Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase.
Turnover of cyclin E is controlled by SCF(Fbw7). 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 E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.
Ye
Xin
X
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nalepa
Grzegorz
G
Welcker
Markus
M
Kessler
Benedikt M
BM
Spooner
Eric
E
Qin
Jun
J
Elledge
Stephen J
SJ
Clurman
Bruce E
BE
Harper
J Wade
JW
D020816:Amino Acid Motifs / Q000502:physiology; D001665:Binding Sites; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010766:Phosphorylation; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism
D019927:Cyclin E; D013912:Threonine; D044843:SKP Cullin F-Box Protein Ligases