Significance: Found in most eukaryotic cells, a centriole is a cylindrically shaped subcellular structure that plays an important role in various cellular processes, including mitotic spindle formation and chromosome segregation. Centriole duplication occurs only once per cell cycle, thus ensuring accurate control of centriole numbers to maintain genomic integrity. Although a growing body of evidence suggests that a Ser/Thr protein kinase, polo-like kinase 4 (Plk4), is a key regulator of centriole duplication, how Plk4 is recruited to centrosomes remains largely unknown. Here we showed that Plk4 dynamically localizes to distinct subcentrosomal regions by interacting with two hierarchically regulated scaffolds, Cep192 and Cep152. Highlighting the importance of these interactions, mutational disruption of either one of these interactions was sufficient to cripple Plk4-dependent centriole biogenesis.

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homologue of Saccharomyces cerevisiae Cdc5. Here, we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5–1 temperature-sensitive mutation and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.

The Plk1 kinase is a major regulator of mitosis that is often overexpressed in human cancers. Studies on phosphopeptide inhibitors specific to the polo-box domain of Plk1 reveal the determinants for specificity and may provide insight into the development of new therapeutics targeting protein-protein interactions. Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Non‐motile primary cilium is an antenna‐like structure whose defect is associated with a wide range of pathologies, including developmental disorders and cancer. Although mechanisms regulating cilia assembly have been extensively studied, how cilia disassembly is regulated remains poorly understood. Here, we report unexpected roles of Dishevelled 2 (Dvl2) and interphase polo‐like kinase 1 (Plk1) in primary cilia disassembly. We demonstrated that Dvl2 is phosphorylated at S143 and T224 in a manner that requires both non‐canonical Wnt5a ligand and casein kinase 1 epsilon (CK1ε), and that this event is critical to interact with Plk1 in early stages of the cell cycle. The resulting Dvl2–Plk1 complex mediated Wnt5a–CK1ε–Dvl2‐dependent primary cilia disassembly by stabilizing the HEF1 scaffold and activating its associated Aurora‐A (AurA), a kinase crucially required for primary cilia disassembly. Thus, via the formation of the Dvl2–Plk1 complex, Plk1 plays an unanticipated role in primary cilia disassembly by linking Wnt5a‐induced biochemical steps to HEF1/AurA‐dependent cilia disassembly. This study may provide new insights into the mechanism underlying ciliary disassembly processes and various cilia‐related disorders.

Plk4 regulates centriole duplication. Two centrosomal scaffold proteins, Cep192 and Cep152, are shown to interact with Plk4 in a temporally and spatially regulated manner, and structural analyses reveal that these interactions are mutually exclusive. Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. We show that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal-structure analyses revealed that Cep192- and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 peptide bound to the CPB markedly better than did the Cep192 peptide and effectively 'snatched' the CPB away from a preformed CPB–Cep192 peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.