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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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Signaling through Enzyme-Linked Cell-Surface Receptors

Enzyme-linked receptors are a second major type of cell-surface receptor. They were recognized initially through their role in responses to extracellular signal proteins that promote the growth, proliferation, differentiation, or survival of cells in animal tissues. These signal proteins are often collectively called growth factors, and they usually act as local mediators at very low concentrations (about 10-9-10-11 M). The responses to them are typically slow (on the order of hours) and usually require many intracellular signaling steps that eventually lead to changes in gene expression. Enzyme-linked receptors have since been found also to mediate direct, rapid effects on the cytoskeleton, controlling the way a cell moves and changes its shape. The extracellular signals that induce these rapid responses are often not diffusible but are instead attached to surfaces over which the cell is crawling. Disorders of cell proliferation, differentiation, survival, and migration are fundamental events that can give rise to cancer, and abnormalities of signaling through enzyme-linked receptors have major roles in this class of disease.

Like G-protein-linked receptors, enzyme-linked receptors are transmembrane proteins with their ligand-binding domain on the outer surface of the plasma membrane. Instead of having a cytosolic domain that associates with a trimeric G protein, however, their cytosolic domain either has an intrinsic enzyme activity or associates directly with an enzyme. Whereas a G-protein-linked receptor has seven transmembrane segments, each subunit of an enzyme-linked receptor usually has only one.

Six classes of enzyme-linked receptors have thus far been identified:

1.

Receptor tyrosine kinases phosphorylate specific tyrosines on a small set of intracellular signaling proteins.

2.

Tyrosine-kinase-associated receptors associate with intracellular proteins that have tyrosine kinase activity.

3.

Receptorlike tyrosine phosphatases remove phosphate groups from tyrosines of specific intracellular signaling proteins. (They are called “receptorlike” because the presumptive ligands have not yet been identified, and so their receptor function has not been directly demonstrated.)

4.

Receptor serine/threonine kinases phosphorylate specific serines or threonines on associated latent gene regulatory proteins.

5.

Receptor guanylyl cyclases directly catalyze the production of cyclic GMP in the cytosol.

6.

Histidine-kinase-associated receptors activate a “two-component” signaling pathway in which the kinase phosphorylates itself on histidine and then immediately transfers the phosphate to a second intracellular signaling protein.

We begin our discussion with the receptor tyrosine kinases, the most numerous of the enzyme-linked receptors. We then consider the other classes in turn.

Activated Receptor Tyrosine Kinases Phosphorylate Themselves

The extracellular signal proteins that act through receptor tyrosine kinases consist of a large variety of secreted growth factors and hormones. Notable examples discussed elsewhere in this book include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), insulin, insulinlike growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), macrophage-colony-stimulating factor (M-CSF), and all the neurotrophins, including nerve growth factor (NGF).

Many cell-surface-bound signal proteins also act through these receptors. The largest class of these membrane-bound ligands is the ephrins, which regulate the cell adhesion and repulsion responses that guide the migration of cells and axons along specific pathways during animal development (discussed in Chapter 21). The receptors for ephrins, called Eph receptors, are also the most numerous receptor tyrosine kinases. The ephrins and Eph receptors are unusual in that they can simultaneously act as both ligand and receptor: on binding to an Eph receptor, some ephrins not only activate the Eph receptor but also become activated themselves to transmit signals into the interior of the ephrin-expressing cell. In this way, an interaction between an ephrin protein on one cell and an Eph protein on another cell can lead to bidirectional reciprocal signaling that changes the behavior of both cells. Such bidirectional signaling between ephrins and Eph receptors is required, for example, to keep cells in particular parts of the developing brain from mixing with cells in neighboring parts.

Receptor tyrosine kinases can be classified into more than 16 structural subfamilies, each dedicated to its complementary family of protein ligands. Several of these families that operate in mammals are shown in Figure 15-49, and some of their ligands and functions are given in Table 15-4. In all cases, the binding of a signal protein to the ligand-binding domain on the outside of the cell activates the intracellular tyrosine kinase domain. Once activated, the kinase domain transfers a phosphate group from ATP to selected tyrosine side chains, both on the receptor proteins themselves and on intracellular signaling proteins that subsequently bind to the phosphorylated receptors.

Figure 15-49. Seven subfamilies of receptor tyrosine kinases.

Figure 15-49

Seven subfamilies of receptor tyrosine kinases. Only one or two members of each subfamily are indicated. Note that the tyrosine kinase domain is interrupted by a “kinase insert region” in some of the subfamilies. The functional roles of (more...)

Table 15-4. Some Signaling Proteins That Act Via Receptor Tyrosine Kinases.

Table 15-4

Some Signaling Proteins That Act Via Receptor Tyrosine Kinases.

How does the binding of an extracellular ligand activate the kinase domain on the other side of the plasma membrane? For a G-protein-linked receptor, ligand binding is thought to change the relative orientation of several of the transmembrane α helices, thereby shifting the position of the cytoplasmic loops relative to each other. It is difficult to imagine, however, how a conformational change could propagate across the lipid bilayer through a single transmembrane α helix. Instead, for the enzyme-linked receptors, two or more receptor chains come together in the membrane, forming a dimer or higher oligomer. In some cases, ligand binding induces the oligomerization. In other cases, the oligomerization occurs before ligand binding, and the ligand causes a reorientation of the receptor chains in the membrane. In either case, the rearrangement induced in cytosolic tails of the receptors initiates the intracellular signaling process. For receptor tyrosine kinases, the rearrangement enables the neighboring kinase domains of the receptor chains to cross-phosphorylate each other on multiple tyrosines, a process referred to as autophosphorylation.

To activate a receptor tyrosine kinase the ligand usually has to bind simultaneously to two adjacent receptor chains. PDGF, for example, is a dimer, which cross-links two receptors together (Figure 15-50A). Even some monomeric ligands, such as EGF, bind to two receptors simultaneously and cross-link them directly. By contrast, FGFs, which are also monomers, first form multimers by binding to heparan sulfate proteoglycans, either on the target cell surface or in the extracellular matrix. In this way, they are able to cross-link adjacent receptors (Figure 15-50B). In contact-dependent signaling, the ligands form clusters in the plasma membrane of the signaling cell and can thereby cross-link the receptors on the target cell (Figure15-50C); thus, whereas membrane-bound ephrins activate Eph receptors, soluble ephrins will do so only if they are aggregated.

Figure 15-50. Three ways in which signaling proteins can cross-link receptor chains.

Figure 15-50

Three ways in which signaling proteins can cross-link receptor chains. When the receptor chains are cross-linked, the kinase domains of adjacent receptors cross-phosphorylate each other, stimulating the kinase activity of the receptor and creating docking (more...)

Because of the requirement for receptor oligomerization, it is relatively easy to inactivate a specific receptor tyrosine kinase to determine its importance for a cell response. For this purpose, cells are transfected with DNA encoding a mutant form of the receptor that oligomerizes normally but has an inactive kinase domain. When coexpressed at a high level with normal receptors, the mutant receptor acts in a dominant-negative way, disabling the normal receptors by forming inactive dimers with them (Figure 15-51).

Figure 15-51. Inhibition of signaling through normal receptor tyrosine kinases by an excess of mutant receptors.

