期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 279, 期 48, 页码 50110-50119出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M409226200
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资金
- NCI NIH HHS [CA102742] Funding Source: Medline
- NIA NIH HHS [AG11095, R01 AG011085] Funding Source: Medline
Turnover of cyclin E is controlled by SCFFbw7. Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E-T380A to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E-S384A associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin ES384A in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.
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