Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is downregulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the ␤-transducin repeat-containing protein 1͞2 (␤-TrCP1͞2) F-box protein-containing SKP1͞Cul1͞F-box protein (SCF) complex (SCF ␤-TrCP1͞2 ) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for ␤-TrCP, recognition of Wee1A by ␤-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for ␤-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by ␤-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCF ␤-TrCP1͞2 in vitro. Mutation of these residues or depletion of ␤-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that ␤-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide.

HIV-1 viral protein R (Vpr) is one of the human immunodeficiency virus type 1 encoded proteins that have important roles in viral pathogenesis. However, no clinical drug for AIDS therapy that targets Vpr has been developed. Here, we have established a screening system to isolate Vpr inhibitors using budding yeast cells. We purified a Vpr inhibitory compound from fungal metabolites and identified it as fumagillin, a chemical already known to be a potent inhibitor of angiogenesis. Fumagillin not only reversed the growth inhibitory activity of Vpr in yeast and human cells, but also inhibited Vpr-dependent viral gene expression upon the infection of human macrophages.

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