19.4: Regulation of the Cell Cycle

Progress through the cell cycle is regulated. The cycle can be controlled or put on “pause” at any one of several phase transitions. Such checkpoints monitor whether the cell is on track to complete a successful cell-division event. Superimposed on these controls are signals that promote cell differentiation. Embryonic cells differentiate as the embryo develops. Even after terminal differentiation of cells that form all adult tissues and organs, adult stem cells will divide and differentiate to replace worn out cells. Once differentiated, cells are typically signaled in \(\rm G_1\) to enter \(\rm G_0\) and to stop cycling. In some circumstances cells in \(\rm G_0\) are recruited to resume cycling. However, if this resumption should occur by mistake, the cells may be transformed to cancer cells. Here we consider how the normal transition between phases of the cell cycle is controlled.

19.4.1 Discovery and Characterization of Maturation Promoting Factor (MPF)

To monitor their progress through the cell cycle, cells produce internal chemical signals that tell them when it’s time to begin replication or mitosis, or even when to enter into \(\rm G_0\) when they reach their terminally differentiated state. The experiment that first demonstrated a chemical regulator of the cell cycle involved fusing very large frog’s eggs! The experiment is described in Figure 19.9.

Figure 19.9: Experiment leading to the discovery of meiosis-promoting factor (MPF), the first known chemical regulator of cell division.

The hypothesis tested here was that frog oocyte cytoplasm from germinal vesicle-stage oocytes (i.e., oocytes in mid meiosis) contain a chemical that causes the cell to lose its nuclear membrane, to condense its chromatin into chromosomes, and to enter meiosis. The test was to withdraw cytoplasm from one of these large, mid meiotic oocytes with a fine hypodermic needle and to inject it into a pre meiotic oocyte. The result of the experiment was that the mid meiotic oocyte cytoplasm induced premature meiosis in the immature oocyte.

Subsequently, a Maturation Promoting Factor (MPF) was isolated from the mid meiotic cells—a factor that when injected into pre meiotic cells, would cause them to enter meiosis. MPF turns out to be a protein kinase made up of two polypeptide subunits (Figure 19.10, below). MPF was subsequently shown to stimulate somatic cells in \(\rm G_2\) to enter premature mitosis. So (conveniently) MPF can also be Mitosis Promoting Factor! Hereafter we will discuss the effects of an MPF as being equivalent in mitosis and meiosis. When active, MPF targets many cellular proteins.

Figure 19.10: MPF was shown to be a two-subunit protein kinase that transfers phosphates from ATP to several different proteins.

342 Discovery of MPF Kinase and Its Role in Meiosis and Mitosis

Assays of MPF activity as well as the actual levels of the two subunits over time during the cell cycle are graphed in Figure 19.11.

Figure 19.11: Graph comparing plots of cellular cdk and cyclin levels over time with one of MPF activity.

One subunit of MPF is cyclin. The other subunit, cyclin-dependent kinase (cdk), contains the kinase-enzyme active site. Both subunits must be bound to make an active kinase. Cyclin was so named because its levels rise gradually after cytokinesis, peak at the next mitosis, and then fall. Levels of the cdk subunit do not change significantly during the life of the cell. Because the kinase activity of MPF requires cyclin, it tracks the rise in cyclin near the end of the \(\rm G_2\), and its fall after mitosis. Cyclin begins to accumulate in \(\rm G_1\), rising gradually and binding to more and more cdk subunits. MPF reaches a threshold concentration in \(\rm G_2\) that triggers entry into mitosis. For their discovery of these central molecules, L. H. Hartwell, R. T. Hunt, and P. M. Nurse won the 2001 Nobel Prize in Physiology or Medicine.

CHALLENGE

What kinds of proteins might be activated by cdk-catalyzed phosphorylation to promote progress through the cell cycle?

19.4.2 Other Cyclins, CDKs, and Cell-Cycle Checkpoints

Other chemical signals accumulate at different points in the cell cycle. For example, when cells in S are fused with cells in \(\rm G_1\), the \(\rm G_1\) cells begin synthesizing DNA (visualized as \({}^3\)H-thymine incorporation). Figure 19.12 describes an experiment showing control of progress to different phases of the cell cycle.

Figure 19.12: Cell-fusion experiments revealed additional chemical regulators of the cell-cycle activity.

An S phase factor could be isolated from the S phase cells. This factor also turns out to be a two-subunit protein kinase, albeit a different one from MPF. Just as MPF signals cells in \(\rm G_2\) to begin mitosis, the S-phase kinase signals cells in \(\rm G_1\) to enter the S phase of the cell cycle and to start replicating DNA. MPF and the S-phase kinase govern activities at two of several cell-cycle checkpoints. In each case, the activity of the kinases is governed by prior progress through the cell cycle. In other words, if the cell is not ready to begin mitosis, active MPF production is delayed until it is. Likewise, the S-phase kinase will not be activated until the cell is ready to begin DNA synthesis.

343 Cell-Cycle Control at Check Points and the \(\rm G_0\) "Phases"

Control of progress through the cell cycle is more intricate than we currently know, but the best-described checkpoints separate \(\rm G_1\), \(\rm G_2\), and M (Figure 19.13).

Figure 19.13: Simplified diagram of cell-cycle checkpoints at which progress through the cycle is assessed. If progress through a phase is incomplete, cell-cycle regulators (kinases) delay onset of the next phase.

We generally envision checkpoints as monitoring and blocking progress until essential events of a current phase of the cell-cycle phase are completed. These kinases are part of molecular sensing mechanisms that act by phosphorylating cytoplasmic and/or nuclear proteins required by upcoming phases of the cycle. Let’s look at some events that are monitored at these checkpoints in more detail.

