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.

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.