M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP.
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.
Watanabe
Nobumoto
N
Antibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. nwatanab@riken.go.jp
Arai
Harumi
H
Nishihara
Yoshifumi
Y
Taniguchi
Makoto
M
Watanabe
Naoko
N
Hunter
Tony
T
Osada
Hiroyuki
H
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*
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