19.4.2.a The \(\rm G_1\) Checkpoint

The \(\rm G_1\) checkpoint controls the transition from the \(\rm G_1\) to the S phase of the cell cycle. If actively dividing cells (e.g., stem cells) in \(\rm G_1\) fail to complete their preparation for replication, the S-phase kinase won’t be produced, and the cells won’t proceed to the S phase until the preparatory biochemistry catches up with the rest of the cycle. To enter S, a cell must be ready to make proteins of replication, like DNA polymerases, helicases, and primases, among others. Only when these molecules have accumulated to (or become active at) appropriate levels is it “safe” to enter S and begin replicating DNA.

Now, what about those cells that are fully differentiated? Terminally differentiated cells stop producing the active \(\rm G_1\)-checkpoint kinase and stop dividing. Thus, they are arrested in \(\rm G_0\) (see section 19.4.2.d).

19.4.2.b The G2 Checkpoint

Passage through the \(\rm G_2\) checkpoint is only possible if DNA made in the prior S phase is not damaged. Or if it was, then that damage has been (or can be) repaired. (Review the proofreading functions of DNA polymerase and the various DNA-repair pathways.) Cells that do successfully complete replication and pass the \(\rm G_2\) checkpoint must prepare to make the proteins necessary for the upcoming mitotic phase. These include nuclear proteins necessary to condense chromatin into chromosomes, tubulins for making microtubules, and more. Only when levels of these and other required proteins reach a threshold can the cell begin mitosis.

Consider the following two tasks required of the \(\rm G_2\) checkpoint (in fact, any checkpoint):

• Sensing whether prior phase activities have been successfully completed
• Delaying transition to the next phase if those activities are unfinished

But what if sensing is imperfect and a checkpoint is leaky? A recent study suggests that either the \(\rm G_2\) checkpoint is leaky, or at least, that incomplete activities in the S phase are tolerated, and that some DNA repair is not resolved until mitosis is underway in M! See more about this at DNA repair at mitosis.

19.4.2.c The M Checkpoint

The M checkpoint is monitored by the original MPF-catalyzed phosphorylation of proteins, which do the following:

a) Bind to chromatin, causing it to condense and to form chromatids

b) Lead to the breakdown of the nuclear envelope

c) Enable spindle-fiber formation and their attachment to chromatids

d) Lead to the onset of mitosis

We have seen that the tension in the spindle apparatus at metaphase tugs at the kinetochores holding the duplicated chromatids together. As this tension reaches a threshold, MPF peaks and an activated separase enzyme causes the chromatids to separate at their centromeres. Starting in anaphase, continuing tension in the spindle apparatus draws the new chromosomes to opposite poles of the cell. Near the end of mitosis and cytokinesis, proteins phosphorylated by MPF initiate the breakdown of cyclin in the cell. Passing the M checkpoint means that the cell will complete mitosis and cytokinesis, and that each daughter cell will enter a new \(\rm G_1\) phase.

During cell division, yeast cells seem to have the three checkpoints discussed here. More-complex eukaryotes use more cyclins and cdks to control the cell cycle at more checkpoints. Different cyclins show cyclic patterns of synthesis, while their cdks remain at constant levels throughout the cell cycle (as in MPF). While the different cdk and cyclin gene families are evolutionarily conserved, each cyclin/cdk pair has been co-opted in evolution to monitor different cell-cycle events and to catalyze phosphorylation of phase-specific proteins. To learn more, see Elledge S. J. (1996) Cell Cycle Checkpoints: Preventing an Identity Crisis. Science 274:1664-1672.

19.4.2.d The \(\rm G_0\) State

\(\rm G_0\) is not really a phase of the cell cycle, since cells in \(\rm G_0\) have reached a terminally differentiated state and have stopped dividing. Terminally differentiated cells in tissues and organs no longer divide. But even these cells have a finite half-life. Recall that our red blood cells lack a nucleus and have a half-life of about 30 days, so they must be replaced every sixty days or so. Because cells in many tissues (that do retain their nuclei) are in \(\rm G_0\) and can’t divide, when they age they must be replaced by stem cells, which can divide and differentiate. Lifetime in G0 can vary. Some cells live so long in \(\rm G_0\) that they are nearly never replaced (muscle cells, neurons). Others live short lives in \(\rm G_0\) (e.g., stem cells and some embryonic cells).

CHALLENGE

Here’s something to ponder - recall that somatic cells are diploid, and that germ cells (sperm, egg) are haploid. When in the life of a somatic cell is it truly diploid? How would you characterize cells in \(\rm G_2\)?

Lymphocytes are differentiated immune-system cells that can reenter \(\rm G_1\). Exposure of lymphocytes to foreign chemicals or pathogens activates mitogens, causing the cells to exit \(\rm G_0\) to start dividing, and to produce the legions of cells that make antibodies that neutralize toxins and fight off pathogens. The retinoblastoma (Rb) protein is mitogen, a transcription factor that turns on genes leading to cell proliferation.

But, what if cells resume cycling when they shouldn’t? What if they are inappropriately signaled to exit \(\rm G_0\)? Such cells are in trouble! Having escaped normal controls on cell division, they can become a focal point of cancer-cell growth. You can guess from its name that the retinoblastoma gene was discovered as a mutation that causes retinal cancer. For more about the normal function of the Rb protein and its interaction with a \(\rm G_1\) cdk, check out the following link.

345-2 Rb Gene Encodes the regulatory subunit of a Transcription Factor

CHALLENGE

What kind of Rb gene mutation might cause cancer?