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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

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Molecular Biology of the Cell. 4th edition.

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Components of the Cell-Cycle Control System

For many years cell biologists watched the puppet show of DNA synthesis, mitosis, and cytokinesis but had no idea of what lay behind the curtain controlling these events. The cell-cycle control system was simply a black box inside the cell. It was not even clear whether there was a separate control system, or whether the processes of DNA synthesis, mitosis, and cytokinesis somehow controlled themselves. A major breakthrough came in the late 1980s with the identification of the key proteins of the control system, along with the realization that they are distinct from the proteins that perform the processes of DNA replication, chromosome segregation, and so on.

We first consider the basic principles upon which the cell-cycle control system operates. Then we discuss the protein components of the system and how they work together to activate the different phases of the cell cycle.

The Cell-Cycle Control System Triggers the Major Processes of the Cell Cycle

The cell-cycle control system operates much like the control system of an automatic clothes-washing machine. The washing machine functions in a series of stages: it takes in water, mixes it with detergent, washes the clothes, rinses them, and spins them dry. These essential processes of the wash cycle are analogous to the essential processes of the cell cycle—DNA replication, mitosis, and so on. In both cases, a central controller triggers each process in a set sequence (Figure 17-13).

Figure 17-13. The control of the cell cycle.

Figure 17-13

The control of the cell cycle. The essential processes of the cell cycle—such as DNA replication, mitosis, and cytokinesis—are triggered by a cell-cycle control system. By analogy with a washing machine, the cell-cycle control system is (more...)

How might one design a control system that safely guides the cell through the events of the cell cycle (or a wash cycle, for that matter)? In principle, one can imagine that the most basic control system should possess the following features:

  • A clock, or timer, that turns on each event at a specific time, thus providing a fixed amount of time for the completion of each event.
  • A mechanism for initiating events in the correct order; entry into mitosis, for example, must always come after DNA replication.
  • A mechanism to ensure that each event is triggered only once per cycle.
  • Binary (on/off) switches that trigger events in a complete, irreversible fashion. It would clearly be disastrous, for example, if events like chromosome condensation or nuclear envelope breakdown were initiated but not completed.
  • Robustness: backup mechanisms to ensure that the cycle can work properly even when parts of the system malfunction.
  • Adaptability, so that the system's behavior can be modified to suit specific cell types or environmental conditions.

We shall see in this chapter that the cell-cycle control system possesses all of these features, and that we are now beginning to understand the molecular mechanisms involved.

The Control System Can Arrest the Cell Cycle at Specific Checkpoints

We can illustrate the importance of an adjustable cell-cycle control system by extending our washing machine analogy. The control system of simple embryonic cell cycles, like the controller in a simple washing machine, is based on a clock. The clock is unaffected by the events it regulates and will progress through the whole sequence of events even if one of those events has not been successfully completed. In contrast, the control system of most cell cycles (and sophisticated washing machines) is responsive to information received back from the processes it is controlling. Sensors, for example, detect the completion of DNA synthesis (or the successful filling of the washtub), and, if some malfunction prevents the successful completion of this process, signals are sent to the control system to delay progression to the next phase. These delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage.

In most cells there are several points in the cell cycle, called checkpoints, at which the cycle can be arrested if previous events have not been completed (Figure 17-14). Entry into mitosis is prevented, for example, when DNA replication is not complete, and chromosome separation in mitosis is delayed if some chromosomes are not properly attached to the mitotic spindle.

Figure 17-14. Checkpoints in the cell-cycle control system.

Figure 17-14

Checkpoints in the cell-cycle control system. Information about the completion of cell-cycle events, as well as signals from the environment, can cause the control system to arrest the cycle at specific checkpoints. The most prominent checkpoints occur (more...)

Progression through G1 and G2 is delayed by braking mechanisms if the DNA in the chromosomes is damaged by radiation or chemicals. Delays at these DNA damage checkpoints provide time for the damaged DNA to be repaired, after which the cell-cycle brakes are released and progress resumes.

Checkpoints are important in another way as well. They are points in the cell cycle at which the control system can be regulated by extracellular signals from other cells. These signals—which can either promote or inhibit cell proliferation—tend to act by regulating progression through a G1 checkpoint, using mechanisms discussed later in the chapter.

Checkpoints Generally Operate Through Negative Intracellular Signals

Checkpoint mechanisms like those just described tend to act through negative intracellular signals that arrest the cell cycle, rather than through the removal of positive signals that normally stimulate cell-cycle progression. The following argument suggests why this is so.

Consider, for example, the checkpoint that monitors the attachment of chromosomes to the mitotic spindle. If a cell proceeds into anaphase and starts to segregate its chromosomes into separate daughter cells before all chromosomes are appropriately attached, one daughter receives an incomplete chromosome set, while the other daughter receives a surplus. The cell therefore needs to be able to detect the attachment of the last unattached chromosome to the microtubules of the spindle. In a cell with many chromosomes, if each chromosome sends a positive signal to the cell-cycle control system once it is attached, the attachment of the last chromosome will be hard to detect, as it will be signaled by only a small fractional change in the total intensity of the “go” signal. On the other hand, if each unattached chromosome sends a negative signal to inhibit progress through the cell cycle, the attachment of the last chromosome will be easily detected because it will cause a change from some “stop” signal to none. A similar argument would imply that unreplicated DNA inhibits the initiation of mitosis, creating a stop signal that persists until the completion of DNA replication.

