Ye, XinNalepa, GrzegorzWelcker, MarkusKessler, Benedikt M.Spooner, EricQin, JunElledge, Stephen J.Clurman, Bruce E.Harper, J. Wade2019-10-122004Ye, Xin, Grzegorz Nalepa, Markus Welcker, Benedikt M. Kessler, Eric Spooner, Jun Qin, Stephen J. Elledge, Bruce E. Clurman, and J. Wade Harper. 2004. “Recognition of Phosphodegron Motifs in Human Cyclin E by the SCF Fbw7Ubiquitin Ligase.” Journal of Biological Chemistry279 (48): 50110–19. doi:10.1074/jbc.M409226200.0021-92581083-351Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41542729Turnover 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.en-USJournal Article2019-10-1210.1074/jbc.M409226200