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Modern Genetic Analysis Chapter 15. Regulation of Cell Number: Normal and Cancer Cells
Controlling the Cell Proliferation and Death Machinery

We have used the term engine to describe the role of the cyclinCDK complex or the caspase cascade in cell proliferation or programmed cell death, respectively. To continue the analogy, ignition switches and accelerators (positive controls) start up the engines and get these processes moving, and brakes (negative controls) slow down or halt the processes when necessary. Like the cell cycle and apoptosis, the positive and negative controls comprise a series of modulations of protein activities through proteinprotein interactions and protein modifications.

Intracellular Signals

Some of the elements of the positive and negative control loops consist of signals that originate within the cell.

The cell cycle: negative intracellular controls.

Through activation of proteins that can inhibit the protein kinase activity of CDKcyclin complexes, the cell cycle can be held in check until various monitoring mechanisms give a "green light," indicating that the cell is properly prepared to proceed to the next phase of the cycle.

One example of how this "checkpoint" system operates begins with damaged DNA (Figure 15-6). When DNA is damaged during G1 (for example, by X-irradiation), CDK activity of CDKcyclin complexes is inhibited. The inhibition seems to be mediated by a protein called p53. Part of the p53 protein recognizes certain kinds of DNA mismatches. In the presence of such mismatches, p53 is able to activate another protein, p21. When its levels are high, p21 binds to the CDKcyclin complex and inhibits its protein kinase enzymatic activity. In the absence of its protein kinase activity, CDK's target proteins are not phosphorylated, and the cell cycle is unable to progress. When the DNA mismatches have been repaired, the inhibiting processes are reversed. This reversal is accomplished by a post-DNA-repair drop in p53 levels and a cessation of inhibition of the CDKcyclin protein kinase activity and leads to removal of the G1-to-S checkpoint block.

In this manner, checkpoints monitor the status of DNA replication, the spindle apparatus, and other key components of the cell cycle and can operate as braking systems when necessary. The key is the existence of regulatory proteins that can modulate the protein kinase activity of the cyclinCDK complex.

MESSAGE
Fail-safe systems (checkpoints) ensure that the cell cycle does not progress until the cell is competent.

The cell cycle: positive intracellular controls.

It is necessary not only to release the cell cycle "brake," but also to engage the "transmission" and the "engine" to advance the cell cycle. Once the brake is released, independent signals from within or outside the cell induce a cascade of protein kinases that phosphorylate the appropriate cyclinCDK complex, thereby activating the complex. This activation in turn allows the complex to phosphorylate its target proteins.

Apoptosis: positive intracellular controls.

It has been known for several years that, in some manner, many forms of cellular damage trigger leakage of mitochondria and that this leakage somehow induces the apoptotic response. Indeed, it now appears that one of the ignition switches is cytochrome c, one of the mitochondrial proteins normally taking part in cell respiration. Leakage of cytochrome c into the cytoplasm is detected and triggers the activation of initiator caspases. This detection is thought to happen through the binding of cytochrome c to another protein called Apaf (apoptotic protease activating factor). The cytochrome cApaf complex then binds to and activates the initiator caspase.

Apoptosis: negative intracellular controls.

The irreversibility of cell death has probably been the compelling factor in the evolution of backup systems to make sure that the apoptosis pathway remains "off" under normal conditions. Proteins such as Bcl-2 and Bcl-x in mammals accomplish this. Among the possible actions for these Bcl proteins is that they block the release of cytochrome c from mitochondria (possibly by making it more difficult for mitochondria to burst) and by binding to Apaf and preventing its interaction with the initiator caspase.

Extracellular Signals

A cell in a multicellular organism continually assesses its own internal status regarding proliferation and survival. Nonetheless, the proliferative and survival abilities of a cell must be subservient to the needs of the population of cells of which it is a member (populations such as the entire early embryo, a tissue, or a body part such as a limb or an organ). For example, in many adult organs, stem cells divide to produce replacement cells only when there is a depletion of cell numbers. Without such homeostatic mechanisms, organs would not be proportioned appropriately for the size of a given individual organism.

Mechanisms for cellcell communication.

