Cyclin E-CDK2 is a regulator of p27Kip1.
CDK inhibitors are thought to prevent cell proliferation by negatively regulating cyclin-CDK complexes. We propose that the opposite is also true, that cyclin-CDK complexes in mammmalian cells can promote cell cycle progression by directly down-regulating CDK inhibitors. We show that expression of cyclin E-CDK2 in murine fibroblasts causes phosphorylation of the CDK inhibitor p27Kip1 on T187, and that cyclin E-CDK2 can directly phosphorylate p27 T187 in vitro. We further show that cyclin E-CDK2-dependent phosphorylation of p27 results in elimination of p27 from the cell, allowing cells to transit from G1 to S phase. Moreover, mutation of T187 in p27 to alanine creates a p27 protein that causes a G1 block resistant to cyclin E and whose level of expression is not modulated by cyclin E. A kinetic analysis of the interaction between p27 and cyclin E-CDK2 explains how p27 can be regulated by the same enzyme it targets for inhibition. We show that p27 interacts with cyclin E-CDK2 in at least two distinct ways: one resulting in p27 phosphorylation and release, the other in tight binding and cyclin E-CDK2 inhibition. The binding of ATP to the CDK governs which state predominates. At low ATP (< 50 microM) p27 is primarily a CDK inhibitor, but at ATP concentrations approaching physiological levels (> 1 mM) p27 is more likely to be a substrate. Thus, we have identified p27 as a biologically relevant cyclin E-CDK2 substrate, demonstrated the physiological consequences of p27 phosphorylation, and developed a kinetic model to explain how p27 can be both an inhibitor and a substrate of cyclin E-CDK2.
Sheaff
R J
RJ
Division of Basic Sciences, Fred Hutchinson Cancer Research Center (FHCRC), Seattle, Washington 98104, USA.
Groudine
M
M
Gordon
M
M
Roberts
J M
JM
Clurman
B E
BE
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D015536:Down-Regulation; D016193:G1 Phase / Q000502:physiology; D006657:Histones / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016196:S Phase / Q000502:physiology; D013912:Threonine / Q000378:metabolism; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D016213:Cyclins; D006657:Histones; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases