We and others have shown that two PCM scaffolds, Cep192 and Cep152, bind to Plk4 to properly recruit the latter to centrosomes. In a detailed analysis using three-dimensional (3-D) structured illumination microscopy (SIM), we found that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Interestingly, the ring-like Plk4 signal becomes a dot-like structure prior to recruiting Sas6, a major component of the so-called centriolar cartwheel. Subsequent studies have shown that Plk4 interacts with and phosphorylates a centrosomal component, STIL, to facilitate the STIL-Sas6 interaction, and this event is critical for the recruitment of Sas6 to the procentriole assembly site. At the biochemical level, Plk4 is known to dimerize primarily through the CPB, and perhaps also through PB3, and this dimerization step may be important for trans-autophosphorylating a degron motif located immediately downstream of the kinase domain (KD) and inducing ubiquitin-mediated degradation. This mechanism is thought to be important for keeping Plk4 in low abundance and preventing centriole overduplication. However, whether the interaction of Plk4 with various scaffolds, such as Cep192, Cep152, or STIL/Sas6, safeguards Plk4 from unscheduled degradation and/or whether Plk4 assembles into an active form of structure (detected as a dot-like appearance in 3-D SIM) prior to inducing procentriole assembly are not known. In addition, how Plk4 locates to a single focus of activity following centriole disengagement and how Plk4 nucleates a single procentriole near the preexisting centriole are fascinating questions that remain unanswered. Although Cep152 has been suggested as a licensing factor that regulates the timing of Plk4-dependent procentriole assembly, how it is organized into a ring-like structure around the proximal ends of centrioles remains a mystery. Interestingly, Cep63 is known to form a complex with Cep152, and this complex formation appears to be important for targeting Cep152 to centrosomes. Furthermore, selective chemical crosslinking suggests that Cep57 forms a stable complex with Cep63 and Cep152 and generates a ring-like structure around centrioles. However, little investigation has been carried out to determine how these proteins form a complex and how they contribute to Plk4-dependent centriole duplication. Given the presence of multiple cancer and microcephaly-associated mutations in these scaffold proteins (Catalogue of Somatic Mutations in Cancer, www.sanger.ac.uk/cosmic/), investigation into whether these three proteins are physically organized into a higher-order structure, and, if so, whether forming the assembly is important to promote Plk4 function may shed new light onto the mechanism of how Plk4 regulates centriole biogenesis. To this end, we aim to investigate how Plk4 becomes an active form to induce procentriole assembly and how Plk4 function is regulated through its interactions with centrosomal scaffold proteins. A defect in this process results in centrosome aberrations that lead to chromosome missegregation and aneuploidy, a hallmark of cancer. We have previously studied the physiological significance of the interactions between Plk4 and two centrosomal scaffold proteins, Cep192 and Cep152. Now, we'd like to apply our knowledge and skill set to investigating the molecular steps of how Plk4 is converted to a biologically active structure capable of inducing centriole biogenesis, and whether and how the scaffold proteins that bind to Plk4 are organized into a higher-order structure around centrioles.
| Nasa, Isha; Trinkle-Mulcahy, Laura; Douglas, P et al. (2017) Recruitment of PP1 to the centrosomal scaffold protein CEP192. Biochem Biophys Res Commun 484:864-870 |
| Park, Suk-Youl; Park, Jung-Eun; Kim, Tae-Sung et al. (2014) Molecular basis for unidirectional scaffold switching of human Plk4 in centriole biogenesis. Nat Struct Mol Biol 21:696-703 |
| Chang, Jaerak; Seo, Sang Gwon; Lee, Kyung Ho et al. (2013) Essential role of Cenexin1, but not Odf2, in ciliogenesis. Cell Cycle 12:655-62 |
| Kim, Tae-Sung; Park, Jung-Eun; Shukla, Anil et al. (2013) Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152. Proc Natl Acad Sci U S A 110:E4849-57 |
