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

Research output: Contribution to journalArticle

99 Citations (Scopus)

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
Externally publishedYes

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SKP Cullin F-Box Protein Ligases
Cyclin E
Alanine
Phosphorylation
Protein Isoforms
Mutation
Mutant Proteins
Degradation
Association reactions
Phosphopeptides
HEK293 Cells
Alternative Splicing
Point Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ye, X., Nalepa, G., Welcker, M., Kessler, B. M., Spooner, E., Qin, J., ... 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

Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase. / Ye, Xin; Nalepa, Grzegorz; Welcker, Markus; Kessler, Benedikt M.; Spooner, Eric; Qin, Jun; Elledge, Stephen J.; Clurman, Bruce E.; Harper, J. Wade.

In: Journal of Biological Chemistry, Vol. 279, No. 48, 26.11.2004, p. 50110-50119.

Research output: Contribution to journalArticle

Ye, X, Nalepa, G, Welcker, M, Kessler, BM, Spooner, E, Qin, J, Elledge, SJ, Clurman, BE & Harper, JW 2004, 'Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase', Journal of Biological Chemistry, vol. 279, no. 48, pp. 50110-50119. https://doi.org/10.1074/jbc.M409226200
Ye, Xin ; Nalepa, Grzegorz ; Welcker, Markus ; Kessler, Benedikt M. ; Spooner, Eric ; Qin, Jun ; Elledge, Stephen J. ; Clurman, Bruce E. ; Harper, J. Wade. / Recognition of phosphodegron motifs in human cyclin E by the SCF Fbw7 ubiquitin ligase. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 48. pp. 50110-50119.
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AU - Welcker, Markus

AU - Kessler, Benedikt M.

AU - Spooner, Eric

AU - Qin, Jun

AU - Elledge, Stephen J.

AU - Clurman, Bruce E.

AU - Harper, J. Wade

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