Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286.
Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.
Germain
D
D
Peter MacCallum Cancer Institute, St. Andrew's Place, East Melbourne, Melbourne, Victoria 3002, Australia. d.germain@pmci.unimelb.edu.au
Russell
A
A
Thompson
A
A
Hendley
J
J
D000818:Animals; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016193:G1 Phase; D006801:Humans; D051379:Mice; D010447:Peptide Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D016196:S Phase; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D019938:Cyclin D1; D013912:Threonine; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease