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.
