Chromosomal Instability (CIN) occurs in over 90% of all sporadic tumors and manifests as whole chromosome loss or gain, gene deletions, amplifications, inversion, and translocations. CIN is not only a hallmark of cancer but promotes tumorigenesis. CIN is caused by errors during mitosis and one major CIN-promoting mechanism is centrosome over-duplication (amplification); another cancer hallmark. Centrosome amplification causes abnormal mitotic spindle assembly, directly promoting chromosome mis-segration with consequent aneuploidy and other forms of CIN. Central to controlling centrosome numbers and function are the Polo kinases, including Polo-like kinase 4 (Plk4). Plk4 is a component of centrosomes and recognized as the master-regulator of centrosome function and duplication. Plk4 is a mitotic kinase whose levels increase throughout S-phase and G2 to peak in mitosis. During late mitosis, Plk4 localizes to a spot on parent centrioles, licensing this single site for future daughter centriole assembly. Plk4 activity initiates the hierarchial recruiment of two conserved essential centriole proteins: Ana2, followed by the cartwheel protein Sas6. By analysis in a yeast-2-hybrid screen, we identified several novel interactions of centriole proteins, including the interaction of Ana2 and Plk4. Plk4 phosphorylates Ana2 to both positively and negatively regulate centriole duplication. Our preliminary data suggests that Plk4 recruits Ana2 by phosphorylating a protein on the outer centriole surface, generating a phospho-landing platform, and that this Plk4 target is Sas4 (CPAP in humans). Notably, the Sas4 pattern on centrioles is complex, forming both a ring and an asymmetric spot during mitotic progression. Like Sas4, Ana2 is a Plk4 substrate, and when mixed with purified Ana2, Sas4 stimulates Ana2 hyperphosphorylation in vitro. Thus, Plk4 influences centriole assembly on multiple platforms.

Centrosomes are organelles that promote microtubule growth. Normally, a single centrosome duplicates once each cell cycle to guide assembly of a bipolar mitotic spindle, ensuring that each daughter cell inherits an equal complement of the genome and a single centrosome. Centrosomes are composed of a pair of ‘mother-daughter’ centrioles and, during duplication, each mother centriole assembles one daughter at a single site. However, mother centrioles can inappropriately assemble multiple daughters, thereby generating centriole amplification (or overduplication), resulting in multipolar spindle assembly and, consequently, chromosome missegration - a driving force for chromosomal instability/aneuploidy which induces birth defects, miscarriage, and tumorigenesis. We have elucidated how the cell cycle control program regulates the centriole duplication machinery to limit centriole duplication to one event per cell cycle via the cell cycle-dependent regulation of Ana2/STIL and PLK4 degradation. In the case of the centrosome licensing factor Plk4, we found that autophosphorylation promotes its own destruction during interphase, which is then counteracted by the Protein Phosphatase 2A (PP2A) in complex with its Twins (tws) regulatory subunit during mitosis. This promotes stabilization of Plk4 and thus allows for the licensing of the mother centriole, making it competent to duplicate during the proceeding S-phase. While PP2Atws plays a positive role in regulating Plk4 to promote centriole duplication, we found that PP2A complexed with the Well-rounded (wrd) and Widerborst (wdb) regulatory subunits negatively regulates Ana2 by promoting its degradation to limit centriole duplication. PP2Awrd/wdb dephosphorylates numerous serine/threonine residues residing in Ana2, including several CDK phosphorylation consensus motifs. We found that CDK1/cycA and CDK2/cycE phosphorylate these residues to promote Ana2 stabilization from S-phase, the start of centriole duplication, to M-phase, the start of centriole duplication licensing. Interestingly, we found that the tumorigenic SV40 virus protein Small Tumor Antigen (ST) amplifies centrioles by targeting the PP2A complex to stabilize Plk4 as well as Ana2, underscoring the oncogenic importance of these newly discovered centriole duplication pathways. Finally, we shed insight into the mechanism for centriole amplification upon Ana2 stabilization by showing that Ana2 associates with Plk4 to promote Plk4 kinase activity as well as Plk4 stabilization.