Formation of p27-CDK complexes during the human mitotic cell cycle.
In eukaryotic cells, the coordinated activation of different cyclin-dependent kinases regulates entry into S-phase. In vitro and in nonproliferating cells, p27 associates with and inhibits cyclin/cycin-dependent kinase (CDK) holoenzymes containing either CDK4, CDK6, or CDK2. Although many different types of proliferating cells contain p27 protein, neither the interactions of p27 with cyclin/CDK complexes nor the consequences of this interaction during the mitotic cycle have been fully explored. We report that, in MANCA cells, the amount of p27 is constant during the cell cycle. In addition, p27 associates with three different CDKs: CDK2, CDK4, and CDK6. Furthermore, the amount of p27 is significantly lower than the amount of cyclin D3 in these cells. The amount of CDK4 and CDK6 associated with p27 does not change in a cell cycle-dependent fashion; in contrast, the amount of CDK2 associated with p27 is lowest in early G1 cells and increases to a maximum in mid-G1 phase, reaching a steady-state level in late G1-phase cells. After mid-G1 phase, the amount of each p27/CDK complex remains constant through the remainder of the cell cycle. p27-immunoprecipitates contain an Rb-kinase activity. The substrate specificity, the expression pattern of this kinase, and the ability to deplete 50% of this kinase activity with a CDK6-specific antibody suggest that the CDK6 protein mediates, in part, the p27-associated Rb-kinase activity. In contrast, p27 complexes containing CDK2 are incapable of phosphorylating histone H1. These data are consistent with a model wherein cyclin D/CDK complexes sequester the CDK2-dependent kinase inhibitory activity of p27.
Soos
T J
TJ
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Research Center, New York, New York 10021, USA.
Kiyokawa
H
H
Yan
J S
JS
Rubin
M S
MS
Giordano
A
A
DeBlasio
A
A
Bottega
S
S
Wong
B
B
Mendelsohn
J
J
Koff
A
A
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000502:physiology; D006224:Cricetinae; D056743:Cyclin D3; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology; D025521:Tumor Suppressor Proteins
C534696:CCND3 protein, human; D018797:Cell Cycle Proteins; D056743:Cyclin D3; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases