SummaryPrimary cilia, which emanate from the cell surface, exhibit assembly and disassembly dynamics along the progression of the cell cycle. However, the mechanism that links ciliary dynamics and cell cycle regulation remains elusive. In the present study, we report that Pololike kinase 1 (Plk1), one of the key cell cycle regulators, which regulate centrosome maturation, bipolar spindle assembly and cytokinesis, acts as a pivotal player that connects ciliary dynamics and cell cycle regulation. We found that the kinase activity of centrosome enriched Plk1 is required for primary cilia disassembly before mitotic entry, wherein Plk1 interacts with and activates histone deacetylase 6 (HDAC6) to promote ciliary deacetylation and resorption. Furthermore, we showed that pericentriolar material 1 (PCM1) acts upstream of Plk1 and recruits the kinase to pericentriolar matrix (PCM) in a dynein-dynactin complex-dependent manner. This process coincides with the primary cilia disassembly dynamics at the onset of mitosis, as depletion of PCM1 by shRNA dramatically disrupted the pericentriolar accumulation of Plk1. Notably, the interaction between PCM1 and Plk1 is phosphorylation dependent, and CDK1 functions as the priming kinase to facilitate the interaction. Our data suggest a mechanism whereby the recruitment of Plk1 to pericentriolar matrix by PCM1 plays a pivotal role in the regulation of primary cilia disassembly before mitotic entry. Thus, the regulation of ciliary dynamics and cell proliferation share some common regulators.

CDK1 and Plk1 sequentially phosphorylate and activate Usp16, which in turn deubiquitinates Plk1 to maintain the kinase’s kinetochore localization and promote proper chromosome alignment in mitosis.

Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 μM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.

Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.