P27Kip1 and p21Cip1 are not required for the formation of active D cyclin-cdk4 complexes.
Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27(Kip1) and p21(Cip1) (p27/p21(-/-)). Such conditions included ectopic expression of cyclin D1 and inhibition of D cyclin degradation by the proteasome inhibitor MG132. However, as determined by treatment of wild-type MEFs with MG132, maximal accumulation of D cyclin-cdk4 complexes required p27(Kip1) and p21(Cip1) and coincided with the formation of inactive D cyclin-cdk4-p27(Kip1) or -p21(Cip1) complexes. p27(Kip1) or p21(Cip1) also increased the abundance of D cyclin-cdk4 complexes and reduced amounts of cdk4 activity when ectopically expressed in p27/p21(-/-) MEFs. Lastly, increases in the stability of the D cyclins accounted for their greater abundance in wild-type MEFs than in p27/p21(-/-) MEFs. We conclude that (i) D cyclin-cdk4 complexes are formed and become active in the absence of p27(Kip1) and p21(Cip1) and (ii) p27(Kip1) and p21(Cip1) maximize the accumulation but inhibit the activity of D cyclin-cdk4 complexes. We suggest that D cyclin-cdk4 complexes are more stable when bound to p27(Kip1) or p21(Cip1) and that formation of ternary complexes also stabilizes the D cyclins.
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000502:physiology*; D002478:Cells, Cultured; D056741:Cyclin D; D056743:Cyclin D3; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism* / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000502:physiology*
C534697:Ccnd3 protein, mouse; C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D056741:Cyclin D; D056743:Cyclin D3; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003513:Cycloheximide; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde