The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.

Plk1 plays a pivotal role in cell proliferation and is considered an attractive target for anti-cancer 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 interacted with the PBD of Plk1, but not the two 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 critical for providing both specificity and affinity to the interaction. Inhibition of the Plk1 PBD by phospho-Thr mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. Thus, the mode of the minimal peptide and PBD interaction may provide a template for designing anti-Plk1 therapeutic agents.