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Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4419-24. Epub 2004 Mar 22.

M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP.

Author information

1
Antibiotics Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. nwatanab@riken.go.jp

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.

PMID:
15070733
PMCID:
PMC384762
DOI:
10.1073/pnas.0307700101
[Indexed for MEDLINE]
Free PMC Article

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