Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway.
The expression of D-type G1 cyclins and their assembly with their catalytic partners, the cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), into active holoenzyme complexes are regulated by growth factor-induced signals. In turn, the ability of cyclin D-dependent kinases to trigger phosphorylation of the retinoblastoma (Rb) protein in the mid- to late G1 phase of the cell cycle makes the inactivation of Rb's growth suppressive function a mitogen-dependent step. The ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their inherent instability, which ensures their precipitous degradation in cells deprived of growth factors. However, the mechanisms governing the turnover of D-type cyclins have not yet been elucidated. We now show that cyclin D1 turnover is governed by ubiquitination and proteasomal degradation, which are positively regulated by cyclin D1 phosphorylation on threonine-286. Although "free" or CDK4-bound cyclin D1 molecules are intrinsically unstable (t1/2 < 30 min), a cyclin D1 mutant (T286A) containing an alanine for threonine-286 substitution fails to undergo efficient polyubiquitination in an in vitro system or in vivo, and it is markedly stabilized (t1/2 approximately 3.5 hr) when inducibly expressed in either quiescent or proliferating mouse fibroblasts. Phosphorylation of cyclin D1 on threonine-286 also occurs in insect Sf9 cells, and although the process is enhanced significantly by the binding of cyclin D1 to CDK4, it does not depend on CDK4 catalytic activity. This implies that another kinase can phosphorylate cyclin D1 to accelerate its destruction and points to yet another means by which cyclin D-dependent kinase activity may be exogenously regulated.
Diehl
J A
JA
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Zindy
F
F
Sherr
C J
CJ
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D018411:Spodoptera; D013912:Threonine / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D016213:Cyclins; D009097:Multienzyme Complexes; D015513:Oncogene Proteins; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D014452:Ubiquitins; D019938:Cyclin D1; D013912:Threonine; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex