Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase

Xin Ye, Grzegorz Nalepa, Markus Welcker, Benedikt M. Kessler, Eric Spooner, Jun Qin, Stephen J. Elledge, Bruce E. Clurman, J. Wade Harper

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Abstract

Turnover of cyclin E is controlled by SCF Fbw7. Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7α and -γ are nuclear and the β-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 82 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 Fbw7β. Cyclin E S384A associated more weakly with Fbw7α and -β isoforms but was not defective in Thr 380 phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E S384A in the nucleus, which may contribute to the inability of cytoplasmic Fbw7β to promote turnover of this cyclin E mutant protein.

Original languageEnglish (US)
Pages (from-to)50110-50119
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number48
DOIs
StatePublished - Nov 26 2004

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ye, X., Nalepa, G., Welcker, M., Kessler, B. M., Spooner, E., Qin, J., Elledge, S. J., Clurman, B. E., & Harper, J. W. (2004). Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase. Journal of Biological Chemistry, 279(48), 50110-50119. https://doi.org/10.1074/jbc.M409226200