There are three major checkpoints for cell cycle control (Figure 1). The first regulates the transition from G1 to S phase. Recall that G1 can be a very long phase, even (in the case of G0) as long as the lifespan of the cell. However, once the cell reaches S phase, it is committed to going through S, G2, and M phases to reproduce. This is because once S phase has begun, there is more than the normal diploid complement of DNA inside the cell. Over time this would confuse the cell (e.g., by overexpression of duplicated genes) as it tried to use the DNA to direct RNA and protein synthesis, and it could become sick and die. The second major checkpoint regulates entry into mitosis. Once mitosis begins, most of the metabolic activity of the cell is shut down, and the cell concentrates its resources on dividing the nuclear and cellular material equally to support the life of both resulting daughter cells. If the cell needs more time to make final repairs on the DNA or even to bulk up a little, this checkpoint can hold the cell in G2 a little longer for those things to happen. Finally, the third major checkpoint occurs during mitosis, and regulates the transition from metaphase into anaphase. Since the sister chromatids are being split apart and moved to opposite poles to form the new nuclei, it is important that all of them are perfectly lined up at metaphase and the proteins holding them together have dropped off. If they do not split evenly, the daughter cells will have abnormal numbers of chromosomes (aneuploidy) usually leading to deleterious consequences.
What is the molecular mechanism that regulates the progress of the cell cycle? While many of the checkpoint sensing mechanisms are still unclear, they seem to converge on two sets of proteins that act together to trigger cell cycle advancement. These proteins are known as the cyclins and the cyclin-dependent kinases (cdk). As the names suggest, the cyclins are proteins that regulate progression through the cell cycle, and must be present in sufficient concentration to help activate the appropriate cdk. The cyclin-dependent kinase is the active, enzymatic, half of the partnership, and activates other enzymes by phosphorylation. Although the cyclins appear to be necessary for cdk activation, they are not sufficient, as there are intermediate phosphorylation and dephosphorylation steps required to activate the cdk after cyclin binding. Both the cyclins and the cdk’s are families of related proteins, and they can combine in different ways to govern particular points in the cell cycle (Figure 2). Interestingly, the intracellular level of cdks is fairly constant. The level of cyclins, on the other hand, uctuates dramatically depending on the state of the cell with respect to the cell cycle.
Figure 2. Cyclins are involved in control of the cell cycle.
The methodology of some of the early experiments is perfectly suited to explaining how this works. The seminal paper in this field was a 1971 paper in J. Exp. Zool. by Masui and Markert. In it, they examined frog (Xenopus laevis) eggs that were arrested at G2. The oocytes arrest for about 8 months naturally in order to build up the mass needed to start a new organism once it has been fertilized. The basic question being asked is what is causing the eggs to come out of G2 and into M phase? It was already known that the hormone progesterone can trigger this transition, but what are the intracellular players in the change in cell state? Masui and Markert decided to test whether there was a cytoplasmic molecule that was responsible. They took a small amount of cytoplasm from an M-phase egg and injected it into a G2-arrested egg. This triggered the maturation of the G2-arrested egg and pushed it into M phase, even without progesterone. The activity was called maturation promoting factor (MPF), and was hypothesized to be a soluble, cytosolic protein.
In later experiments, other investigators attempted to find the specific protein trigger, and from there, presumably, the rest of the mechanism. Fractionating the M-phase oocyte cytoplasm by column chromatography, a protein, named cyclin B, was found to rise and fall in concentration in direct synchronization with MPF activity. Furthermore, addition of cyclin B alone was sufficient to rescue MPF activity from M-phase cytoplasmic extract that had been depleted by RNase treatment (preventing synthesis of any new proteins, including cyclin B, and abolishing MPF activity). This clearly places cyclin B in the forefront of the maturation mechanism, but there was one major issue: cyclin B had no enzymatic activity. How was it effecting the changes needed for progress from G2 to M phase?
This problem was answered by experiments on a very different organism, the fission yeast, Schizosaccharomyces pombe. Because they have a very short cycle time, a relatively small genome, and they can be given random mutations en masse by irradiation or chemical treatment, yeast are excellent model organisms for many types of biological study. After random mutation of a population of yeast, they can be screened for mutations of particular types, such as cell division cycle (cdc). When the mutations are sequenced and identified, they are often named by the type of mutation and order of discovery. Cdc2, it turns out, showed two interesting phenotypes when mutated in opposite directions. Mutations that knocked out function of cdc2 caused the formation of extremely large yeast that do not undergo cell division, while mutations that made cdc2 overactive caused the formation of rapidly dividing very small cells. The interpretation was that when cdc2 is missing or inactive, the cells cannot progress to mitosis, so they stay in G2 accumulating bulk material in preparation for a cell split that never comes. Conversely, when cdc2 is overactive, it drives the cell quickly into mitosis, even if it has not been in G2 long enough to synthesize enough mass to form two normal-sized cells. This ties cdc2 nicely to cell cycle regulation, and it even has an enzymatic activity: it is a kinase. This made it a perfect candidate as a first-order coordinator of cellular events because phosphorylation is fast, phosphorylation usually activates some other enzyme, and kinases usually act on an array of targets, not just one. So we now have a cyclin (identified as cdc13 in S. pombe) and a cyclin-dependent kinase that work together to promote cell cycle progression into M phase.