Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.
Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. We have tested this model in vivo and found it to be an inadequate representation of the pathways that regulate cyclin E degradation. We show that assembly of cyclin E into cyclin E-Cdk2 complexes is required in vivo for turnover by the Fbw7 pathway; that Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384; that phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3; and that two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone.
Welcker
Markus
M
Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Singer
Jeffrey
J
Loeb
Keith R
KR
Grim
Jonathan
J
Bloecher
Andrew
A
Gurien-West
Mark
M
Clurman
Bruce E
BE
Roberts
James M
JM
D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000378:metabolism / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D044783:F-Box Proteins / Q000378:metabolism; D000073979:F-Box-WD Repeat-Containing Protein 7; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism / Q000502:physiology*; D038341:Glycogen Synthase Kinases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012190:Retroviridae / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D018797:Cell Cycle Proteins; D019927:Cyclin E; D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513273:FBXW7 protein, human; C513272:Fbxw7 protein, mouse; D044767:Ubiquitin-Protein Ligases; D038341:Glycogen Synthase Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D038362:Glycogen Synthase Kinase 3