Figure 15-51

Inhibition of signaling through normal receptor tyrosine kinases by an excess of mutant receptors. (A) In this example, the normal receptors dimerize in response to ligand binding. The two kinase domains cross-phosphorylate each other, increasing the (more...)

Autophosphorylation of the cytosolic tail of receptor tyrosine kinases contributes to the activation process in two ways. First, phosphorylation of tyrosines within the kinase domain increases the kinase activity of the enzyme. Second, phosphorylation of tyrosines outside the kinase domain creates high-affinity docking sites for the binding of a number of intracellular signaling proteins in the target cell. Each type of signaling protein binds to a different phosphorylated site on the activated receptor because it contains a specific phosphotyrosine-binding domain that recognizes surrounding features of the polypeptide chain in addition to the phosphotyrosine. Once bound to the activated kinase, the signaling protein may itself become phosphorylated on tyrosines and thereby activated; alternatively, the binding alone may be sufficient to activate the docked signaling protein. In summary, autophosphorylation serves as a switch to trigger the transient assembly of a large intracellular signaling complex, which then broadcasts signals along multiple routes to many destinations in the cell (Figure 15-52). Because different receptor tyrosine kinases bind different combinations of these signaling proteins, they activate different responses.

Figure 15-52. The docking of intracellular signaling proteins on an activated receptor tyrosine kinase.

Figure 15-52

The docking of intracellular signaling proteins on an activated receptor tyrosine kinase. The activated receptor and its bound signaling proteins form a signaling complex that can then broadcast signals along multiple signaling pathways.

The receptors for insulin and IGF-1 act in a slightly different way. They are tetramers to start with (see Figure 15-49), and ligand binding is thought to induce a rearrangement of the transmembrane receptor chains, so that the two kinase domains come close together. Most of the phosphotyrosine docking sites generated by ligand binding are not on the receptor itself, but on a specialized docking protein called insulin receptor substrate-1 (IRS-1). The activated receptor first autophosphorylates its kinase domains, which then phosphorylate IRS-1 on multiple tyrosines, thereby creating many more docking sites than could be accommodated on the receptor alone. Other docking proteins are used in a similar way by some other receptor tyrosine kinases to enlarge the size of the signaling complex.

Phosphorylated Tyrosines Serve as Docking Sites For Proteins With SH2 Domains

A whole menagerie of intracellular signaling proteins can bind to the phosphotyrosines on activated receptor tyrosine kinases (or on special docking proteins such as IRS-1) to help to relay the signal onward. Some docked proteins are enzymes, such as phospholipase C-γ (PLC-γ), which functions in the same way as phospholipase C-β—activating the inositol phospholipid signaling pathway discussed earlier in connection with G-protein-linked receptors. Through this pathway, receptor tyrosine kinases can increase cytosolic Ca2+ levels. Much more often, these receptors depend more on relay chains of protein-protein interactions. For example, another enzyme that docks on these receptors is the cytoplasmic tyrosine kinase Src, which phosphorylates other signaling proteins on tyrosines. Yet another is phosphatidylinositol 3′-kinase (PI 3-kinase), which, as we discuss later, generates specific lipid molecules in the plasma membrane to attract other signaling proteins there.

Although the intracellular signaling proteins that bind to phosphotyrosines on activated receptor tyrosine kinases and docking proteins have varied structures and functions, they usually share highly conserved phosphotyrosine-binding domains. These can be either SH2 domains (for Src homology region, because it was first found in the Src protein) or, less commonly, PTB domains (for phosphotyrosine-binding). By recognizing specific phosphorylated tyrosines, these small domains serve as modules that enable the proteins that contain them to bind to activated receptor tyrosine kinases, as well as to many other intracellular signaling proteins that have been transiently phosphorylated on tyrosines (Figure 15-53). Many signaling proteins also contain other protein modules that allow them to interact specifically with other proteins as part of the signaling process. These include the SH3 domain (again, so named because it was first discovered in Src), which binds to proline-rich motifs in intracellular proteins (see Figure 15-20).

Figure 15-53. The binding of SH2-containing intracellular signaling proteins to an activated PDGF receptor.

Figure 15-53

The binding of SH2-containing intracellular signaling proteins to an activated PDGF receptor. (A) This drawing of a PDGF receptor shows five of the tyrosine autophosphorylation sites, three in the kinase insert region and two on the C-terminal tail, to (more...)

Not all proteins that bind to activated receptor tyrosine kinases via SH2 domains help to relay the signal onward. Some act to decrease the signaling process, providing negative feedback. One example is the c-Cbl protein, which can dock on some activated receptors and catalyze their conjugation with ubiquitin. This ubiquitylation promotes the internalization and degradation of the receptors—a process called receptor down-regulation (see Figure 15-25).

Some signaling proteins are composed almost entirely of SH2 and SH3 domains and function as adaptors to couple tyrosine-phosphorylated proteins to other proteins that do not have their own SH2 domains (see Figure 15-20). Such adaptor proteins help to couple activated receptors to the important downstream signaling protein Ras. As we discuss next, Ras acts as a transducer and bifurcation signaling protein, changing the nature of the signal and broadcasting it along multiple downstream pathways, including a major signaling pathway that can help stimulate cells to proliferate or differentiate. Mutations that activate this pathway, and thereby stimulate cell division inappropriately, are a causative factor in many types of cancer.

Ras Is Activated by a Guanine Nucleotide Exchange Factor

The Ras proteins belong to the large Ras superfamily of monomeric GTPases. The family also contains two other subfamilies: the Rho family, involved in relaying signals from cell-surface receptors to the actin cytoskeleton and elsewhere (discussed in Chapter 16), and the Rab family, involved in regulating the traffic of intracellular transport vesicles (discussed in Chapter 13). Like almost all of these monomeric GTPases, the Ras proteins contain a covalently attached lipid group that helps to anchor the protein to a membrane—in this case, to the cytoplasmic face of the plasma membrane where the protein functions. There are multiple Ras proteins, and different ones act in different cell types. Because they all seem to work in much the same way, we shall refer to them simply as Ras.

Ras helps to broadcast signals from the cell surface to other parts of the cell. It is often required, for example, when receptor tyrosine kinases signal to the nucleus to stimulate cell proliferation or differentiation by altering gene expression. If Ras function is inhibited by the microinjection of neutralizing anti-Ras antibodies or a dominant-negative mutant form of Ras, the cell proliferation or differentiation responses normally induced by the activated receptor tyrosine kinases do not occur. Conversely, if a hyperactive mutant Ras protein is introduced into some cell lines, the effect on cell proliferation or differentiation is sometimes the same as that induced by the binding of ligands to cell-surface receptors. In fact, Ras was first discovered as the hyperactive product of a mutant ras gene that promoted the development of cancer; we now know that about 30% of human tumors have a hyperactive ras mutation.

Like other GTP-binding proteins, Ras functions as a switch, cycling between two distinct conformational states—active when GTP is bound and inactive when GDP is bound (see Figure 15-17). Two classes of signaling proteins regulate Ras activity by influencing its transition between active and inactive states. Guanine nucleotide exchange factors (GEFs) promote the exchange of bound nucleotide by stimulating the dissociation of GDP and the subsequent uptake of GTP from the cytosol, thereby activating Ras. GTPase-activating proteins (GAPs) increase the rate of hydrolysis of bound GTP by Ras, thereby inactivating Ras (Figure 15-54). Hyperactive mutant forms of Ras are resistant to GAP-mediated GTPase stimulation and are locked permanently in the GTP-bound active state, which is why they promote the development of cancer.