The most convincing evidence that checkpoints operate through negative signals comes from studies of cells in which a checkpoint is inactivated by either mutation or chemical treatment. In these cells, the cell cycle continues to progress even if DNA replication or spindle assembly is incomplete, indicating that checkpoints are generally not essential for cell-cycle progression. Checkpoints are best viewed as accessory braking systems that have been added to the cell-cycle control system to provide a more sophisticated form of regulation.

Although most checkpoints are not essential for normal cell-cycle progression under ideal conditions, populations of cells with checkpoint defects often accumulate mutations due to occasional malfunctions in DNA replication, DNA repair, or spindle assembly. Some of these mutations can promote the development of cancer, as we discuss later and in Chapter 23.

The Cell-Cycle Control System Is Based on Cyclically Activated Protein Kinases

At the heart of the cell-cycle control system is a family of protein kinases known as cyclin-dependent kinases (Cdks). The activity of these kinases rises and falls as the cell progresses through the cycle. The oscillations lead directly to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle—DNA replication, mitosis, and cytokinesis. An increase in Cdk activity at the beginning of mitosis, for example, leads to increased phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, and spindle assembly.

Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins. The most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are tightly bound to a cyclin, they have no protein kinase activity (Figure 17-15). Cyclins were originally named as such because they undergo a cycle of synthesis and degradation in each cell cycle. Cdk levels, by contrast, are constant, at least in the simplest cell cycles. Cyclical changes in cyclin levels result in the cyclic assembly and activation of the cyclin-Cdk complexes; this activation in turn triggers cell-cycle events (Figure 17-16).

Figure 17-15. Two key components of the cell-cycle control system.

Figure 17-15

Two key components of the cell-cycle control system. A complex of cyclin with Cdk acts as a protein kinase to trigger specific cell-cycle events. Without cyclin, Cdk is inactive.

Figure 17-16. A simplified view of the core of the cell-cycle control system.

Figure 17-16

A simplified view of the core of the cell-cycle control system. Cdk associates successively with different cyclins to trigger the different events of the cycle. Cdk activity is usually terminated by cyclin degradation. For simplicity, only the cyclins (more...)

There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. Three of these classes are required in all eucaryotic cells:

1.

G1/S-cyclins bind Cdks at the end of G1 and commit the cell to DNA replication.

2.

S-cyclins bind Cdks during S phase and are required for the initiation of DNA replication.

3.

M-cyclins promote the events of mitosis.

In most cells, a fourth class of cyclins, the G1-cyclins, helps promote passage through Start or the restriction point in late G1.

In yeast cells, a single Cdk protein binds all classes of cyclins and drives all cell-cycle events by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are four Cdks. Two interact with G1-cyclins, one with G1/S- and S-cyclins, and one with M-cyclins. In this chapter, we simply refer to the different cyclin-Cdk complexes as G 1-Cdk, G1/S-Cdk, S-Cdk, and M-Cdk. The names of the individual Cdks and cyclins are given in Table 17-1.

Table 17-1. The Major Cyclins and Cdks of Vertebrates and Budding Yeast.

Table 17-1

The Major Cyclins and Cdks of Vertebrates and Budding Yeast.

How do different cyclin-Cdk complexes drive different cell-cycle events? The answer, at least in part, seems to be that the cyclin protein does not simply activate its Cdk partner but also directs it to specific target proteins. As a result, each cyclin-Cdk complex phosphorylates a different set of substrate proteins. The same cyclin-Cdk complex can also induce different effects at different times in the cycle, probably because the accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G2.

Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone blocking the entrance to a cave (Figure 17-17A). Cyclin binding causes the slab to move away from the active site, resulting in partial activation of the Cdk enzyme (Figure 17-17B). Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure 17-17C).

Figure 17-17. The structural basis of Cdk activation.

Figure 17-17

The structural basis of Cdk activation. These drawings are based on three-dimensional structures of human Cdk2, as determined by x-ray crystallography. The location of the bound ATP is indicated. The enzyme is shown in three states. (A) In the inactive (more...)

Cdk Activity Can Be Suppressed Both by Inhibitory Phosphorylation and by Inhibitory Proteins

The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several additional mechanisms, however, are important for fine-tuning Cdk activity at specific stages in the cell cycle.

The activity of a cyclin-Cdk complex can be inhibited by phosphorylation at a pair of amino acids in the roof of the active site. Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity (Figure 17-18). We see later that this regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis.

Figure 17-18. The regulation of Cdk activity by inhibitory phosphorylation.

Figure 17-18

The regulation of Cdk activity by inhibitory phosphorylation. The active cyclin-Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 results (more...)

