At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is downregulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF ␤-TrCP . The F-box protein ␤-TrCP (␤-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to ␤-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved ␤-TrCPbinding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in ␤-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to ␤-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of ␤-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second ␤-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.polo-like kinase 1 ͉ ubiquitin ͉ cell cycle ͉ CK2 ͉ ␤-TrCP

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...