Figure 15-54. The regulation of Ras activity.

Figure 15-54

The regulation of Ras activity. GTPase-activating proteins (GAPs) inactivate Ras by stimulating it to hydrolyze its bound GTP; the inactivated Ras remains tightly bound to GDP. Guanine nucleotide exchange factors (GEFs) activate Ras by stimulating it (more...)

In principle, receptor tyrosine kinases could activate Ras either by activating a GEF or by inhibiting a GAP. Even though some GAPs bind directly (via their SH2 domains) to activated receptor tyrosine kinases (see Figure 15-53), whereas GEFs bind only indirectly, it is the indirect coupling of the receptor to a GEF that is responsible for driving Ras into its active state. In fact, the loss of function of a Ras-specific GEF has a similar effect to the loss of function of that Ras. The activation of the other Ras-like proteins, including those of the Rho family, is also thought to occur through the activation of GEFs.

Genetic studies in flies and worms, and biochemical studies in mammalian cells, indicate that adaptor proteins link receptor tyrosine kinases to Ras. The Grb-2 protein in mammalian cells, for example, binds through its SH2 domain to specific phosphotyrosines on activated receptor tyrosine kinases and through its SH3 domains to proline-rich motifs on a GEF called Sos. Some activated receptor tyrosine kinases, however, do not display the specific phosphotyrosines required for Grb-2 docking; these receptors recruit another adaptor protein called Shc, which binds both to the activated receptor and to Grb-2, thereby coupling the receptor to Sos by a more indirect route. The assembly of the complex of receptor-Grb-2-Sos (or receptor-Shc-Grb-2-Sos) brings Sos into position to activate neighboring Ras molecules by stimulating it to exchange its bound GDP for GTP (Figure 15-55). The importance of Grb-2 is indicated by the finding that Grb-2-deficient mice die early in embryogenesis. Very similar sets of proteins are thought to operate in all animals to activate Ras.

Figure 15-55. The activation of Ras by an activated receptor tyrosine kinase.

Figure 15-55

The activation of Ras by an activated receptor tyrosine kinase. Most of the signaling proteins bound to the activated receptor are omitted for simplicity. The Grb-2 adaptor protein binds to a specific phosphotyrosine on the receptor and to the Ras guanine (more...)

This pathway from receptor tyrosine kinases is not the only means of activating Ras. Other Ras GEFs are activated independently of Sos. One that is found mainly in the brain, for example, is activated by Ca2+ and diacylglycerol and can couple G-protein-linked receptors to Ras activation.

Once activated, Ras in turn activates various other signaling proteins to relay the signal downstream along several pathways. One of the signaling pathways Ras activates is a serine/threonine phosphorylation cascade that is highly conserved in eucaryotic cells from yeasts to humans. As we discuss next, a crucial component in this cascade is a novel type of protein kinase called MAP-kinase.

Ras Activates a Downstream Serine/Threonine Phosphorylation Cascade That Includes a MAP-Kinase

Both the tyrosine phosphorylations and the activation of Ras triggered by activated receptor tyrosine kinases are short-lived. Tyrosine-specific protein phosphatases (discussed later) quickly reverse the phosphorylations, and GAPs induce activated Ras to inactivate itself by hydrolyzing its bound GTP to GDP. To stimulate cells to proliferate or differentiate, these short-lived signaling events must be converted into longer-lasting ones that can sustain the signal and relay it downstream to the nucleus to alter the pattern of gene expression. Activated Ras triggers this conversion by initiating a series of downstream serine/threonine phosphorylations, which are much longer-lived than tyrosine phosphorylations. Many serine/threonine kinases participate in this phosphorylation cascade, but three of them constitute the core module of the cascade. The last of the three is called a mitogen-activated protein kinase (MAP-kinase).

An unusual feature of a MAP-kinase is that its full activation requires the phosphorylation of both a threonine and a tyrosine, which are separated in the protein by a single amino acid. The protein kinase that catalyzes both of these phosphorylations is called a MAP-kinase-kinase, which in the mammalian Ras signaling pathway is called MEK. The requirement for both a tyrosine and a threonine phosphorylation ensures that the MAP-kinase is kept inactive unless specifically activated by a MAP-kinase-kinase, whose only known substrate is a MAP-kinase. MAP-kinase-kinase is itself activated by phosphorylation catalyzed by the first kinase in the three-component module, MAP-kinase-kinase-kinase, which in the mammalian Ras signaling pathway is called Raf. The Raf kinase is activated by activated Ras.

Once activated, the MAP-kinase relays the signal downstream by phosphorylating various proteins in the cell, including gene regulatory proteins and other protein kinases (Figure 15-56). It enters the nucleus, for example, and phosphorylates one or more components of a gene regulatory complex. This activates the transcription of a set of immediate early genes, so named because they turn on within minutes of the time that cells are stimulated by an extracellular signal, even if protein synthesis is experimentally blocked with drugs. Some of these genes encode other gene regulatory proteins that turn on other genes, a process that requires both protein synthesis and more time. In this way the Ras-MAP-kinase signaling pathway conveys signals from the cell surface to the nucleus and alters the pattern of gene expression in significant ways. Among the genes activated by this pathway are those required for cell proliferation, such as the genes encoding G1 cyclins (discussed in Chapter 17).

Figure 15-56. The MAP-kinase serine/threonine phosphorylation pathway activated by Ras.

Figure 15-56

The MAP-kinase serine/threonine phosphorylation pathway activated by Ras. Multiple such pathways involving structurally and functionally related proteins operate in all eucaryotes, each coupling an extracellular stimulus to a variety of cell outputs. (more...)

MAP-kinases are usually activated only transiently in response to extracellular signals, and the period of time they remain active can profoundly influence the nature of the response. When EGF activates its receptors on a neural precursor cell line, for example, MAP-kinase activity peaks at 5 minutes and rapidly declines, and the cells later go on to divide. By contrast, when NGF activates its receptors on the same cells, MAP-kinase activity remains high for many hours, and the cells stop proliferating and differentiate into neurons.

MAP-kinases are inactivated by dephosphorylation, and the specific removal of phosphate from either the tyrosine or the threonine is enough to inactivate the enzyme. In some cases, stimulation by an extracellular signal induces the expression of a dual-specificity phosphatase that removes both phosphates and inactivates the kinase, providing a form of negative feedback. In other cases, stimulation causes the kinase to be switched off more rapidly by phosphatases that are already present.

Three-component MAP-kinase signaling modules operate in all animal cells, as well as in yeasts, with different ones mediating different responses in the same cell. In budding yeast, for example, one such module mediates the mating pheromone response via the βγ complex of a G protein, another the response to starvation, and yet another the response to osmotic shock. Some of these three-component MAP-kinase modules use one or more of the same kinases and yet manage to activate different effector proteins and hence different responses. How do cells avoid cross talk between the different parallel signaling pathways to ensure that each response is specific? One way is to use scaffold proteins that bind all or some of the kinases in a specific module to form a complex, as illustrated in Figure 15-57 and discussed earlier (see Figure 15-19A).

Figure 15-57. The organization of MAP-kinase pathways by scaffold proteins in budding yeast.

Figure 15-57

The organization of MAP-kinase pathways by scaffold proteins in budding yeast. Budding yeast have at least six three-component MAP-kinase modules involved in a variety of biological processes, including the two responses illustrated here—a mating (more...)