Cyclin-Cdk complexes can also be regulated by the binding of Cdk inhibitor proteins (CKIs). There are a variety of CKI proteins, and they are primarily employed in the control of G1 and S phase. The three-dimensional structure of a cyclin-Cdk-CKI complex reveals that CKI binding dramatically rearranges the structure of the Cdk active site, rendering it inactive (Figure 17-19).

Figure 17-19. The inhibition of a cyclin-Cdk complex by a CKI.

Figure 17-19

The inhibition of a cyclin-Cdk complex by a CKI. This drawing is based on the three-dimensional structure of the human cyclin A-Cdk2 complex bound to the CKI p27, as determined by x-ray crystallography. The p27 binds to both the cyclin and Cdk in the (more...)

The Cell-Cycle Control System Depends on Cyclical Proteolysis

Cell-cycle control depends crucially on at least two distinct enzyme complexes that act at different times in the cycle to cause the proteolysis of key proteins of the cell-cycle control system, thereby inactivating them. Most notably, cyclin-Cdk complexes are inactivated by regulated proteolysis of cyclins at certain cell-cycle stages. This cyclin destruction occurs by a ubiquitin-dependent mechanism, like that involved in the proteolysis of many other intracellular proteins (discussed in Chapter 6). An activated enzyme complex recognizes specific amino-acid sequences on the cyclin and attaches multiple copies of ubiquitin to it, marking the protein for complete destruction in proteasomes.

The rate-limiting step in cyclin destruction is the final ubiquitin-transfer reaction catalyzed by enzymes known as ubiquitin ligases (see Figure 6-87B). Two ubiquitin ligases are important in the destruction of cyclins and other cell-cycle regulators. In G1 and S phase, an enzyme complex called SCF (after its three main protein subunits) is responsible for the ubiquitylation and destruction of G1/S-cyclins and certain CKI proteins that control S-phase initiation. In M phase, the anaphase-promoting complex (APC) is responsible for the ubiquitylation and proteolysis of M-cyclins and other regulators of mitosis.

These two large, multisubunit complexes contain some related components, but they are regulated in different ways. SCF activity is constant during the cell cycle. Ubiquitylation by SCF is controlled by changes in the phosphorylation state of its target proteins: only specifically phosphorylated proteins are recognized, ubiquitylated, and destroyed (Figure 17-20A). APC activity, by contrast, changes at different stages of the cell cycle. APC is turned on mainly by the addition of activating subunits to the complex (Figure 17-20B). We discuss the functions of SCF and APC in more detail later.

Figure 17-20. The control of proteolysis by SCF and APC during the cell cycle.

Figure 17-20

The control of proteolysis by SCF and APC during the cell cycle. (A) The phosphorylation of a target protein, such as the CKI shown, allows the protein to be recognized by SCF, which is constitutively active. With the help of two additional proteins called (more...)

Cell-Cycle Control Also Depends on Transcriptional Regulation

In the frog embryonic cell cycle discussed earlier, gene transcription does not occur. Cell-cycle control depends exclusively on post-transcriptional mechanisms that involve the regulation of Cdk activity by phosphorylation and the binding of regulatory proteins such as cyclins, which are themselves regulated by proteolysis. In the more complex cell cycles of most cell types, however, transcriptional control provides an added level of regulation. Cyclin levels in most cells, for example, are controlled not only by changes in cyclin degradation but also by changes in cyclin gene transcription and cyclin synthesis.

In certain organisms, such as budding yeasts, one can use DNA arrays (discussed in Chapter 8) to analyze changes in the expression of all of the genes in the genome as the cell progresses through the cell cycle. The results of these studies are surprising. About 10% of the yeast genes encode mRNAs whose levels oscillate during the cell cycle. Some of these genes encode proteins with known cell-cycle functions, but the functions of many others are unknown. It seems likely that these oscillations in gene expression are controlled by the cyclin-Cdk-dependent phosphorylation of gene regulatory proteins, but the details of this regulation remain unknown.

Summary

Events of the cell cycle are triggered by an independent cell-cycle control system, which ensures that the events are properly timed, occur in the correct order, and occur only once per cell cycle. The control system is responsive to various intracellular and extracellular signals, so that cell-cycle progression can be arrested when the cell either fails to complete an essential cell-cycle process or encounters unfavorable environmental conditions.

The central components of the cell-cycle control system are cyclin-dependent protein kinases (Cdks), whose activity depends on association with regulatory subunits called cyclins. Oscillations in the activities of various cyclin-Cdk complexes leads to the initiation of various cell-cycle events. Thus, activation of S-phase cyclin-Cdk complexes initiates S phase, while activation of M-phase cyclin-Cdk complexes triggers mitosis. The activities of cyclin-Cdk complexes are influenced by several mechanisms, including phosphorylation of the Cdk subunit, the binding of special inhibitory proteins (CKIs), proteolysis of cyclins, and changes in the transcription of genes encoding Cdk regulators. Two enzyme complexes, SCF and APC, are also crucial components of the cell-cycle control system; they induce the proteolysis of specific cell-cycle regulators by ubiquitylating them and thereby trigger several critical events in the cycle.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
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