Many kinds of signals need to be transmitted between cells to coordinate virtually all aspects of the development and physiology of complex multicellular organisms. The major routes of cellcell communication are briefly outlined here.

All systems for intercellular communication have several components. A molecule called a ligand is produced by secretion from signaling cells (Figure 15-7). Some ligands, called hormones, are long-range endocrine signals that are transmitted throughout the body by being released from endocrine organs into the circulatory system. (Recall the discussion of steroid hormones and their receptors in Chapter 14.) Hormones can act as master control switches for many different tissues, which can then respond in a coordinated fashion. Other secreted ligands act as paracrine signals; that is, they do not enter the circulatory system but act only locally, in some cases only on immediately adjacent cells. We shall have more to say about paracrine and endocrine signals in Chapter 16. Some ligands are proteins, whereas others are small molecules such as steroids or vitamin D. Most (but not all) endocrine signals are small molecules, such as the mammalian steroid hormones that are responsible for male (androgen) or female (estrogen) sex-specific phenotypes. In contrast, most paracrine signals are proteins. Here we focus on paracrine signaling through protein ligands.

Protein ligands act as signals by binding to and thereby activating transmembrane receptor proteins that are embedded in the plasma membrane at the surface of the cell. These ligandreceptor complexes initiate chemical signals in the cytoplasm just inside the plasma membrane of the cell. Such signals are passed through a series of intermediary molecules until they finally alter the structure of transcription factors in the nucleus, leading to the activation of transcription of some genes and repression of others.

Transmembrane receptors have one part (the extracellular domain) outside of the cell, a middle part that passes once or several times through the plasma membrane, and another part (the cytoplasmic domain) inside the cell (Figure 15-8).

The extracellular domain of the receptor is the site to which the ligand binds. Many polypeptide ligands are dimers and can simultaneously bind two receptor monomers. This simultaneous binding brings the cytoplasmic domains of the two receptor subunits into close proximity and activates the signaling activity of these cytoplasmic domains. Some receptors for polypeptide ligands are receptor tyrosine kinases (RTKs, Figure 15-8b). Their cytoplasmic domains, when activated, have the ability to phosphorylate certain tyrosine residues on target proteins. Others are receptor serine/threonine kinases. Still other receptors have no enzymatic activity, but conformational changes in a receptor (when a ligand binds to it) cause conformational changes in (and activation of) receptor-bound cytoplasmic proteins.

Perhaps the best understood of the receptors for polypeptide ligands are the receptor tyrosine kinases (Figure 15-9). RTK is a monomer essentially "floating" within the plane of the plasma membrane. When ligand and RTK bind to form a ligandRTK complex, two RTK monomers bind to form a dimer. RTK dimerization activates the protein kinase enzymatic activity of the cytoplasmic domain of the RTK. The first phosphorylation targets of the kinase are several tyrosines in the cytoplasmic domain of the RTK itself; this process is called autophosphorylation because the kinase acts upon itself. Autophosphorylation initiates a signal transduction cascade, in which, sequentially, modifications in the conformations of one protein lead to modifications in the conformations of others. Eventually, the signal transduction cascade leads to the modification of transcriptional activators and repressors and hence to changes in the activities of many genes in the target cell.

RTK autophosphorylation activates signal transduction cascades in two ways. In one process, phosphorylated sites on the RTK are targets for binding by various adaptor proteins (Figure 15-9a). Multiple adaptor proteins "dock" on phosphorylated sites on the RTK in the vicinity of one another. These adaptor proteins in turn have affinity for other proteinselements of signal transduction cascades. By bringing these other signal transduction elements into proximity with one another, proteinprotein interactions lead to activation of these cascades. In the other process, the phosphorylated RTK is conformationally changed so that its tyrosine kinase active site phosphorylates other target proteins (Figure 15-9b). These phosphorylated target proteins are then changed in conformation, allowing them to participate in a signal transduction cascade. By these two processes, activation of one RTK can lead to the simultaneous activation of multiple signal transduction pathways.