Mammalian cells also use this strategy to prevent cross talk between MAP-kinase signaling pathways. At least 5 parallel MAP-kinase modules can operate in a mammalian cell. These modules are composed of at least 12 MAP-kinases, 7 MAP-kinase-kinases, and 7 MAP-kinase-kinase-kinases. Several of these modules are activated by different kinds of cell stresses, such as UV irradiation, heat shock, osmotic stress, and stimulation by inflammatory cytokines. The three kinases in at least some of these stress-activated modules are held together by binding to a common scaffold protein, just as in yeast. The scaffold strategy provides precision, helps to create a large change in MAP-kinase activity in response to small changes in signal molecule concentration, and avoids cross-talk. However, it reduces the opportunities for amplification and spreading of the signal to different parts of the cell, which require at least some of the components to be diffusible (see Figure 15-16).

When Ras is activated by receptor tyrosine kinases, it usually activates more than just the MAP-kinase signaling pathway. It also usually helps activate PI3-kinase, which can signal cells to survive and grow.

PI 3-Kinase Produces Inositol Phospholipid Docking Sites in the Plasma Membrane

Extracellular signal proteins stimulate cells to divide, in part by activating the Ras-MAP-kinase pathway just discussed. If cells continually divided without growing, however, they would get progressively smaller and would eventually disappear. Thus, to proliferate, most cells need to be stimulated to enlarge (grow), as well as to divide. In some cases, one signal protein does both; in others one signal protein (a mitogen) mainly stimulates cell division, while another (a growth factor) mainly stimulates cell growth. One of the major intracellular signaling pathways leading to cell growth involves phosphatidylinositol 3-kinase (PI 3-kinase). This kinase principally phosphorylates inositol phospholipids rather than proteins; it can be activated by receptor tyrosine kinases, as well as by many other types of cell-surface receptors, including some that are G-protein-linked.

Phosphatidylinositol (PI) is unique among membrane lipids because it can undergo reversible phosphorylation at multiple sites to generate a variety of distinct inositol phospholipids. When activated, PI 3-kinase catalyzes the phosphorylation of inositol phospholipids at the 3 position of the inositol ring to generate lipids called PI(3,4)P2 or PI(3,4,5)P3 (Figure 15-58). The PI(3,4)P2 and PI(3,4,5)P3 then serve as docking sites for intracellular signaling proteins, bringing these proteins together into signaling complexes, which relay the signal into the cell from the cytosolic face of the plasma membrane.

Figure 15-58. The generation of inositol phospholipid docking sites by PI 3-kinase.

Figure 15-58

The generation of inositol phospholipid docking sites by PI 3-kinase. PI 3-kinase phosphorylates the inositol ring on carbon atom 3 to generate the inositol phospholipids shown at the bottom of the figure; the two lipids shown in red can serve as docking (more...)

It is important to distinguish this use of inositol phospholipids from their use we discussed earlier. We considered earlier how PI(4,5)P2 is cleaved by PLC-β (in the case of G-protein-linked receptors) or PLC-γ (in the case of receptor tyrosine kinases) to generate soluble IP3 and membrane-bound diacylglycerol. The IP3 releases Ca2+ from the ER, while the diacylglycerol activates PKC (see Figures 15-58 and 15-35). By contrast, PI(3,4)P2 and PI(3,4,5)P3 are not cleaved by PLC. They remain in the plasma membrane until they are dephosphorylated by specific inositol phospholipid phosphatases that remove phosphate from the 3 position of the inositol ring. Mutations that inactivate one such phosphatase (called PTEN), and thereby prolong signaling by PI 3-kinase, promote the development of cancer, and they are found in many human cancers. The mutations result in prolonged cell survival, indicating that signaling through PI 3-kinase normally promotes cell survival, as well as cell growth.

There are various types of PI 3-kinases. The one that is activated by receptor tyrosine kinases consists of a catalytic and regulatory subunit. The regulatory subunit is an adaptor protein that binds to phosphotyrosines on activated receptor tyrosine kinases through its SH2 domains (see Figure 15-53). Another PI 3-kinase has a different regulatory subunit and is activated by the βγ complex of a trimeric G protein when G-protein-linked receptors are activated by their extracellular ligand. The catalytic subunit, which is similar in both cases, also has a binding site for activated Ras, which allows Ras to directly stimulate PI 3-kinases.

Intracellular signaling proteins bind to the PI(3,4)P2 and PI(3,4,5)P3 that are produced by activated PI 3-kinase mainly through their Pleckstrin homology (PH) domain, first identified in the platelet protein Pleckstrin. PH domains are found in about 200 human proteins, including Sos (the GEF discussed earlier that activates Ras), and some atypical PKCs that do not depend on Ca2+ for their activation. The importance of these domains is illustrated dramatically by certain genetic immunodeficiency diseases in both humans and mice, where the PH domain in a cytoplasmic tyrosine kinase called BTK is inactivated by mutation. Normally, when antigen receptors on B lymphocytes (B cells) activate PI 3-kinase, the resulting inositol lipid docking sites recruit both BTK and PLC-γ to the cytoplasmic face of the plasma membrane. There, the two proteins interact: BTK phosphorylates and activates PLC-γ, which then cleaves PI(4,5)P2 to generate IP3 and diacylglycerol to relay the signal onward (Figure 15-59). Because the mutant BTK cannot bind to the lipid docking sites produced after receptor activation, the receptors cannot signal the B cells to proliferate or survive, resulting in a severe deficiency in antibody production.

Figure 15-59. The recruitment of signaling proteins with PH domains to the plasma membrane during B cell activation.

Figure 15-59

The recruitment of signaling proteins with PH domains to the plasma membrane during B cell activation. (A) PI 3-kinase binds to a phosphotyrosine on the activated B cell receptor complex and is thereby activated to phosphorylate the inositol phospholipid (more...)

The PI 3-Kinase/Protein Kinase B Signaling Pathway Can Stimulate Cells to Survive and Grow

One way in which PI 3-kinase signals cells to survive is by indirectly activating protein kinase B (PKB) (also called Akt). This kinase contains a PH domain, which directs it to the plasma membrane when PI 3-kinase is activated there by an extracellular survival signal. After binding to PI(3,4,5)P3 on the cytosolic face of the membrane, the PKB alters its conformation so that it can now be activated in a process that requires phosphorylation by a phosphatidylinositol-dependent protein kinase called PDK1, which is recruited to the membrane in the same way. Once activated, the PKB returns to the cytoplasm and phosphorylates a variety of target proteins. One of these, called BAD, is a protein that normally encourages cells to undergo programmed cell death, or apoptosis (mentioned earlier and discussed in detail in Chapter 17). By phosphorylating BAD, PKB inactivates it, thereby promoting cell survival (Figure 15-60). PKB also promotes cell survival by inhibiting other cell death activators, in some cases by inhibiting the transcription of the genes that encode them.

Figure 15-60. One way in which signaling through PI 3-kinase promotes cell survival.

Figure 15-60

One way in which signaling through PI 3-kinase promotes cell survival. An extracellular survival signal activates a receptor tyrosine kinase, which recruits and activates PI 3-kinase. The PI 3-kinase produces PI(3,4,5)P3 and PI(3,4)P2 (not shown), both (more...)

