Deficiency in Chromosome Congression by the Inhibition of Plk1 Polo Box Domain-dependent Recognition

Abstract

Polo-like kinase 1 (Plk1) is one of the key regulators of mitotic cell division. In addition to an N-terminal protein kinase catalytic domain, Plk1 possesses a phosphopeptide binding domain named polo box domain (PBD) at its C terminus. PBD is postulated to be essential for Plk1 localization and substrate targeting. Here, we developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis but also prolonged the progression of mitosis in HeLa cells. Although apparently normal bipolar spindles were formed even in the presence of PPG, the perturbation of chromosome alignment at metaphase plates activated the spindle assembly checkpoint pathway. These results demonstrate the predominant role of PBD-dependent binding on smooth chromosome congression at metaphase. In eukaryotes, Plk12 (or its orthologs) possesses a wide variety of essential functions during mitosis (1, 2). Although levels of Plk1 increase in late G 2 and rapidly decrease by proteolysis at the end of M phase, Plk1 localization changes dramatically when cells transit through the various mitotic stages. During late G 2 and prophase, Plk1 localizes primarily at the centrosomes, where Plk1 is involved in centrosome maturation, separation, and microtubule nucleation (3-5). At this stage, Plk1 also regulates mitotic entry through the phosphorylation and activation of Cdc25 (6, 7). In parallel, the phosphorylation of Wee1 by Plk1, which is primed by Cdk1, promotes the destruction of Wee1, the inhibitory kinase of mitotic entry (8, 9). By metaphase, a fraction of Plk1 relocalizes to the kinetochores, where it is involved in the regulation of the spindle assembly checkpoint pathway and proper chromosome segregation. Plk1 sensitizes the tension between sister chromatids and regulates the stability of kinetochore-microtubule interactions by phosphorylating kinetochore proteins such as BubR1 and proteins that harbor 3F3/2 epitopes (4, 10 -14). Although Plk1 localization at chromosome arms is required for loss of arm cohesion during M-phase (15, 16), localized Plk1 activity at the kinetochore is important for the proper accumulation of kinetochore proteins such as BubR1, Mad1, Mad2, Cdc20, and centromere/ kinetochore-associated protein-E (CENP-E) (4, 10 -12, 17, 18). During anaphase, Plk1 is concentrated in the spindle midzone, where it may facilitate microtubule sliding. After chromosomal segregation, Plk1 remains in the central spindle and midbody, where it is involved in formation of the cleavage furrow (19 -21).These various localizations of Plk1 are mediated by its noncatalytic C-terminal half, the polo box domain (PBD), which comprises two polo box motifs (22, 23). Recently, Yaffe and co-workers (24, 25) discovered that the PBD constitutes a phosphopeptide binding domain which binds with maximal affin...

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

“…The first identified small-molecule compound, Poloxin, shows its high specificity by aiming at the PBD of Plk1, 16 which is directly followed by another inspiring report that purpurogallin (PPG), a benzotropolone-containing natural compound derived from nutgalls, also blocks the PBD of Plk1 with selectivity. 39 The data demonstrate that inhibition of the PBD is sufficient to specifically interfere with the multiple functions of Plk1.…”

“…Notably, centrosomes were unfocused and distanced in cells treated with another PBD inhibitor, PPG. 39 Moreover, cells treated with a pan-PBD inhibitor, poloxipan, also displayed fragmented centrosomes. 42 However, enforced PBD expression did not impair centrosome maturation/separation.…”

“…47 Furthermore, apoptosis was strongly induced in normal rat kidney cells and NIH/3T3 cells on treatment with the PBD inhibitor, PPG. 39 Therefore, we suppose that Plk1 is required for the proliferation of both normal/nontransformed cells and tumor cells. Thus, the Plk1 inhibitors, targeting either the protein kinase domain, such as BI 2536, or the PBD domain of Plk1, such as Poloxin and PPG, affect both cancer cells and nontransformed but proliferating cells with a comparable efficacy, at least in cell culture system.…”

Trachyspic acid 19-butyl ester, a new inhibitor of Plk1 polo box domain-dependent recognition from uncharacterized fungus RKGS-F2684

“…9,10 We established a screening system for the inhibitors of PBD-dependent binding. 11 In this system, the open reading frame of a green fluorescent protein (GFP) is fused to PBD and expressed in bacteria. Target phosphopeptides for the PBD binding sequence are chemically synthesized and bound covalently to maleimide-activated 96-well plates.…”

Plk1-Targeted Small Molecule Inhibitors: Molecular Basis for Their Potency and Specificity

“…PoloBoxtide effectively interferes with Plk1 PBD-dependent binding in vitro in a concentration-dependent manner with an in vitro IC 50 of 5 µM and Kd of 280 nM (Elia et al, 2003a;2003b;Watanabe et al, 2009). Minimal PoloBoxtides, PMQSpTPL and MQSpTPL, described above have been used for many experiments.…”