On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells.
Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.
Arooz
T
T
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay.
Yam
C H
CH
Siu
W Y
WY
Lau
A
A
Li
K K
KK
Poon
R Y
RY
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002460:Cell Line / Q000201:enzymology* / Q000378:metabolism*; D002474:Cell-Free System / Q000201:enzymology / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004249:DNA Damage; D004317:Doxorubicin / Q000633:toxicity; D004791:Enzyme Inhibitors / Q000378:metabolism; D005260:Female; D006367:HeLa Cells / Q000737:chemistry / Q000166:cytology; D006801:Humans; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism* / Q000473:pathology
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D004317:Doxorubicin; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases