- and M phase-synchronized HeLa cells were measured by western blotting using Phos-tag SDS-PAGE. Slower migration of phosphorylated eIF3F in Phos-tag SDS-PAGE results in separation of fast-migrating under-phosphorylated and slow-migrating hyper-phosphorylated eIF3F ( formula image -eIF3F) bands in the gel. Protein levels of formula image -eIF3F and eIF3F were detected by western blotting using an anti-eIF3F antibody. The ratios of formula image -eIF3F/total eIF3F are depicted. The levels of GAPDH were monitored as a loading control. b Phosphorylation levels of endogenous eIF3F in interphase- or M phase-synchronized HeLa cells with (lanes 3 and 4) or without knockdown (lanes 1 and 2) of CDK11 were monitored by western blotting using Phos-tag SDS-PAGE. Protein levels of formula image -eIF3F and eIF3F were detected by western blotting using an anti-eIF3F antibody. The ratios of formula image -eIF3F/total eIF3F are depicted. The levels of GAPDH were monitored as a loading control
Fig. 5
Fig. 5
Unphosphorylatable mutants of eIF3F do not repress translation. a Schematic diagrams of phospho-mimetic mutants [eIF3F(D1D2) and eIF3F(D3D4)], and unphosphorylatable mutants [eIF3F(A1A2) and eIF3F(A3A4)] of eIF3F. b The effects of ectopic expression of eIF3F(WT) and its derivatives on cap-dependent translation were monitored as described in Fig. 1 except that effector plasmids encoding eIF3F derivatives instead of CDK11 isoforms were used. The relative cap-dependent translation efficiencies, which were normalized to CrPV IRES-dependent translation activity in each sample, are depicted. The cap-dependent translation activity in control vector-transfected cells was set to 1 (lane 1). Experiments were repeated three times with duplicate samples. The columns and bars represent the means and ± standard deviations, respectively. Asterisk (*) depicts P < 0.05, lane 4 compared with lane 2. Asterisks (**) depict P < 0.005, lane 6 compared with lane 2. c The levels of ectopically expressed proteins (eIF3F and its derivatives) in cells were monitored by western blotting using an anti-Flag antibody. The level of GAPDH protein was also monitored using an anti-GAPDH antibody as an endogenous protein control
Fig. 6
Fig. 6
CDK11/p58 phosphorylates Thr255 and/or Ser258 on eIF3F. a eIF3F(A3A4) nullifies the cap-dependent translation repression by CDK11/p58, but eIF3F(A1A2) nullifies the cap-dependent translation repression by CDK11/p46. Plasmids encoding Flag-CDK11/p46, Flag-CDK11/p58 plus Flag-CCND3, myc-eIF3F, or its derivatives were co-transfected into HeLa cells as indicated. The effects of co-expression of eIF3F derivatives and CDK11 isoforms on cap-dependent translation were monitored as described in Fig. 1, except that the effector plasmids encoding both eIF3F derivatives and CDK11 isoforms were used. The relative cap-dependent translation efficiencies, which were normalized to CrPV IRES-dependent translation activity in each sample, are depicted. The cap-dependent translation activity in control vector-transfected cells was set to 1 (lane 1). Experiments were repeated three times with duplicate samples. The columns and bars represent the means and ± standard deviations, respectively. Asterisk (*) depicts P < 0.05, lane 8 compared with lane 6, and lane 11 compared with lane 10; n.s. non-significant (P > 0.05). b The levels of ectopically expressed proteins (CDK11 isoforms, eIF3F derivatives, and CCND3) in cells were monitored by western blotting using anti-Flag and anti-myc antibodies. GAPDH levels were monitored using an anti-GAPDH antibody as an endogenous protein control
Fig. 7
Fig. 7
Phosphorylation of Thr255 and/or Ser258 in eIF3F is required for M phase-specific translational repression. HeLa cell lines ectopically expressing eIF3F(WT), eIF3F(A1A2), or eIF3F(A3A4) were established as described in “Materials and methods”. a Global translation of the established cells, which were synchronized in interphase (I) or M phase (M), was monitored by western blotting using the SUnSET method. b Band densities of newly synthesized proteins in panel (a) were measured and normalized to the amount of total proteins (Fig. S3b). The relative values are depicted as the sum of normalized band intensities in interphase cells established with a control vector is set to 1. Experiments were repeated three times. The columns and bars represent the means and ± standard deviations, respectively. c The levels of Flag-eIF3F(WT), Flag-eIF3F(A1A2), and Flag-eIF3F(A3A4) in the established cells in interphase- and M phase-synchronized cells were monitored by western blotting using an anti-Flag antibody. GAPDH levels were monitored using an anti-GAPDH antibody as an endogenous protein control
Fig. 8
Fig. 8
Unphosphorylatable eIF3F(A3A4) completely nullifies M phase-specific translational repression. a HeLa cells ectopically expressing eIF3F variants were synchronized at G1/S boundary by double thymidine block and resumed cell cycle progression by removing the compound. Cells were harvested at the indicated time points after cell cycle resumption, and the global translation activity of cells was monitored by western blotting using the SUnSET method. b Band densities of newly synthesized proteins in a were measured and normalized to the amount of total proteins (Fig. S4b). The relative values are depicted as the sum of normalized band intensities of each cell line at 1 h after cell cycle resumption is set to 1. Experiments were repeated three times. The columns and bars represent the means and ± standard deviations, respectively. c The levels of CDK11/p110, CDK11/p58, Flag-eIF3F(WT), and Flag-eIF3F(A3A4) in the established cells were monitored by western blotting using anti-CDK11 and anti-Flag antibodies. GAPDH levels were monitored using an anti-GAPDH antibody as an endogenous protein control
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