The pathways by which PI 3-kinase signals cells to grow (and increase their metabolism generally) are complex and still poorly understood. One way in which growth factors stimulate cell growth is by increasing the rate of protein synthesis through enhancing the efficiency with which ribosomes translate certain mRNAs into protein. A protein kinase called S6 kinase is part of one of the signaling pathways from PI 3-kinase to the ribosome. It phosphorylates and thereby activates the S6 subunit of ribosomes, which helps to increase the translation of a subset of mRNAs that encode ribosomal proteins and other components of the translational apparatus. The activation of S6 kinase is itself a complex process that depends on PDK1 and the phosphorylation of many sites on the protein. PDK1 may phosphorylate one of these sites in response to PI 3-kinase activation.

Figure 15-61 summarizes the five parallel intracellular signaling pathways we have discussed so far—one triggered by G-protein-linked receptors, two triggered by receptor tyrosine kinases, and two triggered by both kinds of receptors.

Figure 15-61. Five parallel intracellular signaling pathways activated by G-protein-linked receptors, receptor tyrosine kinases, or both.

Figure 15-61

Five parallel intracellular signaling pathways activated by G-protein-linked receptors, receptor tyrosine kinases, or both. In this schematic example, the five kinases (shaded yellow) at the end of each pathway phosphorylate target proteins (shaded red), (more...)

Tyrosine-Kinase-associated Receptors Depend on Cytoplasmic Tyrosine Kinases for Their Activity

Many cell-surface receptors depend on tyrosine phosphorylation for their activity and yet lack an obvious tyrosine kinase domain. These receptors act through cytoplasmic tyrosine kinases, which are associated with the receptors and phosphorylate various target proteins, often including the receptors themselves, when the receptors bind their ligand. The receptors thus function in much the same way as receptor tyrosine kinases, except that their kinase domain is encoded by a separate gene and is noncovalently associated with the receptor polypeptide chain. As with receptor tyrosine kinases, these receptors must oligomerize to function (Figure 15-62).

Figure 15-62. The three-dimensional structure of human growth hormone bound to its receptor.

Figure 15-62

The three-dimensional structure of human growth hormone bound to its receptor. The hormone (red) has cross-linked two identical receptors (one shown in green and the other in blue). Hormone binding activates cytoplasmic tyrosine kinases that are tightly (more...)

Many of these receptors depend on members of the largest family of mammalian cytoplasmic tyrosine kinases, the Src family of protein kinases (see Figure 3-68). This family includes the following members: Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk. These protein kinases all contain SH2 and SH3 domains and are located on the cytoplasmic side of the plasma membrane, held there partly by their interaction with transmembrane receptor proteins and partly by covalently attached lipid chains. Different family members are associated with different receptors and phosphorylate overlapping but distinct sets of target proteins. Lyn, Fyn, and Lck, for example, are each associated with different sets of receptors in lymphocytes. In each case the kinase is activated when an extracellular ligand binds to the appropriate receptor protein. Src itself, as well as several other family members, can also bind to activated receptor tyrosine kinases; in these cases, the receptor and cytoplasmic kinases mutually stimulate each other's catalytic activity, thereby strengthening and prolonging the signal.

Another type of cytoplasmic tyrosine kinase associates with integrins, the main family of receptors that cells use to bind to the extracellular matrix (discussed in Chapter 19). The binding of matrix components to integrins can activate intracellular signaling pathways that influence the behavior of the cell. When integrins cluster at sites of matrix contact, they help trigger the assembly of cell-matrix junctions called focal adhesions. Among the many proteins recruited into these junctions is the cytoplasmic tyrosine kinase called focal adhesion kinase (FAK), which binds to the cytosolic tail of one of the integrin subunits with the assistance of other cytoskeletal protein. The clustered FAK molecules cross-phosphorylate each other, creating phosphotyrosine docking sites where the Src kinase can bind. Src and FAK now phosphorylate each other and other proteins that assemble in the junction, including many of the signaling proteins used by receptor tyrosine kinases. In this way, the two kinases signal to the cell that it has adhered to a suitable substratum, where the cell can now survive, grow, divide, migrate, and so on. Mice deficient in FAK die early in development, and their cells do not migrate normally in a culture dish.

Cytokine receptors are the subfamily of enzyme-linked receptors that we discuss next. They constitute the largest and most diverse class of receptors that rely on cytoplasmic kinases to relay signals into the cell. They include receptors for many kinds of local mediators (collectively called cytokines), as well as receptors for some hormones, such as growth hormone (see Figure 15-62) and prolactin. As we discuss next, these receptors are stably associated with a class of cytoplasmic tyrosine kinases called Jaks, which activate latent gene regulatory proteins called STATs. The STAT proteins are normally inactive, being located at the cell surface; cytokine or hormone binding causes them to migrate to the nucleus and activate gene transcription.

Cytokine Receptors Activate the Jak-STAT Signaling Pathway, Providing a Fast Track to the Nucleus

Many intracellular signaling pathways lead from cell-surface receptors to the nucleus, where they alter gene transcription. The Jak-STAT signaling pathway, however, provides one of the most direct routes. It was initially discovered in studies on the effects of interferons, which are cytokines secreted by cells (especially white blood cells) in response to viral infection. Interferons bind to receptors on noninfected neighboring cells and induce the cells to produce proteins that increase their resistance to viral infection. When activated, interferon receptors activate a novel class of cytoplasmic tyrosine kinases called Janus kinases (Jaks) (after the two-faced Roman god). The Jaks then phosphorylate and activate a set of latent gene regulatory proteins called STATs (signal transducers and activators of transcription), which move into the nucleus and stimulate the transcription of specific genes. More than 30 cytokines and hormones activate the Jak-STAT pathway by binding to cytokine receptors, some of which are listed in Table 15-5.

Table 15-5. Some Signaling Proteins That Act Through Cytokine Receptors and the Jak-STAT Signaling Pathway.

Table 15-5

Some Signaling Proteins That Act Through Cytokine Receptors and the Jak-STAT Signaling Pathway.

All STATs also have an SH2 domain that enables them to dock onto specific phosphotyrosines on some activated receptor tyrosine kinase receptors. These receptors can directly activate the bound STAT, independently of Jaks. In fact, the nematode C. elegans uses STATs for signaling but does not make any Jaks or cytokine receptors, suggesting that STATs evolved before Jaks and cytokine receptors.

Cytokine receptors are composed of two or more polypeptide chains. Some cytokine receptor chains are specific to a particular cytokine receptor, while others are shared among several such receptors. All cytokine receptors, however, are associated with one or more Jaks. There are four known Jaks—Jak1, Jak2, Jak3, and Tyk2—and each is associated with particular cytokine receptors. The receptors for α-interferon, for example, are associated with Jak1 and Tyk2, whereas the receptors for γ-interferon are associated with Jak1 and Jak2 (see Table 15-5). As expected, mice that lack Jak1 do not respond to either of these interferons. The receptor for the hormone erythropoietin, which stimulates erythrocyte precursor cells to survive, proliferate, and differentiate, is associated with only Jak2. In Jak2-deficient mice, erythrocyte development fails, and the mice die early in development.

Cytokine binding either induces the receptor chains to oligomerize or reorients the chains in a preformed oligomer. In either case, the binding brings the associated Jaks close enough together for them to cross-phosphorylate each other, thereby increasing the activity of their tyrosine kinase domains. The Jaks then phosphorylate tyrosines on the cytokine receptors, creating phosphotyrosine docking sites for STATs and other signaling proteins.

