The existence of different regulators at different stages of the cell cycle was revealed by early experiments, performed in yeast that fused together cells in different stages of the cycle. Fusion of the plasma membranes generates a hybrid cell called a heterokaryon that contains two nuclei in a common cytoplasm. The results can be summarized as follows:
When a cell in S phase is fused with a cell in G1, both nuclei in the heterokaryon replicate DNA. This suggests that the cytoplasm of the S phase cell contains an activator of DNA replication. The regulator identified by these fusions is called the S phase activator. Similarly, when a mitotic cell is fused with a cell at any stage of the interphase, it causes the interphase cell to enter a pseudo-mitosis characterized by a premature condensation of its chromosomes. This suggests that an M phase inducer is present in dividing cells. However, both these inducers are only present transiently, because fusions between G1 and G2 cells do not induce replication or mitosis in either nucleus of the heterokaryon. Furthermore, other fusion experiments (S x G2) also showed that DNA that has replicated becomes refractory to the effects of the S phase activator, a feature to ensure that each sequence of DNA replicates only once.
A striking feature of cell-cycle regulation is that similar regulatory activities are employed in probably all eucaryotic systems. Indeed injection of cytoplasm extracted from arrested frog eggs (equivalent to M phase somatic cells) into arrested oocytes (equivalent to G2 somatic cells) induces the latter to enter the M phase. The active component of the extract, the M phase promoting factor (MPF), was found to be a kinase that can phosphorylate a variety of protein substrates, as we shall soon see l. By phosphorylating target proteins at a specific point in the cell-cycle, MPF controls their ability to function. Similarly, the S phase activator is also a Ser/Thr protein kinase related to this M phase kinase.
M phase kinase has two subunits with different functions. In the S.Pombe yeast, Cdc2 (also referred to as p34) is the kinase catalytic subunit which phosphorylates Ser and Thr residues in target proteins. Its partner is a cyclin; this is a regulatory subunit which is necessary for the kinase to function with appropriate substrates. M phase kinase is hence a cyclin-dependent protein kinase (CDK). This family of protein kinases is at the heart of the eucaryotic cell-cycle control system. However, it is their cyclical activation (and hence regulation) that enables them to mediate such a control. Indeed, their activity rises and falls as the cell progresses through the cycle. The oscillations lead directly to the cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle_ DNA replication, mitosis and cytokinesis.
The prototypic member of the CDK family p34cdc2 controls entry into mitosis in all eukaryotes. The picture that has emerged so far is that p34/cyclin B may be directly responsible for a substantial part of the phosphorylation occurring in mitotic cells. Indeed, there are several lines of evidence indicating that direct phosphorylation of lamins by p34/cyclin B plays a major role in the disassembly of this karyoskeletal system. In addition, chromosome condensation is accompanied by extensive phosphorylation of histone H1, certain high mobility proteins and several transcription factors. For most these proteins, phosphorylation on CDK sites is thought to weaken their interaction with DNA. Moreover, topoisomerase II, which is required for chromosome condensation and segregation of sister chromatids, has also been shown to be phosphorylated by p34/cyclin B in vitro. The events regulated by this M phase kinase are reversible: phosphorylation of substrates is required for the reorganization of the cell into a mitotic spindle, and dephosphorylation of the same substrates is required to return to an interphase organization.
In the S. Pombe yeast, p34cdc2 is also required during the G1 phase. In higher eukaryotes however, the traverse of the G1 controls as well as progression through S phase both appear to be controlled by distinct, structurally related kinases, including cdk2, cdk4 and cdk5, and their associated cyclins. CDK/cyclin complexes are hence also required during interphase. The prime CDK candidate in vertebrates for carrying out an S phase function is the cyclin A/CDK2 complex. Single-stranded DNA-binding replication protein A and perhaps DNA polymerase α are likely to be included amongst the S phase substrates of CDKs.
We have established up to this point how the phosphorylation catalysed by cyclin-dependent kinases and dephosphorylation (catalysed by phophatases) are critical events that regulate the cell cycle. Indeed, they control the activities of the substrates that execute the decisions of the regulatory circuit. However reversible phosphorylation events also control the activities of the regulatory circuit itself. As we mentioned above, checkpoint controls operate to ensure, for example, that DNA replication is complete before mitosis is initiated. Major elements in these checkpoint controls are the kinases and phosphatases that determine the phosphorylation state of the CDKs. This has been best characterized in the context of the G2/M-phase transition.
CDKs, as we have said, are cyclically activated kinases, and one of the mechanisms modulating their activity is their phosphorylation state. Indeed, phosphorylation of a conserved threonine residue at position 161 in yeast Cdc2 is critical for the activity of CDK/cyclin complexes. Thr 161 is phosphorylated by CDK-activating kinase (CAK), resulting in a conformational change that fully exposes the catalytic cleft so that the substrate may bind. Such a phosphorylation may also serve to stabilize the cyclin-CDK complex.
Another mode of CDK regulation is through phosphorylation within the active site of the enzyme at a conserved Tyr 15. Vertebrate CDKs undergo additional phosphorylation at the adjacent Thr 14. However, Tyr 15 phosphorylation (as well as that on Thr 14 in vertebrates) proves to be inhibitory. While it does not interfere with with ATP binding, it sterically hinders substrate access to the catalytic site of the enzyme. Wee 1 tyrosine kinase is responsible for phosphorylating Cdc2 at Tyr 15, and its activity is itself intricately regulated by an upstream network of kinases and phosphatases.
The Cdc25 family of phosphatases are tightly regulated enzymes and are responsible for the dephosphorylation of Tyr 15 (and Thr 14), and hence activation of CDKs. To be fully active, a cyclin/CDK complex must hence be phosphorylated at Thr 161 and dephosphorylated at Tyr 15 (and Thr 14).
It is thus the opposing activities of Wee 1 kinase and Cdc25 phosphatase that govern the timing of mitosis.
Reversible phosphorylation events that control the activities of these CDKs are hence also critical to the eucaryotic cell-cycle. However, these are not the sole mechanisms by which CDK activity is modulated. As is implied by their name, CDKs are cyclin-dependent. Thus cyclin-binding is an essential prerogative for these kinases to be active. It is hence also used to signal the exit of the M phase. Indeed, once division has taken place, the cyclin constituent undergoes ubiquitin-dependent proteolysis, resulting in M phase CDK inactivation, and entry into G1 phase.
Cell-cycle regulation is achieved by controlling the substrates of the regulatory circuit, as well as by controlling the regulatory circuit itself. In both cases, reversible phosphorylation events involving cyclin-dependent kinases greatly contribute to achieving this goal.
This cell-cycle control is crucial seeing as it has a central role in regulating cell numbers in the tissues of the body. When the system malfunctions, excessive cell divisions can result in cancer.