title: M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP.
abstract: 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 down-regulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the beta-transducin repeat-containing protein 1/2 (beta-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCF(beta-TrCP1/2)) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for beta-TrCP, recognition of Wee1A by beta-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for beta-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 beta-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCF(beta-TrCP1/2) in vitro. Mutation of these residues or depletion of beta-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that beta-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.
authors.affiliation: Antibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. nwatanab@riken.go.jp
authors.forename: Nobumoto
authors.initials: N
authors.lastname: Watanabe
authors.forename: Harumi
authors.initials: H
authors.lastname: Arai
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishihara
authors.forename: Makoto
authors.initials: M
authors.lastname: Taniguchi
authors.forename: Naoko
authors.initials: N
authors.lastname: Watanabe
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D017124:Conserved Sequence; D044783:F-Box Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D008938:Mitosis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D044843:SKP Cullin F-Box Protein Ligases; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human