There are seven known STATs, each with an SH2 domain that performs two functions. First, it mediates the binding of the STAT protein to a phosphotyrosine docking site on an activated cytokine receptor (or receptor tyrosine kinase); once bound, the Jaks phosphorylate the STAT on tyrosines, causing it to dissociate from the receptor. Second, the SH2 domain on the released STAT now mediates its binding to a phosphotyrosine on another STAT molecule, forming either a STAT homodimer or heterodimer. The STAT dimer then moves into the nucleus, where, in combination with other gene regulatory proteins, it binds to a specific DNA response element in various genes and stimulates their transcription (Figure 15-63). In response to the hormone prolactin, for example, which stimulates breast cells to produce milk, activated STAT5 stimulates the transcription of genes that encode milk proteins.

Figure 15-63. The Jak-STAT signaling pathway activated by α-interferon.

Figure 15-63

The Jak-STAT signaling pathway activated by α-interferon. The binding of interferon either causes two separate receptor polypeptide chains to dimerize (as shown) or reorients the receptor chains in a preformed dimer. In either case, the associated (more...)

Cytokine receptors activate the appropriate STAT proteins because the SH2 domain of these STATs recognizes only the specific phosphotyrosine docking sites on these receptors. Activated receptors for α-interferon, for example, recruit both STAT1 and STAT2, whereas activated receptors for γ-interferon recruit only STAT1. If the SH2 domain of the α-interferon receptor is replaced with the SH2 domain of the γ-interferon receptor, the activated hybrid receptor recruits both STAT1 and STAT2, just like the α-interferon receptor itself.

The responses mediated by STATs are often regulated by negative feedback. In addition to activating genes that encode proteins mediating the cytokine-induced response, the STAT dimers may also activate genes that encode inhibitory proteins. In some cases, the inhibitor binds to both the activated cytokine receptors and STAT proteins, which blocks further STAT activation and helps to shut off the response; in other cases, the inhibitor achieves the same result by blocking Jak function.

Such negative feedback mechanisms, however, are not enough on their own to turn off the response. The activated Jaks and STATs also have to be inactivated by dephosphorylation of their phosphotyrosines. As in all signaling pathways that use tyrosine phosphorylation, the dephosphorylation is performed by protein tyrosine phosphatases, which are as important in the signaling process as the protein tyrosine kinases that add the phosphates.

Some Protein Tyrosine Phosphatases May Act as Cell-Surface Receptors

As discussed earlier, only a small number of serine/threonine phosphatase catalytic subunits are responsible for removing phosphate groups from phosphorylated serines and threonines on proteins. By contrast, there are about 30 protein tyrosine phosphatases (PTPs) encoded in the human genome. Like tyrosine kinases, they occur in both cytoplasmic and transmembrane forms, none of which are structurally related to serine/threonine protein phosphatases. Individual protein tyrosine phosphatases display exquisite specificity for their substrates, removing phosphate groups from only selected phosphotyrosines on a subset of tyrosine-phosphorylated proteins. Together, these phosphatases ensure that tyrosine phosphorylations are short-lived and that the level of tyrosine phosphorylation in resting cells is very low. They do not, however, simply continuously reverse the effects of protein tyrosine kinases; they are regulated to act only at the appropriate time in a signaling response or in the cell-division cycle (discussed in Chapter 17).

Two cytoplasmic tyrosine phosphatases in vertebrates have SH2 domains and are therefore called SHP-1 and SHP-2 (Figure 15-64). SHP-1 helps to terminate some cytokine responses in blood cells by dephosphorylating activated Jaks: mutant erythropoietin receptors that cannot recruit SHP-1, for example, activate Jak2 for much longer than normal. Moreover, SHP-1-deficient mice have abnormalities in almost all blood cell lineages, emphasizing the importance of SHP-1 in blood cell development. Both SHP-1 and SHP-2 also help terminate responses mediated by some receptor tyrosine kinases.

Figure 15-64. Some protein tyrosine phosphatases.

Figure 15-64

Some protein tyrosine phosphatases. The cytoplasmic tyrosine phosphatases SHP-1 and SHP-2 have similar structures, with two SH2 domains. The three transmembrane receptorlike tyrosine phosphatases have two tandemly arranged intracellular phosphatase domains, (more...)

There are a large number of transmembrane protein tyrosine phosphatases, but the functions of most of them are unknown. At least some are thought to function as receptors; as this has not been directly demonstrated, however, they are referred to as receptorlike tyrosine phosphatases. They all have a single transmembrane segment and usually possess two tyrosine phosphatase domains on the cytosolic side of the plasma membrane. An important example is the CD45 protein (see Figure 15-64), which is found on the surface of all white blood cells and has an essential role in the activation of both T and B lymphocytes by foreign antigens. The ligand that is presumed to bind to the extracellular domain of the CD45 protein has not been identified. However, the role of CD45 in signal transduction has been studied by using recombinant DNA techniques to construct a hybrid protein with an extracellular EGF-binding domain and intracellular CD45 tyrosine phosphatase domains. The surprising result is that EGF binding seems to inactivate the phosphatase activity of the hybrid protein rather than activating it.

This finding raises the possibility that some receptor tyrosine kinases and receptor tyrosine phosphatases may collaborate when they bind their respective cell-surface-bound ligands—with the kinases adding more phosphates and the phosphatase removing fewer—to maximally stimulate the tyrosine phosphorylation of selected intracellular signaling proteins. The significance of ligand-induced inhibition of CD45 phosphatase is still uncertain, however, and it seems unlikely to be the whole story; CD45 requires its phosphatase activity to function in lymphocyte activation.

Some receptorlike tyrosine phosphatases display features of cell-adhesion proteins and can even mediate homophilic cell-cell binding in cell adhesion assays (see Figure 19-26). In the developing nervous system, for example, they may have an important role in guiding the growing tips of developing nerve cell axons to their targets. In Drosophila, the genes encoding several receptorlike tyrosine phosphatases are expressed exclusively in the nervous system, and when some of them are inactivated by mutation, the axons of certain developing neurons fail to find their way to their normal targets. In some cases at least, the phosphatase activity of the protein is required to counteract the action of a cytoplasmic tyrosine kinase for normal axon guidance.

Transmembrane tyrosine phosphatases can also serve as signaling ligands that activate receptors on a neighboring cell. An example is the protein tyrosine phosphatase ζ/β (see Figure 15-64), which is expressed on the surface of certain glial cells in the mammalian brain. It binds to a receptor protein (called contactin) on developing nerve cells, stimulating the cells to extend long processes. It is possible that the phosphatase also conveys a signal to the glial cell in this interaction, but such bidirectional signaling has not been directly demonstrated for transmembrane tyrosine phosphatases.

Having discussed the crucial role of tyrosine phosphorylation and dephosphorylation in the intracellular signaling pathways activated by many enzyme-linked receptors, we now turn to a class of enzyme-linked receptors that rely entirely on serine/threonine phosphorylation. These transmembrane serine/ threonine kinases activate an even more direct signaling pathway to the nucleus than does the Jak-STAT pathway discussed earlier. They directly phosphorylate latent gene regulatory proteins called Smads, which then migrate into the nucleus to activate gene transcription.

