ABSTRACT
Many mechanisms either activate or inhibit the cdks and thereby either promote or arrest progression through the mitotic cell cycle. Since the signal transduction pathways emanating from extracellular mitogens and the agents controlling these pathways are complicated there may yet be novel mechanisms of cell cycle regulation remaining to be elucidated. In this article we outline the different techniques used to study the cell cycle and its regulation. These include: establishing that the cell cycle is arrested by propidium iodide staining followed by FACS analysis or by measuring 3H-thymidine incorporation into DNA; measuring the amount of cyclin/cdk associated kinase activity; assessing the steady-state expression profiles of cyclins, cdks and ckis by immunoblotting; and investigating the formation of complexes between these proteins by coimmunoprecipitations. Caveats and advantages of each technique are discussed. Following this paradigm yielded the discovery of the cell cycle inhibitors p27Kip1 and p21Cip1 and could very well lead to the discovery or novel cell cycle regulatory mechanisms.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Animals , Blotting, Western , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Separation , Coloring Agents/pharmacology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclins/metabolism , DNA/analysis , Enzyme Inhibitors/pharmacology , Flow Cytometry , Glutathione Transferase/metabolism , Immunoblotting , Mice , Models, Biological , Phosphorylation , Precipitin Tests , Propidium/pharmacology , Protein Kinases/metabolism , Retinoblastoma Protein/metabolism , Signal Transduction , Thymidine/metabolism , Tumor Suppressor Proteins/metabolismSubject(s)
Cell Cycle Proteins , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Enzyme Inhibitors/metabolism , Microtubule-Associated Proteins/metabolism , Signal Transduction , Tumor Suppressor Proteins , Animals , Cyclin-Dependent Kinase Inhibitor p27 , HumansABSTRACT
Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.