Quite often, the next step in propagating the signal is to activate a G-protein. G-proteins cycle between being bound by GDP (the inactive state) and being bound by GTP (the activated state). The propagation of the signal from the RTK leads to the activation of a protein that binds to the inactive GDP-bound G-protein, changing its conformation so that it then binds to a molecule of GTP (Figure 15-10). The specific G-protein called Ras is of special importance in carcinogenesis, as discussed later.

The activated GTP-bound G-protein then binds to a cytoplasmic protein kinase, in turn changing its conformation and activating its protein kinase activity. This protein kinase then phosphorylates other proteins, including other protein kinases. (In the example in Figure 15-11, the protein kinases farther down the cascade are called Raf, MEK, and MAP kinase.) The targets of some of these protein kinases are transcriptional activators and repressors. The phosphorylation of the transcription factors changes their conformations, leading to the activation of transcription of some genes and the repression of others (Figure 15-11).

Cellcell signaling depends on conformational changes.

We have seen that the steps in ligand receptor binding and in signaling within the cell depend on conformational changes. For example, the conformational changes caused by the binding of ligands to receptors activate the signaling pathways. Likewise, conformational changes in protein kinases enable them to phosphorylate specific amino acids on specific proteins, and other proteins undergo conformational changes when they bind to GTP. Not only do these conformational changes permit rapid response to an initial signal, but they also are readily reversible, enabling signals to be shut down rapidly and permitting recycling of the components of the signaling system so that they are ready to receive further signals.

The cell cycle: positive extracellular controls.

Cell division is promoted by the action of mitogens, polypeptide ligands released usually from a paracrine (nearby) source. Many mitogens, also called growth factors, such as EGF (epidermal growth factor), activate RTKs and initiate exactly the sort of signal transduction pathway heretofore described.

The cell cycle: negative extracellular controls.

Certain secreted proteins are known to inhibit cells from dividing. One example is TGF- b , a ligand that is thought to be secreted in a variety of tissues under growth inhibitory conditions. The TGF- b ligand binds to and activates the TGF- b receptor's serine/threonine kinase activity. This activation in turn leads to phosphorylation of proteins called SMADs, which cause changes in transcriptional activities, and perhaps to phosphorylation of other substrates as well. As a result of this signal transduction cascade, the phosphorylation and inactivation of the Rb protein are eventually blocked. Recall, from earlier in the chapter, Rb's cell cycle role of preventing activation of the E2F transcription factor. This block to Rb inactivation thus keeps E2F off and blocks progression of the cell cycle.

Apoptosis: positive extracellular controls.

Often, the command for self-destruction comes from a neighboring cell. For example, within the immune system, only a small percentage of B cells and T cells mature to make functional antibody or T cell receptor protein, respectively. If nonfunctional, immature B cells and T cells were not eliminated by induced self-destruction, the vast majority of them would clog up the immune system. The self-destruction signal is activated through the Fas system (Figure 15-12). A cell-surface membrane-bound protein called FasL (Fas ligand) binds to Fas cell-surface receptors on an adjacent cell. This induces trimerization of the ligandreceptor complex and trimerization of a cytoplasmic domain of the Fas transmembrane receptor. This in turn, directly or indirectly, activates a molecule such as Apaf (discussed earlier in this chapter), which activates an initiator caspase and thus the caspase cascade.

Apoptosis: negative extracellular controls.

Negative secreted factors that are necessary to block activation of the apoptosis pathway also exist, and they are sometimes referred to as survival factors. How they impinge on the apoptosis pathway is not clear at present.

MESSAGE
Intercellular signaling systems communicate instructions between cells to proliferate or to arrest the cell cycle and to initiate or postpone self-destruction.

An Integrated View of the Control of Cell Numbers

What we have seen in the preceding sections is that there are numerous ways to modulate cell number. The general theme is that pathways exist for controlling cell proliferation and self-destruction and that activation of these pathways requires the correct array of positive inputs and the absence of negative, or inhibitory, inputs. Not only do cells have mechanisms for self-assessment of their status regarding proliferation ability or viability, but neighboring cells can play instructive roles through cellcell signaling (Figure 15-13).

© 1999 by W. H. Freeman and Company. All rights reserved.