Signal Proteins of the TGF-β Superfamily Act Through Receptor Serine/Threonine Kinases and Smads

The transforming growth factor-β (TGF-β) superfamily consists of a large number of structurally related, secreted, dimeric proteins. They act either as hormones or, more commonly, as local mediators to regulate a wide range of biological functions in all animals. During development, they regulate pattern formation and influence various cell behaviors, including proliferation, differentiation, extracellular matrix production, and cell death. In adults, they are involved in tissue repair and in immune regulation, as well as in many other processes. The superfamily includes the TGF-βs themselves, the activins, and the bone morphogenetic proteins (BMPs). The BMPs constitute the largest family.

All of these proteins act through enzyme-linked receptors that are single-pass transmembrane proteins with a serine/threonine kinase domain on the cytosolic side of the plasma membrane. There are two classes of these receptor serine/threonine kinasestype I and type II—which are structurally similar. Each member of the TGF-β superfamily binds to a characteristic combination of type-I and type-II receptors, both of which are required for signaling. Typically, the ligand first binds to and activates a type-II receptor homodimer, which recruits, phosphorylates, and activates a type-I receptor homodimer, forming an active tetrameric receptor complex.

Once activated, the receptor complex uses a strategy for rapidly relaying the signal to the nucleus that is very similar to the Jak-STAT strategy used by cytokine receptors. The route to the nucleus, however, is even more direct. The type-I receptor directly binds and phosphorylates a latent gene regulatory protein of the Smad family (named after the first two identified, Sma in C. elegans and Mad in Drosophila). Activated TGF-β receptors and activin receptors phosphorylate Smad2 or Smad3, while activated BMP receptors phosphorylate Smad1, Smad5, or Smad8. Once one of these Smads has been phosphorylated, it dissociates from the receptor and binds to Smad4, which can form a complex with any of the above five receptor-activated Smads. The Smad complex then moves into the nucleus, where it associates with other gene regulatory proteins, binds to specific sites in DNA, and activates a particular set of target genes (Figure 15-65).

Figure 15-65. A model for the Smad-dependent signaling pathway activated by TGF-β.

Figure 15-65

A model for the Smad-dependent signaling pathway activated by TGF-β. Note that TGF-β is a dimer and that Smads open up to expose a dimerization surface when they are phosphorylated. Several features of the pathway have been omitted for (more...)

Some TGF-β family members serve as graded morphogens during development, inducing different responses in a developing cell depending on their concentration (discussed in Chapter 21). The different responses can be reproduced by experimentally altering the amount of active Smad complexes in the nucleus, suggesting that the level of these complexes may provide a direct readout of the level of receptor activation. If the DNA-binding sites in different target genes have different affinities for the complexes, then the particular genes activated would reflect the cell's position in the concentration gradient of the morphogen.

As with the Jak-STAT pathway, the Smad pathway is also often regulated by feedback inhibition. Among the target genes activated by Smad complexes are those that encode inhibitory Smads, including Smad6 and Smad7. These Smads act as decoys. They bind to activated type-I receptors and prevent other Smads from binding there. This blocks the formation of active Smad complexes and shuts off the response to the TGF-β family ligand. Other types of extracellular ligands can also stimulate the production of inhibitory Smads to antagonize signaling by a TGF-β ligand; γ-interferon, for example, activates the Jak-STAT pathway, and the resulting activated STAT dimers induce the production of Smad7, which inhibits signaling by TGF-β.

In addition to these intracellular inhibitors, a number of secreted extracellular inhibitory proteins can also neutralize signaling mediated by TGF-β family members. They directly bind to the signal molecules and prevent them from activating their receptors on target cells. Noggin and chordin, for example, inhibit BMPs, and follistatin inhibits activins. Noggin and chordin help to induce the development of the vertebrate nervous system by preventing BMPs from inhibiting this development (discussed in Chapter 21). The TGF-β family members, as well as some of their inhibitors, are usually secreted as inactive precursors that are subsequently activated by proteolytic cleavage.

We turn now to enzyme-linked receptors that are neither kinases nor associated with kinases. We saw earlier that nitric oxide is widely used as a signaling molecule, diffusing through the plasma membrane of a target cell and stimulating a cytoplasmic guanylyl cyclase to produce the intracellular mediator cyclic GMP. The receptors we now consider are transmembrane proteins with guanylyl cyclase activity.

Receptor Guanylyl Cyclases Generate Cyclic GMP Directly

Receptor guanylyl cyclases are single-pass transmembrane proteins with an extracellular binding site for a signal molecule and an intracellular guanylyl cyclase catalytic domain. The binding of the signal molecule activates the cyclase domain to produce cyclic GMP, which in turn binds to and activates a cyclic GMP-dependent protein kinase (PKG), which phosphorylates specific proteins on serine or threonine. Thus, receptor guanylyl cyclases use cyclic GMP as an intracellular mediator in the same way that some G-protein-linked receptors use cyclic AMP, except that the linkage between ligand binding and cyclase activity is a direct one.

Among the signal molecules that use receptor guanylyl cyclase receptors are the natriuretic peptides (NPs), a family of structurally related secreted signal peptides that regulate salt and water balance and dilate blood vessels. There are several types of NPs, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Muscle cells in the atrium of the heart secrete ANP when blood pressure rises. The ANP stimulates the kidneys to secrete Na+ and water and induces the smooth muscle cells in blood vessels walls to relax. Both of these effects tend to lower the blood pressure. When gene targeting is used to inactivate the ANP receptor guanylyl cyclase in mice, the mice have chronically elevated blood pressure, resulting in progressive heart enlargement.

An increasing number of receptor guanylyl cyclases are being discovered, but in most cases they are orphan receptors, where the ligand that normally activates them is unknown. The genome of the nematode C. elegans, for example, encodes 26 of these receptors. Most of those that have been studied are expressed in specific subsets of sensory neurons, suggesting that they may be involved in detecting particular molecules in the worm's environment. Some of the orphan receptors in mammals are found in sensory neurons in the part of the nose involved in detecting pheromones.

All the signaling pathways activated by G-protein-linked and enzyme-linked receptors we have discussed so far depend on serine/threonine-specific protein kinases, tyrosine-specific protein kinases, or both. These kinases are all structurally related, as reviewed in Figure 15-66. Some enzyme-linked receptors, however, depend on an entirely unrelated type of protein kinase, as we now discuss.

Figure 15-66. Some of the protein kinases discussed in this chapter.

Figure 15-66

Some of the protein kinases discussed in this chapter. The size and location of their catalytic domains (dark green) are shown. In each case the catalytic domain is about 250 amino acids long. These domains are all similar in amino acid sequence, suggesting (more...)

Bacterial Chemotaxis Depends on a Two-Component Signaling Pathway Activated by Histidine-Kinase-associated Receptors

As pointed out earlier, many of the mechanisms involved in chemical signaling between cells in multicellular animals are thought to have evolved from mechanisms used by unicellular organisms to respond to chemical changes in their environment. In fact, some of the same intracellular mediators, such as cyclic nucleotides and Ca2+, are used by both types of organisms. Among the best-studied reactions of unicellular organisms to extracellular signals are their chemotactic responses, in which cell movement is oriented toward or away from a source of some chemical in the environment. We conclude this section on enzyme-linked receptors with a brief account of bacterial chemotaxis, which depends on a two-component signaling pathway, involving histidine-kinase-associated receptors. The same type of signaling pathway is used by yeasts and plants, although apparently not by animals.

