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, 51 (4), 1-17

Phosphoregulation of the Oncogenic Protein Regulator of Cytokinesis 1 (PRC1) by the Atypical CDK16/CCNY Complex

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Phosphoregulation of the Oncogenic Protein Regulator of Cytokinesis 1 (PRC1) by the Atypical CDK16/CCNY Complex

Sara Hernández-Ortega et al. Exp Mol Med.

Abstract

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Generation of an Analog-Sensitive (AS) version of CDK16.
a CDK16 phosphorylates CCNY in vitro. ATPγS was used as a phosphodonor, and thiophosphorylation (ThioP) was evaluated by western blotting. CDK16 and CCNY were purified from E. coli. b Identification of the gatekeeper residue in the ATP-binding pocket by sequence alignment with other CDKs. The motif for this conserved sequence was generated using WebLogo. c In vitro kinase assay using WT- or AS-GST-CDK16 with GST-CCNY as a substrate. The indicated N6-substituted ATPγS analog was used as a phosphodonor. d In vitro kinase assay using WT- or AS-GST-CDK16 with ATPγS or N6-Ph-ATPγS, respectively, in the presence of different inhibitors (1–5) to test the ability of these inhibitors to inhibit the AS mutant. e In vitro kinase assay conducted to establish the specific IC50 of 3MBPP1. WT- or AS-GST-CDK16 was incubated with [γ-32P]-ATP in the presence of different concentrations of 3MBPP1 for the indicated time intervals (in min)
Fig. 2
Fig. 2. A double proteomic approach reveals a role for the CDK16/CCNY complex in cytoskeletal regulation.
a Strategy used to identify CCNY interactors using MCF7 cell extracts. The graphical representation of GO terms obtained by STRING analysis for biological processes and cellular components is shown below. Data were filtered with a p-value cutoff of 0.05 and a fold change cutoff of log2(3.23) and seen in all three biological replicates. b Chemical genetic approach based on the labeling of AS-GST-CDK16 substrates in HeLa cell extracts. The corresponding graphical representation of GO terms obtained by STRING analysis is shown below. Peptides that were present in the AS-CDK16 proteomic analysis but in neither the WT-CDK16 nor the negative control analyses were selected. The significance threshold for the false discovery rate (FDR) was set at ≥0.01. MS, mass spectrometry; N6-Ph-ATPγS, bulky ATP analog
Fig. 3
Fig. 3. Identification of PRC1 as a direct substrate of the CDK16/CCNY complex.
a The CDK16 phosphorylation consensus sequence was identified using WebLogo. b The Venn diagram of selected CCNY interactors (Screening A, Scr A) and CDK16/CCNY direct substrates (Screening B, Scr B) illustrates that PRC1 is the only substrate identified by both approaches. c Schematic representation of the human PRC1 locus showing the NLS domain where Thr481 is located. d In vitro kinase assay using WT-GST-CDK16, GST-CCNY and either WT or T481A-mutant of PRC1
Fig. 4
Fig. 4. Generation of a clonal 293T analog-sensitive (AS)-CDK16 cell line by CRISPR-Cas9.
a Schematic representation of the human CDK16 locus showing the target site used for CRISPR/Cas-mediated genome editing in 293T cells and the ssDNA used as repair template. (b, c) Colony formation assay of 293T and 293T AS-CDK16 cells grown in the absence or presence of 3MBPP1. The columns represent the means ± SEMs of 7 independent experiments performed in triplicate. **P < 0.01 vs nontreated cells, Mann–Whitney test. (d, e) 293T AS-CDK16 cells were synchronized and treated with 3MBPP1. After 6 h, cells were fixed and immunostained with a phospho-T481-PRC1 (pPRC1) antibody. Three independent experiments were performed, and 150–200 cells were analyzed per condition. The graph shows the results of one representative experiment, and the columns represent the means ± SEMs. ***P < 0.001 vs control, Student’s t-test
Fig. 5
Fig. 5. CDK16 inhibition promotes PRC1 delocalization to the nucleus and PRC1 accumulation.
a Analysis of PRC1 distribution in 293T AS-CDK16 cells after 3MBPP1 treatment (0, 5 and 10 µM) and subcellular fractionation. The columns represent the means ± SEMs of 9 independent experiments. b Representative images of PRC1 immunofluorescence (red) and the Na+/K+ATPase (membrane marker) in 293T AS-CDK16 cells treated with 0 (control), 5 or 10 µM 3MBPP1. c PRC1 accumulation in 293T and 293T AS-CDK16 cells treated with 3MBPP1 (0, 5 and 10 µM) for 6 h was assessed by western blotting. The columns represent the means ± SEMs of 12 independent experiments. *P < 0.05, **P < 0.01 vs control; Mann–Whitney test. d Analysis of PRC1 distribution in HT-29 colon cancer cells after treatment with dabrafenib (0, 2 and 10 nM) for 24 h and subcellular fractionation. The columns represent the means ± SEMs of 8 independent experiments. e Representative images of PRC1 immunofluorescence (red) and the Na+/K+ ATPase in HT-29 cells treated with 0 (control), 2 or 10 nM dabrafenib for 24 h. f PRC1 accumulation in HT-29 cells treated with 2 nM dabrafenib for 24 h was assessed by western blotting. The columns represent the means ± SEMs of 8 independent experiments. *P < 0.05 vs nontreated (NT) cells, Mann–Whitney test
Fig. 6
Fig. 6. The proliferative action of CDK16 is mediated by PRC1 phosphorylation.
ac Here, 293T AS-CDK16 cells were seeded and transfected the following day with siRNA targeting PRC1 or with scrambled control and treated with 0 or 5 µM 3MBPP1. a Western blot analysis was performed to confirm PRC1 downregulation. b Cell viability was monitored by an MTT assay, and the columns represent the means ± SEMs of 6 independent experiments performed in triplicate. *P < 0.05, **P < 0.01 vs control; Mann–Whitney test. c Cell proliferation was assessed by a BrdU incorporation assay 72 h after transfection. The columns represent the means ± SEMs of 4 independent experiments performed in triplicate. *P < 0.05 vs control, Mann–Whitney test. d Here, 293T AS-CDK16 cells were seeded and transfected the following day with siRNA targeting PRC1 or with scrambled control siRNA. After 3 days, the cells were detached, seeded in 6-well plates and treated with 0 or 5 µM 3MBPP1. The colony formation ability was assessed 2 weeks later. The columns represent the means ± SEMs of 4 independent experiments performed in duplicate. *P < 0.05 vs control, Mann–Whitney test
Fig. 7
Fig. 7. CDK16 promotes cancer cell proliferation via PRC1 phosphorylation.
a Tumor cell lines were seeded and transfected the following day with siRNA targeting PRC1 and/or CDK16 or with scrambled control. Cell viability was assessed by an MTT assay, and the columns represent the means ± SEMs of at least 3 independent experiments performed in triplicate. *P < 0.05 vs control, Mann–Whitney test. b Tumor cell lines were seeded and transfected the following day with siRNA targeting PRC1 and/or CDK16 or with scrambled control. After 3 days, the cells were detached and seeded in 6-well plates. The colony formation ability was assessed 2 weeks later. The columns represent the means ± SEMs of at least 3 independent experiments performed in duplicate. *P < 0.05 vs control, Mann–Whitney test

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