Motile bacteria will swim toward higher concentrations of nutrients (attractants), such as sugars, amino acids, and small peptides, and away from higher concentrations of various noxious chemicals (repellents). They swim by means of flagella, each of which is attached by a short, flexible hook at its base to a small protein disc embedded in the bacterial membrane. This disc is part of a tiny motor that uses the energy stored in the transmembrane H+ gradient to rotate rapidly and turn the helical flagellum (Figure 15-67). Because the flagella on the bacterial surface have an intrinsic “handedness,” different directions of rotation have different effects on movement. Counterclockwise rotation allows all the flagella to draw together into a coherent bundle, so that the bacterium swims uniformly in one direction. Clockwise rotation causes them to fly apart, so that the bacterium tumbles chaotically without moving forward (Figure 15-68). In the absence of any environmental stimulus, the direction of rotation of the disc reverses every few seconds, producing a characteristic pattern of movement in which smooth swimming in a straight line is interrupted by abrupt, random changes in direction caused by tumbling.

Figure 15-67. The bacterial flagellar motor.

Figure 15-67

The bacterial flagellar motor. The flagellum is linked to a flexible hook. The hook is attached to a series of protein rings (shown in red), which are embedded in the outer and inner (plasma) membranes. The rings form a rotor, which rotates with the flagellum (more...)

Figure 15-68. Positions of the flagella on E. coli during swimming.

Figure 15-68

Positions of the flagella on E. coli during swimming. (A) When the flagella rotate counterclockwise, they are drawn together into a single bundle, which acts as a propeller to produce smooth swimming. (B) When the flagella rotate clockwise, they fly apart (more...)

The normal swimming behavior of bacteria is modified by chemotactic attractants or repellents, which bind to specific receptor proteins and affect the frequency of tumbling by increasing or decreasing the time that elapses between successive changes in direction of flagellar rotation. When bacteria are swimming in a favorable direction (toward a higher concentration of an attractant or away from a higher concentration of a repellent), they tumble less frequently than when they are swimming in an unfavorable direction (or when no gradient is present). Since the periods of smooth swimming are longer when a bacterium is traveling in a favorable direction, it will gradually progress in that direction—toward an attractant or away from a repellent.

These responses are mediated by histidine-kinase-associated chemotaxis receptors, which typically are dimeric transmembrane proteins that bind specific attractants and repellents on the outside of the plasma membrane. The cytoplasmic tails of the receptors are stably associated with an adaptor protein CheW and a histidine kinase CheA, which help to couple the receptors to the flagellar motor. Repellent binding activates the receptors, whereas attractant binding inactivates them; a single receptor can bind either type of molecule, with opposite consequences. The binding of a repellent to the receptor activates CheA, which phosphorylates itself on a histidine and almost immediately transfers the phosphate to an aspartic acid on a messenger protein CheY. The phosphorylated CheY dissociates from the receptor, diffuses through the cytosol, binds to the flagellar motor, and causes the motor to rotate clockwise, so that the bacterium tumbles. CheY has intrinsic phosphatase activity and dephosphorylates itself in a process that is greatly accelerated by the CheZ protein (Figure 15-69).

Figure 15-69. The two-component signaling pathway that enables chemotaxis receptors to control the flagellar motor during bacterial chemotaxis.

Figure 15-69

The two-component signaling pathway that enables chemotaxis receptors to control the flagellar motor during bacterial chemotaxis. The histidine kinase CheA is stably bound to the receptor via the adaptor protein CheW. The binding of a repellent increases (more...)

The response to an increase in the concentration of an attractant or repellent is only transient, even if the higher level of ligand is maintained, as the bacteria desensitize, or adapt, to the increased stimulus. Whereas the initial effect on tumbling occurs in less than a second, adaptation takes minutes. The adaptation is a crucial part of the response, as it enables the bacteria to respond to changes in concentration of ligand rather than to steady-state levels. It is mediated by the covalent methylation (catalyzed by a methyl transferase) and demethylation (catalyzed by a methylase) of the chemotaxis receptors, which change their responsiveness to ligand binding when methylated.

All of the genes and proteins involved in this highly adaptive behavior have now been identified. It therefore seems likely that bacterial chemotaxis will be the first signaling system to be completely understood in molecular terms. Even in this relatively simple signaling network, computer-based simulations are required to comprehend how the system works as an integrated network. Cell signaling pathways will provide an especially rich area of investigation for a new generation of computational biologists, as their network properties will not be understandable without powerful computational tools.

There are some cell-surface receptor proteins that do not fit into the three major classes we have discussed thus far—ion-channel-linked, G-protein-linked, and enzyme-linked. In the next section, we consider cell-surface receptors that activate signaling pathways that depend on proteolysis. These pathways have especially important roles in animal development.

Summary

There are five known classes of enzyme-linked receptors: (1) receptor tyrosine kinases, (2) tyrosine-kinase-associated receptors, (3) receptor serine/threonine kinases, (4) transmembrane guanylyl cyclases, and (5) histidine-kinase-associated receptors. In addition, some transmembrane tyrosine phosphatases, which remove phosphate from phosphotyrosine side chains of specific proteins, are thought to function as receptors, although for the most part their ligands are unknown. The first two classes of receptors are by far the most numerous.

Ligand binding to receptor tyrosine kinases induces the receptors to cross-phosphorylate their cytoplasmic domains on multiple tyrosines. The autophosphorylation activates the kinases, as well as producing a set of phosphotyrosines that then serve as docking sites for a set of intracellular signaling proteins, which bind via their SH2 (or PTB) domains. Some of the docked proteins serve as adaptors to couple the receptors to the small GTPase Ras, which, in turn, activates a cascade of serine/threonine phosphorylations that converge on a MAP-kinase, which relays the signal to the nucleus by phosphorylating gene regulatory proteins there. Ras can also activate another protein that docks on activated receptor tyrosine kinases—PI 3-kinase—which generates specific inositol phospholipids that serve as docking sites in the plasma membrane for signaling proteins with PH domains, including protein kinase B (PKB).

Tyrosine-kinase-associated receptors depend on various cytoplasmic tyrosine kinases for their action. These kinases include members of the Src family, which associate with many kinds of receptors, and the focal adhesion kinase (FAK), which associates with integrins at focal adhesions. The cytoplasmic tyrosine kinases then phosphorylate a variety of signaling proteins to relay the signal onward. The largest family of receptors in this class is the cytokine receptors family. When stimulated by ligand binding, these receptors activate Jak cytoplasmic tyrosine kinases, which phosphorylate STATs. The STATs then dimerize, migrate to the nucleus, and activate the transcription of specific genes. Receptor serine/threonine kinases, which are activated by signaling proteins of the TGF-β superfamily, act similarly: they directly phosphorylate and activate Smads, which then oligomerize with another Smad, migrate to the nucleus, and activate gene transcription.

Bacterial chemotaxis is mediated by histidine-kinase-associated chemotaxis receptors. When activated by the binding of a repellent, the receptors stimulate their associated protein kinase to phosphorylate itself on histidine and then transfer that phosphate to a messenger protein, which relays the signal to the flagellar motor to alter the bacterium's swimming behavior. Attractants have the opposite effect on this kinase and therefore on swimming.

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 .
Bookshelf ID: NBK26822

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